Preservation of naive-phenotype CD4+ T cells after vaccination contributes to durable immunity.

Memory T cells are conventionally associated with durable recall responses. In our longitudinal analyses of CD4+ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found CD45RO-CCR7+ virus-specific CD4+ T cells that expanded shortly after vaccination and persisted months to years after immunization. Further phenotypic analyses revealed the presence of stem cell-like memory T cells within this subset. In addition, after vaccination T cells lacking known memory markers and functionally resembling genuine naive T cells were identified, referred to herein as marker-negative T (TMN) cells. Single-cell TCR sequencing detected expanded clonotypes within the TMN subset and identified TMN TCRs shared with memory and effector T cells. Longitudinal tracking of YFV-specific responses over subsequent years revealed superior stability of TMN cells, which correlated with the longevity of the overall tetramer+ population. These findings uncover additional complexity within the post-immune T cell compartment and implicate TMN cells in durable immune responses.

Differentiation marker-negative CD4+ T cells persist after yellow fever virus vaccination and contribute to durable memory.

Factors that contribute to durable immunological memory remain incompletely understood. In our longitudinal analyses of CD4+ T cell responses to the yellow fever virus (YFV) vaccine by peptide-MHC tetramers, we unexpectedly found naïve phenotype virus-specific CD4+ T cells that persisted months to years after immunization. These Marker negative T cells (TMN) lacked CD95, CXCR3, CD11a, and CD49d surface protein expression, distinguishing them from previously discovered stem-cell memory T cells. Functionally, they resembled genuine naïve T cells upon in vitro stimulation. Single-cell TCR sequencing detected expanded clonotypes within the TMN subset and identified a shared repertoire with memory and effector T cells. T cells expressing TMN-associated TCRs were rare before vaccination, suggesting their expansion following vaccination. Longitudinal tracking of YFV-specific responses over the subsequent years revealed superior stability of the TMN subset and their association with the longevity of the overall population. The identification of these long-lived, antigen-experienced T cells may inform the design of durable T cell-based vaccines and engineered T cell therapies.

Pre-existing T Cell Memory to Novel Pathogens.

Immunological experiences lead to the development of specific T and B cell memory, which readies the host for a later pathogen rechallenge. Currently, immunological memory is best understood as a linear process whereby memory responses are generated by and directed against the same pathogen. However, numerous studies have identified memory cells that target pathogens in unexposed individuals. How "pre-existing memory" forms and impacts the outcome of infection remains unclear. In this review, we discuss differences in the composition of baseline T cell repertoire in mice and humans, factors that influence pre-existing immune states, and recent literature on their functional significance. We summarize current knowledge on the roles of pre-existing T cells in homeostasis and perturbation and their impacts on health and disease.

Identification of Human Antigen-Specific T Cells Using Class II MHC Tetramer Staining and Enrichment.

The development of peptide-major histocompatibility complex tetramers has enabled direct characterization and enumeration of antigen-specific T cells. However, the weaker interaction between class II tetramers and CD4+ T cells increases the challenge of using tetramers to analyze CD4+ T cell responses. Here, we provide an optimized class II tetramer staining protocol with a magnetic-bead enrichment strategy for the detection and functional analyses of human antigen-specific CD4+ T cells. This approach enables direct sampling of lymphocytes that recognize specific peptide-MHC complexes, including rare pathogen-specific CD4+ T cells from unexposed individuals.

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens.

The baseline composition of T cells directly affects later response to pathogens, but the complexity of precursor states remains poorly defined. Here, we examined the baseline state of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells in unexposed individuals. SARS-CoV-2-specific CD4+ T cells were identified in prepandemic blood samples by major histocompatibility complex (MHC) class II tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2-specific T cells that expressed memory phenotype markers. Integrated phenotypic analyses demonstrated diverse preexisting memory states that included cells with distinct polarization features and trafficking potential to barrier tissues. T cell clones generated from tetramer-labeled cells cross-reacted with antigens from commensal bacteria in the skin and gastrointestinal tract. Direct ex vivo tetramer staining for one spike-specific population showed a similar level of cross-reactivity to sequences from endemic coronavirus and commensal bacteria. These data highlight the complexity of precursor T cell repertoire and implicate noninfectious exposures to common microbes as a key factor that shapes human preexisting immunity to SARS-CoV-2.

Transcriptome and TCR Repertoire Measurements of CXCR3+ T Follicular Helper Cells Within HIV-Infected Human Lymph Nodes.

Follicular-helper T cells (TFH) are an essential arm of the adaptive immune system. Although TFH were first discovered through their ability to contribute to antibody affinity maturation through co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing TFH in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of TFH receiving attention in chronic infection are CXCR3-expressing TFH cells (CXCR3+TFH) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other TFH subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of TFH within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3+TFH compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3+TFH away from germinal center TFH (GC-TFH), including the altered expression of several important transcription factors and cytokines. CXCR3+TFH also upregulated cell migration transcriptional programs and were clonally related to peripheral TFH populations. In combination, these data suggest that CXCR3+TFH have a greater tendency to enter circulation than their CXCR3- counterparts, potentially functioning through distinct modalities that may lead to enhanced defense.

SARS-CoV-2-specific T cells in unexposed adults display broad trafficking potential and cross-react with commensal antigens.

The baseline composition of T cells directly impacts later response to a pathogen, but the complexity of precursor states remains poorly defined. Here we examined the baseline state of SARS-CoV-2 specific T cells in unexposed individuals. SARS-CoV-2 specific CD4 + T cells were identified in pre-pandemic blood samples by class II peptide-MHC tetramer staining and enrichment. Our data revealed a substantial number of SARS-CoV-2 specific T cells that expressed memory phenotype markers, including memory cells with gut homing receptors. T cell clones generated from tetramer-labeled cells cross-reacted with bacterial peptides and responded to stool lysates in a MHC-dependent manner. Integrated phenotypic analyses revealed additional precursor diversity that included T cells with distinct polarized states and trafficking potential to other barrier tissues. Our findings illustrate a complex pre-existing memory pool poised for immunologic challenges and implicate non-infectious stimuli from commensal colonization as a factor that shapes pre-existing immunity. ONE SENTENCE SUMMARY: Pre-existing immunity to SARS-CoV-2 contains a complex pool of precursor lymphocytes that include differentiated cells with broad tissue tropism and the potential to cross-react with commensal antigens.

Vaccination reshapes the virus-specific T cell repertoire in unexposed adults.

We examined how baseline CD4+ T cell repertoire and precursor states impact responses to pathogen infection in humans using primary immunization with yellow fever virus (YFV) vaccine. YFV-specific T cells in unexposed individuals were identified by peptide-MHC tetramer staining and tracked pre- and post-vaccination by tetramers and TCR sequencing. A substantial number of YFV-reactive T cells expressed memory phenotype markers and contained expanded clones in the absence of exposure to YFV. After vaccination, pre-existing YFV-specific T cell populations with low clonal diversity underwent limited expansion, but rare populations with a reservoir of unexpanded TCRs generated robust responses. These altered dynamics reorganized the immunodominance hierarchy and resulted in an overall increase in higher avidity T cells. Thus, instead of further increasing the representation of dominant clones, YFV vaccination recruits rare and more responsive T cells. Our findings illustrate the impact of vaccines in prioritizing T cell responses and reveal repertoire reorganization as a key component of effective vaccination.

Mass Cytometry Defines Virus-Specific CD4+ T Cells in Influenza Vaccination.

The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-γ, and TNF-α, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.

Mapping the Lineage Relationship between CXCR5+ and CXCR5- CD4+ T Cells in HIV-Infected Human Lymph Nodes.

CXCR5 is a key marker of follicular helper T (TFH) cells. Using primary lymph nodes (LNs) from HIV-infected patients, we identified a population of CXCR5- CD4+ T cells with TFH-cell-like features. This CXCR5- subset becomes expanded in severe HIV infection and is characterized by the upregulation of activation markers and high PD-1 and ICOS surface expression. Integrated analyses on the phenotypic heterogeneity, functional capacity, T cell receptor (TCR) repertoire, transcriptional profile, and epigenetic state of CXCR5-PD-1+ICOS+ T cells revealed a shared clonal relationship with TFH cells. CXCR5-PD-1+ICOS+ T cells retained a poised state for CXCR5 expression and exhibited a migratory transcriptional program. TCR sequence overlap revealed a contribution of LN-derived CXCR5-PD-1+ICOS+ T cells to circulating CXCR5- CD4+ T cells with B cell help function. These data link LN pathology to circulating T cells and expand the current understanding on the diversity of T cells that regulate B cell responses during chronic inflammation.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Identification and characterization of HIV-specific resident memory CD8+ T cells in human lymphoid tissue.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

The receptor repertoire and functional profile of follicular T cells in HIV-infected lymph nodes.

Follicular helper CD4+ T cells (TFH) play an integral role in promoting B cell differentiation and affinity maturation. Whereas TFH cell frequencies are increased in lymph nodes (LNs) from individuals infected with HIV, humoral immunity remains impaired during chronic HIV infection. Whether HIV inhibits TFH responses in LNs remains unclear. Advances in this area have been limited by the difficulty of accessing human lymphoid tissues. Here, we combined high-dimensional mass cytometry with T cell receptor repertoire sequencing to interrogate the composition of TFH cells in primary human LNs. We found evidence for intact antigen-driven clonal expansion of TFH cells and selective utilization of specific complementarity-determining region 3 (CDR3) motifs during chronic HIV infection, but the resulting TFH cells acquired an activation-related TFH cell signature characterized by interleukin-21 (IL-21) dominance. These IL-21+ TFH cells contained an oligoclonal HIV-reactive population that preferentially accumulated in patients with severe HIV infection and was associated with aberrant B cell distribution in the same LN. These data indicate that TFH cells remain capable of responding to HIV antigens during chronic HIV infection but become functionally skewed and oligoclonally restricted under persistent antigen stimulation.

Successive annual influenza vaccination induces a recurrent oligoclonotypic memory response in circulating T follicular helper cells.

T follicular helper (Tfh) CD4 cells are crucial providers of B cell help during adaptive immune responses. A circulating population of CD4 T cells, termed cTfh, have similarity to lymphoid Tfh, can provide B cell help, and responded to influenza vaccination. However, it is unclear whether human vaccination-induced cTfh respond in an antigen-specific manner and whether they form long-lasting memory. Here, we identified a cTfh population that expressed multiple T cell activation markers and could be readily identified by coexpression of ICOS and CD38. This subset expressed more Bcl-6, c-Maf, and IL-21 than other blood CD4 subsets. Influenza vaccination induced a strong response in the ICOS+CD38+ cTfh at day 7, and this population included hemagglutinin-specific cells by tetramer staining and antigen-stimulated Activation Induced Marker (AIM) expression. Moreover, TCRB sequencing identified a clonal response in ICOS+CD38+ cTfh that correlated strongly with the increased circulating ICOS+CD38+ cTfh frequency and the circulating plasmablast response. In subjects who received successive annual vaccinations, a recurrent oligoclonal response was identified in the ICOS+CD38+ cTfh subset at 7 days after every vaccination. These oligoclonal responses in ICOS+CD38+ cTfh after vaccination persisted in the ICOS-CD38- cTfh repertoire in subsequent years, suggesting clonal maintenance in a memory reservoir in the more-stable ICOS-CD38- cTfh subset. These data highlight the antigen-specificity, lineage relationships and memory properties of human cTfh responses to vaccination, providing new avenues for tracking and monitoring cTfh responses during infection and vaccination in humans.

Antigen exposure shapes the ratio between antigen-specific Tregs and conventional T cells in human peripheral blood.

The T-cell receptor (TCR) is required for maturation and function of regulatory T cells (Tregs), but the ligand specificities of Tregs outside the context of transgenic TCRs are largely unknown. Using peptide-MHC tetramers, we isolated rare specific Foxp3+ cells directly ex vivo from adult peripheral blood and defined their frequency and phenotype. We find that a proportion of circulating Tregs recognize foreign antigens and the frequency of these cells are similar to that of self-reactive Tregs in the absence of cognate infection. In contrast, the frequencies of Tregs that recognize some common microbial antigens are significantly reduced in the blood of most adults. Exposure to peripheral antigens likely has a major influence on the balance between Tregs and conventional T-cell subsets because a larger proportion of flu-specific T cells has a regulatory cell phenotype in the cord blood. Consistent with this finding, we show that lymphocytic choriomeningitis virus infection can directly modulate the ratio of virus-specific effectors and Tregs in mice. The resulting change in the balance within an antigen-specific T-cell population further correlates with the magnitude of effector response and the chronicity of infection. Taken together, our data highlight the importance of antigen specificity in the functional dynamics of the T-cell repertoire. Each specific population of CD4+ T cells in human peripheral blood contains a subset of Tregs at birth, but the balance between regulatory and effector subsets changes in response to peripheral antigen exposure and this could impact the robustness of antipathogen immunity.

Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer.

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4(+)and CD8(+)αß T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4(+)CD8(+)double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.

Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.

The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4⁺ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1⁺CXCR5⁺CD4⁺ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV⁺ individuals.

Antiviral memory phenotype T cells in unexposed adults.

αß T cells are an integral part of protective immunity against pathogens. After precursor cells arise in the adult bone marrow or fetal liver, they migrate to the thymus where they rearrange their T-cell receptor genes (TCR) and undergo selection on the basis of their interactions with ligands expressed by thymic stroma and other cells. Those that survive then exit the thymus to populate the peripheral immune compartment, where they patrol the blood and lymphoid systems. The composition of this pre-immune peripheral repertoire is critically important in determining the robustness of an immune response. In both mice and humans, the magnitude and diversity of a response are directly correlated with the frequency of precursor T cells. Equally relevant are the functional characteristics of these lymphocytes. Engagement of a specific antigen to the TCR activates signaling pathways in the naive T cell that result in cellular proliferation and the acquisition of particular effector functions. A portion of these persist following the resolution of infection and become memory cells. These memory cells can mount a faster and stronger response when they encounter the same antigen at a later time. As the molecular basis for TCR ligand interaction has become better defined, it is clear that some T cells can recognize multiple distinct ligands and therefore T-cell memory developed by exposure to one ligand may play a significant role in the response to a different antigen. Thus, there is an increasing focus on understanding how exposure to related or unrelated antigens influences the T-cell repertoire and impacts subsequent immunity. In this review, we discuss the issue of TCR cross-reactivity in the development of memory phenotype CD4(+) T cells and the implications for pathogen-specific responses. We review both the human and mouse data and discuss the therapeutic implications of these findings in the contexts of infection and vaccination.

Virus-specific CD4(+) memory-phenotype T cells are abundant in unexposed adults.

Although T cell memory is generally thought to require direct antigen exposure, we found an abundance of memory-phenotype cells (20%-90%, averaging over 50%) of CD4(+) T cells specific to viral antigens in adults who had never been infected. These cells express the appropriate memory markers and genes, rapidly produce cytokines, and have clonally expanded. In contrast, the same T cell receptor (TCR) specificities in newborns are almost entirely naïve, which might explain the vulnerability of young children to infections. One mechanism for this phenomenon is TCR cross-reactivity to environmental antigens, and in support of this, we found extensive cross-recognition by HIV-1 and influenza-reactive T lymphocytes to other microbial peptides and expansion of one of these after influenza vaccination. Thus, the presence of these memory-phenotype T cells has significant implications for immunity to novel pathogens, child and adult health, and the influence of pathogen-rich versus hygienic environments.

The promised land of human immunology.

Advances in technology and data analysis have made it possible to take a new look at human immunology. These advances run the gamut from systems biology approaches, which are likely in the vanguard of how we can start "to put the pieces together" of immune function, to a deeper understanding of specific diseases and vaccines and the immune repertoire. In our own experience, we have also found that asking simple questions about human immunity has often given us very surprising answers, causing a rethink of established dogma. Thus, we have developed a new perspective on the nature of the αß TCR repertoire and also the likely role of T-cell repertoire (TCR) cross-reactivity in generating T memory independent of specific antigen interactions. These findings show that human immunology is not just a necessary step for "translating" basic immunology to treat diseases or develop better vaccines, but is also an important complement to the inbred mouse model.