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Single-cell RNA sequencing (scRNA-seq) data has been widely used for cell trajectory inference, with the assumption that cells with similar expression profiles share the same differentiation state. However, the inferred trajectory may not reveal clonal differentiation heterogeneity among T cell clones. Single-cell T cell receptor sequencing (scTCR-seq) data provides invaluable insights into the clonal relationship among cells, yet it lacks functional characteristics. Therefore, scRNA-seq and scTCR-seq data complement each other in improving trajectory inference, where a reliable computational tool is still missing. We developed LRT, a computational framework for the integrative analysis of scTCR-seq and scRNA-seq data to explore clonal differentiation trajectory heterogeneity. Specifically, LRT uses the transcriptomics information from scRNA-seq data to construct overall cell trajectories and then utilizes both the TCR sequence information and phenotype information to identify clonotype clusters with distinct differentiation biasedness. LRT provides a comprehensive analysis workflow, including preprocessing, cell trajectory inference, clonotype clustering, trajectory biasedness evaluation, and clonotype cluster characterization. We illustrated its utility using scRNA-seq and scTCR-seq data of CD8+ T cells and CD4+ T cells with acute lymphocytic choriomeningitis virus infection. These analyses identified several clonotype clusters with distinct skewed distribution along the differentiation path, which cannot be revealed solely based on scRNA-seq data. Clones from different clonotype clusters exhibited diverse expansion capability, V-J gene usage pattern and CDR3 motifs. The LRT framework was implemented as an R package 'LRT', and it is now publicly accessible at https://github.com/JuanXie19/LRT. In addition, it provides two Shiny apps 'shinyClone' and 'shinyClust' that allow users to interactively explore distributions of clonotypes, conduct repertoire analysis, implement clustering of clonotypes, trajectory biasedness evaluation, and clonotype cluster characterization. Author summary: Understanding the dynamic changes behind biological processes is important for determining molecular mechanisms underlying normal tissue formulation, developmental disorders and pathologies. Usually, a biological process can be characterized by identifying a trajectory, a path that goes through the various cellular states associated with the process. Since cells in different states may express different sets of genes, researchers often infer cell trajectory via capturing transcriptomics changes. Dozens of methods have been developed for cell trajectory inference, and scRNA-seq data is predominantly utilized. However, cells from different clones may exist distinct differentiation patterns, while methods based only on scRNA-seq data cannot capture this heterogeneity. T cells play a key role in the immune system, and their high antigen recognition specificity is largely determined by their TCR sequences. Thanks to the advent of scTCR-seq technology, we can now identify the group of cells coming from the same clone. This paper describes our novel computational framework, namely LRT, and demonstrates that by complementing scRNA-seq data with the clonal information from scTCR-seq data using LRT, we are able to examine the clonal differentiation heterogeneity that cannot be revealed solely based on scRNA-seq data. [ABSTRACT FROM AUTHOR]

The AIDS is a chronic disease and the researchers still exert their high efforts to reach the cure of HIV infection. The most common treatment is the antiretroviral therapy (cART) and the virus can be more effected if the patients stop using cART. The other problem is that the CD8+ T cells might be exhausted by persistent immune activation by cART. This paper introduces fractional-order into a mathematical model of HIV infection combining with stem cell therapy and control the infection by the immune system cells (CD8+ T cells). The paper introduced the numerical solutions for the mathematical model. The results show that the stem cell therapy with the activation of immune system cells might causes the cure for a HIV patient. This results are consistent with medical studies. Also, we proposed the effect of the fractional order (α) on the figures of the components. [ABSTRACT FROM AUTHOR]

T cells recognize their ligand, the peptide major histocompatibility complex (MHC), via the T-cell receptor (TCR), which is composed of covalently linked α and β or γ and δ chains. This recognition is critical for T-cell ontogeny and controls the selection, activation, and function of T lymphocytes. Specific TCR αβ variable regions have been associated with immunopathogenesis of Chagas disease. Here, we present a systematic review that compiles experimental in vivo and human data regarding the preferential expression of variable alpha (Vα) and variable beta (Vβ) chain regions in Trypanosoma cruzi infection. The original studies indexed in PubMed/Medline, Scopus, and Web of Science databases were screened according to the PRISMA strategy. The analysis showed that expression of TCR Vα subfamilies were evaluated in one human study, and, unlike TCR Vβ, TCR Vα presented a more restricted usage. Despite the great variability in the usage of TCR Vβ regions in human Chagas disease, a down-regulation of TCR Vβ5 expression by T cells from patients in the acute phase of the disease was shown. Opposingly, this TCR region was found overly expressed in CD4+ T cells from chronic Chagas patients. It was also demonstrated that murine Vβ9+ T cells derived from nonlymphoid organs of T. cruzi-infected animals had a modulatory profile, while splenic Vβ9+ T cells produced inflammatory cytokines, indicating that although they display the same TCR Vβ region usage, these cells are functionally distinct. Despite the limitations of few papers and year of publication of the studies, compiling the data derived from them reveals that further investigation of TCR usage will point to their potential role in protective or pathogenic responses, as biomarkers of disease progression, and in the search for dominant peptides potentially useful for the development of vaccines or therapies. Author summary: Chagas disease is a neglected tropical disease, caused by infection with Trypanosoma cruzi. Differential expression of certain T-cell receptor (TCR) variable regions has been associated with the immunopathogenesis of Chagas disease. Here, we present a systematic review that compiled experimental in vivo and human data regarding the preferential expression of TCR alpha and beta chain variable regions in Chagas disease. The original studies indexed in the PubMed/Medline, Scopus, and Web of Science databases were screened according to the PRISMA strategy. Despite the great variability in the use of TCR Vβ in T. cruzi infection, the outcomes indicate that there is a down-regulation of TCR Vβ5 expression in T cells from patients in the acute phase of Chagas disease. However, this region is preferentially expressed by CD4+ T cells from chronic Chagas patients. Additionally, it has been demonstrated that murine Vβ9+ T cells derived from nonlymphoid organs displayed a modulatory profile, while splenic Vβ9+ T cells produced inflammatory cytokines, indicating that although they express the same TCR Vβ region, these cells are functionally distinct. Information on TCR expression, specificity and function have critical impact on vaccine design. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) targets immune cells and tries to inactivate dendritic cells and interferon molecules to continue its replication process. Since EBOV detailed mechanism has not been identified so far, it would be useful to understand the growth and spread of EBOV dynamics based on mathematical methods and simulation approaches. Computational approaches such as Cellular Automata (CA) have the advantage of simplicity over solving complicated differential equations. The spread of Ebola virus in lymph nodes is studied using a simplified Cellular Automata model with only four parameters. In addition to considering healthy and infected cells, this paper also considers T lymphocytes as well as cell movement ability during the simulation in order to investigate different scenarios in the dynamics of an EBOV system. It is shown that the value of the probability of death of T cells affects the number of infected cells significantly in the steady-state. For a special case of parameters set, the system shows oscillating dynamics. The results were in good agreement with an ordinary differential equation-based model which indicated CA method in combination with experimental discoveries could help biologists find out more about the EBOV mechanism and hopefully to control the disease. [ABSTRACT FROM AUTHOR]

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH. Author summary: People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes. [ABSTRACT FROM AUTHOR]

In this essay, we show that 3 distinct approaches to immunological exhaustion coexist and that they only partially overlap, generating potential misunderstandings. Exploring cases ranging from viral infections to cancer, we propose that it is crucial, for experimental and therapeutic purposes, to clarify these approaches and their interconnections so as to make the concept of exhaustion genuinely operational. Author summary: In this essay, we have written a critical review on immunological exhaustion. We believe that this widely used concept often remains in fact imprecise because there exist 3 different approaches to exhaustion, namely, in terms of dysfunction, cause, and marker, and those are not sufficiently well distinguished and articulated in most scientific papers. We also propose to talk about "exhaustion" for and only for the phenomena in which all 3 approaches are aligned. This can be called the "convergence strategy": T cells would be described as "exhausted" if and only if they are simultaneously dysfunctional, express given markers such as programmed cell death protein 1 (PD-1), and induced by a specific cause such as chronicity. This strategy could be perfectly reasonable, and we see much value in adopting it, because it would force everyone to be more specific and rigorous when talking about exhaustion. Clarifying the characteristics of cells defined as "exhausted," and using a convergent strategy to define T-cell exhaustion could have major experimental and clinical consequences, including for viral infections such as the Coronavirus Disease 2019 (COVID-19). [ABSTRACT FROM AUTHOR]

Manipulation of immune cell functions, independently of direct infection of these cells, emerges as a key process in viral pathophysiology. Chronic infection by Human T-cell Leukemia Virus type 1 (HTLV-1) is associated with immune dysfunctions, including misdirected responses of dendritic cells (DCs). Here, we interrogate the ability of transformed HTLV-1-infected T cells to manipulate human DC functions. We show that exposure to transformed HTLV-1-infected T cells induces a biased and peculiar transcriptional signature in monocyte-derived DCs, associated with an inefficient maturation and a poor responsiveness to subsequent stimulation by a TLR4 agonist. This poor responsiveness is also associated with a unique transcriptional landscape characterized by a set of genes whose expression is either conferred, impaired or abolished by HTLV-1 pre-exposure. Induction of this functional impairment requires several hours of coculture with transformed HTLV-1-infected cells, and associated mechanisms driven by viral capture, cell-cell contacts, and soluble mediators. Altogether, this cross-talk between infected T cells and DCs illustrate how HTLV-1 might co-opt communications between cells to induce a unique local tolerogenic immune microenvironment suitable for its own persistence. Author summary: Chronic viral infection is associated with an escape from immune surveillance. This may rely on the induction of inappropriate DC responses, which can contribute to immunopathology. Immune dysfunctions have been repeatedly reported in people living with Human T-cell Leukemia Virus type 1 (HTLV-1), years before fatal clinical symptom onset, including misdirected responses of dendritic cells (DCs). Here, we report that HTLV-1-infected T cells actively manipulate neighboring, uninfected MDDC functions by rewiring their transcriptional response, leading to a biased, pro-tolerogenic responsiveness in MDDCs, induced by the bidirectional release of soluble mediators, in cooperation with mechanisms dependent on cell-cell contacts. This cross-talk illustrate how HTLV-1 might co-opt communications between cells to induce a local tolerogenic immune microenvironment suitable for its own persistence. [ABSTRACT FROM AUTHOR]

The primate-specific chemokine CCL18 is a potent chemoattractant for T cells and is expressed at elevated levels in several inflammatory diseases. However, the cognate receptor for CCL18 remains unconfirmed. Here, we describe attempts to validate a previous report that the chemokine receptor CCR8 is the human CCL18 receptor (Islam et al. J Exp Med. 2013, 210:1889–98). Two mouse pre-B cell lines (4DE4 and L1.2) exogenously expressing CCR8 exhibited robust migration in response to the known CCR8 ligand CCL1 but not to CCL18. Similarly, CCL1 but not CCL18 induced internalization of CCR8 on 4DE4 cells. CCR8 expressed on Chinese hamster ovarian (CHO) cells mediated robust G protein activation, inhibition of cAMP synthesis and β-arrestin2 recruitment in response to CCL1 but not CCL18. Several N- and C-terminal variants of CCL18 also failed to stimulate CCR8 activation. On the other hand, and as previously reported, CCL18 inhibited CCL11-stimulated migration of 4DE4 cells expressing the receptor CCR3. These data suggest that CCR8, at least in the absence of unidentified cofactors, does not function as a high affinity receptor for CCL18. [ABSTRACT FROM AUTHOR]

Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded in <28 days with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low. Author summary: A major challenge to curing HIV is the presence of cells with rebound-competent viral reservoirs (RCVR) capable of re-establishing systemic infection when ART is interrupted. CD4+ T cells harboring RCVR persist for life due to homeostatic mechanisms that allow them to proliferate and expand during ART. In this study, we explored whether pan-lymphocyte depletion with a licensed CD52-depleting monoclonal antibody called alemtuzumab, can eliminate or reduce RCVR in SIV-infected rhesus macaques (RM) when administered either at the time of ART initiation or during chronic ART administration. While alemtuzumab induced massive depletion of lymphocytes in blood, including >95% depletion of CD4+ T cells, there were no measurable effects of alemtuzumab on cell-associated viral loads during ART or SIV rebound dynamics following ART interruption in most RM. Three alemtuzumab-treated RM failed to manifest rebound viremia during extended follow up after ART withdrawal. However, these animals had the lowest levels of pre-ART SIV replication, suggesting that alemtuzumab may have been effective in RM with early and presumably smaller RCVR. Overall, our study highlights the current limitations of lymphocyte depletion strategies in facilitating the permanent disruption of RCVR, particularly once the RCVR is established above levels that induce consistent post-ART viremia. [ABSTRACT FROM AUTHOR]

Activation of the cGAS-STING pathway plays a key role in the innate immune response to cancer through Type-1 Interferon (IFN) production and T cell priming. Accumulation of cytosolic double-stranded DNA (dsDNA) within tumor cells and dying cells is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) to create the secondary messenger cGAMP, which in turn activates STING (STimulator of INterferon Genes), resulting in the subsequent expression of IFN-related genes. This process is regulated by Three-prime Repair EXonuclease 1 (TREX1), a 3' → 5' exonuclease that degrades cytosolic dsDNA, thereby dampening activation of the cGAS-STING pathway, which in turn diminishes immunostimulatory IFN secretion. Here, we characterize the activity of VB-85680, a potent small-molecule inhibitor of TREX1. We first demonstrate that VB-85680 inhibits TREX1 exonuclease activity in vitro in lysates from both human and mouse cell lines. We then show that treatment of intact cells with VB-85680 results in activation of downstream STING signaling, and activation of IFN-stimulated genes (ISGs). THP1-Dual™ cells cultured under low-serum conditions exhibited an enhanced ISG response when treated with VB-85680 in combination with exogenous DNA. Collectively, these findings suggest the potential of a TREX1 exonuclease inhibitor to work in combination with agents that generate cytosolic DNA to enhance the acquisition of the anti-tumor immunity widely associated with STING pathway activation. [ABSTRACT FROM AUTHOR]

We previously demonstrated that glycyrrhizin (GL) suppressed inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer (CC). In this study, we found an accumulation of regulatory T cells (Tregs) in the spleen and suppression by GL in model mice. ICR mice were divided into four groups: Control, GL, CC, and GL-treated CC (CC+GL), and were sacrificed 20 weeks after AOM/DSS treatment. We measured spleen weight, areas of white and red pulp, and CD8+ T cells (cytotoxic T lymphocytes, CTL), and CD11c-positive cells (dendritic cells) in splenic tissues and forkhead box protein 3 (FoxP3)-positive cells (Tregs) in colorectal and splenic tissues. In all cases, the CC group showed a significant increase compared with those in Control group, and GL administration significantly attenuated this increase. These results indicate that Tregs accumulated in the spleen may participate in inflammation-related carcinogenesis by suppressing CTL. We also suggest that GL which binds to high-mobility group box 1 (HMGB1), suppresses carcinogenesis with decreasing Tregs in the spleen. Furthermore, there was an expression of FoxP3 in cancer cells, indicating that it may be involved in the malignant transformation of cancer cells. [ABSTRACT FROM AUTHOR]

CD8 T cells are the predominant effector cells of adaptive immunity in preventing cytomegalovirus (CMV) multiple-organ disease caused by cytopathogenic tissue infection. The mechanism by which CMV-specific, naïve CD8 T cells become primed and clonally expand is of fundamental importance for our understanding of CMV immune control. For CD8 T-cell priming, two pathways have been identified: direct antigen presentation by infected professional antigen-presenting cells (pAPCs) and antigen cross-presentation by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Studies in mouse models using murine CMV (mCMV) and precluding either pathway genetically or experimentally have shown that, in principle, both pathways can congruently generate the mouse MHC/H-2 class-I-determined epitope-specificity repertoire of the CD8 T-cell response. Recent studies, however, have shown that direct antigen presentation is the canonical pathway when both are accessible. This raised the question of why antigen cross-presentation is ineffective even under conditions of high virus replication thought to provide high amounts of antigenic material for feeding cross-presenting pAPCs. As delivery of antigenic material for cross-presentation is associated with programmed cell death, and as CMVs encode inhibitors of different cell death pathways, we pursued the idea that these inhibitors restrict antigen delivery and thus CD8 T-cell priming by cross-presentation. To test this hypothesis, we compared the CD8 T-cell responses to recombinant mCMVs lacking expression of the apoptosis-inhibiting protein M36 or the necroptosis-inhibiting protein M45 with responses to wild-type mCMV and revertant viruses expressing the respective cell death inhibitors. The data reveal that increased programmed cell death improves CD8 T-cell priming in mice capable of antigen cross-presentation but not in a mutant mouse strain unable to cross-present. These findings strongly support the conclusion that CMV cell death inhibitors restrict the priming of CD8 T cells by antigen cross-presentation. Author summary: In patients as well as in experimental mouse models, CD8 T cells represent the most potent antiviral effector cells in preventing CMV disease in immunocompromised recipients of hematopoietic cell transplantation. Despite the clinical relevance of mounting a protective response, the mode of CMV antigen presentation to naïve CD8 T cells was long debated. In principle, naïve CD8 T cells can be sensitized through "direct antigen presentation" on the surface of infected professional antigen-presenting cells (pAPCs) or through "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected cells following cell death. As CMVs are cytopathogenic, eventually killing host cells, and as they encode immune evasion proteins interfering with the MHC/HLA class-I pathway of direct antigen presentation, it seemed reasonable to propose a dominant role for antigen cross-presentation. Mouse models precluding either mode of antigen presentation, however, revealed that both can raise an equivalent CD8 T-cell response, and recent work has identified direct antigen presentation as the canonical pathway taken when both are accessible. Here we show that virus-encoded inhibitors of two programmed cell death modalities, apoptosis and necroptosis, reduce antigen release to below a level required for efficient cross-presentation. This solves an open question in CMV immunology. [ABSTRACT FROM AUTHOR]

Quantifying the kinetics with which memory T cell populations are generated and maintained is essential for identifying the determinants of the duration of immunity. The quality and persistence of circulating CD4 effector memory (TEM) and central memory (TCM) T cells in mice appear to shift with age, but it is unclear whether these changes are driven by the aging host environment, by cell age effects, or both. Here, we address these issues by combining DNA labelling methods, established fate-mapping systems, a novel reporter mouse strain, and mathematical models. Together, these allow us to quantify the dynamics of both young and established circulating memory CD4 T cell subsets, within both young and old mice. We show that that these cells and their descendents become more persistent the longer they reside within the TCM and TEM pools. This behaviour may limit memory CD4 T cell diversity by skewing TCR repertoires towards clones generated early in life, but may also compensate for functional defects in new memory cells generated in old age. The quality and longevity of CD4+ effector and central memory T cells change with age in mice. This study shows that these cells have longer lifespans and become more quiescent when the organism ages, but these properties depend on the age of the cells and not on the age of the host environment. [ABSTRACT FROM AUTHOR]

Membrane-associated RING-CH (MARCH) 2 is a member of the MARCH protein family of RING-CH finger E3 ubiquitin ligases that play important roles in regulating the levels of proteins found on the cell surface. MARCH1, 2 and 8 inhibit HIV-1 infection by preventing the incorporation of the envelope glycoproteins into nascent virions. However, a better understanding on the mechanism utilized by MARCH proteins to restrict HIV-1 infection is needed. In this report, we identify an amino acid in human MARCH2, absent in mouse MARCH2, critical for its antiretroviral function. Moreover, we map the domains of human MARCH2 critical for restricting as well as binding to the HIV-1 envelope glycoproteins. In addition, we demonstrate that MARCH2 is present inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. Finally, we show that MARCH2 acts as an HIV-1 restriction factor only in primary CD4+ T cells and can prevent cell-to-cell transmission of HIV-1. Our findings reveal important new aspects of the antiviral mechanism utilized by human MARCH2 to restrict HIV-1 that have potential implications to all MARCH proteins with antiviral functions and their viral targets. Author summary: Recently, Membrane-associated RING-CH (MARCH) proteins have emerged as important antiviral factors that block virus infection by preventing viral envelope glycoprotein incorporation into newly formed virions in an ill-defined mechanism. Here, we examine in depth the role of MARCH2 during Human Immunodeficiency Virus type 1 (HIV-1) infection and show that it utilizes two mechanisms to inhibit HIV-1: 1) prevents the envelope glycoprotein incorporation into nascent virions in a RING-CH-dependent manner, 2) when incorporated in virions, it blocks virus-cell membrane fusion in a RING-CH independent manner. We also demonstrate that MARCH2 restricts HIV-1 only in CD4+ T cells and can prevent cell-to-cell transmission. In conclusion, our findings reveal new mechanistic aspects regarding the anti-HIV-1 function of MARCH2. [ABSTRACT FROM AUTHOR]

Hantaviruses are single-stranded RNA viruses belonging to the family Bunyaviridae that causes hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) worldwide. Currently, there is no effective vaccination or therapy available for the treatment of hantavirus, hence there is a dire need for research to formulate therapeutics for the disease. Computational vaccine designing is currently a highly accurate, time and cost-effective approach for designing effective vaccines against different diseases. In the current study, we shortlisted highly antigenic proteins i.e., envelope, and nucleoprotein from the proteome of hantavirus and subjected to the selection of highly antigenic epitopes to design of next-generation multi-epitope vaccine constructs. A highly antigenic and stable adjuvant was attached to the immune epitopes (T-cell, B-cell, and HTL) to design Env-Vac, NP-Vac, and Com-Vac constructs, which exhibit stronger antigenic, non-allergenic, and favorable physiochemical properties. Moreover, the 3D structures were predicted and docking analysis revealed robust interactions with the human Toll-like receptor 3 (TLR3) to initiate the immune cascade. The total free energy calculated for Env-Vac, NP-Vac, and Com-Vac was -50.02 kcal/mol, -24.13 kcal/mol, and -62.30 kcal/mol, respectively. In silico cloning, results demonstrated a CAI value for the Env-Vac, NP-Vac, and Com-Vac of 0.957, 0.954, and 0.956, respectively, while their corresponding GC contents were 65.1%, 64.0%, and 63.6%. In addition, the immune simulation results from three doses of shots released significant levels of IgG, IgM, interleukins, and cytokines, as well as antigen clearance over time, after receiving the vaccine and two booster doses. Our vaccines against Hantavirus were found to be highly immunogenic, inducing a robust immune response that demands experimental validation for clinical usage. [ABSTRACT FROM AUTHOR]

Antigen-presenting cells (APCs) play a crucial role in the immune system by breaking down antigens into peptide fragments that subsequently bind to major histocompatibility complex (MHC) molecules. Previous studies indicate that stable proteins can impede CD4+ T cell stimulation by hindering antigen processing and presentation. Conversely, certain proteins require stabilization in order to activate the immune response. Several factors, including the characteristics of the protein and the utilization of different adjuvants in animal experiments, may contribute to this disparity. In this study, we investigated the impact of adjuvants on antigen administration in mice, specifically focusing on the stability of the CH2 domain. Consequently, the CH2 domain induced a stronger IgG response in comparison to the stabilized one when using Alum and PBS (without adjuvant). On the other hand, animal experiment using Freund's adjuvant showed the opposite results. These findings indicate the significance of considering the intrinsic conformational stability of a protein when eliciting its immunogenicity, particularly within the context of vaccine development. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) remains a global public health threat. Understanding the dynamics of host-pathogen interactions within TB granulomas will assist in identifying what leads to the successful elimination of infection. In vitro TB models provide a controllable environment to study these granuloma dynamics. Previously we developed a biomimetic 3D spheroid granuloma model that controls bacteria better than a traditional monolayer culture counterpart. We used agent-based simulations to predict the mechanistic reason for this difference. Our calibrated simulations were able to predict heterogeneous bacterial dynamics that are consistent with experimental data. In one group of simulations, spheroids are found to have higher macrophage activation than their traditional counterparts, leading to better bacterial control. This higher macrophage activation in the spheroids was not due to higher counts of activated T cells, instead fewer activated T cells were able to activate more macrophages due to the proximity of these cells to each other within the spheroid. In a second group of simulations, spheroids again have more macrophage activation but also more T cell activation, specifically CD8+ T cells. This higher level of CD8+ T cell activation is predicted to be due to the proximity of these cells to the cells that activate them. Multiple mechanisms of control were predicted. Simulations removing individual mechanisms show that one group of simulations has a CD4+ T cell dominant response, while the other has a mixed/CD8+ T cell dominant response. Lastly, we demonstrated that in spheroids the initial structure and movement rules work synergistically to reduce bacterial load. These findings provide valuable insights into how the structural complexity of in vitro models impacts immune responses. Moreover, our study has implications for engineering more physiologically relevant in vitro models and advancing our understanding of TB pathogenesis and potential therapeutic interventions. Author summary: Tuberculosis (TB) is a respiratory infection that is associated with granulomas, organized clusters of immune cells that form around the bacteria. Recent work has focused on making in vitro models that better approximate these granulomas, such as our spheroid granuloma model. We found that this spheroid was better able to control bacteria than its traditional culture counterpart, so, in this work, we use agent-based modeling to explore the causes of this differential control. The simulated spheroids have increased macrophage activation. Surprisingly, this is not due to larger numbers of activated T cells, which activate these macrophages. Rather, the increase in activated macrophages is due to their proximity to fewer activated T cells. We also see a subset of simulations where the spheroids have increased cytotoxic CD8+ T cell activation. The simulation is further used to simulate changes in the structure of the in vitro models to explore its impact on immune response and bacterial control. These findings reveal the importance of structural complexity in in vitro models and offer insights into TB pathogenesis. Ultimately, our work paves the way for more realistic in vitro models and a deeper understanding of how our bodies combat TB. [ABSTRACT FROM AUTHOR]

A strong genetic predictor of outcome following untreated HIV-1 infection is the carriage of specific alleles of human leukocyte antigens (HLAs) that present viral epitopes to T cells. Residual variation in outcome measures may be attributed, in part, to viral adaptation to HLA-restricted T cell responses. Variants of the endoplasmic reticulum aminopeptidases (ERAPs) influence the repertoire of T cell epitopes presented by HLA alleles as they trim pathogen-derived peptide precursors to optimal lengths for antigen presentation, along with other functions unrelated to antigen presentation. We investigated whether ERAP variants influence HLA-associated HIV-1 adaptation with demonstrable effects on overall HIV-1 disease outcome. Utilizing host and viral data of 249 West Australian individuals with HIV-1 subtype B infection, we identified a novel association between two linked ERAP2 single nucleotide polymorphisms (SNPs; rs2248374 and rs2549782) with plasma HIV RNA concentration (viral load) (P adjusted = 0.0024 for both SNPs). Greater HLA-associated HIV-1 adaptation in the HIV-1 Gag gene correlated significantly with higher viral load, lower CD4+ T cell count and proportion; P = 0.0103, P = 0.0061, P = 0.0061, respectively). When considered together, there was a significant interaction between the two ERAP2 SNPs and HLA-associated HIV-1 adaptation on viral load (P = 0.0111). In a comprehensive multivariate model, addition of ERAP2 haplotypes and HLA associated adaptation as an interaction term to known HLA and CCR5 determinants and demographic factors, increased the explanatory variance of population viral load from 17.67% to 45.1% in this dataset. These effects were not replicated in publicly available datasets with comparably sized cohorts, suggesting that any true global epistasis may be dependent on specific HLA-ERAP allelic combinations. Our data raises the possibility that ERAP2 variants may shape peptide repertoires presented to HLA class I-restricted T cells to modulate the degree of viral adaptation within individuals, in turn contributing to disease variability at the population level. Analyses of other populations and experimental studies, ideally with locally derived ERAP genotyping and HLA-specific viral adaptations are needed to elucidate this further. Author summary: HIV infection outcome is variable between individuals and understanding the factors that impact this variation is important for efforts towards a HIV cure or vaccine. Here, we found that the level of HIV in the blood is affected by whether an individual carries a specific form of ERAP2, a molecule that influences processing and presentation of the virus to the immune system, as well as the degree to which HIV has mutated to adapt to immune responses. We also show that the interaction of ERAP2 and other known genetic factors explains greater variation in infection outcome than these factors alone. These findings expand our knowledge of the potential importance of viral processing and presentation in the immune response to HIV. [ABSTRACT FROM AUTHOR]

The involvement of ©δ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. ©δ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αβ T cell deficient mice in order to analyze the memory potential of ©δ T cells. As for CMV-specific αβ T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ ©δ TEM that principally localized in infected organ vasculature. Typifying T cell memory, ©δ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking ©δ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed ©δ TCM cells, but not ©δ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two ©δ T cell memory subsets which also revealed their similarity to classically adaptive αβ CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM ©δ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches. Author summary: Cytomegalovirus (CMV) is a widespread, latent virus that can cause severe organ disease in immune-compromised patients. Anti-CMV memory immune responses are essential to control viral reactivation and/or reinfection events that commonly take place in solid organ transplantation. The role of ©δ T-cell receptor bearing lymphocytes could be crucial in this context where immunosuppressive/ablative treatments cause suboptimal and/or delayed αβ T cell responses. Here we asked whether ©δ T cells could compensate for the absence of αβ T cells in the long-term control of mouse CMV infection. Three months post-primary viral challenge in αβ-T cell deficient mice, ©δ T cells displayed similar features as cytolytic, CMV-specific αβ CD8 T cells. We showed that previous priming with CMV endowed ©δ T cells with an enhanced antiviral potential and that long-term maintenance of ©δ T cell-mediated antiviral protection was dependent on ©δ central memory T cells (TCM). As observed in human, the ©δ T cell response to a secondary CMV challenge generated a private TCR δ repertoire. Finally, our gene expression/accessibility single cell analysis revealed that ©δ TCM shared similar features as their αβ T cell counterpart. Our results sustain the adaptive-like properties of these unconventional T cells and reveal the interest of targeting ©δ TCM subset in novel antiviral vaccination approaches. [ABSTRACT FROM AUTHOR]

Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable. Heligmosomoides bakeri (parasitic roundworm, previously Heligmosomoides polygyrus) and Toxoplasma gondii (protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response, respectively. Several studies have demonstrated reduced inflammatory cytokine responses in animals co-infected with such organisms. However, while general cytokine signatures have been examined, the impact of the different cytokine producing lymphocytes on parasite control/clearance is not fully understood. We investigated five different lymphocyte populations (NK, NKT, γδ T, CD4+ T and CD8+ T cells), five organs (small intestine, Peyer's patches, mesenteric lymph nodes, spleen and liver), and 4 cytokines (IFN©, IL-4, IL-10 and IL-13) at two different time points (days 5 and 10 post T. gondii infection). We found that co-infected animals had significantly higher mortality than either single infection. This was accompanied by transient and local changes in parasite loads and cytokine profiles. Despite the early changes in lymphocyte and cytokine profiles, severe intestinal pathology in co-infected mice likely contributed to early mortality due to significant damage by both parasites in the small intestine. Our work demonstrates the importance of taking a broad view during infection research, studying multiple cell types, organs/tissues and time points to link and/or uncouple immunological from pathological findings. Our results provide insights into how co-infection with parasites stimulating different arms of the immune system can lead to drastic changes in infection dynamics. [ABSTRACT FROM AUTHOR]

Background: Many newborn screening programs worldwide have introduced screening for diseases using DNA extracted from dried blood spots (DBS). In Germany, DNA-based assays are currently used to screen for severe combined immunodeficiency (SCID), spinal muscular atrophy (SMA), and sickle cell disease (SCD). Methods: This study analysed the impact of pre-analytic DNA carry-over in sample preparation on the outcome of DNA-based newborn screening for SCID and SMA and compared the efficacy of rapid extraction versus automated protocols. Additionally, the distribution of T cell receptor excision circles (TREC) on DBS cards, commonly used for routine newborn screening, was determined. Results: Contaminations from the punching procedure were detected in the SCID and SMA assays in all experimental setups tested. However, a careful evaluation of a cut-off allowed for a clear separation of true positive polymerase chain reaction (PCR) amplifications. Our rapid in-house extraction protocol produced similar amounts compared to automated commercial systems. Therefore, it can be used for reliable DNA-based screening. Additionally, the amount of extracted DNA significantly differs depending on the location of punching within a DBS. Conclusions: Newborn screening for SMA and SCID can be performed reliably. It is crucial to ensure that affected newborns are not overlooked. Therefore a carefully consideration of potential contaminating factors and the definition of appropriate cut-offs to minimise the risk of false results are of special concern. It is also important to note that the location of punching plays a pivotal role, and therefore an exact quantification of TREC numbers per μl may not be reliable and should therefore be avoided. [ABSTRACT FROM AUTHOR]

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered. Author summary: This study highlights the significance of AXL+ Siglec-6+ dendritic cells (ASDC) in HIV transmission, particularly in inflamed peripheral tissues such as anogenital tissues, where HIV transmission is prevalent. It reveals that ASDCs are present in inflamed human tissues, including psoriasis affected skin, colorectal cancer and anogenital tissues. However, they are absent in uninflamed tissues. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDC migration from blood to inflamed tissues, and their interactions with HIV and CD4 T cells. Notably, different subsets of ASDCs exhibit different expression levels of HIV binding receptors and showed trends of different phases of HIV transmission to T cells. Understanding ASDC involvement in HIV transmission could provide valuable insights for developing strategies to inhibit their recruitment to inflamed tissues and their ability to transmit the virus to CD4 T cells, potentially offering new avenues for HIV prevention and treatment. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies. Author summary: The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS), a lethal condition that necessitates life-long antiretroviral therapy for prevention. The envelope protein (Env) of HIV mediates the viral entry into host cells and is the sole target for the immune system on the surface of the virus. Env's extensive modification with sugars, so called N-glycans, shields crucial epitopes from neutralizing antibodies. Interestingly, key HIV producing cell types, such as CD4+ T cells and macrophages, modify Env with distinct N-glycosylation patterns. However, the specific molecular disparities in N-glycosylation between viruses from these cell types are unexplored, and whether the producer cell type impacts the viral spread and resistance to neutralization is incompletely understood. Utilizing the simian immunodeficiency virus (SIV) as a model for HIV, our study revealed differing N-glycan profiles between SIV Env produced in CD4+ T cells (T-SIV) and macrophages (M-SIV), with T-SIV incorporating more N-glycans of the oligomannose-type into Env than M-SIV. M-SIV displayed enhanced infectivity and transmissibility in in vitro studies. Additionally, M-SIV exhibited greater susceptibility to neutralizing sera compared to T-SIV, while T-SIV showed heightened susceptibility to mannose-specific carbohydrate-binding agents. These findings emphasize the critical role of host cell-dependent attributes in shaping N-glycosylation of Env as well as viral infectivity and spread. These findings have important implications for the development of new biomedical interventions against HIV. [ABSTRACT FROM AUTHOR]

Mutations in the non-structural protein regions of hepatitis C virus (HCV) are a cause of a non-sustained virological response (SVR) to treatment with direct-acting antivirals (DAAs) for chronic hepatitis; however, there are non-SVR cases without these mutations. In this study, we examined immune cell profiles in peripheral blood before and after ombitasvir/paritaprevir/ritonavir treatment and screened for genes that could be used to predict the therapeutic effects of DAAs. Fluorescence-activated cell sorting analysis indicated that the median frequencies of programmed cell death-1-positive (PD-1+) effector regulatory T cells (eTregs), PD-1+CD8+ T cells, and PD-1+Helper T cells were decreased significantly in SVR cases, but without significant changes in non-SVR cases. The frequency of PD-1+ naïve Tregs was significantly higher in the SVR group than in the non-SVR group before and after treatment. Similar results were found in patients treated with other DAAs (e.g., daclatasvir plus asunaprevir) and supported an immune response after HCV therapy. RNA-sequencing analysis indicated a significant increase in the expression of genes associated with the immune response in the SVR group, while genes related to intracellular and extracellular signal transduction were highly expressed in the non-SVR group. Therefore, we searched for genes associated with PD-1+ eTregs and CD8+ T cells that were significantly different between the SVR and non-SVR groups and found that T-box transcription factor 21 was associated with the non-SVR state. These results indicate that PD-1-related signaling pathways are associated with a non-SVR mechanism after DAAs treatment separate from mutation-related drug resistance. [ABSTRACT FROM AUTHOR]

Leprosy has a high rate of cripplehood and lacks available early effective diagnosis methods for prevention and treatment, thus novel effective molecule markers are urgently required. In this study, we conducted bioinformatics analysis with leprosy and normal samples acquired from the GEO database(GSE84893, GSE74481, GSE17763, GSE16844 and GSE443). Through WGCNA analysis, 85 hub genes were screened(GS > 0.7 and MM > 0.8). Through DEG analysis, 82 up-regulated and 3 down-regulated genes were screened(|Log2FC| > 3 and FDR < 0.05). Then 49 intersection genes were considered as crucial and subjected to GO annotation, KEGG pathway and PPI analysis to determine the biological significance in the pathogenesis of leprosy. Finally, we identified a gene-pathway network, suggesting ITK, CD48, IL2RG, CCR5, FGR, JAK3, STAT1, LCK, PTPRC, CXCR4 can be used as biomarkers and these genes are active in 6 immune system pathways, including Chemokine signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, T cell receptor signaling pathway, Natural killer cell mediated cytotoxicity and Leukocyte transendothelial migration. We identified 10 crucial gene markers and related important pathways that acted as essential components in the etiology of leprosy. Our study provides potential targets for diagnostic biomarkers and therapy of leprosy. [ABSTRACT FROM AUTHOR]

Background: In most cases, Zika virus (ZIKV) causes a self-limited acute illness in adults, characterized by mild clinical symptoms that resolve within a few days. Immune responses, both innate and adaptive, play a central role in controlling and eliminating virus-infected cells during the early stages of infection. Aim: To test the hypothesis that circulating T cells exhibit phenotypic and functional activation characteristics during the viremic phase of ZIKV infection. Methods: A comprehensive analysis using mass cytometry was performed on peripheral blood mononuclear cells obtained from patients with acute ZIKV infection (as confirmed by RT-PCR) and compared with that from healthy donors (HD). The frequency of IFN-γ-producing T cells in response to peptide pools covering immunogenic regions of structural and nonstructural ZIKV proteins was quantified using an ELISpot assay. Results: Circulating CD4+ and CD8+ T lymphocytes from ZIKV-infected patients expressed higher levels of IFN-γ and pSTAT-5, as well as cell surface markers associated with proliferation (Ki-67), activation ((HLA-DR, CD38) or exhaustion (PD1 and CTLA-4), compared to those from HD. Activation of CD4+ and CD8+ memory T cell subsets, including Transitional Memory T Cells (TTM), Effector Memory T cells (TEM), and Effector Memory T cells Re-expressing CD45RA (TEMRA), was prominent among CD4+ T cell subset of ZIKV-infected patients and was associated with increased levels of IFN-γ, pSTAT-5, Ki-67, CTLA-4, and PD1, as compared to HD. Additionally, approximately 30% of ZIKV-infected patients exhibited a T cell response primarily directed against the ZIKV NS5 protein. Conclusion: Circulating T lymphocytes spontaneously produce IFN-γ and express elevated levels of pSTAT-5 during the early phase of ZIKV infection whereas recognition of ZIKV antigen results in the generation of virus-specific IFN-γ-producing T cells. [ABSTRACT FROM AUTHOR]

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community. [ABSTRACT FROM AUTHOR]

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic. [ABSTRACT FROM AUTHOR]

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model, we found that Treg cells play a role during the initial stages after T. cruzi infection, restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation, exhaustion and functional markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model. Author summary: Chagas disease, caused by Trypanosoma cruzi, can result in severe health complications. While the exact mechanisms underlying the disease's pathogenesis remain incompletely understood, the host's inflammatory immune response is believed to play a critical role. To shed light on disease mechanisms and potential treatments, we investigated the impact of regulatory T (Treg) cells on the development of effector immune responses against T. cruzi. Our findings reveal that Treg cells dampen parasite-specific CD8+ T cells, a crucial arm of the immune response in counteracting the parasite. Notably, this regulatory influence occurs primarily during the early stages of T. cruzi infection. Furthermore, we observed that while Treg cells have minimal effects on antigen-presenting cells, they modulate the magnitude and phenotype of conventional CD4+ T cells. Importantly, we identified CD39, a molecule involved in the purinergic pathway, as essential for the suppressive functions of Treg cells during T. cruzi infection. Our findings enhance the understanding of the regulatory response during the acute phase of T. cruzi infection and may have implications for the development of novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular parasite that establishes a long-term infection in the brain of many warm-blooded hosts, including humans and rodents. Like all obligate intracellular microbes, Toxoplasma uses many effector proteins to manipulate the host cell to ensure parasite survival. While some of these effector proteins are universal to all Toxoplasma strains, some are polymorphic between Toxoplasma strains. One such polymorphic effector is GRA15. The gra15 allele carried by type II strains activates host NF-κB signaling, leading to the release of cytokines such as IL-12, TNF, and IL-1β from immune cells infected with type II parasites. Prior work also suggested that GRA15 promotes early host control of parasites in vivo, but the effect of GRA15 on parasite persistence in the brain and the peripheral immune response has not been well defined. For this reason, we sought to address this gap by generating a new IIΔgra15 strain and comparing outcomes at 3 weeks post infection between WT and IIΔgra15 infected mice. We found that the brain parasite burden and the number of macrophages/microglia and T cells in the brain did not differ between WT and IIΔgra15 infected mice. In addition, while IIΔgra15 infected mice had a lower number and frequency of splenic M1-like macrophages and frequency of PD-1+ CTLA-4+ CD4+ T cells and NK cells compared to WT infected mice, the IFN-γ+ CD4 and CD8 T cell populations were equivalent. In summary, our results suggest that in vivo GRA15 may have a subtle effect on the peripheral immune response, but this effect is not strong enough to alter brain parasite burden or parenchymal immune cell number at 3 weeks post infection. [ABSTRACT FROM AUTHOR]

Background: Fatigue is a common symptom after viral infection. Chinese herbal medicine (CHM) is thought to be a potential effective intervention in relieving fatigue. Purpose: To assess the effectiveness and safety of CHM for the treatment of post-viral fatigue. Study design: Systematic review and meta-analysis of randomized controlled trials (RCTs). Methods: The protocol of this systematic review was registered on PROSPERO (CRD42022380356). Trials reported changes of fatigue symptom, which compared CHM to no treatment, placebo or drugs, were included. Six electronic databases and three clinical trial registration platforms were searched from inception to November 2023. Literature screening, data extraction, and risk bias assessment were independently carried out by two reviewers. Quality of the included trials was evaluated using Cochrane risk of bias tool, and the certainty of the evidence was evaluated using GRADE. The meta-analysis was performed using Review Manager 5.4, mean difference (MD) and its 95% confidence interval (CI) was used for estimate effect of continuous data. Heterogeneity among trials was assessed through I2 value. Results: Overall, nineteen studies with 1921 patients were included. Results of individual trial or meta-analysis showed that CHM was better than no treatment (MD = -0.80 scores, 95%CI -1.43 to -0.17 scores, P = 0.01, 60 participants, 1 trial), placebo (MD = -1.90 scores, 95%CI -2.38 to -1.42 scores, P<0.00001, 184 participants, 1 trial), placebo on basis of rehabilitation therapy (MD = -14.90 scores, 95%CI -24.53 to -5.27 scores, P = 0.02, 118 participants, 1 trial) or drugs (MD = -0.38 scores, 95%CI -0.48 to -0.27 scores, I2 = 0%, P<0.00001, 498 participants, 4 trials) on relieving fatigue symptoms assessing by Traditional Chinese Medicine fatigue scores. Trials compared CHM plus drugs to drugs alone also showed better effect of combination therapy (average MD = -0.56 scores). In addition, CHM may improve the percentage of CD4 T lymphocytes and reduce the level of serum IL-6 (MD = -14.64 scores, 95%CI 18.36 to -10.91 scores, I2 = 0%, P<0.00001, 146 participants, 2 trials). Conclusion: Current systematic review found that the participation of CHM can improve the symptoms of post-viral fatigue and some immune indicators. However, the safety of CHM remains unknown and large sample, high quality multicenter RCTs are still needed in the future. [ABSTRACT FROM AUTHOR]

Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1. [ABSTRACT FROM AUTHOR]

Biomarkers that could predict the evolution of the graft in transplanted patients and that could allow to adapt the care of the patients would be an invaluable tool. Additionally, certain biomarkers can be target of treatments and help to stratify patients. Potential effective biomarkers have been identified but still need to be confirmed. CD45RC, one of the splicing variants of the CD45 molecule, a tyrosine phosphatase that is critical in negatively or positively regulating the TCR and the BCR signaling, is one marker already described. The frequency of CD8+ T cells expressing high levels of CD45RC before transplantation is increased in patients with an increased risk of acute rejection. However, single biomarkers have limited predictive reliability and the correlation of the expression levels of CD45RC with other cell markers was not reported. In this study, we performed a fluorescent-based high dimensional immunophenotyping of T cells on a cohort of 69 kidney transplant patients either with stable graft function or having experienced acute transplant rejection during the first year after transplantation or at the time of rejection. We identified combinations of markers and cell subsets associated with activation/inflammation or Tregs/tolerance (HLA-DR, PD-1, IFNγ, CD28) as significant biomarkers associated to transplant outcome, and showed the importance of cell segregation based on the CD45RC marker to identify the signature of a stable graft function. Our study highlights potential reliable biomarkers in transplantation to predict and/or monitor easily graft-directed immune responses and adapt immunosuppression treatments to mitigate adverse effects. [ABSTRACT FROM AUTHOR]

Objective: To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. Methods: Participants with chikungunya arthritis were recruited from Colombia from 2019–2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. Results: Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). Conclusion: Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy. [ABSTRACT FROM AUTHOR]

Background: The CD4 T lymphocyte count in people living with HIV (PLHIV) is a predictor for the progression of the disease (AIDS), survival and response to antiretroviral treatment (ART). A CD4 T lymphocyte count of less than 200 cells/mm3 is indicative of a greater risk for the onset of opportunistic diseases and death. Defaulting on treatment for tuberculosis (TB) may impact immune recovery in PLHIV who are taking ART. The aim of this study was to investigate an association of the CD4 lymphocyte with TB treatment Trajectory and with death. Methods: A cohort of PLHIV over eighteen years of age and who were taking ART and who had defaulted on pulmonary TB treatment. Latent Class analysis was used to identify different trajectories of CD4 T lymphocyte counts over time. Results: Latent class 1 (High CD4 trajectory) grouped individuals together who were characterized as maintaining a low probability (0 to 29%) of a CD4 count ≤ 200 cells/mm3over time, while latent class 2 (Low CD4 trajectory) grouped individuals together with a high probability (93% to 60%), and latent class 3 (Fluctuating CD4 trajectory), grouped individuals with a fluctuating probability (66% to 0%). The chance of defaulting on treatment earlier (≤ 90 days) was four times higher in latent class 2 (Low CD4 trajectory). Although there was no statistical significance, there was a higher frequency of deaths in this same latent class. Conclusion: Individuals with a high probability of a CD4 count ≤ 200 cells/ mm3 should be monitored in order to avoid treatment default and thereby prevent death. New studies should be conducted with a larger sample size and a longer follow-up time in PLHIV who initiated ART treatment early so as to support clinical decisions for a better understanding of immune behavior. [ABSTRACT FROM AUTHOR]

Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors. [ABSTRACT FROM AUTHOR]

The advancements in next-generation sequencing have made it possible to effectively detect somatic mutations, which has led to the development of personalized neoantigen cancer vaccines that are tailored to the unique variants found in a patient's cancer. These vaccines can provide significant clinical benefit by leveraging the patient's immune response to eliminate malignant cells. However, determining the optimal vaccine dose for each patient is a challenge due to the heterogeneity of tumors. To address this challenge, we formulate a mathematical dose optimization problem based on a previous mathematical model that encompasses the immune response cascade produced by the vaccine in a patient. We propose an optimization approach to identify the optimal personalized vaccine doses, considering a fixed vaccination schedule, while simultaneously minimizing the overall number of tumor and activated T cells. To validate our approach, we perform in silico experiments on six real-world clinical trial patients with advanced melanoma. We compare the results of applying an optimal vaccine dose to those of a suboptimal dose (the dose used in the clinical trial and its deviations). Our simulations reveal that an optimal vaccine regimen of higher initial doses and lower final doses may lead to a reduction in tumor size for certain patients. Our mathematical dose optimization offers a promising approach to determining an optimal vaccine dose for each patient and improving clinical outcomes. Author summary: The development of neoantigen cancer vaccines have rapidly increased over the past decade with the advancement of next-generation sequencing technologies to determine immunogenic peptides from patient's somatic mutations. However, traditional methods to determine the cancer vaccine dose often produce suboptimal clinical outcomes. This work use our previous mathematical model to represent the immunological cascade at the cellular and subcellular levels elicited by the vaccine dose, and focuses on developing a mathematical optimization approach to identify the optimal vaccine dose to minimize two objective functionals, (i) minimize the amount of peptide dose and tumor size, and (ii) minimize the number of activated T cells in addition to the objective functional i. The optimization approach allows the identification of optimal vaccine doses among a set of tested doses for a higher clinical benefit in tumor reduction. To demonstrate and validate our optimization approach, we perform in silico experiments on six patients with melanoma from a clinical trial study. The results show that the predicted optimal vaccine doses can provide higher clinical benefit in tumor reduction when compared to the clinical trial doses for some patients. [ABSTRACT FROM AUTHOR]

A typical HTLV-1-infected individual carries >104 different HTLV-1-infected T cell clones, each with a single-copy provirus integrated in a unique genomic site. We previously showed that the HTLV-1 provirus causes aberrant transcription in the flanking host genome and, by binding the chromatin architectural protein CTCF, forms abnormal chromatin loops with the host genome. However, it remained unknown whether these effects were exerted simply by the presence of the provirus or were induced by its transcription. To answer this question, we sorted HTLV-1-infected T-cell clones into cells positive or negative for proviral plus-strand expression, and then quantified host and provirus transcription using RNA-seq, and chromatin looping using quantitative chromosome conformation capture (q4C), in each cell population. We found that proviral plus-strand transcription induces aberrant transcription and splicing in the flanking genome but suppresses aberrant chromatin loop formation with the nearby host chromatin. Reducing provirus-induced host transcription with an inhibitor of transcriptional elongation allows recovery of chromatin loops in the plus-strand-expressing population. We conclude that aberrant host transcription induced by proviral expression causes temporary, reversible disruption of chromatin looping in the vicinity of the provirus. Author summary: The human T cell leukemia virus HTLV-1 causes an aggressive leukemia or lymphoma in ~5% of people infected with the virus, and a further 1–4% develop a chronic inflammatory disease that leads to progressive paralysis of the legs. HTLV-1 is a retrovirus, like HIV, and these viruses insert themselves–as a 'provirus'—into the DNA of the T lymphocytes that they infect: this is largely why the viruses are very difficult to eradicate. The provirus remains dormant for most of the time, but it can be reactivated when the T cell meets a change in temperature or pH, or a number of other conditions. We recently discovered that HTLV-1, when it is integrated into the host DNA, changes the 3D structure of the genome in the infected cell, and interferes with the normal function of the host genes that lie near HTLV-1 in the genome. What we have now found is that the change in the 3D structure of the genome is caused simply by the presence of the provirus, but that when the provirus is reactivated the 3D structural changes are temporarily removed, and nearby host genes can be abnormally activated by the provirus. [ABSTRACT FROM AUTHOR]

Fusobacterium nucleatum (Fn) and enterotoxigenic Bacteroides fragilis (ETBF) are two pathobionts consistently enriched in the gut microbiomes of patients with colorectal cancer (CRC) compared to healthy counterparts and frequently observed for their direct association within tumors. Although several molecular mechanisms have been identified that directly link these organisms to features of CRC in specific cell types, their specific effects on the epithelium and local immune compartment are not well-understood. To fill this gap, we leveraged single-cell RNA sequencing (scRNA-seq) on wildtype mice and mouse model of CRC. We find that Fn and ETBF exacerbate cancer-like transcriptional phenotypes in transit-amplifying and mature enterocytes in a mouse model of CRC. We also observed increased T cells in the pathobiont-exposed mice, but these pathobiont-specific differences observed in wildtype mice were abrogated in the mouse model of CRC. Although there are similarities in the responses provoked by each organism, we find pathobiont-specific effects in Myc-signaling and fatty acid metabolism. These findings support a role for Fn and ETBF in potentiating tumorigenesis via the induction of a cancer stem cell-like transit-amplifying and enterocyte population and the disruption of CTL cytotoxic function. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells. Author summary: Cytomegalovirus (CMV) infection is a very common cause of complications in transplant recipients. These patients suffer from opportunistic viruses because their immune system is weakened by immunosuppressive drugs, which are required to avoid transplant rejection by T cells, a key lymphocyte population involved in antiviral defenses. The commonly used immunosuppressive drugs target gene products in a signaling pathway inside T lymphocytes involving two Nuclear Factors of Activated T cells (NFAT) called NFATc1 and NFATc2. We tested the effects of NFAT signaling on T cell responses to CMV in mice lacking either of these genes, or both of them. We show that the T cell responses to CMV are compromised in absence of NFATc1 in a peculiar manner. While early responses upon infection are robust, they are not maintained in the long term. A detailed analysis of these cells showed that cells lacking NFAT display a deficit in T-cell maturation upon the initial activation. Our data may have relevance for the design of future immunosuppressive drugs that may protect patients from organ rejection, while retaining antiviral immune defenses. [ABSTRACT FROM AUTHOR]

PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01–0.3 mg/kg/dose) and GLP toxicity study (0.1–3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 μg/mL and 37.9 μg*h/mL, respectively. [ABSTRACT FROM AUTHOR]

The breadth of pathogens to which T cells can respond is determined by the T cell receptors (TCRs) present in an individual's repertoire. Although more than 90% of the sequence diversity among TCRs is generated by terminal deoxynucleotidyl transferase (TdT)-mediated N-nucleotide addition during V(D)J recombination, the benefit of TdT-altered TCRs remains unclear. Here, we computationally and experimentally investigated whether TCRs with higher N-nucleotide diversity via TdT make distinct contributions to acute or chronic pathogen control specifically through the inclusion of TCRs with lower antigen binding strengths (i.e., lower reactivity to peptide-major histocompatibility complex (pMHC)). When T cells with high pMHC reactivity have a greater propensity to become functionally exhausted than those of low pMHC reactivity, our computational model predicts a shift toward T cells with low pMHC reactivity over time during chronic, but not acute, infections. This TCR-affinity shift is critical, as the elimination of T cells with lower pMHC reactivity in silico substantially increased the time to clear a chronic infection, while acute infection control remained largely unchanged. Corroborating an affinity-centric benefit for TCR diversification via TdT, we found evidence that TdT-deficient TCR repertoires possess fewer T cells with weaker pMHC binding strengths in vivo and showed that TdT-deficient mice infected with a chronic, but not an acute, viral pathogen led to protracted viral clearance. In contrast, in the case of a chronic fungal pathogen where T cells fail to clear the infection, both our computational model and experimental data showed that TdT-diversified TCR repertoires conferred no additional protection to the hosts. Taken together, our in silico and in vivo data suggest that TdT-mediated TCR diversity is of particular benefit for the eventual resolution of prolonged pathogen replication through the inclusion of TCRs with lower foreign antigen binding strengths. Although more than 90% of the sequence diversity among T cell receptors (TCRs) is generated by terminal deoxynucleotidyl transferase (TdT)-mediated nucleotide addition, the benefit of TdT-altered TCRs remains unclear. This study shows that low-affinity T cells generated by TdT are important for curtailing chronic pathogen replication. [ABSTRACT FROM AUTHOR]

Background: ATL1102 is a 2'MOE gapmer antisense oligonucleotide to the CD49d alpha subunit of VLA-4, inhibiting expression of CD49d on lymphocytes, reducing survival, activation and migration to sites of inflammation. Children with DMD have dystrophin deficient muscles susceptible to contraction induced injury, which triggers the immune system, exacerbating muscle damage. CD49d is a biomarker of disease severity in DMD, with increased numbers of high CD49d expressing T cells correlating with more severe and progressive weakess, despite corticosteroid treatment. Methods: This Phase 2 open label study assessed the safety, efficacy and pharmacokinetic profile of ATL1102 administered as 25 mg weekly by subcutaneous injection for 24 weeks in 9 non-ambulatory boys with DMD aged 10–18 years. The main objective was to assess safety and tolerability of ATL1102. Secondary objectives included the effect of ATL1102 on lymphocyte numbers in the blood, functional changes in upper limb function as assessed by Performance of Upper Limb test (PUL 2.0) and upper limb strength using MyoGrip and MyoPinch compared to baseline. Results: Eight out of nine participants were on a stable dose of corticosteroids. ATL1102 was generally safe and well tolerated. No serious adverse events were reported. There were no participant withdrawals from the study. The most commonly reported adverse events were injection site erythema and skin discoloration. There was no statistically significant change in lymphocyte count from baseline to week 8, 12 or 24 of dosing however, the CD3+CD49d+ T lymphocytes were statistically significantly higher at week 28 compared to week 24, four weeks past the last dose (mean change 0.40x109/L 95%CI 0.05, 0.74; p = 0.030). Functional muscle strength, as measured by the PUL2.0, EK2 and Myoset grip and pinch measures, and MRI fat fraction of the forearm muscles were stable throughout the trial period. Conclusion: ATL1102, a novel antisense drug being developed for the treatment of inflammation that exacerbates muscle fibre damage in DMD, appears to be safe and well tolerated in non-ambulant boys with DMD. The apparent stabilisation observed on multiple muscle disease progression parameters assessed over the study duration support the continued development of ATL1102 for the treatment of DMD. Trial registration: Clinical Trial Registration. Australian New Zealand Clinical Trials Registry Number: ACTRN12618000970246. [ABSTRACT FROM AUTHOR]

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8—an anti-Ly6G antibody, or RB6-8C5—an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers—CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections. [ABSTRACT FROM AUTHOR]

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells. Author summary: Functional memory T cells are critical to ultimately control chronic viral infection. We show that cytomegalovirus (CMV) induces a population of unique memory CD4 T cells that expand late during infection and resolve viral persistence much better than conventional T memory cells. Mutation of the genomic CMV epitope recognized by these T cells results in markedly enhanced persistent replication, and introduction of the epitope into a CMV strain that lacks it induces much quicker resolution of persistence. When induced via vaccination, we show these T cells provide significantly enhanced protection against acute viral challenge. Together our study identifies a novel population of CD4 T cells that utilize distinct effector function(s) to rapidly resolve persistent viral replication. [ABSTRACT FROM AUTHOR]

It has been well established that cancer cells can evade immune surveillance by mutating themselves. Understanding genetic alterations in cancer cells that contribute to immune regulation could lead to better immunotherapy patient stratification and identification of novel immune-oncology (IO) targets. In this report, we describe our effort of genome-wide association analyses across 22 TCGA cancer types to explore the associations between genetic alterations in cancer cells and 76 immune traits. Results showed that the tumor microenvironment (TME) is shaped by different gene mutations in different cancer types. Out of the key genes that drive multiple immune traits, top hit KEAP1 in lung adenocarcinoma (LUAD) was selected for validation. It was found that KEAP1 mutations can explain more than 10% of the variance for multiple immune traits in LUAD. Using public scRNA-seq data, further analysis confirmed that KEAP1 mutations activate the NRF2 pathway and promote a suppressive TME. The activation of the NRF2 pathway is negatively correlated with lower T cell infiltration and higher T cell exhaustion. Meanwhile, several immune check point genes, such as CD274 (PD-L1), are highly expressed in NRF2-activated cancer cells. By integrating multiple RNA-seq data, a NRF2 gene signature was curated, which predicts anti-PD1 therapy response better than CD274 gene alone in a mixed cohort of different subtypes of non-small cell lung cancer (NSCLC) including LUAD, highlighting the important role of KEAP1-NRF2 axis in shaping the TME in NSCLC. Finally, a list of overexpressed ligands in NRF2 pathway activated cancer cells were identified and could potentially be targeted for TME remodeling in LUAD. Author summary: Recent studies have found that some genetic changes help cancer cells to evade the immune surveillance. To systematically understand the impact of cancer cell genetic alterations to immune regulation, we examined 76 immune traits across 22 cancer types. The tumor microenvironment (TME), crucial for cancer development, varies based on gene mutations in different cancers. Notably, the KEAP1 gene in lung adenocarcinoma (LUAD) emerged as a key player, explaining over 10% of immune trait variations. KEAP1 mutations activate the NRF2 pathway, creating a suppressive TME in LUAD with lower T cell infiltration and heightened T cell exhaustion. Additionally, genes such as CD274 (PD-L1), associated with immune checkpoints, exhibit high expression in NRF2-activated cancer cells. By developing a NRF2 gene signature, we found that it more effectively predicts anti-PD1 therapy responses than CD274 alone in non-small cell lung cancer. Lastly, we identified ligands overexpressed in NRF2-activated cancer cells, suggesting potential targets for reshaping the LUAD microenvironment. In essence, understanding these genetic interactions helps improve lung cancer treatment and enhance the efficacy of immunotherapy. [ABSTRACT FROM AUTHOR]

Stable microbial colonization of the skin depends on tight control by the host immune system. The lipid-dependent yeast Malassezia typically colonizes skin as a harmless commensal and is subject to host type 17 immunosurveillance, but this fungus has also been associated with diverse skin pathologies in both humans and animals. Using a murine model of Malassezia exposure, we show that Vγ4+ dermal γδ T cells expand rapidly and are the major source of IL-17A mediating fungal control in colonized skin. A pool of memory-like Malassezia-responsive Vγ4+ T cells persisted in the skin, were enriched in draining lymph nodes even after fungal clearance, and were protective upon fungal re-exposure up to several weeks later. Induction of γδT17 immunity depended on IL-23 and IL-1 family cytokine signalling, whereas Toll-like and C-type lectin receptors were dispensable. Furthermore, Vγ4+ T cells from Malassezia-exposed hosts were able to respond directly and selectively to Malassezia-derived ligands, independently of antigen-presenting host cells. The fungal moieties detected were shared across diverse species of the Malassezia genus, but not conserved in other Basidiomycota or Ascomycota. These data provide novel mechanistic insight into the induction and maintenance of type 17 immunosurveillance of skin commensal colonization that has significant implications for cutaneous health. Author summary: Malassezia is the most abundant fungus living on our skin and is usually harmless, but this microbe has also been shown to play a role in pathological conditions such as eczema and dermatitis. Here, we investigated how a population of Vγ4+ γδ T cells protect mouse skin against fungal overgrowth. While generally considered part of the innate immune system, we found that γδ T cells were maintained long after Malassezia was cleared from the skin of experimentally-infected animals. These cells displayed memory-like features and were highly efficient at fighting the fungus after a secondary challenge. We observed that classic fungal pattern recognition receptors were not involved, and that antigen-presenting cells were not required to generate Malassezia-protective γδ T cells. Finally, we confirmed that murine γδ T cells can directly recognise Malassezia and that the Malassezia-derived structure that triggers these responses was not conserved among other fungi. Our results highlight for the first time an important role for γδ T cells in preventing uncontrolled growth of the most abundant skin fungus. These findings have implications for several Malassezia-associated pathologies including eczema, dermatitis, and potentially even cancer. [ABSTRACT FROM AUTHOR]

This article explores the relationship between certain cancer therapies and the risk of fungal infections. It focuses on three types of therapies: BTK inhibitors, PI3K inhibitors, and CD-52 monoclonal antibodies. These therapies have been found to increase susceptibility to invasive fungal infections (IFIs) by impairing the immune response or depleting T cells. The article suggests that repurposing existing cancer therapeutics could be a strategy to address the limited options for antifungal treatments. It also discusses the potential of using targeted therapies, immunotherapy, and interferon-gamma in combination with antifungals to improve outcomes. Further research is needed to evaluate the effectiveness of these approaches. [Extracted from the article]

Immunotherapy is changing the Head and Neck Squamous Cell Carcinoma (HNSCC) landscape and improving outcomes for patients with recurrent or metastatic HNSCC. A deeper understanding of the tumor microenvironment (TME) is required in light of the limitations of patients' responses to immunotherapy. Here, we aimed to examine how Nivolumab affects infiltrating Tregs in the HNSCC TME. We used single-cell RNA sequencing data from eight tissues isolated from four HNSCC donors before and after Nivolumab treatment. Interestingly, the study found that Treg counts and suppressive activity increased following Nivolumab therapy. We also discovered that changes in the CD44-SSP1 axis, NKG2C/D-HLA-E axis, and KRAS signaling may have contributed to the increase in Treg numbers. Furthermore, our study suggests that decreasing the activity of the KRAS and Notch signaling pathways, and increasing FOXP3, CTLA-4, LAG-3, and GZMA expression, may be mechanisms that enhance the killing and suppressive capacity of Tregs. Additionally, the result of pseudo-temporal analysis of the HNSCC TME indicated that after Nivolumab therapy, the expression of certain inhibitory immune checkpoints including TIGIT, ENTPD1, and CD276 and LY9, were decreased in Tregs, while LAG-3 showed an increased expression level. The study also found that Tregs had a dense communication network with cluster two, and that certain ligand-receptor pairs, including SPP1/CD44, HLA-E/KLRC2, HLA-E/KLRK1, ANXA1/FPR3, and CXCL9/FCGR2A, had notable changes after the therapy. These changes in gene expression and cell interactions may have implications for the role of Tregs in the TME and in response to Nivolumab therapy. [ABSTRACT FROM AUTHOR]

Engineered T cells hold great promise to become part of an effective HIV cure strategy, but it is currently unclear how best to redirect T cells to target HIV. To gain insight, we generated engineered T cells using lentiviral vectors encoding one of three distinct HIV-specific T cell receptors (TCRs) or a previously optimized HIV-targeting chimeric antigen receptor (CAR) and compared their functional capabilities. All engineered T cells had robust, antigen-specific polyfunctional cytokine profiles when mixed with artificial antigen-presenting cells. However, only the CAR T cells could potently control HIV replication. TCR affinity enhancement did not augment HIV control but did allow TCR T cells to recognize common HIV escape variants. Interestingly, either altering Nef activity or adding additional target epitopes into the HIV genome bolstered TCR T cell anti-HIV activity, but CAR T cells remained superior in their ability to control HIV replication. To better understand why CAR T cells control HIV replication better than TCR T cells, we performed a time course to determine when HIV-specific T cells were first able to activate Caspase 3 in HIV-infected targets. We demonstrated that CAR T cells recognized and killed HIV-infected targets more rapidly than TCR T cells, which correlates with their ability to control HIV replication. These studies suggest that the speed of target recognition and killing is a key determinant of whether engineered T cell therapies will be effective against infectious diseases. Author summary: Infusion of HIV-specific engineered T cells is a promising approach to eliminate HIV infected cells that reverse latency and begin to replicate HIV after ART withdrawal. However, it is unclear whether chimeric antigen receptors (CAR) or TCRs are the best approach to generate HIV-specific T cells for adoptive T cell therapy. We generated CD8 T cells expressing one of three HIV-specific TCRs (TCR-T) or a previously optimized HIV-specific CAR and interrogated which population controlled HIV replication more effectively. HIV-specific CAR T cells were far superior to any of the TCR-T cells. Increasing TCR affinity or interfering with the ability of Nef to downregulate MHC class I had modest effects on the ability TCR-T cells to control HIV. Inserting additional cognate epitopes within the HIV genome did improve the ability of TCR-T cells to control HIV but HIV CAR T cells were still superior. The ability to control HIV replication best correlated with the ability of engineered T cell to rapidly induce cell death in HIV-infected T cells, suggesting that the speed of recognition and initiation of T cell mediated killing is a key determinant on whether engineered T cells can control viral infections. [ABSTRACT FROM AUTHOR]