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CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

Mouse models have contributed significantly to our knowledge of the pathogenesis of autoimmune diseases. However, differences in mouse and human immune systems limit the translation of the knowledge in mechanism and therapeutic outcomes from mouse to human. Therefore, a human model of autoimmune diseases is urgently needed. Recently, our lab has developed a functional tonsil organotypic system that can recapitulate key T cells responses and germinal center response to influenza vaccine in vitro. We now want to utilize this tonsil system to study autoimmunity. Regulatory T cells (Tregs) have been suggested to play a central role in maintaining self-tolerance. Reduced Treg cell frequencies and impaired suppressive function have been reported in a wide range of autoimmune diseases. In this study, we show that we can efficiently knock out FOXP3 in T cells from human tonsil organoids by using CRISPR technology. We also show that tonsil organoids with FOXP3 knocked-out T cells are able to induce humoral immune responses towards autoantigens stimulation and influenza vaccination. This system could be useful for studying underlying mechanisms in the functions of Tregs in the autoimmune condition in humans and provides insights into the therapeutic strategy for autoimmune diseases.

Despite clear differences in immune system responses and in the prevalence of autoimmune diseases between males and females, there is little understanding of the processes involved. In this study, we identified a gene signature of immature-like neutrophils, characterized by the overexpression of genes encoding for several granule-containing proteins, which was found at higher levels (up to 3-fold) in young (20–30 y old) but not older (60 to >89 y old) males compared with females. Functional and phenotypic characterization of peripheral blood neutrophils revealed more mature and responsive neutrophils in young females, which also exhibited an elevated capacity in neutrophil extracellular trap formation at baseline and upon microbial or sterile autoimmune stimuli. The expression levels of the immature-like neutrophil signature increased linearly with pregnancy, an immune state of increased susceptibility to certain infections. Using mass cytometry, we also find increased frequencies of immature forms of neutrophils in the blood of women during late pregnancy. Thus, our findings show novel sex differences in innate immunity and identify a common neutrophil signature in males and in pregnant women.

T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

Narcolepsy is a sleeping disorder, caused by selective loss of neurons secreting a wake-promoting hormone, hypocretin (HCRT). Increased narcolepsy incidence was noted in China after the peak of 2009 H1N1 pandemic influenza (pH1N1) cases and in Europe after Pandemrix vaccine administration. The causes of HCRT neuron depletion and its relationship with pH1N1 are unknown. As narcolepsy susceptibility is associated with HLA-DQ6 and with a TCR α gene (J24) polymorphism, we hypothesized that abnormally expanded J24+/CD4+ T cells in DQ6+ subjects mediate autoimmunity against HCRT neurons and that molecular mimicry (epitopes in pH1N1 and self-antigens) drives T cell expansion via DQ6 presentation. We reported the in vivo expansion of DQ6/HCRT87–97–reactive J24+/CD4+ clones using a J24 allele and an un-conventional phenotype in patients compared to related clones in DQ6+ controls. Here, DQ6/peptide binding and crystal structure identify an epitope, HA274–287 from pH1N1 hemagglutinin, with homology to HCRT87–97 and another peptide, HCRT1–13. DQ6/HA274–287 tetramers isolated >2000 tetramer+/CD4+ T cells in DQ6+ subjects. Single-cell sequencing of TCRs and 25 phenotypic transcripts identify a DQ6/HA274–287 tetramer+/J24+ clone with the identical TCR clonotype (TCR27) and phenotype as the expanded DQ6/HCRT87–97–reactive J24+ clones previously found in the same patient. Control-derived TCR27+ clones identified by DQ6/HCRT1–13 or DQ6/HCRT25–37 tetramers are unexpanded and lack expression of any of the phenotypic genes. Our findings show that DQ6+ subjects carry J24+/CD4+ cells, both public and cross-reactive (e.g TCR27). TCR27 expansion in patients argues for molecular mimicry as a factor in narcolepsy immunopathogenesis.

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder in which neuroinflammation plays a critical function. Yet, little is known about the contribution of the adaptive immune response in AD. Here we used integrated analyses of multiple cohorts to identify peripheral and central adaptive immune changes in AD. First, we performed mass cytometry of peripheral blood mononuclear cells and discovered an immune signature of AD consisting of increased numbers of CD8+ T effector memory CD45RA+ (TEMRA) cells. In a second cohort, we found that CD8+ TEMRA cells were negatively associated with cognition. Furthermore, single cell RNA sequencing revealed their enhanced T cell receptor (TCR) signaling. Notably, by utilizing multiple single cell TCR sequencing strategies on a third cohort, we discovered clonally expanded CD8+ TEMRA cells in patient cerebrospinal fluid (CSF). Finally, we used machine learning, cloning, and peptide screens to demonstrate specificity of clonally expanded AD CSF TCRs to two separate Epstein-Barr virus antigens. These results reveal a novel blood-CSF adaptive immune response in AD and provide evidence of clonal, antigen-experienced T cells patrolling the intrathecal space of brains affected by age-related neurodegeneration.

A hallmark of CD4+ T cell activation and immunological synapse (IS) formation is the migration of the microtubule organization center and associated organelles toward the APCs. In this study, we found that when murine CD4+ T cells were treated with a microtubule-destabilizing agent (vinblastine) after the formation of IS, the microtubule organization center dispersed and all of the major cellular organelles moved away from the IS. Cytokines were no longer directed toward the synapse but were randomly secreted in quantities similar to those seen in synaptic secretion. However, if the actin cytoskeleton was disrupted at the same time with cytochalasin D, the organelles did not shift away from the IS. These findings suggest that there is a complex interplay between the microtubules and actin cytoskeleton, where microtubules are important for directing particular cytokines into the synapse, but they are not involved in the amount of cytokines that are produced for at least 1 h after IS formation. In addition, we found that they play a critical role in mobilizing organelles to reorient toward the synapse during T cell activation and in stabilizing organelles against the force that is generated through actin polymerization so that they move toward the APCs. These findings show that there is a complex interplay between these major cytoskeletal components during synapse formation and maintenance.

Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.

Identification of the specific HLA locus and allele presenting an epitope for recognition by specific TCRs (HLA restriction) is necessary to fully characterize the immune response to Ags. Experimental determination of HLA restriction is complex and technically challenging. As an alternative, the restricting HLA locus and allele can be inferred by genetic association, using response data in an HLA-typed population. However, simple odds ratio (OR) calculations can be problematic when dealing with large numbers of subjects and Ags, and because the same epitope can be presented by multiple alleles (epitope promiscuity). In this study, we develop a tool, denominated Restrictor Analysis Tool for Epitopes, to extract inferred restriction from HLA class II–typed epitope responses. This automated method infers HLA class II restriction from large datasets of T cell responses in HLA class II–typed subjects by calculating ORs and relative frequencies from simple data tables. The program is validated by: 1) analyzing data of previously determined HLA restrictions; 2) experimentally determining in selected individuals new HLA restrictions using HLA-transfected cell lines; and 3) predicting HLA restriction of particular peptides and showing that corresponding HLA class II tetramers efficiently bind to epitope-specific T cells. We further design a specific iterative algorithm to account for promiscuous recognition by calculation of OR values for combinations of different HLA molecules while incorporating predicted HLA binding affinity. The Restrictor Analysis Tool for Epitopes program streamlines the prediction of HLA class II restriction across multiple T cell epitopes and HLA types.

Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by cytomegalovirus (CMV) is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or Epstein-Barr virus (EBV) infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of immunoglobulin heavy chain (IGH) gene rearrangements to study the B cell receptor repertoires over two successive years in 27 individuals ranging in age from 20 to 89 years. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG immunoglobulin genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, while CMV infection correlates with the proportion of highly mutated antibody genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires, and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.

We describe a novel gene, Blimp-1 (for B lymphocyte-induced maturation protein), transcripts of which are rapidly induced during the differentiation of B lymphocytes into immunoglobulin secretory cells and whose expression is characteristic of late B and plasma cell lines. The 856 amino acid open reading frame contains five Kruppel-type zlnc finger motifs and proline-rich and acidic regions similar to those of known transcription factors. Serological studies show an ∼ 100 kd protein that localizes to the nucleus. Stable or transient transfection of Blimp-1 into B cell lymphoma lines leads to the expression of many of the phenotypic changes associated with B cell differentiation into an early plasma cell stage, including induction of J chain message and immunoglobulin secretion, up-regulation of Syndecan-1, and increased cell size and granularity. Thus, Blimp-1 appears to be a pleiotropic regulatory factor capable of at least partially driving the terminal differentiation of B cells.

In 2016, there were 216 million malaria cases – 445,000 of which resulted in deaths. Despite overwhelming evidence that γδ T cells strongly respond during malaria infection and vaccination, their functional and phenotypic characteristics remain the least understood facets of the adaptive immune response. Therefore, we studied the role of these cells in human and mouse malaria. In both Plasmodium falciparum-infected subjects and in P. chabaudi-infected mice, we found γδ T cells expanding rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Silencing the murine γδ T cells led to recurrent rounds of Plasmodium parasitemia. Single-cell T cell receptor sequencing of the expanded mouse cells revealed oligoclonal γδ T cells restricted to the TRAV15N-1 (Vδ6.3) V-region and converging complementarity-determining region 3 (CDR3) motifs. Also, RNA-seq of the expanded γδ T cells showed an unexpected transcriptional profile characterized by myeloid-modulating factors, previously unseen in γδ T cells. The expanded TRAV15N-1 γδ T cells abundantly produced M-CSF, which was necessary for preventing parasitemic recurrence. Interestingly, αβ T cells were the major source of M-CSF during acute infection, while γδ T cells filled that role during the post-acute stage. We have uncovered a novel γδ T cell subset that fills a protective role in the late stage of malaria. These cells could provide the mechanism for other observed correlations between γδ T cell and myeloid activity in cancer and infectious disease.

Antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe autoimmunity. To investigate this, we combined anti-CTLA-4 treatment with a standard colitis model to give mice a more severe form of the disease. Pre-treatment with an antibiotic, Vancomycin provoked an even more severe, largely fatal form, suggesting that a gram-positive component of the microbiota had a mitigating effect. We then found that a commonly used probiotic, Bifidobacterium could largely rescue the mice from immunopathology without an apparent effect on anti-tumor immunity, and this effect may be dependent on regulatory T cells.

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.

The recent outbreak of Zika virus (ZIKV) infection across the Americas has raised dire concerns about its potential long-term health effects including its negative impact on neuronal, psychological and motor development in affected individuals. Emerging evidence pointing to Alzheimer’s-like damage to the adult brain further highlights the urgent need for efficacious prophylactic measures. Zika-affected regions are often endemic to the related Dengue virus (DENV). Antibodies against the two viruses are crossreactive and cause antibody-mediated enhancement (ADE) of infection. This confounds the design of traditional vaccines that elicit humoral antibody-mediated responses. T cell vaccines mediated by cytotoxic CD8+ T cells that recognize conserved viral epitopes presented on the major histocompatibility complex (MHC-I) of infected cells are unencumbered by ADE and can supplement or substitute traditional vaccines. In order to identify robust T cell vaccine candidates, we first used an immunoproteomic approach and isolated over 100 novel DENV and ZIKA epitopes presented by infected B lymphocytes in vitro. These viral peptides were not predicted to be epitopes by computational programs typically used for epitope discovery, which discard >95% of all predictions. Focussing on epitopes derived from polyprotein regions strictly conserved between the two viruses, we validated the presentation of these epitopes in primary dendritic cells and assayed their ability to stimulate robust T cell responses in ex vivo ELISpot assays. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DENV and ZIKV antigens which have the potential to serve as broad acting and effective Zika vaccine candidates.

Presentation of novel antigenic peptides (neoantigens) that distinguish malignant from normal cells by major histocompatibility complexes (MHC) can serve as potent substrates for specific anti-tumor immune responses. We sought to identify mantle cell lymphoma (MCL) neoantigens by taking an integrated genomic and proteomic strategy that interrogates antigen peptides presented by MHC-class I and class II. Peptides bound to MHC were purified via immunoprecipitation followed by identification using mass spectrometry. Mass spectra were searched against patient-specific proteome databases generated by whole exome sequencing and targeted immunoglobulin gene sequencing. This approach was applied to systematically characterize over 36,000 immunopeptides from 17 patients’ tumor specimens. Interestingly, 52 neoantigenic peptides were derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we identified MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. Furthermore, somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC-II. T-cells specific for Ig-derived neoantigens were found in two patients. Following ex vivo activation and expansion, the T-cells were remarkably able to mediate killing of autologous lymphoma cells. These results demonstrate that combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to MCL implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

We describe the generation of three mAbs that recognize the complex of the class II MHC molecule IEk bound to a peptide derived from the carboxyl terminus of moth cytochrome c (residues 95–103). Reactivities of these mAbs are sensitive to single alterations in the sequence of both helices of the MHC molecule and to the bound peptide. The epitopes of these reagents are distinct but overlap substantially. One of these mAbs specifically blocks lymphokine release by T cells responsive to this complex but not others. We have used another to examine how the number of complexes on an APC is related to its ability to stimulate T cells. We find that 200–400 complexes per cell are necessary and sufficient to induce a degree of stimulation, whereas maximum stimulation is achieved only if more than 5000 complexes are present. The analysis indicates that T cell activation is a stochastic process.

Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77–85) and reverse transcriptase (amino acids 464–472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50–1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals

Dengue (DEN) virus infection is a leading cause of hospitalization and death in the developing world and broadly effective vaccines and therapies remain elusive. Identifying infection-specific peptide antigens would open new avenues for developing T cell based interventions. Past efforts towards mapping viral antigens used computational predictions that only partially reflect actual antigens presented by the major histocompatibility complex (MHC). To identify DEN-specific antigens without relying on error prone predictions, we developed an immuno-proteomics approach for interrogating antigen presentation in DEN infected B-lymphocytes. This approach enabled three fundamental findings; First, we identified 86 viral MHC-I antigens (59 novel), and mapped them to presentation hotspots in the DEN genome. Second, we discovered post-translationally modified viral and host antigens and antigens derived from alternate reading frames. Predicting these antigens using computational methods alone would be infeasible. Third, we found antigens responsible for HLA-haplotype dependent immune responses against DEN infection: we genetically engineered B-lymphocytes to express HLA alleles associated with either strong or weak DEN-immune responses and identified corresponding allele specific antigens. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DEN-specific antigens. Ex-vivo assays including ELISPOT and HLA tetramer staining supported our identification of immunogenic antigens in DEN-specific CD8+ T cells. These antigens have potential as both diagnostic tools to characterize DEN-specific T cell immunity, and serve as candidates for designing effective DEN vaccines.

There are many billions of chronic viral infections in humans, as each of the ~7.4 billion individuals alive today each host ~10 chronic infections. The clinical significance of these infections vary widely, yet all individuals with chronic viral infections rely on persistent antiviral immunity to keep these infections in check. Over time, chronically infected individuals exhibit impaired immune function as evidenced by their poor response to vaccination and progressive T cell dysfunction – a process known as exhaustion. The impact of chronic viral infection on other immune cell types is understudied, and whether the immune landscape can be restored upon viral eradication is unknown. To address these questions, we have prospectively recruited a cohort of HCV-infected patients who underwent standard of care treatment leading to viral clearance in 12 weeks, a cohort of virologically suppressed HIV-infected patients, and an aged-matched cohort of healthy individuals. With the use of single-cell mass cytometry (CyTOF) and systems bioinformatics analyses, we comprehensively characterized the molecular pathways and functional signature underlying immune exhaustion. Here we report an immune signaling signature that differentiates individuals with chronic viral infections from uninfected individuals. While this signature is largely unaltered following viral clearance, we identified a set of functional parameters that are restorable upon successful viral eradication which represent a previously unobserved level of plasticity of the human immune system.