Early life is essential for establishing memory T cells, which rapidly populate mucosal sites during infancy, although these nascent memory T cells are less protective than their adult counterparts. Here we used single-cell RNA sequencing of resting and CD3+CD28 antibody-stimulated T cells from lymphoid and mucosal tissues of infant (2–9 months) and adult (40–63 years) organ donors to investigate age-dependent mechanisms for functional regulation of human memory T cells. Infant CCL5+ effector memory T cells exhibited reduced effector function compared to adults. Transcription factor network analysis identified HELIOS and KLF6 as regulators of memory T cell states in infant and adult tissues, respectively. Using single-nucleus RNA sequencing, assay for transposase-accessible chromatin sequencing and CRISPR–Cas9 knockout, we defined HELIOS (IKZF2) as a critical regulator of the infant-specific transcriptional program in CCL5+ effector memory T cells and restricted effector function in SELL+CCR7+ naive and/or central memory T cells. Our findings reveal key mechanisms controlling T cell functional states in early life. Sims, Farber and colleagues analyze T cells from lymphoid and mucosal tissues of infant and adult human organ donors to show that infant T cells have a unique stem-like transcriptional profile and tissue adaptation program.

Obesity is a known risk factor for diseases of the pancreas, including diabetes, pancreatic cancer and pancreatitis, but mechanisms remain unclear. To elucidate how obesity impacts pancreatic immune homeostasis, we performed spatial, transcriptomic and functional profiling of human pancreatic immune cells from obese and non-obese organ donors. Obesity was associated with higher density of tissue resident memory T-cells (TRM) in the exocrine pancreas which display high cytotoxic functions and aggregated around macrophages. Single cell sequencing of pancreatic macrophages revealed two main subsets - FOLR2 + CD11c - fetal-derived macrophages with pro-repair and immunoregulatory function and a FOLR2 - CD11c + monocyte-derived macrophages with greater T-cell interactions and pro-inflammatory function. In obesity, the pancreatic macrophage landscape shifts to lower predominance of FOLR2 + CD11c - macrophages and higher FOLR2 - CD11c + macrophages which interact selectively with the TRM and inflamed exocrine epithelium. Together, these results identify macrophage-T cell circuits and immune epithelial interactions that fuel chronic pancreatic inflammation in obesity - a potential unifying mechanism for obesity-related pancreatic diseases.

Cytomegalovirus (CMV), a ubiquitous herpesvirus, establishes persistent infection that is controlled by both NK and CD8 T cells. While immunity to CMV is primarily studied in adult mice and humans, most individuals acquire CMV during the early years of life and virus-specific NK and T cell responses during this vulnerable life stage are understudied. Here, we show distinct responses by infant and adult NK cells to CMV infection in both mice and humans resulting from cell-intrinsic features and host T cell responses. Infant mice sustained higher viral loads compared to adults following MCMV infection and exhibited non-redundant requirements for NK cells. Infant MCMV-reactive (Ly49H+) NK cells preferentially expanded adaptive-like subsets which were maintained in tissues and exhibited a distinct transcriptional profile relative to adult NK cells. This biased differentiation of adaptive-like NK cells was altered over age and controlled in part, by competition with memory T cells. We demonstrate similar dynamics with human NK cells; distinct adaptive-profiles for HCMV-reactive NK cells in early life and childhood that change over age and are inversely associated with anti-viral T cell responses. Together, our results reveal that NK cells develop adaptive-like responses and seed tissues in early life to provide protective memory when T cell immunity is limited.

Asthma is an immune-mediated lung disease causing airway constriction that is fatal in rare cases, though the immune mechanisms underlying asthma severity are poorly understood. Here, we present a comprehensive immunological profiling of lymphoid organs, lungs, and intestines from human organ donors who died of fatal asthma (FA) compared to donors who died of unrelated causes with or without a history of asthma. Compared to control donors, FA donors exhibit elevated plasma IgE along with enhanced and aberrant immune responses in mucosal-associated lymph nodes (LN) and lungs, respectively. In particular, FA donors show increased memory T and B cells and decreased Treg cells with age in the gut- and lung-associated LN, increased Th2 and Th1 resident memory cells in the lungs, and increased associations between gut and lung immune responses compared to control donors. Our findings reveal mucosal immune dysregulation underlying asthma exacerbation through site-specific and inter-tissue disruption of immune homeostasis. Immune mechanisms driving severe asthma and fatal outcomes remain elusive. Through cross-tissue immune profiling of organ donors who died of fatal asthma versus other causes, the authors here reveal mucosal immune dysregulation marked by enhanced T- and B-cell memory, reduced Tregs, and increased lung Th2 and Th1 effectors linked to priming in gut-associated lymph nodes.

Rejection is a barrier to intestinal transplantation (ITx). ITx rejection may be associated with changes in the ileal microbiome. We sought to analyze whether shifts in the microbiome were associated with intestinal transplant rejection. Ileal effluent samples were collected from ITx patients (n = 8) with multiple samples taken from each patient at times of no (n = 83), mild (n = 39), or moderate (n = 3) rejection, Crohn’s disease (n = 20), and noninflamed control patients (n = 25). Ileal microbiota were quantified using 16S rRNA gene sequencing. Compared to nontransplant samples (noninflamed control, Crohn’s disease), ITx samples had lower alpha diversity (Shannon and Chao1, P < .001) and different beta diversity (Bray-Curtis, P < .005). Beta diversity differed between samples with and without rejection (P = .002). Differential abundance analyses showed enrichment of pathogenic taxa and depletion of commensals in ITx rejection samples. ITx rejection is associated with ileal microbiome dysbiosis, which is a potential target for diagnostic and therapeutic interventions.

Infections and vaccinations elicit coordinated humoral and cellular adaptive immune responses that together provide protection. In addition to antibodies, pathogen-specific memory T and B cells persist in blood and tissues, but it remains unclear how their composition and spatial distribution relate to serum antibody titers, the most common correlate of vaccine-induced protection. Understanding these relationships is essential for predicting vaccine efficacy and optimizing immunization strategies. We analyzed tissues from 58 adult human organ donors vaccinated against SARS-CoV-2, including individuals with and without prior infection. Using multivariate imputation, dimensionality reduction, and correlation, regression, and causal analyses, we identified immune signatures linking memory B cell, CD4 T cell, and CD8 T cell subsets in spleen, lung, and lung-draining lymph nodes with antibody titers and neutralizing activity. Our analyses indicate that humoral immunity is driven primarily by virus-specific B cells and CD4 T cells in lymphoid tissues rather than blood, whereas tissue-localized CD8 T cell responses, although correlated with antibody levels, develop independently. These findings demonstrate that cross-sectional immune profiling across multiple tissues recapitulates established immunological principles and reveal that serum antibody responses emerge from coordinated cellular immune responses distributed throughout the body.

The perinatal period of immune ontogeny reflects prenatal, maternal and postnatal cues that converge to establish durable immune trajectories. Fetal and infant immunity follows distinct, ontogeny-specific programs optimized for tissue protection, tolerance and rapid effector function. Herein, we integrate recent insights into prenatal hematopoiesis, tissue seeding by innate and adaptive lymphocytes, and the establishment of immune specialization across barriers and systemic compartments. We examine how cellular and humoral factors in blood, tissues and/or breast milk calibrate immune development, and how postnatal microbial colonization and environmental exposures further refine immune function in a tissue- and time-dependent manner. Together, these findings redefine early life as a critical window during which immune set points are established, with lasting immunological imprinting linked to host defense, vaccine responsiveness and risk for atopy. A mechanistic understanding of these processes provides a framework for rational, age-adapted strategies to promote immune health across the life course.

Human macrophages (MΦs) reside in tissues and develop tissue-specific identities. While studies in mice have identified molecular signatures for site-specific MΦ differentiation, less is known about the transcriptional profiles of human MΦs in distinct sites, including mucosal tissues and lymphoid organs during homeostasis and activation. Here, we use multimodal single-cell sequencing and ex vivo stimulation assays to define tissue signatures for populations of human MΦs isolated from lungs, small intestine, spleen, bone marrow, and lymph nodes obtained from individual organ donors. Our results reveal distinct tissue-adapted gene and protein profiles of metabolic, adhesion, and immune interaction pathways, which are specific to MΦs and not monocytes isolated from the same sites and exhibit homology to murine MΦs from the same sites. Tissue-adapted MΦs remained responsive to polarizing cytokine stimuli ex vivo, with upregulation of expected transcripts and secreted proteins, while retaining tissue-specific profiles. Patterns of chromatin accessibility in tissue MΦs identified from single-nucleus assay for transposase-accessible chromatin by sequencing reflected gene expression signatures and indicate that differential utilization of transcription factors may drive stable tissue-adapted profiles. Together, our findings show how human MΦ identity is coupled to their site of residence for mucosal and lymphoid organs and is intrinsically maintained during activation and polarization.

The first years of life are essential for the development of memory T cells, which rapidly populate the body's diverse tissue sites during infancy. However, the degree to which tissue memory T cell responses in early life reflect those during adulthood is unclear. Here, we use single cell RNA-sequencing of resting and ex vivo activated T cells from lymphoid and mucosal tissues of infant (aged 2-9 months) and adult (aged 40-65 years) human organ donors to dissect the transcriptional programming of memory T cells over age. Infant memory T cells demonstrate a unique stem-like transcriptional profile and tissue adaptation program, yet exhibit reduced activation capacity and effector function relative to adults. Using CRISPR-Cas9 knockdown, we define Helios (IKZF2) as a critical transcriptional regulator of the infant-specific tissue adaptation program and restricted effector state. Our findings reveal key transcriptional mechanisms that control tissue T cell fate and function in early life.

This study examines the expression of CD45 isoforms on human yellow fever virus vaccine (YFV-17D) specific CD8 T cells longitudinally after vaccination. As expected, effector CD8 T cells at day 14 express CD45RO but within 4 to 6 wk these virus-specific CD8 T cells become CD45RA positive and remain CD45RA for >10 y. The journey for these YFV-specific CD8 T cells goes from naive (CD45RA+ CCR7+) to effector/effector memory (CD45RO+ CCR7-) to Temra (CD45RA+ CCR7-) to stem-cell memory (CD45RA+ CCR7+). These YFV-specific CD8 T cells rarely acquire the canonical Tcm phenotype (CD45RO+ CCR7+). This CD45RO to RA switch coincides with clearance of YFV, so we hypothesized that antigen may be playing a role in regulating CD45 expression. We addressed this issue by ex vivo analysis and provide evidence that this switch is indeed regulated by antigen. Sorted YFV-specific CD45RO effector CD8 T cells reexpress CD45RA when cultured ex vivo in the absence of antigen and retain CD45RO in the presence of cognate peptide. We also extended these ex vivo analysis to human cytomegalovirus (CMV)-specific CD8 T cells and show that CD45RO cells transition to CD45RA in the absence of antigen and CD45RA cells become CD45RO when stimulated with CMV peptide. We then show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific CD8 T cells can repeatedly undergo the same CD45RA to RO to RA transition in vivo after the SARS-CoV-2 mRNA vaccination. Again, the canonical Tcm phenotype spike-specific memory CD8 T cells were not readily detectable. These studies warrant a reevaluation of how human memory CD8 T cells are defined.

The metabolic landscape of cancer greatly influences antitumor immunity, yet it remains unclear how organ-specific metabolites in the tumor microenvironment influence immunosurveillance. We found that accumulation of primary conjugated and secondary bile acids (BAs) are metabolic features of human hepatocellular carcinoma and experimental liver cancer models. Inhibiting conjugated BA synthesis in hepatocytes through deletion of the BA-conjugating enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT) enhanced tumor-specific T cell responses, reduced tumor growth, and sensitized tumors to anti-programmed cell death protein 1 (anti-PD-1) immunotherapy. Furthermore, different BAs regulated CD8+ T cells differently; primary BAs induced oxidative stress, whereas the secondary BA lithocholic acid inhibited T cell function through endoplasmic reticulum stress, which was countered by ursodeoxycholic acid. We demonstrate that modifying BA synthesis or dietary intake of ursodeoxycholic acid could improve tumor immunotherapy in liver cancer model systems.

Vaccines have long been pivotal for protection against a host of pathogens, conferring both individual and population-level protection leading to reduced mortality and morbidity across the world. However, vaccine efficacy is highly variable and early predictors of immune response and vaccine-induced memory remain unclear. Here we investigate the early innate-driven mechanisms leading to different grades of induction of adaptive immunity and protection. We vaccinated mice subcutaneously with protective doses of influenza (Sanofi) and SARS-CoV-2 (Pfizer) vaccines and analyzed their immune response in their draining lymph node (inguinal LN) at early time points (6 and 16 hours). When compared to the contralateral LNs, both vaccines led to increased inguinal LN cellularity at rapid times post-vaccination with the influenza vaccine showing earlier peak response at 6 hours compared to SARS-CoV-2 at 16 hours. Moreover, influenza vaccination led to an increase in DCs and B cell numbers, while the SARS-CoV-2 vaccine showed higher numbers of monocytes and NK cells post-vaccination. Preliminary data showed an increase of IFN-induced cytokines (IP-10, MCP-1, MIP-1β, and MIG) at 16 hours post-vaccination in both the serum and draining LN supernatant of SARS-CoV-2-vaccinated mice. Together, these distinct early innate responses induced by vaccination serve as early potential indicators of vaccine efficacy, providing novel correlates of protection for clinical evaluation. DARPA W911NF-23-2-0018 Vaccines and Immunotherapy (VAC)

Plasmacytoid dendritic cells (pDCs) mount powerful antiviral type I interferon (IFN-I) responses, yet only a fraction of pDCs produces high levels of IFN-I. Here we report that peripheral pDCs in naive mice comprise three subsets (termed A, B and C) that represent progressive differentiation stages. This heterogeneity was generated by tonic IFN-I signaling elicited in part by the cGAS/STING and TLR9 DNA-sensing pathways. A small 'IFN-I-naive' subset (pDC-A) could give rise to other subsets; it was expanded in STING deficiency or after the IFN-I receptor blockade, but was abolished by exogenous IFN-I. In response to RNA viruses, pDC-A showed increased Bcl2-dependent survival and superior IFN-I responses, but was susceptible to virus infection. Conversely, the majority of pDCs comprised the 'IFN-I-primed' subsets (pDC-B/C) that showed lower IFN-I responses and poor survival, but did not support virus replication. Thus, tonic IFN-I signaling decreases the cytokine-producing capacity and survival of pDCs but increases their virus resistance, facilitating optimal antiviral responses.

Early life is a critical period for generating robust immune response against harmful pathogens while simultaneously inducing tolerance to innocuous antigen. The gut is a major site for these early immune exposures, particularly in Peyer’s patches (PP), though the process in humans remains uncharacterized. Here, we examined human small intestine and associated PPs obtained from pediatric organ donors aged 0 – 10 years to investigate immune development, focusing on the T and B cell interactions, using a combination of immunofluorescence microscopy, flow cytometry, and single-cell RNA transcriptome profiling. We identified robust follicular activity characterized by early formation of germinal centers (GC) in the PP and mesenteric lymph nodes (MLN), which peaked at 2 years of age before declining. The proportion of class-switched memory B cells (MBC) increased in the tissues over early life. Although PP and MLN are primary sources of IgA-producing cells, a significant amount of IgG-expressing B cells was also generated. Interestingly, flow cytometry showed increases in follicular helper T cells (Tfh) and regulatory T cells (Treg) prior to the peak in GC response. Our data indicate that, in situ, adaptive immune responses in the infants are predominantly active in the early years of life, with important implications for designing strategies for targeting mucosal responses in vaccines and immunotherapies. Supported by NIH AI168634, grants from the Helmsley Charitable Trust, and 2018PGV1D071. Hematopoiesis and Immune System Development (HEM)

The immune system comprises multiple cell lineages and subsets maintained in tissues throughout the lifespan, with unknown effects of tissue and age on immune cell function. Here we comprehensively profiled RNA and surface protein expression of over 1.25 million immune cells from blood and lymphoid and mucosal tissues from 24 organ donors aged 20–75 years. We annotated major lineages (T cells, B cells, innate lymphoid cells and myeloid cells) and corresponding subsets using a multimodal classifier and probabilistic modeling for comparison across tissue sites and age. We identified dominant site-specific effects on immune cell composition and function across lineages; age-associated effects were manifested by site and lineage for macrophages in mucosal sites, B cells in lymphoid organs, and circulating T cells and natural killer cells across blood and tissues. Our results reveal tissue-specific signatures of immune homeostasis throughout the body, from which to define immune pathologies across the human lifespan. Wells et al. profile RNA and surface protein expression to describe dominant tissue-specific effects on immune cell composition and function across lineages in the human tissues across age.

Although identification and characterization of antigen-specific CD4+ T cells remains crucial for understanding immune responses, several technique hurdles have made it challenging to do so. Building upon our TetTCR-SeqHD platform, we developed a high-throughput and high-dimensional profiling technology, TetTCRII-SeqHD, for antigen-specific CD4+ T cells. The technology leverages the recently developed affinity enhanced class II MHC tetramer and optimizes the staining conditions to address the inherently lower TCR-pMHC class II binding avidity. Multiple tetramers can be multiplexed to simultaneously track T cells with different antigen specificities. Validation using multiple TCRs with known antigens demonstrated an average of > 90% precision and >80% recall rate, comparable to conventional methods while providing comprehensive transcriptional and phenotypic information at single-cell resolution. This advancement of TetTCRII-SeqHD provides valuable insights into helper T cell responses with broad applications in studying infection, vaccination, autoimmune diseases, and cancer immunotherapy. Supported by NIH U19AI128949 and R33CA256086. Technological Innovations in Immunology (TECH)

Advances in T cell biology have revealed heterogeneity among T cell populations that is not captured by existing general nomenclature. This issue has caused an ad hoc broadening of core T cell subset definitions and the invention of new subset designations that have not been uniformly delineated. To address this issue, in this Consensus Statement, we propose guidelines that serve three goals. First, they advocate that primary research reports define the experimental basis by which relevant subsets are designated in the methods section of each study. Second, they provide standardized definitions for existing subset designations in popular use, and common experimental criteria for defining each subset are noted. Last, they present an alternative ‘modular nomenclature’ paradigm. The newly proposed modular nomenclature eschews conceptualization of antigen-experienced T cells as belonging to a few idealized subsets, and the nomenclature instead simply indicates individual biological properties present in a T cell population with brief descriptors. Collectively, these guidelines intend to enhance transparency in the literature while facilitating clearer communication of findings and concepts to researchers, students and clinicians. This Consensus Statement clarifies the existing subset-based nomenclature for T cells. Furthermore, it proposes an alternative modular nomenclature that is designed to be brief and flexible and to avoid ambiguity and unwanted implications. The authors also provide guidance on how T cell nomenclature should be described in research papers.

NK cells, like B and T cells, acquire immune memory; however, our understanding of memory NK cells is primarily limited to specific subsets generated during CMV infection and lacks generalized memory markers. We hypothesized a universal memory signature exists across B, T, and NK cells; thus, we performed whole transcriptional profiling of B, CD4 T, CD8 T, and NK cells over the course of MCMV infection. Indeed, a shared transcriptional signature delineated naïve and memory cells across all lymphocyte populations; including CD55 (DAF, decay accelerating factor), the ectonucleotidase CD39, and the transcription factor Aiolos which were all validated by flow cytometry. As such, naïve cells are CD55hiCD39-Aioloshi and memory are any other combination thereof. The capacity of these markers to delineate memory cells extended to multiple other infectious scenarios and were recapitulated in human memory B and T cells. Single cell transcriptional analysis of human lymphocytes also demonstrated functional distinction between naïve and memory cells. To solidify this memory signature for NK cells, we performed in vitro stimulation and observed upregulation of cytolytic effectors only in memory NK cells. Notably, naïve and memory NK cells exhibit distinct localization throughout the human body. These findings establish a core signature, conserved across cell lineage and species, for identifying memory broadly and provides a foundation for designing strategies to modulate immune memory. NIH AI168634, AI128949, T32AI148099 Viral Immunology (VIR)

Respiratory viral infections establish tissue-resident memory T cells (TRM) in the lung, which provide optimal protection against subsequent infections, though the underlying mechanisms are incompletely understood. Here, we demonstrate in a mouse model of heterosubtypic influenza infection that lung TRM attenuate inflammation by macrophages during secondary versus primary responses, in part, through production of the immunoregulatory cytokine IL-10. During secondary infections, lung TRM were the predominant producers of early IL-10; inhibiting early IL-10 signaling resulted in increased macrophage-mediated inflammation, morbidity, and lung pathology. Moreover, lung TRM were shown to directly modulate lung macrophage responses and polarization in depletion experiments. Finally, IL-10 enhanced IFN-γ production by lung memory CD8+ T cells. Human influenza-specific TRM isolated from lungs recapitulated robust IL-10 expression associated with augmented effector responses of murine TRM. These data support a dual role of TRM in coordinating in situ secondary responses-augmenting effector responses for robust viral clearance while dampening inflammation to limit tissue damage.