Plasticity of intragraft alloreactive T cell clones in human gut correlates with transplant outcomes.

The site of transition between tissue-resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity, and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single-cell level after human intestinal transplantation. Host-versus-graft (HvG)-reactive T cells were mainly distributed to TRM, effector T (Teff)/TRM, and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to Teff/TRM clusters in association with rejection. By integrating pre- and post-transplantation (Tx) mixed lymphocyte reaction-determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in the human gut, providing novel insight into TRM biology.

Guselkumab Modulates Differentially Expressed Genes in Blood of Patients With Psoriatic Arthritis: Results from Two Phase 3, Randomized, Placebo-Controlled Trials.

OBJECTIVE: To evaluate gene expression in blood of patients with psoriatic arthritis (PsA) versus healthy controls and identify changes associated with guselkumab treatment. METHODS: Whole blood transcriptome profiling via paired-end RNA sequencing was conducted using samples from DISCOVER-1 and DISCOVER-2 at baseline (n = 673) and at weeks 4 and 24 from a representative subgroup that received placebo or guselkumab (n = 227 [longitudinal PsA cohort]). Baseline samples were compared with demographically matched healthy controls (n = 21). Guselkumab-mediated changes in gene expression were assessed in participants from the longitudinal PsA cohort who did versus did not achieve at least 20% improvement in American College of Rheumatology response criteria (ACR20) or at least 75% improvement in Psoriasis Area and Severity Index (PASI75). Differential gene expression was analyzed using edgeR. RESULTS: At baseline, 355 upregulated and 314 downregulated genes (PsA-associated genes) were identified in patients with PsA versus healthy controls. Upregulated genes were related to neutrophil, mononuclear cell, and CD11b+ gene sets. No cell type-specific gene sets were identified among downregulated genes. Most PsA-associated genes were modulated by guselkumab treatment. At week 24, genes downregulated by guselkumab were enriched with neutrophil, monocyte, eosinophil, and macrophage gene sets; genes upregulated by guselkumab were enriched with B cell, T cell, and natural killer cell gene sets. Reductions in expression of upregulated PsA-associated gene sets were more pronounced in ACR20 and PASI75 responders than in nonresponders. CONCLUSION: These findings suggest a dysregulation of immune cell profiles in blood from patients in the baseline PsA cohort that approached levels in healthy controls after guselkumab treatment.

Maintenance of the human memory T cell repertoire by subset and tissue site.

BACKGROUND: Immune-mediated protection is mediated by T cells expressing pathogen-specific T cell antigen receptors (TCR) that are maintained at diverse sites of infection as tissue-resident memory T cells (TRM) or that disseminate as circulating effector-memory (TEM), central memory (TCM), or terminal effector (TEMRA) subsets in blood and tissues. The relationship between circulating and tissue resident T cell subsets in humans remains elusive, and is important for promoting site-specific protective immunity. METHODS: We analyzed the TCR repertoire of the major memory CD4+ and CD8+T cell subsets (TEM, TCM, TEMRA, and TRM) isolated from blood and/or lymphoid organs (spleen, lymph nodes, bone marrow) and lungs of nine organ donors, and blood of three living individuals spanning five decades of life. High-throughput sequencing of the variable (V) portion of individual TCR genes for each subset, tissue, and individual were analyzed for clonal diversity, expansion and overlap between lineage, T cell subsets, and anatomic sites. TCR repertoires were further analyzed for TRBV gene usage and CDR3 edit distance. RESULTS: Across blood, lymphoid organs, and lungs, human memory, and effector CD8+T cells exhibit greater clonal expansion and distinct TRBV usage compared to CD4+T cell subsets. Extensive sharing of clones between tissues was observed for CD8+T cells; large clones specific to TEMRA cells were present in all sites, while TEM cells contained clones shared between sites and with TRM. For CD4+T cells, TEM clones exhibited the most sharing between sites, followed by TRM, while TCM clones were diverse with minimal sharing between sites and subsets. Within sites, TRM clones exhibited tissue-specific expansions, and maintained clonal diversity with age, compared to age-associated clonal expansions in circulating memory subsets. Edit distance analysis revealed tissue-specific biases in clonal similarity. CONCLUSIONS: Our results show that the human memory T cell repertoire comprises clones which persist across sites and subsets, along with clones that are more restricted to certain subsets and/or tissue sites. We also provide evidence that the tissue plays a key role in maintaining memory T cells over age, bolstering the rationale for site-specific targeting of memory reservoirs in vaccines and immunotherapies.

Single-cell transcriptomics of human T cells reveals tissue and activation signatures in health and disease.

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8+ T cells and an interferon-response state for CD4+ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8+ compared to CD4+ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.

A Pilot Study Evaluating a Binge-Eating Screener in Children: Development of the Children's Brief Binge-Eating Questionnaire in a Pediatric Obesity Clinic.

Children referred to pediatric obesity programs are at high risk of binge-eating disorder (BED) symptoms. Our goal was to develop and evaluate the Children's Brief Binge-Eating Questionnaire (CBBEQ) as a rapid screen of BED symptomatology. Seventy patients between the ages of 7 and 18 years (57% females) completed the CBBEQ and underwent the Eating Disorder Assessment for DSM-5 (EDA). Reliability and validity were assessed by examining results of the CBBEQ compared with the EDA, as well as measures of sleep, depression, and anxiety. Twenty-four of seventy (34%) children met full diagnostic interview criteria for BED and 12 (17%) met subclinical criteria. The CBBEQ demonstrated 100% specificity, 93% sensitivity, and a 100% negative predictive value for BED at a cutoff total score of 9. If confirmed in larger sample, this questionnaire could be a quick and accurate clinical tool for non-mental health providers to identify children at risk for BED.

Location, location, location: Tissue resident memory T cells in mice and humans.

The discovery of T cells resident in diverse tissues has altered our understanding of adaptive immunity to encompass site-specific responses mediated by tissue-adapted memory T cells throughout the body. Here, we discuss the key phenotypic, transcriptional, and functional features of these tissue-resident memory T cells (TRM) as established in mouse models of infection and translated to humans by novel tissue sampling approaches. Integration of findings from mouse and human studies may hold the key to unlocking the potential of TRM for promoting tissue immunity and preventing infection.

Microanatomical dissection of human intestinal T-cell immunity reveals site-specific changes in gut-associated lymphoid tissues over life.

Defining adaptive immunity with the complex structures of the human gastrointestinal (GI) tract over life is essential for understanding immune responses to ingested antigens, commensal and pathogenic microorganisms, and dysfunctions in disease. We present here an analysis of lymphocyte localization and T cell subset composition across the human GI tract including mucosal sites (jejunum, ileum, colon), gut-associated lymphoid tissues (isolated lymphoid follicles (ILFs), Peyer's patches (PPs), appendix), and mesenteric lymph nodes (MLNs) from a total of 68 donors spanning eight decades of life. In pediatric donors, ILFs and PP containing naïve T cells and regulatory T cells (Tregs) are prevalent in the jejunum and ileum, respectively; these decline in frequency with age, contrasting stable frequencies of ILFs and T cell subsets in the colon. In the mucosa, tissue resident memory T cells develop during childhood, and persist in high frequencies into advanced ages, while T cell composition changes with age in GALT and MLN. These spatial and temporal features of human intestinal T cell immunity define signatures that can be used to train predictive machine learning algorithms. Our findings demonstrate an anatomic basis for age-associated alterations in immune responses, and establish a quantitative baseline for intestinal immunity to define disease pathologies.

Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that Are Maintained by a Circulating Pool.

Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.

Functional heterogeneity of human tissue-resident memory T cells based on dye efflux capacities.

Tissue-resident memory T cells (TRMs) accelerate pathogen clearance through rapid and enhanced functional responses in situ. TRMs are prevalent in diverse anatomic sites throughout the human lifespan, yet their phenotypic and functional diversity has not been fully described. Here, we identify subpopulations of human TRMs based on the ability to efflux fluorescent dyes [efflux(+) TRMs] located within mucosal and lymphoid sites with distinct transcriptional profiles, turnover, and functional capacities. Compared with efflux(-) TRMs, efflux(+) TRMs showed transcriptional and phenotypic features of quiescence including reduced turnover, decreased expression of exhaustion markers, and increased proliferative capacity and signaling in response to homeostatic cytokines. Moreover, upon activation, efflux(+) TRMs secreted lower levels of inflammatory cytokines such as IFN-γ and IL-2 and underwent reduced degranulation. Interestingly, analysis of TRM subsets following activation revealed that both efflux(+) and efflux(-) TRMs undergo extensive transcriptional changes following TCR ligation but retain core TRM transcriptional properties including retention markers, suggesting that TRMs carry out effector function in situ. Overall, our results suggest a model for tissue-resident immunity wherein heterogeneous subsets have differential capacities for longevity and effector function.

Human Lymph Nodes Maintain TCF-1hi Memory T Cells with High Functional Potential and Clonal Diversity throughout Life.

Translating studies on T cell function and modulation from mouse models to humans requires extrapolating in vivo results on mouse T cell responses in lymphoid organs (spleen and lymph nodes [LN]) to human peripheral blood T cells. However, our understanding of T cell responses in human lymphoid sites and their relation to peripheral blood remains sparse. In this study, we used a unique human tissue resource to study human T cells in different anatomical compartments within individual donors and identify a subset of memory CD8+ T cells in LN, which maintain a distinct differentiation and functional profile compared with memory CD8+ T cells in blood, spleen, bone marrow, and lungs. Whole-transcriptome and high-dimensional cytometry by time-of-flight profiling reveals that LN memory CD8+ T cells express signatures of quiescence and self-renewal compared with corresponding populations in blood, spleen, bone marrow, and lung. LN memory T cells exhibit a distinct transcriptional signature, including expression of stem cell-associated transcription factors TCF-1 and LEF-1, T follicular helper cell markers CXCR5 and CXCR4, and reduced expression of effector molecules. LN memory T cells display high homology to a subset of mouse CD8+ T cells identified in chronic infection models that respond to checkpoint blockade immunotherapy. Functionally, human LN memory T cells exhibit increased proliferation to TCR-mediated stimulation and maintain higher TCR clonal diversity compared with memory T cells from blood and other sites. These findings establish human LN as reservoirs for memory T cells with high capacities for expansion and diverse recognition and important targets for immunotherapies.

Human Tissue-Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites.

Tissue-resident memory T cells (TRMs) in mice mediate optimal protective immunity to infection and vaccination, while in humans, the existence and properties of TRMs remain unclear. Here, we use a unique human tissue resource to determine whether human tissue memory T cells constitute a distinct subset in diverse mucosal and lymphoid tissues. We identify a core transcriptional profile within the CD69+ subset of memory CD4+ and CD8+ T cells in lung and spleen that is distinct from that of CD69- TEM cells in tissues and circulation and defines human TRMs based on homology to the transcriptional profile of mouse CD8+ TRMs. Human TRMs in diverse sites exhibit increased expression of adhesion and inhibitory molecules, produce both pro-inflammatory and regulatory cytokines, and have reduced turnover compared with circulating TEM, suggesting unique adaptations for in situ immunity. Together, our results provide a unifying signature for human TRM and a blueprint for designing tissue-targeted immunotherapies.

Reduced generation of lung tissue-resident memory T cells during infancy.

Infants suffer disproportionately from respiratory infections and generate reduced vaccine responses compared with adults, although the underlying mechanisms remain unclear. In adult mice, lung-localized, tissue-resident memory T cells (TRMs) mediate optimal protection to respiratory pathogens, and we hypothesized that reduced protection in infancy could be due to impaired establishment of lung TRM. Using an infant mouse model, we demonstrate generation of lung-homing, virus-specific T effectors after influenza infection or live-attenuated vaccination, similar to adults. However, infection during infancy generated markedly fewer lung TRMs, and heterosubtypic protection was reduced compared with adults. Impaired TRM establishment was infant-T cell intrinsic, and infant effectors displayed distinct transcriptional profiles enriched for T-bet-regulated genes. Notably, mouse and human infant T cells exhibited increased T-bet expression after activation, and reduction of T-bet levels in infant mice enhanced lung TRM establishment. Our findings reveal that infant T cells are intrinsically programmed for short-term responses, and targeting key regulators could promote long-term, tissue-targeted protection at this critical life stage.

Dendritic Cells Display Subset and Tissue-Specific Maturation Dynamics over Human Life.

Maturation and migration to lymph nodes (LNs) constitutes a central paradigm in conventional dendritic cell (cDC) biology but remains poorly defined in humans. Using our organ donor tissue resource, we analyzed cDC subset distribution, maturation, and migration in mucosal tissues (lungs, intestines), associated lymph nodes (LNs), and other lymphoid sites from 78 individuals ranging from less than 1 year to 93 years of age. The distribution of cDC1 (CD141hiCD13hi) and cDC2 (Sirp-α+CD1c+) subsets was a function of tissue site and was conserved between donors. We identified cDC2 as the major mature (HLA-DRhi) subset in LNs with the highest frequency in lung-draining LNs. Mature cDC2 in mucosal-draining LNs expressed tissue-specific markers derived from the paired mucosal site, reflecting their tissue-migratory origin. These distribution and maturation patterns were largely maintained throughout life, with site-specific variations. Our findings provide evidence for localized DC tissue surveillance and reveal a lifelong division of labor between DC subsets, with cDC2 functioning as guardians of the mucosa.

Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection.

T cell responses to viruses are initiated and maintained in tissue sites; however, knowledge of human antiviral T cells is largely derived from blood. Cytomegalovirus (CMV) persists in most humans, requires T cell immunity to control, yet tissue immune responses remain undefined. Here, we investigated human CMV-specific T cells, virus persistence and CMV-associated T cell homeostasis in blood, lymphoid, mucosal and secretory tissues of 44 CMV seropositive and 28 seronegative donors. CMV-specific T cells were maintained in distinct distribution patterns, highest in blood, bone marrow (BM), or lymph nodes (LN), with the frequency and function in blood distinct from tissues. CMV genomes were detected predominantly in lung and also in spleen, BM, blood and LN. High frequencies of activated CMV-specific T cells were found in blood and BM samples with low virus detection, whereas in lung, CMV-specific T cells were present along with detectable virus. In LNs, CMV-specific T cells exhibited quiescent phenotypes independent of virus. Overall, T cell differentiation was enhanced in sites of viral persistence with age. Together, our results suggest tissue T cell reservoirs for CMV control shaped by both viral and tissue-intrinsic factors, with global effects on homeostasis of tissue T cells over the lifespan.

Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, receptor protein tyrosine phosphatase sigma (PTPRS) is expressed specifically on pDCs. Surface PTPRS was rapidly downregulated after pDC activation, and only PTPRS(-) pDCs produced IFN-α. Antibody-mediated PTPRS crosslinking inhibited pDC activation, whereas PTPRS knockdown enhanced IFN response in a pDC cell line. Similarly, murine Ptprs and the homologous receptor phosphatase Ptprf were specifically co-expressed in murine pDCs. Haplodeficiency or DC-specific deletion of Ptprs on Ptprf-deficient background were associated with enhanced IFN response of pDCs, leukocyte infiltration in the intestine and mild colitis. Thus, PTPRS represents an evolutionarily conserved pDC-specific inhibitory receptor, and is required to prevent spontaneous IFN production and immune-mediated intestinal inflammation.