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CD8 T cell responses to vaccinia virus (VV) and a virus-encoded ovalbumin peptide (OVAP) epitope were examined using adoptively transferred OT-I T cells. The results demonstrate that upon intra-peritoneal challenge with ovalbumin-expressing VV (VV-OVAP), OT-I T cell proliferation occurs initially in lymph nodes and spleens followed by migration of the divided cells to the peritoneal cavity. Massive clonal expansion occurs in response to both the virus and the virus-encoded ovalbumin (OVA) epitope, as demonstrated using low numbers of adoptively transferred cells, and the responding OT-I cells display marked site-dependent functional heterogeneity with respect to IFN-γ and tumor necrosis factor-α (TNF-α) production and granzyme B expression. OT-I cells responding to VV-OVAP develop the capacity to produce IFN-γ in response to antigen as they proliferate and differentiate. In marked contrast, naive OT-I cells rapidly produce TNF-α upon antigen recognition, and this capacity declines as the cells proliferate in response to the virus, suggesting that this potent inflammatory cytokine may be important primarily during initiation of the response. At the peak of clonal expansion, a large fraction (30–60%) of the OT-I cells responding to the virus express high IL-7Rα levels, and the majority of these cells is subsequently lost. While high IL-7Rα expression may be necessary for a CD8 T cell to transition to memory, it is clearly not sufficient. Thus, OT-I cells responding to VV infection exhibit a high degree of heterogeneity within the responding population that differs depending on their anatomical location, despite the specificity and affinity of the TCR being identical on all of the cells. [ABSTRACT FROM PUBLISHER]

CD8+ T cells only require a brief stimulation with antigen in vitro to divide and differentiate into effector and memory cells upon transfer in vivo. The efficiency of clonal expansion and the functional characteristics of memory cells derived from briefly stimulated cells are poorly defined. We developed a system that allowed us to examine programming entirely in vivo. This was achieved by rapidly killing peptide-pulsed DCs carrying a diphtheria toxin receptor transgene with timed injections of diphtheria toxin without altering the course of an accompanying infection. The magnitude of clonal expansion, but not the functionality of the effector cells, correlated directly with the duration of antigen exposure. Furthermore, memory T cells were capable of mounting a secondary response, regardless of the length of antigen encounter during the primary response. These results indicate that the duration of initial antigen encounter influences the magnitude of the primary response, but does not program responsiveness during the secondary challenge. [ABSTRACT FROM AUTHOR]

Tissue-resident CD8+ T cells (Trm) can rapidly eliminate virally infected cells, but their heterogeneous spatial distribution may leave gaps in protection within tissues. Although Trm patrol prior sites of viral replication, murine studies suggest they do not redistribute to adjacent uninfected sites to provide wider protection. We perform mathematical modeling of HSV-2 shedding in and predict that infection does not induce enough Trm in many genital tract regions to eliminate shedding; a strict spatial distribution pattern of mucosal CD8+ T cell density is maintained throughout chronic infection, and trafficking of Trm across wide genital tract areas is unlikely. These predictions are confirmed with spatial analysis of CD8+ T cell distribution in histopathologic specimens from human genital biopsies. Further simulations predict that the key mechanistic correlate of protection following therapeutic HSV-2 vaccination would be an increase in total Trm rather than spatial reassortment of these cells. The fixed spatial structure of Trm induced by HSV-2 is sufficient for rapid elimination of infected cells but only in a portion of genital tract microregions. [ABSTRACT FROM AUTHOR]

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4+ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4+ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4+ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood. [ABSTRACT FROM AUTHOR]

Despite continued progress in the development of novel antiretroviral therapies, it has liecome increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV+ patients. In 2009, an HIV+ patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5-/-donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4+ T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4+ T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4+ T cells, an increase in nonmodified CD4+ T cells, enhanced cytotoxic. T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer; however, the dose-dependent effects of TGF-β on memory CD8 T cell (T mem ) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of T mem reactivation. We found that TGF-β could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or proinflammatory signals. In contrast, even high doses of TGF-β had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 T mem may not always receive TGF-β signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-β signals is of importance. We found that exposure to TGF-β before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-β altered ~10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-β-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-β is not simply suppressing CD8 T mem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner., Competing Interests: Competing interests statement:The authors declare no competing interest.

Tissue-resident memory T cells (T RM ) are considered to be central to maintaining mucosal barrier immunity and tissue homeostasis. Most of this knowledge stems from murine studies, which provide access to all organs. These studies also allow for a thorough assessment of the T RM compartment for each tissue and across tissues with well-defined experimental and environmental variables. Assessing the functional characteristics of the human T RM compartment is substantially more difficult; thus, notably, there is a paucity of studies profiling the T RM compartment in the human female reproductive tract (FRT). The FRT is a mucosal barrier tissue that is naturally exposed to a wide range of commensal and pathogenic microbes, including several sexually transmitted infections of global health significance. We provide an overview of studies describing T cells within the lower FRT tissues and highlight the challenges of studying T RM cells in the FRT: different sampling methods of the FRT greatly affect immune cell recovery, especially of T RM cells. Furthermore, menstrual cycle, menopause, and pregnancy affect FRT immunity, but little is known about changes in the T RM compartment. Finally, we discuss the potential functional plasticity of the T RM compartment during inflammatory episodes in the human FRT to maintain protection and tissue homeostasis, which are required to ensure reproductive fitness., (© 2023 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Mucosal infections pose a significant global health burden. Antigen-specific tissue-resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8+ T cells may also provide innate-like protection against antigenically unrelated pathogens independent of T cell receptor engagement. Whether bystander T cell activation is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated whether innate-like memory CD8+ T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8+ T cells may mediate protection despite the lack of antigen specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-nonspecific CD8+ T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8+ T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8+ T cells from mice and humans. Altogether, our findings suggest that local bystander activation of CD8+ memory T cells contributes a fast and effective innate-like response to infection in mucosal tissue.

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The HIV reservoir is a population of 1-10 million anatomically dispersed, latently infected memory CD4 + T cells in which HIV DNA is quiescently integrated into human chromosomal DNA. When antiretroviral therapy (ART) is stopped and HIV replication initiates in one of these cells, systemic viral spread resumes, rekindling progression to AIDS. Therefore, HIV latency prevents cure. The detection of many populations of identical HIV sequences at unique integration sites implicates CD4 + T cell proliferation as the critical driver of reservoir sustainment after a prolonged period of effective ART. Initial reservoir formation occurs during the first week of primary infection usually before ART is started. While empirical data indicates that both de novo infection and cellular proliferation generate latently infected cells during early untreated infection, it is not known which of these mechanisms is predominant. We developed a mathematical model that recapitulates the profound depletion and brisk recovery of CD4 + T cells, reservoir creation, and viral load trajectory during primary HIV infection. We extended the model to stochastically simulate individual HIV reservoir clones. This model predicts the first detection of HIV infected clones approximately 5 weeks after infection as has recently been shown in vivo and suggests that substantial, uneven proliferation among clones during the recovery from CD4 + lymphopenia is the most plausible explanation for the observed clonal reservoir distribution during the first year of infection., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Published by Elsevier Ltd.)

Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design., (© 2022. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

Immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain 1,2 . An inherent weakness of current treatment approaches is that therapeutically targeted pathways are not restricted to tumours, but are also found in other tissue microenvironments, complicating treatment 3,4 . Despite great efforts to define inflammatory processes in the tumour microenvironment, the understanding of tumour-unique immune alterations is limited by a knowledge gap regarding the immune cell populations in inflamed human tissues. Here, in an effort to identify such tumour-enriched immune alterations, we used complementary single-cell analysis approaches to interrogate the immune infiltrate in human head and neck squamous cell carcinomas and site-matched non-malignant, inflamed tissues. Our analysis revealed a large overlap in the composition and phenotype of immune cells in tumour and inflamed tissues. Computational analysis identified tumour-enriched immune cell interactions, one of which yields a large population of regulatory T (T reg ) cells that is highly enriched in the tumour and uniquely identified among all haematopoietically-derived cells in blood and tissue by co-expression of ICOS and IL-1 receptor type 1 (IL1R1). We provide evidence that these intratumoural IL1R1 + T reg cells had responded to antigen recently and demonstrate that they are clonally expanded with superior suppressive function compared with IL1R1 - T reg cells. In addition to identifying extensive immunological congruence between inflamed tissues and tumours as well as tumour-specific changes with direct disease relevance, our work also provides a blueprint for extricating disease-specific changes from general inflammation-associated patterns., (© 2022. The Author(s).)

Memory CD4 T cells in tissues fulfill numerous functions that are critical for local immune homeostasis and protection against pathogens. Previous studies have highlighted the phenotypic and functional heterogeneity of circulating and tissue-resident memory CD4 T cells across different human tissues such as skin, lung, liver, and colon. Comparatively little is known in regard to memory CD4 T cells across tissues of the female reproductive tract (FRT). We examined CD4 T cells in donor-matched vaginal, ecto- and endocervical tissues, which differ in mucosal structure and exposure to external environmental stimuli. We hypothesized that this could be reflected by tissue-specific differences in the memory CD4 T cell compartment. We found differences in CD4 subset distribution across these tissues. Specifically, CD69 + CD103 + CD4 T cells were significantly more abundant in vaginal than cervical tissues. In contrast, the transcriptional profiles of CD4 subsets were fairly conserved across FRT tissues. CD69 + CD103 + CD4 T cells showed a T H 17 bias independent of tissue niche. Our data suggest that FRT tissues affect T cell subset distribution but have limited effects on the transcriptome of each subset. We discuss the implications for barrier immunity in the FRT.

The mechanisms underlying rapid elimination of herpes simplex virus-2 (HSV-2) in the human genital tract despite low CD8+ and CD4+ tissue-resident T cell (Trm cell) density are unknown. We analyzed shedding episodes during chronic HSV-2 infection; viral clearance always predominated within 24 hours of detection even when viral load exceeded 1 × 107 HSV DNA copies, and surges in granzyme B and IFN-γ occurred within the early hours after reactivation and correlated with local viral load. We next developed an agent-based mathematical model of an HSV-2 genital ulcer to integrate mechanistic observations of Trm cells in in situ proliferation, trafficking, cytolytic effects, and cytokine alarm signaling from murine studies with viral kinetics, histopathology, and lesion size data from humans. A sufficiently high density of HSV-2-specific Trm cells predicted rapid elimination of infected cells, but our data suggest that such Trm cell densities are relatively uncommon in infected tissues. At lower, more commonly observed Trm cell densities, Trm cells must initiate a rapidly diffusing, polyfunctional cytokine response with activation of bystander T cells in order to eliminate a majority of infected cells and eradicate briskly spreading HSV-2 infection.

The immune system of the cervicovaginal tract (CVT) must balance immunosurveillance and active immunity against pathogens with maintenance of tolerance to resident microbiota and to fetal and partner antigens for reproductive purposes. Thus, we predicted that CVT immunity is characterized by distinctive features compared to blood and other tissue compartments. Indeed, we found that CVT CD8+ T-cells had unique transcriptional profiles, particularly in their cytokine signature, compared to that reported for CD8+ T-cells in other tissue sites. Among these CVT CD8+ T-cells, we identified a CD69- CD103- subset that was characterized by reduced migration in response to tissue-exit signals and higher pro-inflammatory potential as compared to their blood counterpart. These inflammatory mucosal CD8+ T-cells (Tim) were increased in frequency in the CVT of individuals with chronic infection, pointing to a potential role in perpetuating inflammation. Our findings highlight the specialized nature of immunity within the CVT and identify Tim cells as potential therapeutic targets to tame tissue inflammation upon chronic infection.

Tissue-resident CD8 + T cells (T rm ) can rapidly eliminate virally infected cells, but their heterogeneous spatial distribution may leave gaps in protection within tissues. Although T rm patrol prior sites of viral replication, murine studies suggest they do not redistribute to adjacent uninfected sites to provide wider protection. We perform mathematical modeling of HSV-2 shedding in Homo sapiens and predict that infection does not induce enough T rm in many genital tract regions to eliminate shedding; a strict spatial distribution pattern of mucosal CD8 + T cell density is maintained throughout chronic infection, and trafficking of T rm across wide genital tract areas is unlikely. These predictions are confirmed with spatial analysis of CD8 + T cell distribution in histopathologic specimens from human genital biopsies. Further simulations predict that the key mechanistic correlate of protection following therapeutic HSV-2 vaccination would be an increase in total T rm rather than spatial reassortment of these cells. The fixed spatial structure of T rm induced by HSV-2 is sufficient for rapid elimination of infected cells but only in a portion of genital tract microregions., (Copyright © 2018 by The American Association of Immunologists, Inc.)

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4 + memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4 + T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4 + memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood., (Copyright © 2017 by The American Association of Immunologists, Inc.)