A guide to adaptive immune memory.

Immune memory - comprising T cells, B cells and plasma cells and their secreted antibodies - is crucial for human survival. It enables the rapid and effective clearance of a pathogen after re-exposure, to minimize damage to the host. When antigen-experienced, memory T cells become activated, they proliferate and produce effector molecules at faster rates and in greater magnitudes than antigen-inexperienced, naive cells. Similarly, memory B cells become activated and differentiate into antibody-secreting cells more rapidly than naive B cells, and they undergo processes that increase their affinity for antigen. The ability of T cells and B cells to form memory cells after antigen exposure is the rationale behind vaccination. Understanding immune memory not only is crucial for the design of more-efficacious vaccines but also has important implications for immunotherapies in infectious disease and cancer. This 'guide to' article provides an overview of the current understanding of the phenotype, function, location, and pathways for the generation, maintenance and protective capacity of memory T cells and memory B cells.

Exhausted intratumoral Vδ2- γδ T cells in human kidney cancer retain effector function.

Gamma delta (γδ) T cells reside within human tissues including tumors, but their function in mediating antitumor responses to immune checkpoint inhibition is unknown. Here we show that kidney cancers are infiltrated by Vδ2- γδ T cells, with equivalent representation of Vδ1+ and Vδ1- cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2- T cells can express the transcriptional program of exhausted αß CD8+ T cells as well as canonical markers of terminal T-cell exhaustion including PD-1, TIGIT and TIM-3. Although Vδ2- γδ T cells have reduced IL-2 production, they retain expression of cytolytic effector molecules and co-stimulatory receptors such as 4-1BB. Exhausted Vδ2- γδ T cells are composed of three distinct populations that lack TCF7, are clonally expanded and express cytotoxic molecules and multiple Vδ2- T-cell receptors. Human tumor-derived Vδ2- γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2- T cells in pretreatment tumor biopsies was used to predict subsequent clinical responses to PD-1 blockade in patients with cancer. Thus, Vδ2- γδ T cells within the tumor microenvironment can contribute to antitumor efficacy.

Inhaled particulate accumulation with age impairs immune function and architecture in human lung lymph nodes.

Older people are particularly susceptible to infectious and neoplastic diseases of the lung and it is unclear how lifelong exposure to environmental pollutants affects respiratory immune function. In an analysis of human lymph nodes (LNs) from 84 organ donors aged 11-93 years, we found a specific age-related decline in lung-associated, but not gut-associated, LN immune function linked to the accumulation of inhaled atmospheric particulate matter. Increasing densities of particulates were found in lung-associated LNs with age, but not in the corresponding gut-associated LNs. Particulates were specifically contained within CD68+CD169- macrophages, which exhibited decreased activation, phagocytic capacity, and altered cytokine production compared with non-particulate-containing macrophages. The structures of B cell follicles and lymphatic drainage were also disrupted in lung-associated LNs with particulates. Our results reveal that the cumulative effects of environmental exposure and age may compromise immune surveillance of the lung via direct effects on immune cell function and lymphoid architecture.

The cell-surface 5'-nucleotidase CD73 defines a functional T memory cell subset that declines with age.

Memory T cells exhibit considerable diversity that determines their ability to be protective. Here, we examine whether changes in T cell heterogeneity contribute to the age-associated failure of immune memory. By screening for age-dependent T cell-surface markers, we identify CD4 and CD8 memory T cell subsets that are unrelated to previously defined subsets of central and effector memory cells. Memory T cells expressing the ecto-5'-nucleotidase CD73 constitute a functionally distinct subset of memory T cells that declines with age. They resemble long-lived, polyfunctional memory cells but are also poised to display effector functions and to develop into cells resembling tissue-resident memory T cells (TRMs). Upstream regulators of differential chromatin accessibility and transcriptomes include transcription factors that facilitate CD73 expression and regulate TRM differentiation. CD73 is not just a surrogate marker of these regulatory networks but is directly involved in T cell survival.

Molecular programs of fibrotic change in aging human lung.

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

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.

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.

Tissue-resident memory T cells in tumor immunity and immunotherapy.

Tissue-resident memory T cells (TRM) represent a heterogeneous T cell population with the functionality of both effector and memory T cells. TRM express residence gene signatures. This feature allows them to traffic to, reside in, and potentially patrol peripheral tissues, thereby enforcing an efficient long-term immune-protective role. Recent studies have revealed TRM involvement in tumor immune responses. TRM tumor infiltration correlates with enhanced response to current immunotherapy and is often associated with favorable clinical outcome in patients with cancer. Thus, targeting TRM may lead to enhanced cancer immunotherapy efficacy. Here, we review and discuss recent advances on the nature of TRM in the context of tumor immunity and immunotherapy.

Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype.

Although human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Tissue-Resident Memory T Cells Mediate Immune Homeostasis in the Human Pancreas through the PD-1/PD-L1 Pathway.

Non-recirculating tissue-resident memory T cells (TRMs) are the predominant T cell subset in diverse tissue sites, where they mediate protective immune responses in situ. Here, we reveal a role for TRM in maintaining immune homeostasis in the human pancreas through interactions with resident macrophages and the PD-1/PD-L1 inhibitory pathway. Using tissues obtained from organ donors, we identify that pancreas T cells comprise CD8+PD-1hi TRMs, which are phenotypically, functionally, and transcriptionally distinct compared to TRMs in neighboring jejunum and lymph node sites. Pancreas TRMs cluster with resident macrophages throughout the exocrine areas; TRM effector functions are enhanced by macrophage-derived co-stimulation and attenuated by the PD-1/PD-L1 pathways. Conversely, in samples from chronic pancreatitis, TRMs exhibit reduced PD-1 expression and reduced interactions with macrophages. These findings suggest important roles for PD-1 and TRM-macrophage interactions in controlling tissue homeostasis and immune dysfunctions underlying inflammatory disease, with important implications for PD-1-based 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.

Human lung tissue resident memory T cells in health and disease.

The human lung contains a heterogeneous population of immune cells which mediate protective responses, maintain tissue homeostasis, but can also promote immunopathology in disease. The majority of T cells in the human lung are tissue resident memory T cells (TRM) which have been shown in mouse models to provide vital roles in the protection against multiple respiratory pathogens, and contribute to heterosubtypic protection in the context of vaccination. In this review, we will discuss recent studies in humans identifying lung TRM, their role in maintaining tissue homeostasis, and emerging evidence implicating TRM in anti-tumor immunity and immune surveillance as well as their potential for immunopathology in chronic airway inflammation.

The Role of the Thymus in the Immune Response.

The thymus is a primary lymphoid organ essential for the development of T lymphocytes, which orchestrate adaptive immune responses. T-cell development in the thymus is spatially regulated; key checkpoints in T-cell maturation and selection occur in cortical and medullary regions to eliminate self-reactive T cells, establish central tolerance, and export naïve T cells to the periphery with the potential to recognize diverse pathogens. Thymic output is also temporally regulated due to age-related involution of the thymus accompanied by loss of epithelial cells. This review discusses the structural and age-related control of thymus function in humans.

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.

Computational Evaluation of B-Cell Clone Sizes in Bulk Populations.

B cell clones expand and contract during adaptive immune responses and can persist or grow uncontrollably in lymphoproliferative disorders. One way to monitor and track B cell clones is to perform large-scale sampling of bulk cell populations, amplifying, and sequencing antibody gene rearrangements by next-generation sequencing (NGS). Here, we describe a series of computational approaches for estimating B cell clone size in NGS immune repertoire profiling data of antibody heavy chain gene rearrangements. We define three different measures of B cell clone size-copy numbers, instances, and unique sequences-and show how these measures can be used to rank clones, analyze their diversity, and study their distribution within and between individuals. We provide a detailed, step-by-step procedure for performing these analyses using two different data sets of spleen samples from human organ donors. In the first data set, 19 independently generated biological replicates from a single individual are analyzed for B cell clone size, diversity and sampling sufficiency for clonal overlap analysis. In the second data set, B cell clones are compared in eight different organ donors. We comment upon frequently encountered pitfalls and offer practical advice with alternative approaches. Overall, we provide a series of pragmatic analytical approaches and show how different clone size measures can be used to study the clonal landscape in bulk B cell immune repertoire profiling data.

Human T Cell Development, Localization, and Function throughout Life.

Throughout life, T cells coordinate multiple aspects of adaptive immunity, including responses to pathogens, allergens, and tumors. In mouse models, the role of T cells is studied in the context of a specific type of pathogen, antigen, or disease condition over a limited time frame, whereas in humans, T cells control multiple insults simultaneously throughout the body and maintain immune homeostasis over decades. In this review, we discuss how human T cells develop and provide essential immune protection at different life stages and highlight tissue localization and subset delineation as key determinants of the T cell functional role in immune responses. We also discuss how anatomic compartments undergo distinct age-associated changes in T cell subset composition and function over a lifetime. It is important to consider age and tissue influences on human T cells when developing targeted strategies to modulate T cell-mediated immunity in vaccines and immunotherapies.

An atlas of B-cell clonal distribution in the human body.

B-cell responses result in clonal expansion, and can occur in a variety of tissues. To define how B-cell clones are distributed in the body, we sequenced 933,427 B-cell clonal lineages and mapped them to eight different anatomic compartments in six human organ donors. We show that large B-cell clones partition into two broad networks-one spans the blood, bone marrow, spleen and lung, while the other is restricted to tissues within the gastrointestinal (GI) tract (jejunum, ileum and colon). Notably, GI tract clones display extensive sharing of sequence variants among different portions of the tract and have higher frequencies of somatic hypermutation, suggesting extensive and serial rounds of clonal expansion and selection. Our findings provide an anatomic atlas of B-cell clonal lineages, their properties and tissue connections. This resource serves as a foundation for studies of tissue-based immunity, including vaccine responses, infections, autoimmunity and cancer.