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Context: Flaviviruses cause severe encephalitic or hemorrhagic diseases in humans. Its members, Kyasanur forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (ALKV), cause hemorrhagic fever and are prevalent in India and Saudi Arabia, respectively, while the tick-borne encephalitis virus (TBEV) causes a dangerous encephalitic infection in Europe and Asia. However, little information is available about the targets of immune responses for these deadly viruses. Here, we predict potential antigenic peptide epitopes of viral envelope protein for inducing a cell-mediated and humoral immune response. Methods: Using the Immune Epitope Database and Analysis Resource (IEDB-AR), we identified 13 MHC-I and two MHC-II dominant conserved epitopes in KFDV and ALKV and six MHC-I and three MHC-II epitopes in TBEV envelope proteins. Parallelly, we also predicted B-cell linear and discontinuous envelope protein epitopes for these viruses. Interestingly, the epitopes are conserved in all three viral envelope proteins. Further, the discontinuous epitopes are structurally compared with the available DENV, ZIKV, WNV, TBEV, and LIV envelope protein antibody structures. Overall structural comparison analyses highlight (i) lateral ridge epitope in the ED-III domain of E protein, and (ii) envelope dimer epitope (EDE) could be targeted for developing potent vaccine candidates as well as therapeutic antibody production. Moreover, existing structural and biochemical functions of the same epitopes in homologous viruses are predicted to have a reduced antibody-dependent enhancement (ADE) effect on flaviviral infection. [ABSTRACT FROM AUTHOR]

Abstract Background Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of pulmonary diseases, even after accounting for their smoking status. Cytotoxic CD8 T-cells are likely implicated in this phenomenon and may act as a double-edged sword. While being essential in viral infection control, their hyperactivation can also contribute to lung mucosal tissue damage. The effects of HIV and smoking on pulmonary mucosal CD8 T-cell dynamics has been a neglected area of research, which we address herein. Methods Bronchoalveolar lavage (BAL) fluid were obtained from ART-treated PLWH (median duration of supressed viral load: 9 years; smokers: n = 14; non-smokers: n = 21) and HIV-uninfected controls (smokers: n = 11; non-smokers: n = 20) without any respiratory symptoms or active infection. Lymphocytes were isolated and CD8 T-cell subsets and homing markers were characterized by multiparametric flow cytometry. Results Both smoking and HIV infection were independently associated with a significant increase in frequencies of total pulmonary mucosal CD8 T-cell. BAL CD8 T-cells were primarily CD69 + expressing CD103 and/or CD49a, at least one of the two granzymes (GzmA/GzmB), and little Perforin. Higher expression levels of CD103, CD69, and GzmB were observed in smokers versus non-smokers. The ex vivo phenotype of GzmA + and GzmB + cells revealed increased expression of CD103 and CXCR6 in smokers, while PLWH displayed elevated levels of CX3CR1 compared to controls. Conclusion Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. Smoking likely increases recruitment and retention of GzmB + CD8 Trm via CXCR6 and CD103. Heightened CX3CR1 expression could be associated with CD8 non-Trm recruitment from the periphery in PLWH.

Background: Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of pulmonary diseases, even after accounting for their smoking status. Cytotoxic CD8 T-cells are likely implicated in this phenomenon and may act as a double-edged sword. While being essential in viral infection control, their hyperactivation can also contribute to lung mucosal tissue damage. The effects of HIV and smoking on pulmonary mucosal CD8 T-cell dynamics has been a neglected area of research, which we address herein. Methods: Bronchoalveolar lavage (BAL) fluid were obtained from ART-treated PLWH (median duration of supressed viral load: 9 years; smokers: n = 14; non-smokers: n = 21) and HIV-uninfected controls (smokers: n = 11; non-smokers: n = 20) without any respiratory symptoms or active infection. Lymphocytes were isolated and CD8 T-cell subsets and homing markers were characterized by multiparametric flow cytometry. Results: Both smoking and HIV infection were independently associated with a significant increase in frequencies of total pulmonary mucosal CD8 T-cell. BAL CD8 T-cells were primarily CD69 + expressing CD103 and/or CD49a, at least one of the two granzymes (GzmA/GzmB), and little Perforin. Higher expression levels of CD103, CD69, and GzmB were observed in smokers versus non-smokers. The ex vivo phenotype of GzmA + and GzmB + cells revealed increased expression of CD103 and CXCR6 in smokers, while PLWH displayed elevated levels of CX3CR1 compared to controls. Conclusion: Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. Smoking likely increases recruitment and retention of GzmB + CD8 Trm via CXCR6 and CD103. Heightened CX3CR1 expression could be associated with CD8 non-Trm recruitment from the periphery in PLWH. [ABSTRACT FROM AUTHOR]

Establishment of cellular diversity is a basic requirement for the development of multicellular organisms. Cellular diversification can be induced by asymmetric cell division (ACD), during which the emerging two daughter cells unequally inherit lineage specific cargo (including transcription factors, receptors for specific signaling inputs, metabolic platforms, and possibly different epigenetic landscapes), resulting in two daughter cells endowed with different fates. While ACD is strongly involved in lineage choices in mammalian stem cells, its role in fate diversification in lineage committed cell subsets that still exhibit plastic potential, such as T‐cells, is currently investigated. In this review, we focus predominantly on the role of ACD in fate diversification of CD8 T‐cells. Further, we discuss the impact of differential T‐cell receptor stimulation strengths and differentiation history on ACD‐mediated fate diversification and highlight a particular importance of ACD in the development of memory CD8 T‐cells upon high‐affinity stimulation conditions. [ABSTRACT FROM AUTHOR]

CD8 T-cells play an important role in interferon-gamma (IFN-γ) production as a host defense against tuberculosis (TB) infection. Therefore, QuantiFERON-TB Gold Plus (QFT-Plus) was developed by adding a TB2 tube beside the TB1 tube. This study aimed to compare and analyze the difference in IFN-γ production between the two tubes in general and specific populations. PubMed, Web of Science, and EBSCO were searched for studies reporting IFN-γ production levels in the TB1 and TB2 tubes. Statistical analysis was performed using RevMan 5.3. A total of 17 studies met the inclusion criteria. The IFN-γ production in the TB2 tube was statistically higher than that in the TB1 tube (mean difference (MD)=0.02, 95 % confidence interval (95 % CI): 0.01–0.03). Further subgroup analysis in specific populations revealed that the MD of IFN-γ production between the TB2 and TB1 tubes was significantly higher in active TB subjects than in latent TB infection (LTBI) subjects (MD=1.13, 95 % CI: 0.49–1.77, and MD=0.30, 95 % CI: 0.00–0.60, respectively). A similar finding was found in immune-mediated inflammatory disease subjects, but not statistically significant. Interestingly, IFN-γ production capacity was lower in active TB subjects than in LTBI subjects in each of the TB1 and TB2 tubes. This study is the first to systematically compare IFN-γ production between the TB1 and TB2 tubes. The IFN-γ production was higher in the TB2 tube than in the TB1 tube, representing the host's CD8 T-cell response magnitude to TB infection. [ABSTRACT FROM AUTHOR]

Introduction: Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. Methods: A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Results: Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. Conclusion: The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM, that result in effective immune responses and thereby improve patient survival. [ABSTRACT FROM AUTHOR]

Background: We evaluated the relevance of PD-1+CD8+ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME). Methods: Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the "3G" chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs. Results: In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r = 0.714, p < 0.001) and proliferative [Ki-67+] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1high compared to CD8PD-1low tumors. Conclusion: This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1+CD8+ T-cells being associated with improved OS. CD8PD-1high tumors have distinct features of an immunologically active, T-cell inflamed TME. [ABSTRACT FROM AUTHOR]

IntroductionCharacterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification.MethodsA comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM.ResultsAcross all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis.ConclusionThe presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

IntroductionImmunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control.MethodsThe present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects.Results & discussionCD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials.

Background: In early stage clinical trials, changes to levels of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are critical biomarkers of the mechanism of action of novel immunotherapies. However, baseline heterogeneity of tumor samples, both between and within patients, and the resultant impact on the validity of clinical trial data is not well defined. Here we identify and quantify the impact of baseline variables on the heterogeneity of FoxP3+ and proliferating CD8+ T-cells levels (MKi67+CD8A+) in the TME both between and within patients for the purpose of informing clinical trial design and analysis. Methods: We compared levels of FoxP3+ and MKi67+CD8+ cell densities (counts/mm2) from >1000 baseline tumor samples from clinical trials and commercially available sources. Using multivariate hierarchical regression techniques, we investigated whether inter-person heterogeneity of activated or regulatory T-cells could be attributed to baseline characteristics including demographics, indication, lesion type, tissue of excision, biopsy method, prior cancer treatment, and tissue type i.e., "fresh" or "archival" status. We also sought to characterize within-patient heterogeneity by lesion type and tissue type. Results: Prior cancer treatment with hormone therapy or chemotherapy that induces immunogenic cell death may alter the TME. Archival tissue is an unreliable substitute for fresh tissue for determining baseline TIL levels. Baseline and on treatment biopsies should be matched by lesion type to avoid bias. [ABSTRACT FROM AUTHOR]

BackgroundIn early stage clinical trials, changes to levels of tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are critical biomarkers of the mechanism of action of novel immunotherapies. However, baseline heterogeneity of tumor samples, both between and within patients, and the resultant impact on the validity of clinical trial data is not well defined. Here we identify and quantify the impact of baseline variables on the heterogeneity of FoxP3+ and proliferating CD8+ T-cells levels (MKi67+CD8A+) in the TME both between and within patients for the purpose of informing clinical trial design and analysis.MethodsWe compared levels of FoxP3+ and MKi67+CD8+ cell densities (counts/mm2) from >1000 baseline tumor samples from clinical trials and commercially available sources. Using multivariate hierarchical regression techniques, we investigated whether inter-person heterogeneity of activated or regulatory T-cells could be attributed to baseline characteristics including demographics, indication, lesion type, tissue of excision, biopsy method, prior cancer treatment, and tissue type i.e., “fresh” or “archival” status. We also sought to characterize within-patient heterogeneity by lesion type and tissue type.ResultsPrior cancer treatment with hormone therapy or chemotherapy that induces immunogenic cell death may alter the TME. Archival tissue is an unreliable substitute for fresh tissue for determining baseline TIL levels. Baseline and on treatment biopsies should be matched by lesion type to avoid bias.

Background A persistently low CD4/CD8 ratio has been reported to inversely correlate with the risk of non-AIDS defining cancer in people living with human immunodeficiency virus (HIV; PLWH) efficiently treated by combination antiretroviral therapy (cART). We evaluated the impact of the CD4/CD8 ratio on the risk of Kaposi sarcoma (KS) or non-Hodgkin lymphoma (NHL), still among the most frequent cancers in treated PLWH. Methods PLWH from the Collaboration of Observational HIV Epidemiological Research Europe (COHERE) were included if they achieved virological control (viral load ≤ 500 copies/mL) within 9 months following cART and without previous KS/LNH diagnosis. Cox models were used to identify factors associated with KS or NHL risk, in all participants and those with CD4 ≥ 500/mm3 at virological control. We analyzed the CD4/CD8 ratio, CD4 count and CD8 count as time-dependent variables, using spline transformations. Results We included 56 708 PLWH, enrolled between 2000 and 2014. At virological control, the median (interquartile range [IQR]) CD4 count, CD8 count, and CD4/CD8 ratio were 414 (296–552)/mm3, 936 (670–1304)/mm3, and 0.43 (0.28–0.65), respectively. Overall, 221 KS and 187 NHL were diagnosed 9 (2–37) and 18 (7–42) months after virological control. Low CD4/CD8 ratios were associated with KS risk (hazard ratio [HR] = 2.02 [95% confidence interval {CI } = 1.23–3.31]) when comparing CD4/CD8 = 0.3 to CD4/CD8 = 1) but not with NHL risk. High CD8 counts were associated with higher NHL risk (HR = 3.14 [95% CI = 1.58–6.22]) when comparing CD8 = 3000/mm3 to CD8 = 1000/mm3). Similar results with increased associations were found in PLWH with CD4 ≥ 500/mm3 at virological control (HR = 3.27 [95% CI = 1.60–6.56] for KS; HR = 5.28 [95% CI = 2.17–12.83] for NHL). Conclusions Low CD4/CD8 ratios and high CD8 counts despite effective cART were associated with increased KS/NHL risks respectively, especially when CD4 ≥ 500/mm3. [ABSTRACT FROM AUTHOR]

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage.

The role of Ly49+CD8 T-cells in the immune system is not clear. Previously, several papers suggested Ly49+CD8 T-cells as immunosuppressors, while multiple studies also suggested their role as potent participants of the immune response. The mechanism of Ly49 expression on CD8 T-cells is also not clear. We investigated phenotype, functions, and regulation of Ly49 expression on murine CD8 T-cells in both normal state and during LCMV infection. CD8 T-cells express different Ly49 receptors compared with NK-cells. In intact mice, Ly49+CD8 T-cells have a phenotype similar to resting central memory CD8 T-cells and do not show impaired proliferation and cytokine production. Conventional CD8 T-cells upregulate Ly49 receptors during TCR-induced stimulation, and IL-2, as well as IL-15, affect it. At the same time, Ly49+CD8 T-cells change the Ly49 expression profile dramatically upon re-stimulation downregulating inhibitory and upregulating activating Ly49 receptors. We observed the expression of Ly49 receptors on the virus-specific CD8 T-cells during LCMV infection, especially marked in the early stages, and participation of Ly49+CD8 T-cells in the anti-viral response. Thus, CD8 T-cells acquire Ly49 receptors during the T-cell activation and show dynamic regulation of Ly49 receptors during stimulation.

Predictive models are becoming more and more commonplace as tools for candidate antigen discovery to meet the challenges of enabling epitope mapping of cohorts with diverse HLA properties. Here we build on the concept of using two key parameters, diversity metric of the HLA profile of individuals within a population and consideration of sequence diversity in the context of an individual's CD8 T-cell immune repertoire to assess the HIV proteome for defined regions of immunogenicity. Using this approach, analysis of HLA adaptation and functional immunogenicity data enabled the identification of regions within the proteome that offer significant conservation, HLA recognition within a population, low prevalence of HLA adaptation and demonstrated immunogenicity. We believe this unique and novel approach to vaccine design as a supplement to vitro functional assays, offers a bespoke pipeline for expedited and rational CD8 T-cell vaccine design for HIV and potentially other pathogens with the potential for both global and local coverage. [ABSTRACT FROM AUTHOR]

The role of Ly49+CD8 T-cells in the immune system is not clear. Previously, several papers suggested Ly49+CD8 T-cells as immunosuppressors, while multiple studies also suggested their role as potent participants of the immune response. The mechanism of Ly49 expression on CD8 T-cells is also not clear. We investigated phenotype, functions, and regulation of Ly49 expression on murine CD8 T-cells in both normal state and during LCMV infection. CD8 T-cells express different Ly49 receptors compared with NK-cells. In intact mice, Ly49+CD8 T-cells have a phenotype similar to resting central memory CD8 T-cells and do not show impaired proliferation and cytokine production. Conventional CD8 T-cells upregulate Ly49 receptors during TCR-induced stimulation, and IL-2, as well as IL-15, affect it. At the same time, Ly49+CD8 T-cells change the Ly49 expression profile dramatically upon re-stimulation downregulating inhibitory and upregulating activating Ly49 receptors. We observed the expression of Ly49 receptors on the virus-specific CD8 T-cells during LCMV infection, especially marked in the early stages, and participation of Ly49+CD8 T-cells in the anti-viral response. Thus, CD8 T-cells acquire Ly49 receptors during the T-cell activation and show dynamic regulation of Ly49 receptors during stimulation. [ABSTRACT FROM AUTHOR]

The first SARS-CoV-2 vaccine(s) will likely be licensed based on neutralizing antibodies in Phase 2 trials, but there are significant concerns about using antibody response in coronavirus infections as a sole metric of protective immunity. Antibody response is often a poor marker of prior coronavirus infection, particularly in mild infections, and is shorter-lived than virus-reactive T-cells; strong antibody response correlates with more severe clinical disease while T-cell response is correlated with less severe disease; and antibody-dependent enhancement of pathology and clinical severity has been described. Indeed, it is unclear whether antibody production is protective or pathogenic in coronavirus infections. Early data with SARS-CoV-2 support these findings. Data from coronavirus infections in animals and humans emphasize the generation of a high-quality T cell response in protective immunity. Yellow Fever and smallpox vaccines are excellent benchmarks for primary immune response to viral vaccination and induce long-lived virus-reactive CD8 T-cells, which are present and measurable within 1–4 months of vaccination. Progress in laboratory markers for SARS-CoV2 has been made with identification of epitopes on CD4 and CD8 T-cells in convalescent blood. These are much less dominated by spike protein than in previous coronavirus infections. Although most vaccine candidates are focusing on spike protein as antigen, natural infection by SARS-CoV-2 induces broad epitope coverage, cross-reactive with other betacoronviruses. It will be important to understand the relation between breadth, functionality and durability of T-cell responses and resulting protective immunity. It would be a public health and general trust-in-medicine nightmare - including a boost to anti-vaccine forces - if immune protection wears off or new disease patterns develop among the immunized. Data correlating clinical outcomes with laboratory markers of cell-mediated immunity, not only with antibody response, after SARS-CoV-2 natural infection and vaccines may prove critically valuable if protective immunity fades or if new patterns of disease emerge.

Calcifying pseudoneoplasm of the neuraxis (CAPNON) is a rare tumor-like fibro-osseous lesion that can develop anywhere in the neuraxis. Approximately a half of reported CAPNONs developed in the spinal region, mostly close to the facet joint (FJ). The diagnosis of spinal CAPNONs is challenging given the existence of mimics and associated pathologies including calcific degeneration of the FJ ligaments (DFJL) and synovial cysts (SCs). The pathogenesis of CAPNON remains elusive, although there have been a few hypotheses including degenerative, reactive, proliferative and immune-mediated processes. Our present study examined clinical, radiological and pathological features of 12 spinal CAPNONs in comparison to 9 DFJL foci, and diagnostic and pathogenic relationship between CAPNONs and FJ pathologies. On imaging, CAPNONs were all tumor-like and typically bigger than DFJL foci. All CAPNONs showed pathologically diagnostic features including characteristic cores, consistently identifiable core-surrounding/peripheral palisading of macrophages and other cells including multinucleated giant cells, variable infiltration of CD8 + T-cells, and multifocal immunopositivity of neurofilament light chain (NF-L). These features were absent or limited in the DFJL foci with statistically significant differences from CAPNONs, except calcifications. Spinal CAPNONs co-existed with DFJL foci in all cases; some had transitional foci with overlapping focal CAPNON and DFJL-like features. These findings, along with our previously reported relationship between CAPNONs and SCs, suggest that spinal CAPNONs may occur in association with or in transition from calcifying/calcified degenerative lesions of FJ ligaments and/or SCs when a reactive proliferative process is complemented by other pathogenic changes such as immune-mediated pathology and NF-L deposition/expression., Competing Interests: Declaration of competing interest Dr. Faiha Fareez has received the Resident Research Grant from Department of Pathology and Molecular Medicine, McMaster University. The other authors receive no funding specified for this work and report no conflicts of interest. Parts of this study have been presented at the 20th International Congress of Neuropathology. Berlin, Germany (September 13–16, 2023), and as an abstract, published in Brain Pathology 2023; 33(S1):189., (Copyright © 2024 Elsevier Inc. All rights reserved.)

IL15 is a key cytokine for the activation and survival of anti-tumor effectors CD8+ T and NK cells. Recently published preclinical studies demonstrate that the therapeutic activity of IL15 requires conventional dendritic cells type 1 (cDC1). Radiotherapy cooperates with IL15 by enhancing cDC1 tumor infiltration via interferon type 1 activation.

CD8 T-cell response efficiency critically depends on the TCR binding strength to peptide-MHC, i.e., the TCR binding avidity. A current challenge in onco-immunology lies in the evaluation of vaccine protocols selecting for tumor-specific T-cells of highest avidity, offering maximal immune protection against tumor cells and clinical benefit. Here, we investigated the impact of peptide and CpG/adjuvant doses on the quality of vaccine-induced CD8 T-cells in relation to binding avidity and functional responses in treated melanoma patients. Using TCR-pMHC binding avidity measurements combined to phenotype and functional assays, we performed a comprehensive study on representative tumor antigen-specific CD8 T-cell clones (n = 454) from seven patients vaccinated with different doses of Melan-A/ELA peptide (0.1 mg vs. 0.5 mg) and CpG-B adjuvant (1–1.3 mg vs. 2.6 mg). Vaccination with high peptide dose favored the early and strong in vivo expansion and differentiation of Melan-A-specific CD8 T-cells. Consistently, T-cell clones generated from those patients showed increased TCR binding avidity (i.e., slow off-rates and CD8 binding independency) readily after 4 monthly vaccine injections (4v). In contrast, the use of low peptide or high CpG-B doses required 8 monthly vaccine injections (8v) for the enrichment of anti-tumor T-cells with high TCR binding avidity and low CD8 binding dependency. Importantly, the CD8 binding-independent vaccine-induced CD8 T-cells displayed enhanced functional avidity, reaching a plateau of maximal function. Thus, T-cell functional potency following peptide/CpG/IFA vaccination may not be further improved beyond a certain TCR binding avidity limit. Our results also indicate that while high peptide dose vaccination induced the early selection of Melan-A-specific CD8 T-cells of increased functional competence, continued serial vaccinations also promoted such high-avidity T-cells. Overall, the systematic assessment of T-cell binding avidity may contribute to optimize vaccine design for improving clinical efficacy.

IL15 is a key cytokine for the activation and survival of anti-tumor effectors CD8+ T and NK cells. Recently published preclinical studies demonstrate that the therapeutic activity of IL15 requires conventional dendritic cells type 1 (cDC1). Radiotherapy cooperates with IL15 by enhancing cDC1 tumor infiltration via interferon type 1 activation. [ABSTRACT FROM AUTHOR]

Background: Notch intercellular communication instructs tissue-specific T-cell development and function. In this study, we explored the roles of dendritic cell (DC)-expressed Notch ligands in the regulation of T-cell effector function. Methods: We generated mice with CD11c lineage-specific deletion of Notch Delta-like ligand (Dll)1 and Jagged (Jag)2. Using these genetically-ablated mice and engineered pharmacological Notch ligand constructs, the roles of various Delta-like and Jagged ligands in the regulation of T-cell-mediated immunity were investigated. We assessed tumor growth, mouse survival, cytokine production, immunophenotyping of myeloid and lymphoid populations infiltrating the tumors, expression of checkpoint molecules and T-cell function in the experimental settings of murine lung and pancreatic tumors and cardiac allograft rejection. Correlative studies were also performed for the expression of NOTCH ligands, NOTCH receptors and PD-1 on various subsets of myeloid and lymphoid cells in tumor-infiltrating immune cells analyzed from primary human lung cancers. Results: Mice with CD11c lineage-specific deletion of Notch ligand gene Dll1, but not Jag2, exhibited accelerated growth of lung and pancreatic tumors concomitant with decreased antigen-specific CD8+T-cell functions and effector-memory (Tem) differentiation. Increased IL-4 but decreased IFN-γ production and elevated populations of T-regulatory and myeloid-derived suppressor cells were observed in Dll1-ablated mice. Multivalent clustered DLL1-triggered Notch signaling overcame DC Dll1 deficiency and improved anti-tumor T-cell responses, whereas the pharmacological interference by monomeric soluble DLL1 construct suppressed the rejection of mouse tumors and cardiac allograft. Moreover, monomeric soluble JAG1 treatment reduced T-regulatory cells and improved anti-tumor immune responses by decreasing the expression of PD-1 on CD8+Tem cells. A significant correlation was observed between DC-expressed Jagged and Delta-like ligands with Tem-expressed PD-1 and Notch receptors, respectively, in human lung tumor-infiltrates. Conclusion: Our data show the importance of specific expression of Notch ligands on DCs in the regulation of Tcell effector function. Thus, strategies incorporating selectively engineered Notch ligands could provide a novel approach of therapeutics for modulating immunity in various immunosuppressive conditions including cancer. [ABSTRACT FROM AUTHOR]

Seasonal influenza viruses cause significant morbidity and mortality in the global population every year. Although seasonal vaccination limits disease, mismatches between the circulating strain and the vaccine strain can severely impair vaccine effectiveness. Because of this, there is an urgent need for a universal vaccine that induces broad protection against drifted seasonal and emerging pandemic influenza viruses. Targeting the conserved stalk region of the influenza virus hemagglutinin (HA), the major glycoprotein on the surface of the virus, results in the production of broadly protective antibody responses. Furthermore, replication deficient viral vectors based on Chimpanzee Adenovirus Oxford 1 (ChAdOx1) and modified vaccinia Ankara (MVA) virus expressing the influenza virus internal antigens, the nucleoprotein (NP) and matrix 1 (M1) protein, can induce strong heterosubtypic influenza virus-specific T cell responses in vaccinated individuals. Here, we combine these two platforms to evaluate the efficacy of a viral vectored vaccination regimen in protecting ferrets from H3N2 influenza virus infection. We observed that viral vectored vaccines expressing both stalk-targeting, chimeric HA constructs, and the NP+M1 fusion protein, in a prime-boost regimen resulted in the production of antibodies toward group 2 HAs, the HA stalk, NP and M1, as well as in induction of influenza virus-specific—IFNγ responses. The immune response induced by this vaccination regime ultimately reduced viral titers in the respiratory tract of influenza virus infected ferrets. Overall, these results improve our understanding of vaccination platforms capable of harnessing both cellular and humoral immunity with the goal of developing a universal influenza virus vaccine.

We report here the rational design and validation of a peptide inhibitor to the PD-1/PD-L1 interaction as an attempt to develop a viable alternative to current inhibitory antibodies. We demonstrated, by biolayer interferometry and in silico docking simulations, that a PD-L1 peptide mimetic (PL120131) can interfere with the PD-1/PD-L1 interaction by binding to PD-1. We show that PL120131 is capable of inhibiting PD-1 mediated apoptotic signaling pathway and rescuing Jurkat cells and primary lymphocytes from apoptosis. Additionally, we show that PL120131 treatment allows for CTL anti-tumor activity. Furthermore, PL120131 can maintain co-culture survivability and activity of T Cells in a 3D co-culture model better than the anti-PD-1 blocking antibody. Together, the characterization of this PD-1/PD-L1 inhibiting peptide provides insight regarding the ability to inhibit PD-L1 binding while maintaining CTL viability and activity that can further the development of alternatives to antibody based immunotherapies. [ABSTRACT FROM AUTHOR]

Lipid catabolism represents an Achilles heel in prostate cancer (PCa) that can be exploited for therapy. CPT1A regulates the entry of fatty acids into the mitochondria for beta-oxidation and its inhibition has been shown to decrease PCa growth. In this study, we examined the pharmacological blockade of lipid oxidation with ranolazine in TRAMPC1 PCa models. Oral administration of ranolazine (100 mg/Kg for 21 days) resulted in decreased tumor CD8+ T-cells Tim3 content, increased macrophages, and decreased blood myeloid immunosuppressive monocytes. Using multispectral staining, drug treatments increased infiltration of CD8+ T-cells and dendritic cells compared to vehicle. Functional studies with spleen cells of drug-treated tumors co-cultured with TRAMPC1 cells showed increased ex vivo T-cell cytotoxic activity, suggesting an anti-tumoral response. Lastly, a decrease in CD4+ and CD8+ T-cells expressing PD1 was observed when exhausted spleen cells were incubated with TRAMPC1 Cpt1a-KD compared to the control cells. These data indicated that genetically blocking the ability of the tumor cells to oxidize lipid can change the activation status of the neighboring T-cells. This study provides new knowledge of the role of lipid catabolism in the intercommunication of tumor and immune cells, which can be extrapolated to other cancers with high CPT1A expression.

CD8 T-cells are critical in controlling infection by intracellular pathogens. Upon encountering antigen presenting cells, T-cell receptor activation promotes the differentiation of naïve CD8 T-cells into strongly proliferating activated and effector stages. We propose a 2D-multiscale computational model to study the maturation of CD8 T-cells in a lymph node controlled by their molecular profile. A novel molecular pathway is presented and converted into an ordinary differential equation model, coupled with a cellular Potts model to describe cell-cell interactions. Key molecular players such as activated IL2 receptor and Tbet levels control the differentiation from naïve into activated and effector stages, respectively, while caspases and Fas-Fas ligand interactions control cell apoptosis. Coupling this molecular model to the cellular scale successfully reproduces qualitatively the evolution of total CD8 T-cell counts observed in mice lymph node, between Day 3 and 5.5 post-infection. Furthermore, this model allows us to make testable predictions of the evolution of the different CD8 T-cell stages.

Objectives Antibody–cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2- and TNF-based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens. Materials and methods We evaluated the therapeutic efficacy of the F8-IL2 immunocytokine utilizing a K-ras mutant, p53 deficient metastatic mouse model of NSCLC derived from the latest generation of genetically engineered and conditional tumor models. In parallel, we assessed the presence of the EDA domain of fibronectin by immunofluorescence in lung biopsies obtained from patients with NSCLC. Results The EDA domain of fibronectin was broadly expressed in lung metastases obtained from our model. Treatment with F8-IL2 induced substantial local changes within immune effector cell populations and demonstrated promising therapeutic efficacy as monotherapy. The target of F8-IL2, the EDA domain of fibronectin, was present in all human lung adenocarcinoma specimens tested. Conclusion Both the therapeutic efficacy in a metastatic mouse model of NSCLC and the extensive presence of the EDA domain of fibronectin in human NSCLC biopsies support the rational development of therapies based on the F8-IL2 immunocytokine for the treatment of NSCLC. [ABSTRACT FROM AUTHOR]

Introduction: Immunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control., Methods: The present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects., Results & Discussion: CD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fernandez, Hayes, Makinde, Hare, King, Xu, Rehawi, Mezzell, Kato, Mugaba, Serwanga, Chemweno, Nduati, Price, Osier, Ochsenbauer, Yue, Hunter, Gilmour and The IAVI protocol C investigators.)

Calcifying pseudoneoplasm of the neuraxis (CAPNON) is a rare tumour-like fibro-osseous lesion in the neuraxis including the spine. It is diagnosed by the presence of the following histological features: granular amorphous to chondromyxoid fibrillary cores with calcification/ossification, peripheral palisading of spindle to epithelioid cells, variable fibrous stroma, and foreign body reaction with multinucleated giant cells, as well as positive NF-L immunostaining. Spinal CAPNON is sometimes named as tumoural calcinosis that is tumour-like dystrophic calcification usually in the periarticular tissue and also described in calcified synovial cyst (CSC). We examined clinical, radiological and pathological features of five spinal CAPNONs and 21 spinal CSCs including three recurrent lesions. The results demonstrated some radiological and pathological overlaps between these two entities, as well as distinct features of each entity to be diagnosed. All CAPNONs showed the diagnostic histological features with NF-L positivity mainly in lesion cores and variable CD8+ T-cells. In contrast, CSCs exhibited the synovial lining and variable degenerative/reactive changes with some CAPNON-like features, but mostly no to occasionally limited NF-L positivity and less CD8+ T-cells with statistically significant differences between groups of CAPNONs and CSCs. Four CSCs contained CAPNON-like foci with the CAPNON diagnostic features including prominent NF-L positivity, and some transitional features from CSC to CAPNON. As the pathogenesis of CAPNON is likely reactive/degenerative in association with an inflammatory/immunological process involving NF-L protein deposition, our findings suggest the link between spinal CAPNON and CSC, with possible transition from CSC to CAPNON or CAPNON developing in reaction to CSC., (Copyright © 2022 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Host protection against several intracellular pathogens requires the induction of CD8+ T cell responses. CD8+ T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8+ T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8+ T cells during various types Leishmania infections, following vaccination, and as potential immunotherapeutic targets.

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8 T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8 central memory T-cells. [ABSTRACT FROM AUTHOR]

Abstract: Cancer immunotherapy requires potent tumor-specific CD8+ and CD4+ T-cell responses, initiated by dendritic cells (DCs). Tumor antigens can be specifically targeted to DCs in vivo by exploiting their expression of C-type lectin receptors (CLR), which bind carbohydrate structures on antigens, resulting in internalization and antigen presentation to T-cells. We explored the potential of glycan-modified liposomes to target antigens to DCs to boost murine and human T-cell responses. Since DC-SIGN is a CLR expressed on DCs, liposomes were modified with DC-SIGN-binding glycans Lewis (Le)B or LeX. Glycan modification of liposomes resulted in increased binding and internalization by BMDCs expressing human DC-SIGN. In the presence of LPS, this led to 100-fold more efficient presentation of the encapsulated antigens to CD4+ and CD8+ T-cells compared to unmodified liposomes or soluble antigen. Similarly, incubation of human moDC with melanoma antigen MART-1-encapsulated liposomes coated with LeX in the presence of LPS led to enhanced antigen-presentation to MART-1-specific CD8+ T-cell clones. Moreover, this formulation drove primary CD8+ T-cells to differentiate into high numbers of tetramer-specific, IFN-γ-producing effector T-cells. Together, our data demonstrate the potency of a glycoliposome-based vaccine targeting DC-SIGN for CD4+ and CD8+ effector T-cell activation. This approach may offer improved options for treatment of cancer patients and opens the way to in situ DC-targeted vaccination. [Copyright &y& Elsevier]

Background: A Giardia outbreak was associated with development of post-infectious functional gastrointestinal disorders (PI-FGID) and chronic fatigue syndrome (PI-CFS). Markers of immune dysfunction have given conflicting results in CFS and FGID patient populations. The aim of this study was to evaluate a wide selection of markers of immune dysfunction in these two co-occurring post-infectious syndromes. Methods: 48 patients, reporting chronic fatigue in a questionnaire study, were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS (n=19) and idiopathic chronic fatigue (n=5) and Rome II criteria for FGIDs (n=54). 22 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Results: In peripheral blood we found significantly higher CD8 T-cell levels in PI-FGID, and significantly lower NK-cell levels in PI-CFS patients. Severity of abdominal and fatigue symptoms correlated negatively with NK-cell levels. A tendency towards lower T-cell CD26 expression in FGID was seen. Conclusion: Patients with PI-CFS and/or PI-FGID 5 years after Giardia lamblia infection showed alterations in NK-cell and CD8-cell populations suggesting a possible immunological abnormality in these conditions. We found no significant changes in other markers examined in this well-defined group of PI-CFS and PI-FGID elicited by a gastrointestinal infection. Controlling for co-morbid conditions is important in evaluation of CFS-biomarkers. [ABSTRACT FROM AUTHOR]

Older individuals (≥50 years of age) are increasingly becoming a new at-risk group for HIV-1 infection and, together with those surviving longer due to the introduction of anti-retroviral therapy (ART), it is predicted that more than half of all HIV-1-infected individuals in the United States will be greater than 50 years of age in the year 2015. Older individuals diagnosed with HIV-1 are prone to faster disease progression and reduced T-cell reconstitution despite successful virologic control with anti-retroviral therapy (ART). There is also growing evidence that the T-cell compartment in HIV-1 adults displays an aged phenotype, and HIV-1-infected individuals are increasingly diagnosed with clinical conditions more commonly seen in older uninfected persons. As aging in the absence of HIV infection is associated with alterations in T-cell function and immunosenescence, the combined impact of both HIV-1 infection and aging may provide an explanation for poorer clinical outcomes observed in older HIV-1-infected individuals. Thus, the development of novel therapeutics to stimulate immune function and delay immunosenescence is critical and would be beneficial to both the elderly and HIV-1-infected individuals. [ABSTRACT FROM AUTHOR]

Abstract: The antibody (Ab) array format provides a unique opportunity to pan and characterize multiple leukocyte subsets in parallel. However, the questions of reproducibility and robustness of leukocyte panning on Ab arrays need to be answered for this technology to become an immunophenotyping tool. The present study sought to address several of these questions, including: (1) purity of leukocyte subsets captured on Ab regions, (2) dynamics of leukocyte binding, (3) elimination of non-specific cell adhesion, and (4) standardization of cell washing conditions. Abs for CD4 T-cells, CD8 T-cells, CD36 monocytes, and CD16b neutrophils were dispensed onto standard glass slides containing a thin film of poly(ethylene glycol) (PEG) hydrogel. PEG gel coating was highly effective in eliminated non-specific cell adhesion on the surface. Incubation of the Ab arrays with red blood cell (RBC) depleted whole blood resulting in antigen-specific panning of leukocyte subsets on the respective Ab domains. A flow through chamber was employed to determine optimal shear stress conditions for removal of non-specifically attached cells. The purity of the four subsets remaining on the surface after washing was determined by Wright staining and immunofluorescence, and was found to be as follows: CD4 T-cells (99.2±0.3%), CD8 T-cells (98.7±0.3%), CD36 monocytes (97.2±0.9%), and CD16b neutrophils (99.1±0.6%). In conclusion, the methods described in this study allow to separate whole blood into pure leukocyte subsets with minimal sample preparation and handling. These approaches will be valuable in the future development of Ab arrays as tools for quantitative immunophenotyping of leukocytes. [Copyright &y& Elsevier]

Lipid catabolism represents an Achilles heel in prostate cancer (PCa) that can be exploited for therapy. CPT1A regulates the entry of fatty acids into the mitochondria for beta-oxidation and its inhibition has been shown to decrease PCa growth. In this study, we examined the pharmacological blockade of lipid oxidation with ranolazine in TRAMPC1 PCa models. Oral administration of ranolazine (100 mg/Kg for 21 days) resulted in decreased tumor CD8+ T-cells Tim3 content, increased macrophages, and decreased blood myeloid immunosuppressive monocytes. Using multispectral staining, drug treatments increased infiltration of CD8+ T-cells and dendritic cells compared to vehicle. Functional studies with spleen cells of drug-treated tumors co-cultured with TRAMPC1 cells showed increased ex vivo T-cell cytotoxic activity, suggesting an anti-tumoral response. Lastly, a decrease in CD4+ and CD8+ T-cells expressing PD1 was observed when exhausted spleen cells were incubated with TRAMPC1 Cpt1a-KD compared to the control cells. These data indicated that genetically blocking the ability of the tumor cells to oxidize lipid can change the activation status of the neighboring T-cells. This study provides new knowledge of the role of lipid catabolism in the intercommunication of tumor and immune cells, which can be extrapolated to other cancers with high CPT1A expression. [ABSTRACT FROM AUTHOR]