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The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1–CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2–22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Supplementary Figure 16: 8 gene score is predominately expressed by CD8+ T cells and NK cells in both patients.

Supplementary Figure 12: TCRβ repertoires in the post-NAC residual disease or tumor scar are most like PD-1HI CD8+ peripheral repertoires.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Supplementary Figure 11: Cytokine secretion is markedly enriched in PD-1HI T cells after NAC in a patient with pCR to NAC in TNBC.

Supplementary Figure 7: Changes in immunologic signatures in response to NAC are not associated with outcome in non-TNBC tumors with residual disease.

Supplementary Figure 3: The prognostic value of sTILs is primarily confined to the post-NAC specimen in TNBC patients with residual disease.

Supplementary Figure 6: Neither time of sample nor institution drive significant gene expression changes.

Supplementary Tables

Supplementary Figure 4: sTILs are not prognostic in ER+ or HER2+ disease.

Supplementary Figure 14: Differentially expressed genes by cluster in whole blood single cell RNA sequencing.

Supplementary Figure 1: Representative images of high and low sTILs.

Supplementary Figure 10: Example FACS gating to identify PD-1HI CD8+ T cells from peripheral blood.

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Supplementary Figure 15: Differential analysis to identify candidate genes for blood-based detection.

Supplementary Figure 13: Purity-of-sort analysis.

Supplementary Figure 17: An 8-gene activated T cell signature derived from whole blood at surgery is associated with residual disease burden and recurrence.

Supplementary Figure 8: Changes in immune-related genes in response to NAC are not associated with outcome in ER+ or HER2+ tumors with residual disease.

SummaryDevelopment of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection againstin vivochallenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Lower CD4The aim of this study was to explore the association between peripherally circulating CD4Data from 1,860 participants (1,270 PWH) without prevalent CVD from the VACS (Veterans Aging Cohort Study), a prospective, observational cohort of veterans with and without HIV infection, were analyzed. T cell subsets were quantified in baseline samples using flow cytometry. Incident CVD events were identified using International Classification of Diseases-9th Revision and International Classification of Diseases-10th Revision diagnosis and procedure codes. Participants were followed from baseline date (2005-2006) to the first of CVD incidence, death, or September 30, 2016. Cox proportional hazards regression was used to model associations between these T cell subsets and the risk for incident CVD while adjusting for demographics and other CVD risk factors.The median participant age at baseline was 51.6 years. Most were male (94%) and of Black race (69.1%). There were 344 incident CVD events (219 in PWH) during follow-up (median 9.8 years). In PWH, higher proportions (per SD increment) of T helper type 17 cells (adjusted HR: 1.19; 95% CI: 1.08-1.31), T effector memory cells re-expressing CD45RA (adjusted HR: 1.19; 95% CI: 1.07-1.34), and CD28Among PWH, T helper type 17 cells, senescent cells, and CD4

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigenssup1/sup. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigenssup2-22/sup. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypessup23,24/sup. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Half a billion years of evolutionary battle forged the vertebrate adaptive immune system, an astonishingly versatile factory for molecules that can adapt to arbitrary attacks. The history of an individual encounter is chronicled within a clonotype: the descendants of a single fully rearranged adaptive immune cell. For B cells, reading this immune history for an individual remains a fundamental challenge of modern immunology. Identification of such clonotypes is a magnificently challenging problem for three reasons:The cell history is inferred rather than directly observed: the only available data are the sequences of V(D)J molecules occurring in a sample of cells.Each immune receptor is a pair of V(D)J molecules. Identifying these pairs at scale is a technological challenge and cannot be done with perfect accuracy—real samples are mixtures of cells and fragments thereof.These molecules can be intensely mutatedduring the optimization of the response to particular antigens, blurring distinctions between kindred molecules.It is thus impossible to determine clonotypes exactly. All solutions to this problem make a trade-off between sensitivity and specificity; useful solutions must address actual artifacts found in real data.We presentenclone1, a system for computing approximate clonotypes from single cell data, and demonstrate its use and value with the 10x Genomics Immune Profiling Solution. To test it, we generate data for 1.6 million individual B cells, from four humans, including deliberately enriched memory cells, to tax the algorithm and provide a resource for the community. We analytically determine the specificity ofenclone’s clonotyping algorithm, showing that on this dataset the probability of co-clonotyping two unrelated B cells is around 10−9. We prove that using only heavy chains increases the error rate by two orders of magnitude.enclonecomprises a comprehensive toolkit for the analysis and display of immune receptor data. It is ultra-fast, easy to install, has public source code, comes with public data, and is documented atbit.ly/enclone. It has three “flavors” of use: (1) as a command-line tool run from a terminal window, that yields visual output; (2) as a command-line tool that yields parseable output that can be fed to other programs; and (3) as a graphical version (GUI).

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

OBJECTIVES/HYPOTHESIS Characterization of the localized adaptive immune response in the airway scar of patients with idiopathic subglottic stenosis (iSGS). STUDY DESIGN Basic Science. METHODS Utilizing 36 patients with subglottic stenosis (25 idiopathic subglottic stenosis [iSGS], 10 iatrogenic post-intubation stenosis [iLTS], and one granulomatosis with polyangiitis [GPA]) we applied immunohistochemical and immunologic techniques coupled with RNA sequencing. RESULTS iSGS, iLTS, and GPA demonstrate a significant immune infiltrate in the subglottic scar consisting of adaptive cell subsets (T cells along with dendritic cells). Interrogation of T cell subtypes showed significantly more CD69+ CD103+ CD8+ tissue resident memory T cells (TRM ) in the iSGS airway scar than iLTS specimens (iSGS vs. iLTS; 50% vs. 28%, P = .0065). Additionally, subglottic CD8+ clones possessed T-cell receptor (TCR) sequences with known antigen specificity for viral and intracellular pathogens. CONCLUSIONS The human subglottis is significantly enriched for CD8+ tissue resident memory T cells in iSGS, which possess TCR sequences proven to recognize viral and intracellular pathogens. These results inform our understanding of iSGS, provide a direction for future discovery, and demonstrate immunologic function in the human proximal airway. Laryngoscope, 131:610-617, 2021.

The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-15.

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8+ T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8+ T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8+ T cells were predominantly dual tetramer+, confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8+ T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8+ T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an ‘effective’ immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8+ T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8+ T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8+ T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8+ T cells were predominantly dual tetramer+, confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8+ T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8+ T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an ‘effective’ immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8+ T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-programmed cell death receptor 1 therapy in a patient with metastatic melanoma. Histologic analyses revealed robust T cell infiltration and prominent programmed death ligand 1 expression. We identified 209 reported cases in global pharmacovigilance databases (across multiple cancer types) of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4+ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor repertoire, which we localized to activated memory cytotoxic (CD45RO+GZMB+Ki67+) CD4 cells. We also identified Epstein-Barr virus-specific T cell receptors and EBV+ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the three cases studied here identify CD4+ and CD8+ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

Cellular immune responses to Gag correlate with improved HIV control. The full extent of cellular immune responses comprises both the number of epitopes recognized by CD4(+) and CD8(+) T cells and the diversity of the T cell receptor (TCR) repertoire directed against each epitope. The optimal diversity of the responsive TCR repertoire is unclear. Therefore, we evaluated the TCR diversity of CD4(+) and CD8(+) T cells responding to HIV-1 Gag to determine if TCR diversity correlates with clinical or virologic metrics. Previous studies of TCR repertoires have been limited primarily to CD8(+) T cell responses directed against a small number of well-characterized T cell epitopes restricted by specific human leukocyte antigens. We stimulated peripheral blood mononuclear cells from 21 chronic HIV-infected individuals overnight with a pool of HIV-1 Gag peptides, followed by sorting of activated CD4(+) and CD8(+) T cells and TCR deep sequencing. We found Gag-reactive CD8(+) T cells to be more oligoclonal, with a few dominant TCRs comprising the bulk of the repertoire, compared with the highly diverse TCR repertoires of Gag-reactive CD4(+) T cells. HIV sequencing of the same donors revealed that high CD4(+) T cell TCR diversity was strongly associated with lower HIV Gag genetic diversity. We conclude that the TCR repertoire of Gag-reactive CD4(+) T helper cells displays substantial diversity without a clearly dominant circulating TCR clonotype, in contrast to a hierarchy of dominant TCR clonotypes in the Gag-reactive CD8(+) T cells, and may serve to limit HIV diversity during chronic infection. IMPORTANCE Human T cells recognize portions of viral proteins bound to host molecules (human leukocyte antigens) on the surface of infected cells. T cells recognize these foreign proteins through their T cell receptors (TCRs), which are formed by the assortment of several available V, D, and J genes to create millions of combinations of unique TCRs. We measured the diversity of T cells responding to the HIV Gag protein. We found that the CD8(+) T cell response is primarily made up of a few dominant unique TCRs, whereas the CD4(+) T cell subset has a much more diverse repertoire of TCRs. We also found there was less change in the virus sequences in subjects with more diverse TCR repertoires. HIV has a high mutation rate, which allows it to evade the immune response. Our findings describe the characteristics of a virus-specific T cell response that may allow it to limit viral evolution.

Select CMV epitopes drive life-long CD8+ T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4+ T cells specific for human CMV (HCMV) are elevated in HIV+ HCMV+ subjects. To determine whether HCMV epitope–specific CD4+ T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4+ T cells in coinfected HLA-DR7+ long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4+ T cells were inflated among these HIV+ subjects compared with those from an HIV− HCMV+ HLA-DR7+ cohort or with HLA-DR7–restricted CD4+ T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4+ T cells consisted of effector memory or effector memory–RA+ subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell–transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX3CR1, CD38, or HLA-DR but less often coexpressed CD38+ and HLA-DR+. The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4+ T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease.

Continued advances in single cell gene detection sensitivity and the ability to analyze data quickly with biological context are critical for discoveries in therapeutic research. The second version of the Chromium Single Cell Immune Profiling Solution by 10x Genomics enables highly sensitive detection of gene expression, full-length paired T-cell alpha- and beta-chain and immunoglobulin sequences, T-cell antigen specificity, and cell surface protein expression from the same single cells, allowing a comprehensive view of the immune response at the cellular level. The new workflow provides a 45% increase in the number of genes detected per cell and up to a 25% increase in the cells detected with paired full-length V(D)J receptor sequences in melanoma tumor derived cells. Using a new version of Cell Ranger (v5.0), clonotypes were grouped from >30,000 cells, increasing the power to detect small clonotype expansions with fewer total cells. In addition, the new software enabled comparison of pre- and post-influenza vaccination B-cell receptor sequences from a single donor, identifying post-vaccination specific clonotypes. These technological and informatics advancements enhance a researcher’s ability to perform a broad characterization of immune cell populations at unprecedented throughput and resolution.

Background Depletion of naïve CD4+ T cells and elevated adaptive immune activation are hallmarks of HIV infection. Higher proportions of memory CD4+ T cells are associated with prevalent diabetes in the general population, but few studies of persons with HIV (PWH) exist. Methods We analyzed data from 1532 PWH and 836 uninfected veterans in the longitudinal Veterans Aging Cohort Study (VACS), which archived peripheral mononuclear cells from these veterans between 2005 and 2007. We used flow cytometry to phenotype CD4+ and CD8+ T cells, including naïve, activated CD38+, senescent CD57+, total memory, and memory subsets. Prevalent diabetes (at blood collection) was identified in the VA electronic medical record using random glucose, hemoglobin A1c, ICD-9 codes, and medication. Cases were validated by two-physician chart review. We used multivariate logistic regression models adjusted for age, gender, body mass index, race/ethnicity, unhealthy alcohol use, hepatitis C, CMV status, and viral suppression stratified by HIV status to identify T-cell subsets associated with diabetes in PWH and uninfected. Results The cohort was 95% male, 68% African-American, and 22% diabetic. Higher CD4+CD45RO+ memory T cells were associated with prevalent diabetes in the uninfected and in PWH (P = 0.03 and P = 0.07, respectively; Figure A). Among subsets, diabetes was associated with higher transitional memory CD4+ T cells in the uninfected (P = 0.01), but higher central memory cells (P = 0.02) and lower effector memory cells (P = 0.04) in PWH. T effector memory RA+ cells were not associated with diabetes. Lower senescent CD4+CD57+ T cells were associated with diabetes in both PWH and uninfected (P = 0.03 and P = 0.04, respectively; Figure B), but results for naïve CD8+ T cells diverged: diabetes was associated with higher naïve CD8+cells in PWH but lower in uninfected (P = 0.01 and P < 0.01, respectively; Figure C). We assessed interaction by HIV status in a pooled model, which was only significant for the naïve CD8+ T cells (P = 0.01). Conclusion The adaptive immune profile associated with prevalent diabetes was similar by HIV status and characterized by a shift in CD4+ T cells from senescent to memory phenotypes, suggesting that chronic immune activation contributes to the higher risk of diabetes in PWH. Disclosures All authors: No reported disclosures.

Checkpoint inhibitors produce durable responses in numerous metastatic cancers, but immune-related adverse events (irAEs) complicate and limit their benefit. IrAEs can affect organ systems idiosyncratically; presentations range from mild and self-limited to fulminant and fatal. The molecular mechanisms underlying irAEs are poorly understood. Here, we report a fatal case of encephalitis arising during anti-PD-1 therapy. Histologic analyses revealed robust T-cell infiltration and prominent PD-L1 expression. We identified 209 reported cases in global pharmacovigilance databases—across multiple cancer types—of encephalitis associated with checkpoint inhibitor regimens, with a 19% fatality rate. We performed further analyses from the index case and two additional cases to shed light on this recurrent and fulminant irAE. Spatial and multi-omic analyses pinpointed activated memory CD4+ T cells as highly enriched in the inflamed, affected region. We identified a highly oligoclonal T cell receptor (TCR) repertoire, which we localized to activated memory cytotoxic (CD45RO+GZMB+) CD4 cells. We also identified Epstein-Barr-virus-specific T cell receptors and EBV+ lymphocytes in the affected region, which we speculate contributed to neural inflammation in the index case. Collectively, the 3 cases studied here identify CD4+ and CD8+ T cells as culprits of checkpoint inhibitor-associated immune encephalitis.

Persons with HIV (PWH) are at high risk for diabetes mellitus. Adipose tissue T cells are a central regulator of inflammation and adipocyte function, and the known establishment of a HIV reservoir in adipose could contribute to metabolic dysregulation. Here, we show that CD4+ T cell subsets increased in the adipose of diabetic PWH, specifically CD69+ and CD69lo CD57+ GPR56+ CX3CR1+, are clonally expanded with shared T cell receptors, suggesting a common lineage. We observed that both CD69+ and CX3CR1+ CD4+ T cells have transcriptomes consistent with a TH1 profile in diabetics versus TH2 in non-diabetics. CX3CR1+ CD4+ T cells from diabetic PWH also have increased expression of genes in innate immune pathways, not present in CD69+ cells, suggesting functional differences. This study sets the stage for future investigations to determine whether viral antigens in adipose tissue may promote clonal expansion of pro-inflammatory CX3CR1+ and CD69+ CD4+ T cells.

Adipose tissue comprises one of the largest organs in the body and performs diverse functions including energy storage and release, regulation of appetite and other neuroendocrine signaling, and modulation of immuity, among others. Adipocytes reside in a complex compartment where antigen, antigen presenting cells, innate immune cells, and adaptive immune cells interact locally and exert systemic effects on inflammation, circulating immune cell profiles, and metabolic homeostasis. T lymphocytes are a major component of the adipose tissue milieu which are altered in disease states such as obesity and human immunodeficiency virus (HIV) infection. While obesity, HIV infection, and simian immunodeficiency virus (SIV; a non-human primate virus similar to HIV) infection are accompanied by enrichment of CD8+ T cells in the adipose tissue, major phenotypic differences in CD4+ T cells and other immune cell populations distinguish HIV/SIV infection from obesity. Furthermore, DNA and RNA species of HIV and SIV can be detected in the stromal vascular fraction of visceral and subcutaneous adipose tissue, and replication-competent HIV resides in local CD4+ T cells. Here, we review studies of adipose tissue CD4+ and CD8+ T cell populations in HIV and SIV, and contrast the findings with those reported in obesity.

Chronic T cell activation and accelerated immune senescence are hallmarks of HIV infection, which may contribute to the increased risk of cardiometabolic diseases in people living with HIV (PLWH). T lymphocytes play a central role in modulating adipose tissue inflammation and, by extension, adipocyte energy storage and release. Here, we assessed the CD4+ and CD8+ T cell profiles in the subcutaneous adipose tissue (SAT) and blood of non-diabetic (n = 9; fasting blood glucose [FBG] < 100 mg/dL), pre-diabetic (n = 8; FBG = 100–125 mg/dL) and diabetic (n = 9; FBG ≥ 126 mg/dL) PLWH, in addition to non- and pre-diabetic, HIV-negative controls (n = 8). SAT was collected by liposuction and T cells were extracted by collagenase digestion. The proportion of naïve (TNai) CD45RO−CCR7+, effector memory (TEM) CD45RO+CCR7−, central memory (TCM) CD45RO+CCR7+, and effector memory revertant RA+(TEMRA) CD45RO−CCR7− CD4+ and CD8+ T cells were measured by flow cytometry. CD4+ and CD8+ TEM and TEMRA were significantly enriched in SAT of PLWH compared to blood. The proportions of SAT CD4+ and CD8+ memory subsets were similar across metabolic status categories in the PLWH, but CD4+ T cell expression of the CD69 early-activation and tissue residence marker, particularly on TEM cells, increased with progressive glucose intolerance. Use of t-distributed Stochastic Neighbor Embedding (t-SNE) identified a separate group of predominantly CD69lo TEM and TEMRA cells co-expressing CD57, CX3CR1, and GPR56, which were significantly greater in diabetics compared to non-diabetics. Expression of the CX3CR1 and GPR56 markers indicate these TEM and TEMRA cells may have anti-viral specificity. Compared to HIV-negative controls, SAT from PLWH had an increased CD8:CD4 ratio, but the distribution of CD4+ and CD8+ memory subsets was similar irrespective of HIV status. Finally, whole adipose tissue from PLWH had significantly higher expression of TLR2, TLR8, and multiple chemokines potentially relevant to immune cell homing compared to HIV-negative controls with similar glucose tolerance.

Summary Persons with HIV are at increased risk for diabetes mellitus compared with individuals without HIV. Adipose tissue is an important regulator of glucose and lipid metabolism, and adipose tissue T cells modulate local inflammatory responses and, by extension, adipocyte function. Persons with HIV and diabetes have a high proportion of CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells in adipose tissue, a subset of which are cytomegalovirus specific, whereas individuals with diabetes but without HIV have predominantly CD69+ CD4+ T cells. Adipose tissue CD69+ and C-G-C+ CD4+ T cell subsets demonstrate higher receptor clonality compared with the same cells in blood, potentially reflecting antigen-driven expansion, but C-G-C+ CD4+ T cells have a more inflammatory and cytotoxic RNA transcriptome. Future studies will explore whether viral antigens have a role in recruitment and proliferation of pro-inflammatory C-G-C+ CD4+ T cells in adipose tissue of persons with HIV., Graphical Abstract, Highlights Adipose tissue memory CD4+ T cells are frequently CD69+ in persons with diabetes Persons with HIV and diabetes have more CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells Adipose tissue C-G-C+ CD4+ T cells and CD69+ CD4+ T cells are clonally expanded C-G-C+ CD4+ T cells are often CMV specific and have more inflammatory transcriptomes, Wanjalla et al. demonstrate that adipose tissue in persons with HIV and diabetes includes a large proportion of clonally expanded, inflammatory, and frequently CMV-specific CX3CR1+ GPR56+ CD57+ (C-G-C+) CD4+ T cells. These cells may contribute to the altered adipocyte function and elevated risk of metabolic disease observed among persons with HIV.

Background & Aims Severe injury to the lining of the stomach leads to changes in the epithelium (reprogramming) that protect and promote repair of the tissue, including development of spasmolytic polypeptide-expressing metaplasia (SPEM) and tuft and foveolar cell hyperplasia. Acute gastric damage elicits a type-2 inflammatory response that includes production of type-2 cytokines and infiltration by eosinophils and alternatively activated macrophages. Stomachs of mice that lack interleukin 33 (IL33) or interleukin 13 (IL13) did not undergo epithelial reprogramming after drug-induced injury. We investigated the role of group 2 innate lymphoid cells (ILC2s) in gastric epithelial repair. Methods Acute gastric injury was induced in C57BL/6J mice (wild-type and RAG1 knockout) by administration of L635. We isolated ILC2s by flow cytometry from stomachs of mice that were and were not given L635 and performed single-cell RNA sequencing. ILC2s were depleted from wild-type and RAG1-knockout mice by administration of anti-CD90.2. We assessed gastric cell lineages, markers of metaplasia, inflammation, and proliferation. Gastric tissue microarrays from patients with gastric adenocarcinoma were analyzed by immunostaining. Results There was a significant increase in the number of GATA3-positive ILC2s in stomach tissues from wild-type mice after L635-induced damage, but not in stomach tissues from IL33-knockout mice. We characterized a marker signature of gastric mucosal ILC2s and identified a transcription profile of metaplasia-associated ILC2s, which included changes in expression of Il5, Il13, Csf2, Pd1, and Ramp3; these changes were validated by quantitative polymerase chain reaction and immunocytochemistry. Depletion of ILC2s from mice blocked development of metaplasia after L635-induced injury in wild-type and RAG1-knockout mice and prevented foveolar and tuft cell hyperplasia and infiltration or activation of macrophages after injury. Numbers of ILC2s were increased in stomach tissues from patients with SPEM compared with patients with normal corpus mucosa. Conclusions In analyses of stomach tissues from mice with gastric tissue damage and patients with SPEM, we found evidence of type 2 inflammation and increased numbers of ILC2s. Our results suggest that ILC2s coordinate the metaplastic response to severe gastric injury.

Profiling the complex interactions of immune infiltrate with tumor cells in a tumor microenvironment is critical for advancing our understanding of tumor biology for developing personalized cancer therapies. Using a droplet-based single cell RNA sequencing (scRNA-seq) platform, we profiled the transcriptome and immune repertoire of gastric, kidney, and lung cancer cells that are primarily immune cells from three different donors. scRNA-seq analysis also identified similar fractions of CD45+ immune cells, CD4+ and CD8+ T cells, CD19+ B cells, and myeloid cells compared to flow. Targeted scRNA-seq was also used to identify paired, full length B-cell (BCR) and T-cell (TCR) receptors. In the gastric adenocarcinoma tumor cells, gene expression analysis identified a large population of B cells, but with no clonal expansion. T cells expressing CD8A constituted about 10% of cells with the top clonotype being 1% of all clones. The clear cell RCC sample had a modest fraction of infiltrating T cells with the top clonotype representing 7.4% of all clones, and no B cell infiltrate. The NSCLC cells were mainly T and B cells but limited clonal expansion was observed; the top TCR clonotype was present on 2.2% of all T cells, no expansion was seen in any of the B cell clonotypes. These findings highlight the value of profiling of tumor immune cells holistically and not relying on the presence of B or T cells alone to understand the immune dynamics of the tumor microenvironment. The presence of tumor-infiltrating lymphocytes is associated with favorable clinical outcomes in some cancers, but understanding their cellular subtype and clonality by high resolution profiling is key in the development of immune-based cancer treatments

B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.

Adipose tissue (AT) CD4+ and CD8+ T cells contribute to obesity-associated insulin resistance. Prior studies identified conserved T-cell receptor (TCR) chain families in obese AT, but the presence and clonal expansion of specific TCR sequences in obesity has not been assessed. We characterized AT and liver CD8+ and CD4+ TCR repertoires of mice fed a low-fat diet (LFD) and high-fat diet (HFD) using deep sequencing of the TCRβ chain to quantify clonal expansion, gene usage, and CDR3 sequence. In AT CD8+ T cells, HFD reduced TCR diversity, increased the prevalence of public TCR clonotypes, and selected for TCR CDR3 regions enriched in positively charged and less polarized amino acids. Although TCR repertoire alone could distinguish between LFD- and HFD-fed mice, these properties of the CDR3 region of AT CD8+ T cells from HFD-fed mice led us to examine the role of negatively charged and nonpolar isolevuglandin (isoLG) adduct-containing antigen-presenting cells within AT. IsoLG-adducted protein species were significantly higher in AT macrophages of HFD-fed mice; isoLGs were elevated in M2-polarized macrophages, promoting CD8+ T-cell activation. Our findings demonstrate that clonal TCR expansion that favors positively charged CDR3s accompanies HFD-induced obesity, which may be an antigen-driven response to isoLG accumulation in macrophages.

Characterization of single antibody lineages within infected individuals has provided insights into the development of Env-specific antibodies. However, a systems-level understanding of the humoral response against HIV-1 is limited. Here, we interrogated the antibody repertoires of multiple HIV-infected donors from an infection-naive state through acute and chronic infection using next-generation sequencing. This analysis revealed the existence of "public" antibody clonotypes that were shared among multiple HIV-infected individuals. The HIV-1 reactivity for representative antibodies from an identified public clonotype shared by three donors was confirmed. Furthermore, a meta-analysis of publicly available antibody repertoire sequencing datasets revealed antibodies with high sequence identity to known HIV-reactive antibodies, even in repertoires that were reported to be HIV naive. The discovery of public antibody clonotypes in HIV-infected individuals represents an avenue of significant potential for better understanding antibody responses to HIV-1 infection, as well as for clonotype-specific vaccine development.

Helicobacter pylorirequires genetic agility to infect new hosts and establish long-term colonization of changing gastric environments. In this study, we analyzedH. pylorigenetic adaptation in the Mongolian gerbil model. This model is of particular interest becauseH. pylori-infected gerbils develop a high level of gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. We analyzed the whole genome sequences ofH. pyloristrains cultured from experimentally infected gerbils, in comparison to the genome sequence of the input strain. The mean annualized single nucleotide polymorphism (SNP) rate per site was 1.5e−5, which is similar to the rates detected previously inH. pylori-infected humans. Many of the mutations occurred within or upstream of genes associated with iron-related functions (fur,tonB1,fecA2,fecA3, andfrpB3) or encoding outer membrane proteins (alpA, oipA, fecA2, fecA3, frpB3andcagY). Most of the SNPs within coding regions (86%) were non-synonymous mutations. Several deletion or insertion mutations led to disruption of open reading frames, suggesting that the corresponding gene products are not required or are deleterious during chronicH. pyloricolonization of the gerbil stomach. Five variants (three SNPs and two deletions) were detected in isolates from multiple animals, which suggests that these mutations conferred a selective advantage. One of the mutations (FurR88H) detected in isolates from multiple animals was previously shown to confer increased resistance to oxidative stress, and we now show that this SNP also confers a survival advantage whenH. pyloriis co-cultured with neutrophils. Collectively, these analyses allow the identification of mutations that are positively selected duringH. pyloricolonization of the gerbil model.

Tumor-specific CD8 T cells (TST) encountering cognate antigen in tumors differentiate to a dysfunctional state characterized by expression of multiple inhibitory receptors and failure to make effector cytokines. We demonstrated that TST differentiate through two discrete chromatin states; TST were initially in a plastic chromatin state and could be functionally rescued but then transitioned to a fixed chromatin state resistant to therapeutic reprogramming. T cells harboring these discrete chromatin states could be identified with distinct, novel membrane protein expression profiles, permitting prospective identification of reprogrammable tumor-specific T cells from bulk tumor-infiltrating T cell populations. Importantly, human tumor-infiltrating PD1-high CD8 T cells were in a similar chromatin accessibility state as murine fixed dysfunctional T cells. Based on our studies, two models could describe TST differentiation: (i) TST progress en-masse from the plastic to fixed state, potentially through a transitional state or (ii) individual TST within a population exist in either the plastic or fixed dysfunctional state; initially, most TST are in the plastic state, however over time plastic T cells fail to proliferate or die while the fixed dysfunctional TST preferentially proliferate and/or survive. To test these models, we carried out single-cell RNA-Sequencing on TST at various time points during tumor progression. Determining the population dynamics of TST differentiation through dysfunctional states in tumors could inform our strategies for TST rescue, for example, by selecting rare plastic TST from bulk populations versus novel strategies to reprogram the epigenome of fixed dysfunctional TST.