Vaccination with nanoparticles displaying gH/gL from Epstein-Barr virus elicits limited cross-protection against rhesus lymphocryptovirus.

Epstein-Barr virus (EBV) is associated with infectious mononucleosis, cancer, and multiple sclerosis. A vaccine that prevents infection and/or EBV-associated morbidity is an unmet need. The viral gH/gL glycoprotein complex is essential for infectivity, making it an attractive vaccine target. Here, we evaluate the immunogenicity of a gH/gL nanoparticle vaccine adjuvanted with the Sigma Adjuvant System (SAS) or a saponin/monophosphoryl lipid A nanoparticle (SMNP) in rhesus macaques. Formulation with SMNP elicits higher titers of neutralizing antibodies and more vaccine-specific CD4+ T cells. All but one animal in the SMNP group were infected after oral challenge with the EBV ortholog rhesus lymphocryptovirus (rhLCV). Their immune plasma had a 10- to 100-fold lower reactivity against rhLCV gH/gL compared to EBV gH/gL. Anti-EBV neutralizing monoclonal antibodies showed reduced binding to rhLCV gH/gL, demonstrating that EBV gH/gL neutralizing epitopes are poorly conserved on rhLCV gH/gL. Prevention of rhLCV infection despite antigenic disparity supports clinical development of gH/gL nanoparticle vaccines against EBV.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

Signals that control MAIT cell function in healthy and inflamed human tissues.

Mucosal-associated invariant T (MAIT) cells have a semi-invariant T-cell receptor that allows recognition of antigen in the context of the MHC class I-related (MR1) protein. Metabolic intermediates of the riboflavin synthesis pathway have been identified as MR1-restricted antigens with agonist properties. As riboflavin synthesis occurs in many bacterial species, but not human cells, it has been proposed that the main purpose of MAIT cells is antibacterial surveillance and protection. The majority of human MAIT cells secrete interferon-gamma (IFNg) upon activation, while some MAIT cells in tissues can also express IL-17. Given that MAIT cells are present in human barrier tissues colonized by a microbiome, MAIT cells must somehow be able to distinguish colonization from infection to ensure effector functions are only elicited when necessary. Importantly, MAIT cells have additional functional properties, including the potential to contribute to restoring tissue homeostasis by expression of CTLA-4 and secretion of the cytokine IL-22. A recent study provided compelling data indicating that the range of human MAIT cell functional properties is explained by plasticity rather than distinct lineages. This further underscores the necessity to better understand how different signals regulate MAIT cell function. In this review, we highlight what is known in regards to activating and inhibitory signals for MAIT cells with a specific focus on signals relevant to healthy and inflamed tissues. We consider the quantity, quality, and the temporal order of these signals on MAIT cell function and discuss the current limitations of computational tools to extrapolate which signals are received by MAIT cells in human tissues. Using lessons learned from conventional CD8 T cells, we also discuss how TCR signals may integrate with cytokine signals in MAIT cells to elicit distinct functional states.

A Noninvasive Method to Sample Immune Cells in the Lower Female Genital Tract Using Menstrual Discs.

T cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection, including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT, but current sampling methods require a healthcare provider and are expensive, limiting the ability to study these populations longitudinally. To address these challenges, we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive, self-applied, and low in cost. To demonstrate reproducibility, we sampled the cervicovaginal fluid of healthy, reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells, and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live, CD45+ leukocytes, as well as distinct populations of T cells and B cells. Within the T cell compartment, activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches, including identification of both tissue-resident and migratory populations. In addition, the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies.

A non-invasive method to sample immune cells in the lower female genital tract using menstrual discs.

T cells in the human female genital tract (FGT) 2 are key mediators of susceptibility to and protection from infection, including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT, but current sampling methods require a healthcare provider and are expensive, limiting the ability to study these populations longitudinally. To address these challenges, we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are non-invasive, self-applied, and low-cost. To demonstrate reproducibility, we sampled the cervicovaginal fluid (CVF) 3 of healthy, reproductive-aged individuals using menstrual discs over three sequential days. CVF was processed for cervicovaginal cells, and high parameter flow cytometry was used to characterize immune populations. We identified large numbers of live, CD45+ leukocytes, as well as distinct populations of T cells and B cells. Within the T cell compartment, activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches, including identification of both tissue resident and migratory populations. In addition, the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies.

TGF-ß broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner.

Transforming growth factor ß (TGF-ß) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-ß signaling. TGF-ß availability is altered by infections and cancer; however, the dose-dependent effects of TGF-ß on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-ß signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-ß 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-ß had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-ß signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-ß signals is of importance. We found that exposure to TGF-ß before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-ß 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-ß-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-ß is not simply suppressing CD8 Tmem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.

TGF-ß broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner.

Transforming growth factor ß (TGF-ß) directly acts on naïve, effector and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-ß signaling. TGF-ß availability is altered by infections and cancer, however the dose-dependent effects of TGF-ß on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-ß signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-ß could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or pro-inflammatory signals. In contrast, even high doses of TGF-ß had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-ß signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-ß signals is of importance. We found that exposure to TGF-ß prior to as well as after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-ß 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-ß-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-ß is not simply suppressing CD8 Tmem, but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner.

Recurrent infection transiently expands human tissue T cells while maintaining long-term homeostasis.

Chronic viral infections are known to lead to T cell exhaustion or dysfunction. However, it remains unclear if antigen exposure episodes from periodic viral reactivation, such as herpes simplex virus type-2 (HSV-2) recrudescence, are sufficient to induce T cell dysfunction, particularly in the context of a tissue-specific localized, rather than a systemic, infection. We designed and implemented a stringent clinical surveillance protocol to longitudinally track both viral shedding and in situ tissue immune responses in a cohort of HSV+ volunteers that agreed to avoid using anti-viral therapy for the course of this study. Comparing lesion to control skin biopsies, we found that tissue T cells expanded immediately after reactivation, and then returned numerically and phenotypically to steady state. T cell responses appeared to be driven at least in part by migration of circulating T cells to the infected tissue. Our data indicate that tissue T cells are stably maintained in response to HSV reactivation, resembling a series of acute recall responses.

Advances and challenges in studying the tissue-resident T cell compartment in the human female reproductive tract.

Tissue-resident memory T cells (TRM ) 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 TRM compartment for each tissue and across tissues with well-defined experimental and environmental variables. Assessing the functional characteristics of the human TRM compartment is substantially more difficult; thus, notably, there is a paucity of studies profiling the TRM 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 TRM cells in the FRT: different sampling methods of the FRT greatly affect immune cell recovery, especially of TRM cells. Furthermore, menstrual cycle, menopause, and pregnancy affect FRT immunity, but little is known about changes in the TRM compartment. Finally, we discuss the potential functional plasticity of the TRM compartment during inflammatory episodes in the human FRT to maintain protection and tissue homeostasis, which are required to ensure reproductive fitness.

Memory T cells possess an innate-like function in local protection from mucosal infection.

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.

Pyrimidine de novo synthesis inhibition selectively blocks effector but not memory T cell development.

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.

50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in tissue biopsies and other human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

Substantial uneven proliferation of CD4+ T cells during recovery from acute HIV infection is sufficient to explain the observed expanded clones in the HIV reservoir.

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.

Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues.

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.

Extricating human tumour immune alterations from tissue inflammation.

Immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain1,2. 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 treatment3,4. 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 (Treg) 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+ Treg cells had responded to antigen recently and demonstrate that they are clonally expanded with superior suppressive function compared with IL1R1- Treg 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.

Convergent clonal selection of donor- and recipient-derived CMV-specific T cells in hematopoietic stem cell transplant patients.

Competition between antigen-specific T cells for peptide:MHC complexes shapes the ensuing T cell response. Mouse model studies provided compelling evidence that competition is a highly effective mechanism controlling the activation of naïve T cells. However, assessing the effect of T cell competition in the context of a human infection requires defined pathogen kinetics and trackable naïve and memory T cell populations of defined specificity. A unique cohort of nonmyeloablative hematopoietic stem cell transplant patients allowed us to assess T cell competition in response to cytomegalovirus (CMV) reactivation, which was documented with detailed virology data. In our cohort, hematopoietic stem cell transplant donors and recipients were CMV seronegative and positive, respectively, thus providing genetically distinct memory and naïve T cell populations. We used single-cell transcriptomics to track donor versus recipient-derived T cell clones over the course of 90 d. We found that donor-derived T cell clones proliferated and expanded substantially following CMV reactivation. However, for immunodominant CMV epitopes, recipient-derived memory T cells remained the overall dominant population. This dominance was maintained despite more robust clonal expansion of donor-derived T cells in response to CMV reactivation. Interestingly, the donor-derived T cells that were recruited into these immunodominant memory populations shared strikingly similar TCR properties with the recipient-derived memory T cells. This selective recruitment of identical and nearly identical clones from the naïve into the immunodominant memory T cell pool suggests that competition is in place but does not interfere with rejuvenating a memory T cell population. Instead, it results in selection of convergent clones to the memory T cell pool.

A regulatory T cell signature distinguishes the immune landscape of COVID-19 patients from those with other respiratory infections.

Despite recent studies of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little is known about how the immune response against SARS-CoV-2 differs from other respiratory infections. We compare the immune signature from hospitalized SARS-CoV-2­infected patients to patients hospitalized prepandemic with influenza or respiratory syncytial virus (RSV). Our in-depth profiling indicates that the immune landscape in SARS-CoV-2 patients is largely similar to flu or RSV patients. Unique to patients infected with SARS-CoV-2 who had the most critical clinical disease were changes in the regulatory T cell (Treg) compartment. A Treg signature including increased frequency, activation status, and migration markers was correlated COVID-19 severity. These findings are relevant as Tregs are considered for therapy to combat the severe inflammation seen in COVID-19 patients. Likewise, having defined the overlapping immune landscapes in SARS-CoV-2, existing knowledge of flu and RSV infections could be leveraged to identify common treatment strategies.

Metabolic regulation by PD-1 signaling promotes long-lived quiescent CD8 T cell memory in mice.

Inhibitory signaling in dysfunctional CD8 T cells through the programmed cell death 1 (PD-1) axis is well established in chronic viral infections and cancers. PD-1 is also transiently induced to high concentrations during priming of acute infections and immunizations, yet its impact on the development of long-lived antigen-independent T cell memory remains unclear. In addition to its expected role in restraining clonal effector expansion, here, we show that PD-1 expression on antigen-specific CD8 T cells is required for the development of a durable CD8 T cell memory pool after antigen clearance. Loss of T cell­specific PD-1 signaling led to increased contraction and a defect in antigen-independent renewal of memory CD8 T cells in response to homeostatic cytokine signals, thus resulting in attrition of the memory pool over time. Whereas exhausted CD8 T cells regain function after PD-1 checkpoint blockade during chronic viral infection, the preexisting pool of resting functional bystander memory CD8 T cells established in response to a previously administered immunogen decreased. Metabolically, PD-1 signals were necessary for regulating the critical balance of mTOR-dependent anabolic glycolysis and fatty acid oxidation programs to meet the bioenergetic needs of quiescent CD8 T cell memory. These results define PD-1 as a key metabolic regulator of protective T cell immunity. Furthermore, these results have potential clinical implications for preexisting CD8 T cell memory during PD-1 checkpoint blockade therapy.