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Background: The T cell-mediated delayed-type hypersensitivity (DTH) response is critical for elucidating cellular immune mechanisms, especially the role of memory T cells upon antigen re-exposure. This study aimed to investigate the specific effects of the immunosuppressive drugs Cyclophosphamide (CY) and Dexamethasone (DEX) on intestinal immunity and microbiota in a DTH mouse model, contributing to a more nuanced understanding of their immunomodulatory mechanisms. Methods: Female BALB/c mice were sensitized to 2,4-dinitrofluorobenzene (DNFB) and randomly allocated into control, CY, and DEX groups. The impact of CY and DEX on immune function was assessed through measurement of thymus and spleen indices, lymphocyte proliferation in mesenteric lymph nodes (MLNs) using MTT assay, and flow cytometric analysis of T cell subsets and TCR expression. Intestinal secretory IgA (sIgA) was quantified by ELISA, and gut microbiota diversity was evaluated using 16S rRNA gene sequencing. Results: CY and DEX significantly reduced the immune function in DNFB-induced sensitized mice, as indicated by decreased thymus and spleen indices, MLN enlargement, intestinal sIgA content, and ear swelling degree. Flow cytometry revealed that CY increased the proportion of total CD3+ T cells but reduced CD3+CD69+ activated T cells and CD3+TCRγ/δ+ T cells, while DEX increased CD3+CD4+ helper T cells. Both drugs induced distinct changes in gut microbiota diversity and structure, with CY enhancing α diversity and DEX reducing it. Conclusions: The study demonstrates that CY and DEX have distinct regulatory effects on the immune organ index, distribution of T cell subsets, and diversity and structure of gut microbiota on DTH-induced immune responses mice, suggesting their differential influence on intestinal mucosal immunity. These findings have implications for the development of targeted immunotherapies and understanding the interplay between immunosuppressive drugs and gut microbiota. [ABSTRACT FROM AUTHOR]

Background: The relationship between dysregulation of the immune system and reproductive health, particularly in the context of abortion, is an area of critical research. Identifying the immunological factors that contribute to abortion could provide valuable insights into its prevention and management. Methods: This study used bidirectional two-sample Mendelian Randomization (MR) approach to evaluate the causal link between 731 immune cell features and the risk of abortion. The study analyzed GWAS data from 257,561 Europeans, including 7,069 cases and 250,492 controls, by utilizing genetic variation as instrumental variables. The immune phenotypes included several cell types, including B cells, T cells, TBNK cells, Treg cells, and monocytes. These were analyzed using the 'TwoSampleMR' package in R software. Results: The study identified 34 immune phenotypes that have a significant causal relationship with abortion risk. Notably, Results from the B cell group showed a positive correlation between abortion and certain phenotypes, including Unsw mem %B cell, PB/PC %B cell, IgD+ CD24+ %B cell and Naive-mature B cell %lymphocyte. In the T cell group, certain maturation stages such as Naive CD8br %T cell and CD4 on CD45RA+ CD4+ exhibited negative causal links, whereas CCR7 on naive CD8br showed a positive association. The group of Treg cells showed both positive and negative causal relationships with abortion, highlighting the complexity of immune regulation in reproductive health. Conclusions: This study reflects the causal relationship between different subtypes of different immune cells and abortion. The results underscore the importance of the immune system in reproductive health and suggest potential therapeutic interventions targeting these immunological pathways. [ABSTRACT FROM AUTHOR]

There is considerable evidence that the immune system plays a role in hypertension, however this role is not fully characterized. Our previous studies demonstrated that mesenteric perivascular adipose tissue (mPVAT) harbors a large T cell population, which is a cell type identified as contributing to hypertension. In the present study, we tested the hypothesis that soluble mediators in mPVAT influence T cell function just prior to the development of hypertension. Toward this end, we utilized a unique model of hypertension in which Dahl S rats on a high fat (HF) diet develop hypertension. We found that conditioned media (CM) from mPVAT from healthy Dahl S rats on control diet buffers T cell activation, however, mPVAT-CM from Dahl S rats on a HF diet markedly increased inflammatory cytokine induction (IFNγ, GM-CSF and IL-17a) by activated T cells. These cytokines are known to promote activation of macrophages and neutrophils, among other effects. Conversely, the anti-inflammatory cytokine, IL-10, was not different between the groups, suggesting the effect is selective for inflammatory cytokines. Furthermore, we conducted bulk RNA-seq on activated T cells cultured in mPVAT-CM from Dahl S rats on either control (CTL) or HF diet for 10 weeks. In accordance with the cytokine analysis, mPVAT-CM from HF diet-fed rats significantly upregulated many genes associated with IFNγ/IL-17 induction, whereas Th2/Treg-associated genes were downregulated. Taken together, these data strongly suggest soluble mediators from mPVAT influence T cell inflammatory status and may promote Th1/Th17 differentiation preceding the development of hypertension triggered by HF diet. [ABSTRACT FROM AUTHOR]

Antiretroviral treatment of HIV infected individuals cannot eliminate the HIV reservoir and immune control of HIV is rarely seen upon treatment interruption. In long-term non-progressors (LTNP), an effective CD8 T cell response is thought to contribute to be immune control of HIV. Here we studied the transcriptional profile of virus specific CD8 T cells during the asymptomatic phase of disease, to gain molecular insights in CD8 T cell functionality in HIV progressors and different groups of LTNP: HLA-B*57 LTNP, non-HLA-B*57 LTNP and individuals carrying the MAVS minor genotype (rs7262903/rs7269320). Principal component analysis revealed distinct overall transcriptional profiles between the groups. The transcription profile of HIV-specific CD8 T cells of LTNP groups was associated with increased cytokine/IL-12 signaling and protein/RNA metabolism pathways, indicating an increased CD8 T cell functionality. Although the transcription profile of CMV-specific CD8 T cells differed from that of HIV-specific CD8 T cells, with mainly an upregulation of gene expression in progressors, similar affected pathways were identified. Moreover, CMV-specific CD8 T cells from progressors showed increased expression of genes related to effector functions and suggests recent antigen exposure. Our data shows that changes in cytokine signaling and the energy demanding RNA and protein metabolism are related to CD8 T cell dysfunction, which may indicate that mitochondrial dysfunction is an important driver of T cell dysfunctionality during chronic HIV infection. Indeed, improvement of mitochondrial function by IL-12 and mitoTempo treatment, enhanced in vitro IFNγ release by PBMC from PWH upon HIV gag and CMV pp65 peptide stimulation. Our study provides new insights into the molecular pathways associated with CD8 T cell mediated immune control of chronic HIV infection which is important for the design of novel treatment strategies to restore or improve the HIV-specific immune response. [ABSTRACT FROM AUTHOR]

B-cell lymphoma, clinically, comprises a heterogeneous group of malignancies that encompass various subtypes. CD20 is an optimal target for therapeutic antibodies in B-cell lymphoma immunotherapy since approximately 90% of B-cell malignancies typically exhibit CD20 expression on their surface, while its presence is limited in normal tissues. In this study, we have developed a series of novel non-IgG-like T cell-dependent bispecific antibodies by constructing Fab-FabCH3, referred to as Tandem Antigen-binding Fragment 002 (TFAB002), which specifically target CD20 for the treatment of malignant B-cell lymphoma. TFAB002s display strong binding affinity with CD20 and moderate binding affinity with CD3, thereby triggering target-specific T-cell activation, cytokine release, and tumor cell lysis in vitro. Furthermore, TFAB002s exhibit potent cytotoxicity against B-cell malignancies that express varying levels of CD20. Besides, the TFAB002s show potent pharmacodynamic activity in vivo in the WIL2-S cells CDX mouse model. Collectively, these results underscore the potential of TFAB002s as a highly promising therapeutic approach for selectively depleting CD20-positive B cells, thereby warranting further clinical evaluation as a viable treatment option for CD20-expressing B-cell malignancies. [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]

Adenosine to inosine (A-to-I) RNA editing by ADAR1 has been implicated in maintaining self-tolerance, preventing autoimmunity, and mediating antiviral immunity. Foreign viral double-stranded RNA triggers rapid interferon response and activates ADAR1 in the host immune system. Emerging data points to a role of ADAR1 A-to-I editing in the inflammatory response associated with severe COVID-19 disease. We identify A-to-I editing events within human whole transcriptome data from SARS-CoV-2 infected individuals, non-infected individuals, and individuals with other viral illnesses from nasopharyngeal swabs. High levels of RNA editing in host cells are associated with low SARS-CoV-2 viral load (p = 9.27 E-06), suggesting an inhibitory effect of ADAR1 on viral infection. Additionally, we find differentially expressed genes associated with RNA-modifications and interferon response. Single cell RNA-sequencing analysis of SARS-CoV-2 infected nasopharyngeal swabs reveals that cytotoxic CD8 T cells upregulate ADAR1 in COVID-19 positive samples (p = 0.0269). We further reveal ADAR1 expression increases with CD4 and CD8 T cell activation, and knockdown of ADAR1 leads to apoptosis and aberrant IL-2 secretion. Together, our data suggests A-to-I RNA editing is required to maintain healthy homeostasis of activated T cells to combat SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

While computational epitope prediction methods have found broad application, their use, specifically in allergy-related contexts, remains relatively less explored. This study benchmarks several publicly available epitope prediction tools, focusing on the allergenic IgE and T-cell epitopes of Fel d 1, an extensively studied allergen. Using a variety of tools accessible via the Immune Epitope Database (IEDB) and other resources, we evaluate their ability to identify the known linear IgE and T-cell epitopes of Fel d 1. Our results show a limited effectiveness for B-cell epitope prediction methods, with most performing only marginally better than random selection. We also explored the general predictive abilities on other allergens, and the results were largely random. When predicting T-cell epitopes, ProPred successfully identified all known Fel d 1 T-cell epitopes, whereas the IEDB approach missed two known epitopes and demonstrated a tendency to over-predict. However, when applied to a larger test set, both methods performed only slightly better than random selection. Our findings show the limitations of current computational epitope prediction methods in accurately identifying allergenic epitopes, emphasizing the need for methodological advancements in allergen research. [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]

The inability of existing vaccines to cope with the mutation rate has highlighted the need for effective preventative strategies for COVID-19. Through the secretion of immunoglobulin A, mucosal delivery of vaccines can effectively stimulate mucosal immunity for better protection against SARS-CoV-2 infection. In this study, various immunoinformatic tools were used to design a multi-epitope oral vaccine against SARS-CoV-2 based on its receptor-binding domain (RBD) and heptad repeat (HR) domains. T and B lymphocyte epitopes were initially predicted from the RBD and HR domains of SARS-CoV-2, and potential antigenic, immunogenic, non-allergenic, and non-toxic epitopes were identified. Epitopes that are highly conserved and have no significant similarity to human proteome were selected. The epitopes were joined with appropriate linkers, and an adjuvant was added to enhance the vaccine efficacy. The vaccine 3D structure constructs were docked with toll-like receptor 4 (TLR-4) and TLR1-TLR2, and the binding affinity was calculated. The designed multi-epitope vaccine construct (MEVC) consisted of 33 antigenic T and B lymphocyte epitopes. The results of molecular dockings and free binding energies confirmed that the MEVC effectively binds to TLR molecules, and the complexes were stable. The results suggested that the designed MEVC is a potentially safe and effective oral vaccine against SARS-CoV-2. This in silico study presents a novel approach for creating an oral multi-epitope vaccine against the rapidly evolving SARS-CoV-2 variants. These findings offer valuable insights for developing an effective strategy to combat COVID-19. Further preclinical and clinical studies are required to confirm the efficacy of the MEVC vaccine. [ABSTRACT FROM AUTHOR]

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes. [ABSTRACT FROM AUTHOR]

Cancer control and treatment remain a significant challenge in cancer therapy and recently immune checkpoints has considered as a novel treatment strategy to develop anti-cancer drugs. Many cancer types use the immune checkpoints and its ligand, PD-1/PD-L1 pathway, to evade detection and destruction by the immune system, which is associated with altered effector function of PD-1 and PD-L1 overexpression on cancer cells to deactivate T cells. In recent years, mAbs have been employed to block immune checkpoints, therefore normalization of the anti-tumor response has enabled the scientists to develop novel biopharmaceuticals. In vivo affinity maturation of antibodies in targeted therapy has sometimes failed, and current experimental methods cannot accommodate the accurate structural details of protein-protein interactions. Therefore, determining favorable binding sites on the protein surface for modulator design of these interactions is a major challenge. In this study, we used the in silico methods to identify favorable binding sites on the PD-1 and PD-L1 and to optimize mAb variants on a large scale. At first, all the binding areas on PD-1 and PD-L1 have been identified. Then, using the RosettaDesign protocol, thousands of antibodies have been generated for 11 different regions on PD-1 and PD-L1 and then the designs with higher stability, affinity, and shape complementarity were selected. Next, molecular dynamics simulations and MM-PBSA analysis were employed to understand the dynamic, structural features of the complexes and measure the binding affinity of the final designs. Our results suggest that binding sites 1, 3 and 6 on PD-1 and binding sites 9 and 11 on PD-L1 can be regarded as the most appropriate sites for the inhibition of PD-1-PD-L1 interaction by the designed antibodies. This study provides comprehensive information regarding the potential binding epitopes on PD-1 which could be considered as hotspots for designing potential biopharmaceuticals. We also showed that mutations in the CDRs regions will rearrange the interaction pattern between the designed antibodies and targets (PD-1 and PD-L1) with improved affinity to effectively inhibit protein-protein interaction and block the immune checkpoint. [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]

Objective: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies. Methods: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10. Results: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases. Conclusions: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk. [ABSTRACT FROM AUTHOR]

Recurrent implantation failure (RIF) presents a significant clinical challenge due to the lack of established diagnostic and therapeutic guidelines. Emerging evidence underscores the crucial role of competitive endogenous RNA (ceRNA) regulatory networks in non-cancerous female reproductive disorders, yet the intricacies and operational characteristics of these networks in RIF are not fully understood. This study aims to demystify the ceRNA regulatory network and identify potential biomarkers for its diagnosis. We analyzed expression profiles of three RNA types (long noncoding RNAs [lncRNAs], microRNAs [miRNAs], and mRNAs) sourced from the GEO database, leading to the identification of the H19-hsa-miR-301a-3p-GAS1 ceRNA network. This network demonstrates significant diagnostic relevance for RIF. Notably, the H19/GAS1 axis within this ceRNA network, identified through correlation analysis, emerged as a promising diagnostic marker, as evidenced by operating receiver operator characteristic (ROC) curve analysis. Further investigation into the binding potential of miR-301a-3p with H19 and GAS1 revealed a close association of these genes with endometrial disorders and embryo loss, as per the Comparative Toxicogenomics Database. Additionally, our immune infiltration analysis revealed a lower proportion of T cells gamma delta (γδ) in RIF, along with distinct differences in the expression of immune cell type-specific markers between fertile patients and those with RIF. We also observed a correlation between aberrant expression of H19/GAS1 and these immune markers, suggesting that the H19/GAS1 axis might play a role in modifying the immune microenvironment, contributing to the pathogenesis of RIF. In conclusion, the ceRNA-based H19/GAS1 axis holds promise as a novel diagnostic biomarker for RIF, potentially enhancing our understanding of its underlying mechanisms and improving the success rates of implantation. [ABSTRACT FROM AUTHOR]

Salmonella infections pose a significant global public health concern due to the substantial expenses associated with monitoring, preventing, and treating the infection. In this study, we explored the core proteome of Salmonella to design a multi-epitope vaccine through Subtractive Proteomics and immunoinformatics approaches. A total of 2395 core proteins were curated from 30 different isolates of Salmonella (strain NZ CP014051 was taken as reference). Utilizing the subtractive proteomics approach on the Salmonella core proteome, Curlin major subunit A (CsgA) was selected as the vaccine candidate. csgA is a conserved gene that is related to biofilm formation. Immunodominant B and T cell epitopes from CsgA were predicted using numerous immunoinformatics tools. T lymphocyte epitopes had adequate population coverage and their corresponding MHC alleles showed significant binding scores after peptide-protein based molecular docking. Afterward, a multi-epitope vaccine was constructed with peptide linkers and Human Beta Defensin-2 (as an adjuvant). The vaccine could be highly antigenic, non-toxic, non-allergic, and have suitable physicochemical properties. Additionally, Molecular Dynamics Simulation and Immune Simulation demonstrated that the vaccine can bind with Toll Like Receptor 4 and elicit a robust immune response. Using in vitro, in vivo, and clinical trials, our findings could yield a Pan-Salmonella vaccine that might provide protection against various Salmonella species. [ABSTRACT FROM AUTHOR]

Introduction: Pancreatic adenocarcinoma is an extremely aggressive neoplasm, with many challenges to be overcome in order to achieve a truly effective treatment. It is characterized by a mostly immunosuppressed environment, with dysfunctional immune cells and active immunoinhibitory pathways that favor tumor evasion and progression. Thus, the study and understanding of the tumor microenvironment and the various cells subtypes and their functional capacities are essential to achieve more effective treatments, especially with the use of new immunotherapeutics. Methods: Seventy cases of pancreatic adenocarcinoma divided into two groups 43 with resectable disease and 27 with unresectable disease were analyzed using immunohistochemical methods regarding the expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), and human leukocyte antigen G (HLA-G) molecules as well as the populations of CD4+ and CD8+ T lymphocytes, regulatory T cells (Tregs), and M2 macrophages (MM2). Several statistical tests, including multivariate analyses, were performed to examine how those immune cells and immunoinhibitory molecules impact the evolution and prognosis of pancreatic adenocarcinoma. Results: CD8+ T lymphocytes and M2 macrophages predominated in the group operated on, and PD-L2 expression predominated in the unresectable group. PD-L2 was associated with T stage, lymph node metastasis, and clinical staging, while in survival analysis, PD-L2 and HLA-G were associated with a shorter survival. In the inoperable cases, Tregs cells, MM2, PD-L1, PD-L2, and HLA-G were positively correlated. Conclusions: PD-L2 and HLA-G expression correlated with worse survival in the cases studied. Tumor microenvironment was characterized by a tolerant and immunosuppressed pattern, mainly in unresectable lesions, where a broad positive influence was observed between immunoinhibitory cells and immune checkpoint proteins expressed by tumor cells. [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]

The development of a cancer vaccine has become an essential focus in the field of medical biotechnology and immunology. In our study, the NY-SAR-35 cancer/testis antigen was targeted to design a novel peptide vaccine using bioinformatics tools, and BALB/c mice were used to evaluate the vaccine's immunological function. This evaluation involved assessing peptide-specific IgG levels in the serum via ELISA and measuring the levels of IFN-γ, IL-4, and granzyme B in the supernatant of cultured splenocytes. The final vaccine construct consisted of two T lymphocyte epitopes linked by the AAY linker. This construct displayed high antigenicity, non-allergenicity, non-toxicity, stability, and ability to induce IFN-γ and IL-4. It showed stable dynamics with both human MHC-I and II molecules, as well as mouse MHC-II molecules, and revealed strong Van der Waals and electrostatic energies. Emulsifying our peptide vaccine in incomplete Freund's adjuvant resulted in a remarkable increase in the levels of IgG. The splenocytes of mice that received the combination of peptide and adjuvant displayed a noteworthy increase in IFN-γ, IL-4, and granzyme B secretion. Additionally, their lymphocytes exhibited higher proliferation rates compared to the control group. Our data demonstrated that our vaccine could stimulate a robust immune response, making it a promising candidate for cancer prevention. However, clinical trials are necessary to assess its efficacy in humans. [ABSTRACT FROM AUTHOR]

Inhibiting the functional role of negative regulators in immune cells is an effective approach for developing immunotherapies. The serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1) involved in the T-cell receptor signaling pathway attenuates T-cell activation by inducing the degradation of SLP-76 through its phosphorylation at Ser-376, reducing the immune response. Interestingly, several studies have shown that the genetic ablation or pharmacological inhibition of HPK1 kinase activity improves the immune response to cancers by enhancing T-cell activation and cytokine production; therefore, HPK1 could be a promising druggable target for T-cell-based cancer immunotherapy. To increase the immune response against cancer cells, we designed and synthesized KHK-6 and evaluated its cellular activity to inhibit HPK1 and enhance T-cell activation. KHK-6 inhibited HPK1 kinase activity with an IC50 value of 20 nM and CD3/CD28-induced phosphorylation of SLP-76 at Ser-376 Moreover, KHK-6 significantly enhanced CD3/CD28-induced production of cytokines; proportion of CD4+ and CD8+ T cells that expressed CD69, CD25, and HLA-DR markers; and T-cell-mediated killing activity of SKOV3 and A549 cells. In conclusion, KHK-6 is a novel ATP-competitive HPK1 inhibitor that blocks the phosphorylation of HPK1 downstream of SLP-76, enhancing the functional activation of T cells. In summary, our study showed the usefulness of KHK-6 in the drug discovery for the HPK1-inhibiting immunotherapy. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea (OSA) is characterized by cyclic normoxic and hypoxic conditions (intermittent hypoxia, IH) induced by the repeated closure of the upper-airway respiratory tract. As a pathomechanism of OSA, IH results in various comorbidities via chronic inflammation and related pathways. However, the role of other inflammatory cells, such as lymphocytes, has not been well-explored. This study aimed to examine the effects of IH on the distribution and balance of T cell subsets and other related cytokines, and mechanisms in the immune system. We modified OSA mouse model (male C57BL/6N male) using our customized chamber that controls specific sleep and oxygenic cycles. To induce hypoxia, the IH group was repeatedly exposed to 5% O2 and 21% O2 lasting for 120 s each for 7 h daily for 4 weeks. Mice were then subjected to a recovery period of 4 weeks, in which IH stimulation was ceased. T cells and related cytokines were analyzed using flow cytometry and immunohistochemistry. Compared with the control group, the IH group had significantly lower levels of CD4+CD25+Foxp3+ regulatory T cells but higher levels of Th 17, IL-4, HIF-1, and inflammatory cytokines. After the recovery period, these altered changes in the immune cells were recovered, and we found no significant difference in their levels between the control and recovery groups. This study revealed that the Th17/Treg ratio is increased by intermittent hypoxia, and this imbalance can explain immune-related diseases, including recently reported allergies, autoimmune, and even cancer diseases, arising from OSA. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) and glioblastoma (GBM) are CNS diseases in whose development and progression immune privilege is intimately important, but in a relatively opposite manner. Maintenance and strengthening of immune privilege have been shown to be an important mechanism in glioblastoma immune evasion, while the breakdown of immune privilege leads to MS initiation and exacerbation. We hypothesize that molecular signaling pathways can be oppositely regulated in peripheral blood CD8+ T cells of MS and glioblastoma patients at a transcriptional level. We analyzed publicly available data of the peripheral blood CD8+ T cell MS vs. control (MSvsCTRL) and GBM vs. control (GBMvsCTRL) differentially expressed gene (DEG) contrasts with Qiagen's Ingenuity pathway analysis software (IPA). We have identified sphingolipid signaling pathway which was significantly downregulated in the GBMvsCTRL and upregulated in the MSvsCTRL. As the pathway is important for the CD8+ T lymphocytes CNS infiltration, this result is in line with our previously stated hypothesis. Comparing publicly available lists of differentially expressed serum exosomal miRNAs from MSvsCTRL and GBMvsCTRL contrasts, we have identified that hsa-miR-182-5p has the greatest potential effect on sphingolipid signaling regarding the number of regulated DEGs in the GBMvsCTRL contrast, while not being able to find any relevant potential sphingolipid signaling target transcripts in the MSvsCTRL contrast. We conclude that the sphingolipid signaling pathway is a top oppositely regulated pathway in peripheral blood CD8+ T cells from GBM and MS, and might be crucial for the differences in CNS immune privilege maintenance of investigated diseases, but further experimental research is necessary. [ABSTRACT FROM AUTHOR]

Signal regulatory protein alpha (SIRPα) is an immune inhibitory receptor on myeloid cells including macrophages and dendritic cells, which binds to CD47, a ubiquitous self-associated molecule. SIRPα-CD47 interaction is exploited by cancer cells to suppress anti-tumor activity of myeloid cells, therefore emerging as a novel immune checkpoint for cancer immunotherapy. In blood cancer, several SIRPα-CD47 blockers have shown encouraging monotherapy activity. However, the anti-tumor activity of SIRPα-CD47 blockers in solid tumors seems limited, suggesting the need for combination therapies to fully exploit the myeloid immune checkpoint in solid tumors. Here we tested whether combination of SIRPα-CD47 blocker with antibody-drug conjugate bearing a topoisomerase I inhibitor DXd (DXd-ADC) would enhance anti-tumor activity in solid tumors. To this end, DS-1103a, a newly developed anti-human SIRPα antibody (Ab), was assessed for the potential combination benefit with datopotamab deruxtecan (Dato-DXd) and trastuzumab deruxtecan (T-DXd), DXd-ADCs targeting human trophoblast cell-surface antigen 2 and human epidermal growth factor receptor 2, respectively. DS-1103a inhibited SIRPα-CD47 interaction and enhanced antibody-dependent cellular phagocytosis of Dato-DXd and T-DXd against human cancer cells. In a whole cancer cell vaccination model, vaccination with DXd-treated cancer cells led to activation of tumor-specific T cells when combined with an anti-mouse SIRPα (anti-mSIRPα) Ab, implying the benefit of combining DXd-ADCs with anti-SIRPα Ab on anti-tumor immunity. Furthermore, in syngeneic mouse models, both Dato-DXd and T-DXd combination with anti-mSIRPα Ab showed stronger anti-tumor activity over the monotherapies. Taken together, this study provides a preclinical rationale of novel therapies for solid tumors combining SIRPα-CD47 blockers with DXd-ADCs. [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]

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1− CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1−E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection. [ABSTRACT FROM AUTHOR]

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings. [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]

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte–non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics. [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]

In the monitoring of human Toxoplasma gondii infection, it is crucial to confirm the development of a specific Th1/Th17 immune response memory. The use of a simple, specific, and sensitive assay to follow the T-cell activation is thus required. Current protocols are not always specific as stimulation with peptides is Human Leukocyte Antigen (HLA)-dependent, while stimulation with total-lysis antigens tends to stimulate seronegative donors resulting to false positives. Here, an improved ELISPOT protocol is reported, using peripheral blood mononuclear cells (PBMC) of T.gondii-infected donors, incubated with the inactivated parasite. The results showed that, contrary to standard protocols, a pre-incubation step at high cell density in presence of the inactivated parasite allowed a specific Th1/Th17 response with the secretion of IFN-γ, IL-2, IL-12 and IL-17 cytokines. This protocol allows to evaluate precisely the immune response after a T.gondii infection. [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]

Background and aims: Infection is a serious complication in patients with cirrhosis. Mucosal-associated invariant T (MAIT) cells are involved in the immune defense against infections and known to be impaired in several chronic conditions, including cirrhosis. Here, we evaluated if MAIT cell levels in peripheral blood are associated with risk of bacterial infections in patients with cirrhosis. Methods: Patients with cirrhosis seen at the Karolinska University Hospital, Stockholm, Sweden, between 2016 and 2019 were included. Levels of MAIT cells in peripheral blood were determined using flow cytometry. Baseline and follow-up data after at least two years of follow-up were collected by chart review for the primary outcome (bacterial infection) and secondary outcomes (decompensation and death). Competing risk and Cox regression were performed. Results: We included 106 patients with cirrhosis. The median MAIT cells fraction in the circulation was 0.8% in cirrhosis compared to 6.1% in healthy controls. In contrast to our hypothesis, we found an association in the adjusted analysis between relatively preserved MAIT cell levels, and a slightly higher risk to develop bacterial infections (adjusted subdistribution hazard ratio (aSHR) 1.15 (95%CI = 1.01–1.31). However, MAIT cell levels were not associated with the risk of hepatic decompensation (aSHR 1.19 (95%CI = 0.91–1.56)) nor with death (adjusted hazard ratio 1.10 (95%CI = 0.97–1.22)). Conclusions: Relatively preserved MAIT cell levels in blood of patients with cirrhosis were associated with a somewhat higher risk of bacterial infections. The clinical relevance of this might not be strong. MAIT cells might however be an interesting biomarker to explore in future studies. [ABSTRACT FROM AUTHOR]

A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/β receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 μg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses. [ABSTRACT FROM AUTHOR]

Background: Canonical α/β T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell−mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/β TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. Methods: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. Results: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. Conclusion: HiTs can be isolated from fully human TCR–displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens. [ABSTRACT FROM AUTHOR]

Aims: In patients with ulcerative colitis (UC), no biomarker is available to help the physician to choose the most suitable biotherapy. The primary objective of this pilot study was to assess the feasibility of identification of α4β7- and TNF-expressing cells, to predict the response to vedolizumab using confocal laser endoscopy (CLE). Methods: Patients with moderate-to-severe UC, naïve of biotherapy, received vedolizumab. Clinical evaluation was performed at each infusion. Endoscopic evaluation was performed before inclusion and at week 22. Fresh colonic biopsies were stained using FITC-labelled vedolizumab and Alexa fluor-labelled adalimumab and ex vivo dual-band CLE images were acquired. Blood samples were collected to measure trough concentrations of vedolizumab and to determine absolute counts of T and B cells subpopulations, NK cells and monocytes. Results: Nineteen patients were enrolled in the study and received at least one dose of vedolizumab. Clinical remission and endoscopic improvement were observed in 58% of whom 5 patients (45%) had an endoscopic subscore of 0. In terms of clinical response and remission, endoscopic improvement and histologic response, FITC-conjugated vedolizumab staining tended to be higher in responder patients compared to non-responders at week 22. A threshold value of 6 positive FITC-vedolizumab staining areas detected by CLE seemed informative to discriminate the responders and non-responders. The results were similar in terms of clinical remission and endoscopic improvement with a sensitivity of 78% and a specificity of 85% (p = 0.05). Trough concentrations and blood immune cells were not associated with responses to vedolizumab. Conclusion: This pilot study demonstrate that dual-band CLE is feasible to detect α4β7- and TNF-expressing cells. Positive α4β7 staining seems to be associated with clinical and endoscopic remission in UC patients treated by anti-α4β7-integrin, subject to validation by larger-scale studies. Clinical-trial.gov: NCT02878083 [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]

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies. [ABSTRACT FROM AUTHOR]

Symptoms in people with carpal tunnel syndrome (CTS) are traditionally attributed to neural tissue, but recent studies suggest that the subsynovial connective tissue (SSCT) may also play a role in CTS. The SSCT undergoes fibrotic thickening which is generally described as "non-inflammatory" based on basic histology. This study uses immunohistochemistry to determine the presence of macrophages and T-cells within SSCT and their relationship with symptoms in people with CTS. SSCT was collected from twenty people with CTS and eight controls undergoing wrist fracture surgery. Immunohistochemical quantification of CD3+ T-cells and CD68+ macrophage densities as well as CD4+/CD8+ T-cell subpopulations were compared between groups using independent t-tests. Spearman correlations were used to identify associations between immune cell densities and CTS symptom scores. The density of CD3+ T-cells was significantly higher in SSCT of people with CTS compared to controls (CTS mean 26.7 (SD 13.7); controls 6.78 (6.3), p = 0.0005) while the density of CD68+ macrophages was lower (CTS mean 9.5 (SD 6.0); controls 17.7 (8.2), p = 0.0058). Neither CD68+ nor CD3+ cell densities correlated with symptom scores. In contrast to previous assumptions, our data show that the SSCT in the carpal tunnel in both people with CTS and controls is not devoid of immune cells. Whereas the higher density of CD68+ macrophages in control participants may be associated with their early recruitment after acute fracture, CD3+ cells within the SSCT may play a role in chronic CTS. [ABSTRACT FROM AUTHOR]

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations. [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]