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Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods., Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi)., Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes., Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Khan, Müller, Kasmapour, Ynga-Durand, Eiz-Vesper, von Einem, Busch and Cicin-Sain.)

Endoplasmic reticulum stress occurs due to large amounts of misfolded proteins, hypoxia, nutrient deprivation, and more. The unfolded protein is a complex intracellular signaling network designed to operate under this stress. Composed of three individual arms, inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor-6, the unfolded protein response looks to resolve stress and return to proteostasis. The CD8 + T cell is a critical cell type for the adaptive immune system. The unfolded protein response has been shown to have a wide-ranging spectrum of effects on CD8 + T cells. CD8 + T cells undergo cellular stress during activation and due to environmental insults. However, the magnitude of the effects this response has on CD8 + T cells is still understudied. Thus, studying these pathways is important to unraveling the inner machinations of these powerful cells. In this review, we will highlight the recent literature in this field, summarize the three pathways of the unfolded protein response, and discuss their roles in CD8 + T cell biology and functionality., 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 © 2024 Nair and Liu.)

Introduction: Mucosal immunization eliciting local T-cell memory has been suggested for improved protection against respiratory infections caused by viral variants evading pre-existing antibodies. However, it remains unclear whether T-cell targeted vaccines suffice for prevention of viral transmission and to which extent local immunity is important in this context., Methods: To study the impact of T-cell vaccination on the course of viral respiratory infection and in particular the capacity to inhibit viral transmission, we used a mouse model involving natural murine parainfluenza infection with a luciferase encoding virus and an adenovirus based nucleoprotein targeting vaccine., Results and Discussion: Prior intranasal immunization inducing strong mucosal CD8+ T cell immunity provided an almost immediate shut-down of the incipient infection and completely inhibited contact based viral spreading. If this first line of defense did not operate, as in parentally immunized mice, recirculating T cells participated in accelerated viral control that reduced the intensity of inter-individual transmission. These observations underscore the importance of pursuing the development of mucosal T-cell inducing vaccines for optimal protection of the individual and inhibition of inter-individual transmission (herd immunity), while at the same time explain why induction of a strong systemic T-cell response may still impact viral transmission., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Zhou, Uddback, Kohlmeier, Christensen and Thomsen.)

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues., 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 © 2023 Paskeviciute, Chen, Xu, Honoré, Vorum, Sørensen, Christensen, Thomsen, Nissen and Steffensen.)

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8 + T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene ( TRAC ) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8 + T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201 + human β-cell line by HLA-A*02:01 restricted CD8 + T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8 + T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells., 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 © 2023 Peters, Yeh, Arnoletti, Brown, Posgai, Mathews and Brusko.)

Formation of long-lasting memory lymphocytes is one of the foundational characteristics of adaptive immunity and the basis of many vaccination strategies. Following the rapid expansion and contraction of effector CD8 T cells, the surviving antigen (Ag)-specific cells give rise to the memory CD8 T cells that persist for a long time and are phenotypically and functionally distinct from their naïve counterparts. Significant heterogeneity exists within the memory CD8 T cell pool, as different subsets display distinct tissue localization preferences, cytotoxic ability, and proliferative capacity, but all memory CD8 T cells are equipped to mount an enhanced immune response upon Ag re-encounter. Memory CD8 T cells demonstrate numerical stability under homeostatic conditions, but sepsis causes a significant decline in the number of memory CD8 T cells and diminishes their Ag-dependent and -independent functions. Sepsis also rewires the transcriptional profile of memory CD8 T cells, which profoundly impacts memory CD8 T cell differentiation and, ultimately, the protective capacity of memory CD8 T cells upon subsequent stimulation. This review delves into different aspects of memory CD8 T cell subsets as well as the immediate and long-term impact of sepsis on memory CD8 T cell biology., 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 © 2023 Heidarian, Griffith and Badovinac.)

Despite the therapeutic success of immune checkpoint blockade (ICB) therapy against multiple tumors, many patients still do not benefit from ICB. In particular, high-grade brain tumors, such as glioblastoma multiforme (GBM), have a very low response rate to ICB, resulting in several failed clinical trials. This low response rate might be caused by a lack of understanding of the unique characteristics of brain immunity. To overcome this knowledge gap, macroscopic studies of brain immunity are needed. We use single cell RNA sequencing to analyze the immune landscape of the tumor microenvironment (TME) under anti-PD-1 antibody treatment in a murine GBM model. We observe that CD8 T cells show a mixed phenotype overall that includes reinvigoration and re-exhaustion states. Furthermore, we find that CCL5 induced by anti-PD-1 treatment might be related to an increase in the number of anti-inflammatory macrophages in the TME. Therefore, we hypothesize that CCL5-mediated recruitment of anti-inflammatory macrophages may be associated with re-exhaustion of CD8 T cells in the TME. We compare our observations in the murine GBM models with publicly available data from human patients with recurrent GBM. Our study provides critical information for the development of novel immunotherapies to overcome the limitations of anti-PD-1 therapy., 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 Park, Kang and Lee.)

Background: Lymphocytic thyroiditis (LT) is frequently seen in the tumor microenvironment (TME) of papillary thyroid carcinomas (PTCs). However, the characteristic of these tumor-infiltrating lymphocytes (TILs) is not well understood., Objective: We aim to define the TME of PTC cases by characterizing the TILs., Design: This is a cross-sectional observational study., Patients: We enrolled 29 PTC (23 having concurrent LT), 14 LT, and 13 hyperplastic nodules with LT (HN) patients from January 2016 to December 2020., Measurements: Immunohistochemical (IHC) expression of CD8, FoxP3, PD-1, and PD-L1 was studied in PTC with LT and compared with HN. PD-1 and PD-L1 expression was correlated at the mRNA level by quantitative real-time PCR. Immunophenotyping of TILs was done in FNAC samples of PTC and LT by flow cytometry., Results: IHC revealed the presence of CD8 + cytotoxic T lymphocytes (CTLs) and FoxP3 + T regulatory cells (Tregs) in 83% and 52% of PTC with LT cases, respectively. Flow cytometric analysis of the PTC samples revealed a significant abundance of CTL compared with Treg and a higher CTL with lower Treg counts compared with LT. On IHC, PD-1 positivity was noted in 56.5% of PTC with LT cases, while intermediate PD-L1 positivity was found in 70% of the cases. There was a significant upregulation of PD-1 mRNA in PTC with LT. A significant correlation was noted with PD-L1 expression with lymph node metastasis and presence of Treg cells., Conclusions: Increased expression of PD-1 and PD-L1 in the TME of PTC may provide a potential molecular mechanism for tumor survival despite the predominance of CTLs, possibly through their inactivation or exhaustion., 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 Banerjee, Nahar, Dahiya, Mukherjee, Dey, Gupta, Radotra, Sachdeva, Sood, Bhadada and Bhansali.)

The advent of technologies that can characterize the phenotypes, functions and fates of individual cells has revealed extensive and often unexpected levels of diversity between cells that are nominally of the same subset. CD8 + T cells, also known as cytotoxic T lymphocytes (CTLs), are no exception. Investigations of individual CD8 + T cells both in vitro and in vivo have highlighted the heterogeneity of cellular responses at the levels of activation, differentiation and function. This review takes a broad perspective on the topic of heterogeneity, outlining different forms of variation that arise during a CD8 + T cell response. Specific attention is paid to the impact of T cell receptor (TCR) stimulation strength on heterogeneity. In particular, this review endeavors to highlight connections between variation at different cellular stages, presenting known mechanisms and key open questions about how variation between cells can arise and propagate., Competing Interests: The author declares 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 Richard.)

Rejection is still a critical barrier to the long-term survival of graft after liver transplantation, requiring clinicians to unveil the underlying mechanism of liver transplant rejection. The cellular diversity and the interplay between immune cells in the liver graft microenvironment remain unclear. Herein, we performed single-cell RNA sequencing analysis to delineate the landscape of immune cells heterogeneity in liver transplantation. T cells, NK cells, B cells, and myeloid cell subsets in human liver and blood were enriched to characterize their tissue distribution, gene expression, and functional modules. The proportion of CCR6+CD4+ T cells increased within an allograft, suggesting that there are more memory CD4+ T cells after transplantation, in parallel with exhausted CTLA4+CD8+ T and actively proliferating MKI67+CD8+ T cells increased significantly, where they manifested heterogeneity, distinct function, and homeostatic proliferation. Remarkably, the changes of CD1c+ DC, CADM+ DC, MDSC, and FOLR3+ Kupffer cells increase significantly, but the proportion of CD163+ Kupffer, APOE+ Kupffer, and GZMA+ Kupffer decreased. Furthermore, we identified LDLR as a novel marker of activated MDSC to prevent liver transplant rejection. Intriguingly, a subset of CD4+CD8+FOXP3+ T cells included in CTLA4+CD8+ T cells was first detected in human liver transplantation. Furthermore, intercellular communication and gene regulatory analysis implicated the LDLR+ MDSC and CTLA4+CD8+ T cells interact through TIGIT-NECTIN2 signaling pathway. Taken together, these findings have gained novel mechanistic insights for understanding the immune landscape in liver transplantation, and it outlines the characteristics of immune cells and provides potential therapeutic targets in liver transplant rejection., 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 Li, Li, Wu, Xu, Teng, Yang, Sun, Zhao, Li, Liu, Yang, Gong and Cai.)

There is currently a lack of effective systemic treatment for patients with advanced pleomorphic rhabdomyosarcoma (PRMS). Although programmed death protein 1 (PD-1) inhibitors have shown efficacy in various solid tumors, their effects on PRMS have not been well established. Here, we present a case of a 12-year-old Chinese male adolescent with metastatic PRMS who benefited from the PD-1 inhibitor nivolumab. The patient initially underwent primary tumor resection but failed to respond to subsequent first-line chemotherapy and second-line pazopanib treatment. Pathological examination showed positive PD-L1 expression and tumor-infiltrating lymphocytes in the tumor tissue, and the patient was administered nivolumab as a posterior-line treatment. After attaining a clinically partial response (PR), surgical resection was performed, which was followed by adjuvant nivolumab. At the time of the submission of this manuscript, the patient achieved recurrence-free survival (RFS) lasting 45 months and counting. This is the first clinical evidence that a patient with refractory PRMS was controlled by anti-PD-1 antibody, with an RFS lasting more than 3 years. This case suggests that PD-L1 expression and T-cell infiltration could be used as potential biomarkers for PRMS immunotherapy., Competing Interests: Authors FG, YT and XZ are employed by 3DMed Clinical Laboratory Inc. The remaining 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 Liu, Liu, Gong, Tian and Zhao.)

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity., Competing Interests: Authors SR, NT-S, AS, MS, EP and AC were employed by Merck & Co., Inc., Kenilworth, NJ, USA. CK and RW were employed by Merck & Co., Inc., Kenilworth, NJ, USA at the time of the study and are currently employed by Synthekine. WB, EM, and TR are currently employed by Merck & Co., Inc., Kenilworth, NJ, USA., (Copyright © 2021 Raghavan, Tovbis-Shifrin, Kochel, Sawant, Mello, Sathe, Blumenschein, Muise, Chackerian, Pinheiro, Rosahl, Luche and de Waal Malefyt.)

Regulatory immunity that provides resistance to relapse emerges during resolution of experimental autoimmune uveitis (EAU). This post-EAU regulatory immunity requires a melanocortin 5 receptor (MC5r)-dependent suppressor antigen presenting cell (APC), as shown using a MC5r single knock-out mouse. The MC5r-dependent APC activates an adenosine 2A receptor (A2Ar)-dependent regulatory Treg cell, as shown using an A2Ar single knock-out mouse. Unexpectedly, when MC5r -/- post-EAU APC were used to activate A2Ar -/- post-EAU T cells the combination of cells significantly suppressed EAU, when transferred to EAU mice. In contrast, transfer of the reciprocal activation scheme did not suppress EAU. In order to explain this finding, MC5r -/- A2Ar -/- double knock-out (DKO) mice were bred. Naïve DKO mice had no differences in the APC populations, or inflammatory T cell subsets, but did have significantly more Treg cells. When we examined the number of CD4 and CD8 T cell subsets, we found significantly fewer CD8 T cells in the DKO mice compared to WT and both single knock-out mice. DKO mice also had significantly reduced EAU severity and accelerated resolution. In order to determine if the CD8 T cell deficiency contributed to the resistance to EAU in the DKO mice, we transferred naïve CD8 T cells from WT mice, that were immunized for EAU. Susceptibility to EAU was restored in DKO mice that received a CD8 T cell transfer. While the mechanism that contributed to the CD8 T cell deficiency in the DKO mice remains to be determined, these observations indicate an importance of CD8 T cells in the initiation of EAU. The involvement of CD4 and CD8 T cells suggests that both class I and class II antigen presentation can trigger an autoimmune response, suggesting a much wider range of antigens may trigger autoimmune disease., 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 © 2021 McDonald, Muhammad, Peters and Lee.)

Classical dendritic cells (cDCs) in mice have been divided into 2 major subsets based on the expression of nuclear transcription factors: a CD8 + Irf8 + Batf3 dependent (DC1) subset, and a CD8 - Irf4 + (DC2) subset. We found that the CD8 + DC1 subset can be further divided into CD8 + DC1a and CD8 + DC1b subsets by differences in surface receptors, gene expression, and function. Whereas all 3 DC subsets can act alone to induce potent Th1 cytokine responses to class I and II MHC restricted peptides derived from ovalbumin (OVA) by OT-I and OT-II transgenic T cells, only the DC1b subset could effectively present glycolipid antigens to natural killer T (NKT) cells. Vaccination with OVA protein pulsed DC1b and DC2 cells were more effective in reducing the growth of the B16-OVA melanoma as compared to pulsed DC1a cells in wild type mice. In conclusion, the Batf3-/- dependent DC1 cells can be further divided into two subsets with different immune functional profiles in vitro and in vivo ., 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 © 2021 Hongo, Zheng, Dutt, Pawar, Meyer, Engleman and Strober.)

CD8 T cell infiltration of the central nervous system (CNS) is necessary for host protection but contributes to neuropathology. Antigen presenting cells (APCs) situated at CNS borders are thought to mediate T cell entry into the parenchyma during neuroinflammation. The identity of the CNS-resident APC that presents antigen via major histocompatibility complex (MHC) class I to CD8 T cells is unknown. Herein, we characterize MHC class I expression in the naïve and virally infected brain and identify microglia and macrophages (CNS-myeloid cells) as APCs that upregulate H-2K b and H-2D b upon infection. Conditional ablation of H-2K b and H-2D b from CNS-myeloid cells allowed us to determine that antigen presentation via H-2D b , but not H-2K b , was required for CNS immune infiltration during Theiler's murine encephalomyelitis virus (TMEV) infection and drives brain atrophy as a consequence of infection. These results demonstrate that CNS-myeloid cells are key APCs mediating CD8 T cell brain infiltration., 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 © 2021 Goddery, Fain, Lipovsky, Ayasoufi, Yokanovich, Malo, Khadka, Tritz, Jin, Hansen and Johnson.)

Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections., 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 © 2021 Smyth, Khamina, Popa, Gudipati, Agerer, Lercher, Kosack, Endler, Baazim, Viczenczova, Huppa and Bergthaler.)

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8 + T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2 + cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2 + cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2 + CD8 + T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice., 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 © 2021 Prasad, Wojciech, Brzostek, Hu, Chua, Tung, Yap, Rybakin and Gascoigne.)

Hepatocytes compose up to 80% of the total liver and have been indicated as important players in the induction of immunologic tolerance in this organ. We show that hepatocytes possess the molecular machinery required for the cross-presentation of extracellular antigens. Using a derivative of the model antigen ovalbumin (OVA) covalently modified with a polymer containing multiple N-acetylgalactosamine residues (pGal-OVA) that enhance extracellular antigen uptake by mimicking the glycome of apoptotic debris, we show efficient hepatocyte-dependent induction of cross-tolerance of both adoptively transferred OT-I cells and endogenous OVA-specific CD8 + T lymphocytes, for example inducing tolerance to OVA-expressing skin transplants. Our study confirms that hepatocytes are capable of inducing peripheral tolerogenesis and provides proof of concept that they may be a valuable candidate for in vivo targeted tolerogenic treatments., Competing Interests: The EPFL has filed for patent protection on the pGal-antigen delivery platform, and MD, DW, and JH are named as inventors on those patents. Anokion SA and Kanyos Bio, Inc., have licensed those patents, and JH and DW participate in equity in those companies. The remaining author declares 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 © 2021 Damo, Wilson, Watkins and Hubbell.)

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo . Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses., 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 © 2021 Dölen, Gileadi, Chen, Valente, Creemers, Van Dinther, van Riessen, Jäger, Hruby, Cerundolo, Diken, Figdor and de Vries.)

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. The most efficacious vaccine, CVI988/Rispens (CVI988), against MD has been used for several decades. However, the mechanisms leading to protective immunity following vaccination are not fully understood. In this study, employing multi-parameter flow cytometry, we performed a comprehensive analysis of T cell responses in CVI988-vaccinated chickens. CVI988 vaccination induced significant expansion of γδ T cells and CD8α + T cells but not CD4 + T cells in spleen, lung and blood at early time-points. The expansion of these cells was CVI988-specific as infection with very virulent MDV RB1B did not elicit expansion of either γδ or CD8α + T cells. Phenotypic analysis showed that CVI988 vaccination elicited preferential proliferation of CD8α + γδ T cells and CD8αα co-receptor expression was upregulated on γδ T cells and CD8α + T cells after immunization. Additionally, cell sorting and quantitative RT-PCR showed that CVI988 vaccination activated γδ T cells and CD8α + T cells which exhibited differential expression of cytotoxic and T cell-related cytokines. Lastly, secondary immunization with CVI988 induced the expansion of CD8 + T cells but not γδ T cells at higher magnitude, compared to primary immunization, suggesting CVI988 did induce memory CD8 + T cells but not γδ T cells in chickens. Our results, for the first time, reveal a potential role of γδ T cells in CVI988-induced immune protection and provide new insights into the mechanism of immune protection against oncogenic MDV., 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 © 2021 Hao, Li, Li, Yang, Qin and Shang.)

The nuclear orphan receptors NR4A1, NR4A2, and NR4A3 are immediate early genes that are induced by various signals. They act as transcription factors and their activity is not regulated by ligand binding and are thus regulated via their expression levels. Their expression is transiently induced in T cells by triggering of the T cell receptor following antigen recognition during both thymic differentiation and peripheral T cell responses. In this review, we will discuss how NR4A family members impact different aspects of the life of a T cell from thymic differentiation to peripheral response against infections and cancer., 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 © 2021 Odagiu, May, Boulet, Baldwin and Labrecque.)

Immunological tolerance has evolved to curtail immune responses against self-antigens and prevent autoimmunity. One mechanism that contributes to immunological tolerance is the expression of inhibitory receptors by lymphocytes that signal to dampen immune responses during the course of an infection and to prevent immune-mediated collateral damage to the host. The understanding that tumors exploit these physiological mechanisms to avoid elimination has led to remarkable, but limited, success in the treatment of cancer through the use of biologics that interfere with the ability of cancers to suppress immune function. This therapy, based on the understanding of how T lymphocytes are normally activated and suppressed, has led to the development of therapeutic blocking antibodies, referred to as immune checkpoint blockade, which either directly or indirectly promote the activation of CD8 T cells to eradicate cancer. Here, we highlight the distinct signaling mechanisms, timing and location of inhibition used by the CTLA-4 and PD-1 inhibitory receptors compared to a novel inhibitory signaling axis comprised of the bioactive lipid, lysophosphatidic acid (LPA), signaling via the LPA5 receptor expressed by CD8 T cells. Importantly, abundant evidence indicates that an LPA-LPA5 signaling axis is also exploited by diverse cancers to suppress T cell activation and function. Clearly, a thorough molecular and biochemical understanding of how diverse T cell inhibitory receptors signal to suppress T cell antigen receptor signaling and function will be important to inform the choice of which complimentary checkpoint blockade modalities might be used for a given cancer., 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 © 2021 Mathew and Torres.)

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro , thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation., 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 © 2020 Puiffe, Dupont, Sako, Gatineau, Cohen, Mestivier, Lebon, Prévost-Blondel, Castellano and Molinier-Frenkel.)

Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark malignant and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1 ∗ 063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8 + T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs., (Copyright © 2020 Weinfurter, Graham, Ericsen, Matschke, Llewellyn-Lacey, Price, Wiseman and Reynolds.)

Memory T cells persist for long term to mediate robust recall response upon rechallenging with previous encountered pathogens. The memory T cell pool is highly heterogeneous based on distinct phenotypic, functional, and locational properties, and contains discrete subsets, which contribute to diverse immune responses. In this mini-review, we will briefly discuss the distinct subsets of memory T cells and then focus on mitochondria-related metabolic and epigenetic regulations of CD8 + T cell memory formation. In particular, we discuss many aspects of mitochondrial quality control systems (biogenesis, dynamics, etc.) in regulating CD8 + T cell fate decision and antitumor immunity. Importantly, targeting mitochondrial metabolism to boost T cell memory formation and metabolic fitness might represent an attractive strategy to improve cancer immunotherapy including CAR-T therapy., (Copyright © 2020 Li and Zhang.)

YAP is a transcriptional coactivator of the Hippo signaling pathway that has largely been studied for its role in the regulation of organ size during development. Several studies have shown that YAP is upregulated in cancer cells, and more recently in the T regulatory (Treg) subset of CD4+ cells. These observations suggest that the transcriptional co-activator may promote tumor persistence and progression. Here, we report that YAP also plays an immunoinhibitory role in CD8 T cells, especially in activated cytotoxic cells usually found in the tumor microenvironment. Our findings add further rationale for the development and use of pharmacologic inhibitors of YAP to treat cancer., (Copyright © 2020 Lebid, Chung, Pardoll and Pan.)

The cytotoxicity of epitope-specific CD8 + T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8 + T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8 + T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8 + T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8 + T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8 + T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8 + T cell cytotoxicity in a practical manner., (Copyright © 2020 Poh, Zheng, Channappanavar, Chang, Nguyen, Rénia, Kedzierska, Perlman and Poon.)

Epigenetic programs that control posttranslational modifications of histone proteins and DNA itself tightly regulate transcriptional networks determining the identity and function of CD8 + T cells. Chromatin-modifying enzymes such as histone acetyltransferases and deacetylases, represent key molecular determinants of the epigenetic imprinting of CD8 + T cells. The functions of these enzymes highly depend on the availability of key products of cellular metabolism pathways such as acetyl-CoA, NAD (Nicotinamide adenine dinucleotide) and SEM (S-adenosylmethionine), suggesting that there is a close crosstalk between the metabolic and the epigenetic regulation of CD8 + T cells. In this review, we will discuss the metabolic regulation of CD8 + T cell epigenetics during activation and differentiation. We will furthermore summarize how metabolic signals from the tumor microenvironment (TME) shape the epigenetic landscape of CD8 + T cells to better understand the mechanism underlying CD8 + T cell exhaustion in anti-tumor and anti-viral immunity, which might help to overcome limitations of current CD8 + T cell-based therapies., (Copyright © 2019 Yerinde, Siegmund, Glauben and Weidinger.)

Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ~30% of those chronically infected. By then, the drugs' efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8 + T cell, CD4 + T cell, and B cell epitopes that have <70% identity to human or human microbiome protein sequences and represent the basis toward the development of an epitope-based vaccine against T. cruzi ., (Copyright © 2019 Michel-Todó, Reche, Bigey, Pinazo, Gascón and Alonso-Padilla.)

Our previous studies of protective immunity and pathology against blood stage malaria parasites have shown that not only CD4 + T cells, but also CD8 + T cells and macrophages, are important for host defense against blood stage malaria infection. Furthermore, we found that Plasmodium yoelii 17XNL (PyNL) parasitizes erythroblasts, the red blood cell (RBC) precursor cells, which then express MHC class I molecules. In the present study, we analyzed spleen cytokine production. In CD8 + T cell-depleted mice, IL-10 production in early stage infection was increased over two-fold relative to infected control animals and IL-10 + CD3 - cells were increased, whereas IFN-γ production in the late stage of infection was decreased. At day 16 after PyNL infection, CD8 + T cells produced more IFN-γ than CD4 + T cells. We evaluated the involvement of the immunoproteasome in induction of immune CD8 + T cells, and the role of Fas in protection against PyNL both of which are downstream of IFN-γ. In cell transfer experiments, at least the single molecules LMP7, LMP2, and PA28 are not essential for CD8 + T cell induction. The Fas mutant LPR mouse was weaker in resistance to PyNL infection than WT mice, and 20% of the animals died. LPR-derived parasitized erythroid cells exhibited less externalization of phosphatidylserine (PS), and phagocytosis by macrophages was impaired. Furthermore, we tried to identify the cause of death in malaria infection. Blood lactate concentration was increased in the CD8 + T cell-depleted PyNL-infected group at day 19 (around peak parasitemia) to similar levels as day 7 after infection with a lethal strain of Py. When we injected mice with lactate at day 4 and 6 of PyNL infection, all mice died at day 8 despite demonstrating low parasitemia, suggesting that hyperlactatemia is one of the causes of death in CD8 + T cell-depleted PyNL-infected mice. We conclude that CD8 + T cells might control cytokine production to some extent and regulate hyperparasitemia and hyperlactatemia in protection against blood stage malaria parasites., (Copyright © 2019 Imai, Suzue, Ngo-Thanh, Ono, Orita, Suzuki, Shimokawa, Olia, Obi, Taniguchi, Ishida, Van Kaer, Murata, Tanaka and Hisaeda.)

Malaria, a disease caused by parasites of the Plasmodium genus, begins when Plasmodium-infected mosquitoes inject malaria sporozoites while searching for blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes, and form liver stages which in mice 48 h later escape into blood and cause clinical malaria. Vaccine-induced activated or memory CD8 T cells are capable of locating and eliminating all liver stages in 48 h, thus preventing the blood-stage disease. However, the rules of how CD8 T cells are able to locate all liver stages within a relatively short time period remains poorly understood. We recently reported formation of clusters consisting of variable numbers of activated CD8 T cells around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental data and mathematical models we now provide additional insights into mechanisms of formation of these clusters. First, we show that a model in which cluster formation is driven exclusively by T-cell-extrinsic factors, such as variability in "attractiveness" of different liver stages, cannot explain distribution of cluster sizes in different experimental conditions. In contrast, the model in which cluster formation is driven by the positive feedback loop (i.e., larger clusters attract more CD8 T cells) can accurately explain the available data. Second, while both Py-specific CD8 T cells and T cells of irrelevant specificity (non-specific CD8 T cells) are attracted to the clusters, we found no evidence that non-specific CD8 T cells play a role in cluster formation. Third and finally, mathematical modeling suggested that formation of clusters occurs rapidly, within few hours after adoptive transfer of CD8 T cells, thus illustrating high efficiency of CD8 T cells in locating their targets in complex peripheral organs, such as the liver. Taken together, our analysis provides novel insights into and attempts to discriminate between alternative mechanisms driving the formation of clusters of antigen-specific CD8 T cells in the liver., (Copyright © 2019 Kelemen, Rajakaruna, Cockburn and Ganusov.)

DENV is a major public health problem worldwide, thus underlining the overall significance of the proposed Program. The four dengue virus (DENV) serotypes (1-4) cause the most common mosquito-borne viral disease of humans, with 3 billion people at risk for infection and up to 100 million cases each year, most often affecting children. The protective role of T cells during viral infection is well-established. Generally, CD8 T cells can control viral infection through several mechanisms, including direct cytotoxicity, and production of pro-inflammatory cytokines such as IFN-γ and TNF-α. Similarly, CD4 T cells are thought to control viral infection through multiple mechanisms, including enhancement of B and CD8 T cell responses, production of inflammatory and anti-viral cytokines, cytotoxicity, and promotion of memory responses. To probe the phenotype of virus-specific T cells, epitopes derived from viral sequences need to be known. Here we discuss the identification of CD4 and CD8 T cell epitopes derived from DENV and how these epitopes have been used by researchers to interrogate the phenotype and function of DENV-specific T cell populations.

HIV-infected adults are at an increased risk to lower respiratory tract infections (LRTIs). CD8 + CD161 ++ TCRvα7.2 + T cells are an innate-like T cell subset that are thought to play an important role in early defense against pathogens in the respiratory tract. HIV infection leads to irreversible depletion of these cells in peripheral blood, however, its impact on this subset in the human airway is still unclear. Here, we show presence of CD103 expressing CD8 + CD161 ++ TCRvα7.2 + T cells in the airway that exhibited a distinct cytokine functional profile compared to their CD103 - airway counterparts and those from peripheral blood. These CD103 expressing airway CD8 + CD161 ++ TCRvα7.2 + T cells were selectively depleted in untreated HIV-infected adults compared to healthy controls. Their frequency was positively correlated with frequency of airway CD4 + T cells. Furthermore, the frequency of airway CD8 + CD161 ++ TCRvα7.2 + T cells was also inversely correlated with HIV plasma viral load, while suppressive antiretroviral therapy (ART) resulted in restoration of airway CD8 + CD161 ++ TCRvα7.2 + T cells. Our findings show that CD103 expressing airway CD8 + CD161 ++ TCRvα7.2 + T cells are functionally distinct and are preferentially depleted during untreated asymptomatic HIV infection. Depletion of CD103 expressing airway CD8 + CD161 ++ TCRvα7.2 + T cells, at a major portal of pathogen entry, could partly contribute to the increased propensity for opportunistic LRTIs observed in untreated HIV-infected adults.

In the past years, there have been significant advances in the understanding of how environmental conditions alone or in conjunction with pathogen invasion affect the metabolism of T cells, thereby influencing their activation, differentiation, and longevity. Detailed insights of the interlinked processes of activation and metabolism can contribute to major advances in immunotherapies. Naive and memory T cells circulate the body. In a quiescent state with low metabolic demands, they predominantly use oxidative phosphorylation for their energy needs. Recognition of cognate antigen combined with costimulatory signals results in a proliferative burst and effector molecule production, requiring rapid release of energy, achieved via dynamically reprogramming metabolic pathways. After activation, most T cells succumb to activation induced cell death, but few differentiate into memory T cells. Of note, some memory T cells permanently occupy tissues without circulating. These, tissue resident T cells are predominantly CD8 T cells, maintained in a metabolic state distinct from naïve and circulating memory CD8 T cells with elements similar to effector CD8 T cells but without undergoing proliferative burst or secreting immune mediators. They continually interact with tissue cells as part of an immune surveillance network, are well-adapted to the tissues they have made their home and where they may encounter different metabolic environments. In this review, we will discuss recent insights in metabolic characteristics of CD8 T cell biology, with emphasis on tissue resident CD8 T cells at the epithelial barriers.

Antiretroviral therapy (ART) has transformed HIV from a fatal disease to a chronic condition. In recent years there has been considerable interest in strategies to enable HIV-infected individuals to cease ART without viral rebound, either by purging all cells infected harboring replication-competent virus (HIV eradication), or by boosting immune responses to allow durable suppression of virus without rebound (HIV remission). Both of these approaches may need to harness HIV-specific CD8 + T cells to eliminate infected cells and/or prevent viral spread. In untreated infection, both HIV-specific and total CD8 + T cells are dysfunctional. Here, we review our current understanding of both global and HIV-specific CD8 + T cell immunity in HIV-infected individuals with durably suppressed viral load under ART, and its implications for HIV cure, eradication or remission. Overall, the literature indicates significant normalization of global T cell parameters, including CD4/8 ratio, activation status, and telomere length. Global characteristics of CD8 + T cells from HIV + ART + individuals align more closely with those of HIV-seronegative individuals than of viremic HIV-infected individuals. However, markers of senescence remain elevated, leading to the hypothesis that immune aging is accelerated in HIV-infected individuals on ART. This phenomenon could have implications for attempts to prime de novo , or boost existing HIV-specific CD8 + T cell responses. A major challenge for both HIV cure and remission strategies is to elicit HIV-specific CD8 + T cell responses superior to that elicited by natural infection in terms of response kinetics, magnitude, breadth, viral suppressive capacity, and tissue localization. Addressing these issues will be critical to the success of HIV cure and remission attempts.

The long-term persistence of viral antigens drives virus-specific CD8 T cell exhaustion during chronic viral infection. Yet exhausted, CD8 T cells are still endowed with certain levels of effector function, by which they can keep viral replication in check in chronic infection. However, the regulatory factors involved in regulating the effector function of exhausted CD8 T cell are largely unknown. Using mouse model of chronic LCMV infection, we found that the deletion of transcription factor TCF-1 in LCMV-specific exhausted CD8 T cells led to the profound reduction in cytokine production and degranulation. Conversely, ectopic expression of TCF-1 or using agonist to activate TCF-1 activities promotes the effector function of exhausted CD8 T cells. Mechanistically, TCF-1 fuels the functionalities of exhausted CD8 T cells by promoting the expression of an array of key effector function-associated transcription regulators, including Foxo1, Zeb2, Id3, and Eomes. These results collectively indicate that targeting TCF-1 mediated transcriptional pathway may represent a promising immunotherapy strategy against chronic viral infections by reinvigorating the effector function of exhausted virus-specific CD8 T cells.

Immune protection and lasting memory are accomplished through the generation of phenotypically and functionally distinct CD8 T cell subsets. Understanding how these effector and memory T cells are formed is the first step in eventually manipulating the immune system for therapeutic benefit. In this review, we will summarize the current understanding of CD8 T cell differentiation upon acute infection, with a focus on the transcriptional and epigenetic regulation of cell fate decision and memory formation. Moreover, we will highlight the importance of high throughput sequencing approaches and single cell technologies in providing insight into genome-wide investigations and the heterogeneity of individual CD8 T cells.

Effector and memory CD8 T cells have an intrinsic difference in the way they must approach antigen; effector cells need to address the pathogen at hand and therefore favor outgrowth of only high-affinity clones. In contrast, the memory pool benefits from greater clonal diversity to recognize and eliminate pathogens with mutations in their immunogenic epitopes. Effector and memory fates are ultimately the result of the same three signals that control T cell activation; T cell receptor (TCR) engagement together with co-stimulation and cytokines. Great progress has been made in our understanding of the transcriptional programs that drive effector or memory differentiation. However, how these two different programs result from the same initial cues is still a matter of debate. An emerging image is that not only the classical three signals determine T cell differentiation, but also the ability of cells to access these signals relative to that of other activated clones. Inter-clonal competition is therefore not only a selective force, but also a mediator of CD8 T cell fate. How this is regulated on a transcriptional level, especially in the context of a selective "hunger game" based on antigen-affinity in which only cells of high-affinity are supposed to survive, is still poorly defined. In this review, we discuss recent literature that illustrates how antigen-affinity dependent inter-clonal competition shapes effector and memory populations in an environment of antigen affinity-driven selection. We argue that fine-tuning of TCR signal intensity presents an attractive target for regulating the scope of CD8 T cell vaccines.

CD8 T cells comprising the memory pool display considerable heterogeneity, with individual cells differing in phenotype and function. This review will focus on our current understanding of heterogeneity within the antigen-specific memory CD8 T cell compartment and classifications of memory CD8 T cell subsets with defined and discrete functionalities. Recent data suggest that phenotype and/or function of numerically stable circulatory memory CD8 T cells are defined by the age of memory CD8 T cell (or time after initial antigen-encounter). In addition, history of antigen stimulations has a profound effect on memory CD8 T cell populations, suggesting that repeated infections (or vaccination) have the capacity to further shape the memory CD8 T cell pool. Finally, genetic background of hosts and history of exposure to diverse microorganisms likely contribute to the observed heterogeneity in the memory CD8 T cell compartment. Extending our tool box and exploring alternative mouse models (i.e., "dirty" and/or outbred mice) to encompass and better model diversity observed in humans will remain an important goal for the near future that will likely shed new light into the mechanisms that govern biology of memory CD8 T cells.

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.

Recent studies indicated that CXCR5 + CD8 + T cells in lymph nodes could eradicate virus-infected target cells. However, in the current study we found that a subset of CXCR5 + CD8 + T cells in the germinal centers from human tonsils or lymph nodes are predominately memory cells that express CD45RO and CD27. The involvement of CXCR5 + CD8 + T cells in humoral immune responses is suggested by their localization in B cell follicles and by the concomitant expression of costimulatory molecules, including CD40L and ICOS after activation. In addition, CXCR5 + CD8 + memory T cells produced significantly higher levels of IL-21, IFN-γ, and IL-4 at mRNA and protein levels compared to CXCR5 - CD8 + memory T cells, but IL-21-expressing CXCR5 + CD8 + T cells did not express Granzyme B and perforin. When cocultured with sorted B cells, sorted CXCR5 + CD8 + T cells promoted the production of antibodies compared to sorted CXCR5 - CD8 + T cells. However, fixed CD8 + T cells failed to help B cells and the neutralyzing antibodies against IL-21 or CD40L inhibited the promoting effects of sorted CXCR5 + CD8 + T cells on B cells for the production of antibodies. Finally, we found that in the germinal centers of lymph nodes from HIV-infected patients contained more CXCR5 + CD8 + T cells compared to normal lymph nodes. Due to their versatile functional capacities, CXCR5 + CD8 + T cells are promising candidate cells for immune therapies, particularly when CD4 + T cell help are limited.

Persistent antigen stimulation in chronic infections has been associated with antigen-specific T cell dysfunction and upregulation of inhibitory receptors, including programmed cell death protein 1 (PD-1). Pulmonary tuberculosis (TB) disease is characterized by high levels of Mycobacterium tuberculosis (Mtb), yet the relationship between bacterial load, PD-1 expression, and Mtb-specific T cell function in human TB has not been well-defined. Using peripheral blood samples from adults with LTBI and with pulmonary TB disease, we tested the hypothesis that PD-1 expression is associated with bacterial load and functional capacity of Mtb-specific T cell responses. We found that PD-1 was expressed at significantly higher levels on Th1 cytokine-producing Mtb-specific CD4 T cells from patients with smear-positive TB, compared with smear-negative TB and LTBI, which decreased after completion of anti-TB treatment. By contrast, expression of PD-1 on Mtb-specific CD8 T cells was significantly lower than on Mtb-specific CD4 T cells and did not differ by Mtb infection and disease status. In vitro stimulation of PBMC with Mtb antigens demonstrated that PD-1 is induced on proliferating Mtb-specific CD4 T cells and that Th1 cytokine production capacity is preferentially maintained within PD-1 + proliferating CD4 T cells, compared with proliferating Mtb-specific CD4 T cells that lack PD-1 expression. Together, these data indicate that expression of PD-1 on Mtb-specific CD4 T cells is indicative of mycobacterial antigen exposure and identifies a population of effector cells with Th1 cytokine production capacity. These studies provide novel insights into the role of the PD-1 pathway in regulating CD4 and CD8 T cell responses in Mtb infection and provide rationale for future studies to evaluate PD-1 expression on antigen-specific CD4 T cells as a potential biomarker for bacterial load and treatment response in human TB.

Humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (RSV), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. While some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. Despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. Nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. However, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. CD8 T cells are critical for mediating clearance following many acute viral infections in the lung. In addition, memory CD8 T cells are capable of providing protection against secondary infections. Therefore, the combined induction of virus-specific CD8 T cells and antibodies may provide optimal protective immunity. Herein, we review the current literature on CD8 T cell responses induced by respiratory virus infections. Additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on RSV vaccination.

Mast cells (MCs) are recognized to participate in the regulation of innate and adaptive immune responses. Owing to their strategic location at the host-environment interface, they control tissue homeostasis and are key cells for starting early host defense against intruders. Upon degranulation induced, e.g., by immunoglobulin E (IgE) and allergen-mediated engagement of the high-affinity IgE receptor, complement or certain neuropeptide receptors, MCs release a wide variety of preformed and newly synthesized products including proteases, lipid mediators, and many cytokines, chemokines, and growth factors. Interestingly, increasing evidence suggests a regulatory role for MCs in inflammatory diseases via the regulation of T cell activities. Furthermore, rather than only serving as effector cells, MCs are now recognized to induce T cell activation, recruitment, proliferation, and cytokine secretion in an antigen-dependent manner and to impact on regulatory T cells. This review synthesizes recent developments in MC-T cell interactions, discusses their biological and clinical relevance, and explores recent controversies in this field of MC research.

Our newly generated murine tumor dendritic cell (MuTuDC) lines, generated from tumors developing in transgenic mice expressing the simian virus 40 large T antigen (SV40LgT) and GFP under the DC specific promoter CD11c, reproduce the phenotypic and functional properties of splenic wild type CD8α(+) conventional DCs. They have an immature phenotype with low co-stimulation molecule expression (CD40, CD70, CD80, and CD86) that is upregulated after activation with toll-like receptor ligands. We observed that after transfer into syngeneic C57BL/6 mice, MuTuDC lines were quickly rejected. Tumors grew efficiently in large T transgene-tolerant mice. To investigate the immune response toward the large T antigen that leads to rejection of the MuTuDC lines, they were genetically engineered by lentiviral transduction to express luciferase and tested for the induction of DC tumors after adoptive transfer in various gene deficient recipient mice. Here, we document that the MuTuDC line was rejected in C57BL/6 mice by a CD4 T cell help-independent, perforin-mediated CD8 T cell response to the SV40LgT without pre-activation or co-injection of adjuvants. Using depleting anti-CD8β antibodies, we were able to induce efficient tumor growth in C57BL/6 mice. These results are important for researchers who want to use the MuTuDC lines for in vivo studies.

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed "exhaustion." Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of "exhaustion." Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term ("chronic") antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.