SUMMARY Tissue-resident memory T cells (TRM) in mice mediate optimal protective immunity to infection and vaccination, while in humans, the existence and properties of TRM remain unclear. Here, we use a unique human tissue resource to determine whether human tissue memory T cells comprise 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 TRM based on homology to the transcriptional profile of mouse CD8+TRM. Human TRM in diverse sites exhibit increased expression of adhesion and inhibitory molecules, produce both pro-inflammatory and regulatory cytokines, and have reduced proliferation 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.

The type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 play critical roles in stimulating innate and adaptive immune responses required for resistance to helminth infection and promotion of allergic inflammation, metabolic homeostasis and tissue repair1–3. Group 2 innate lymphoid cells (ILC2s) are a potent source of type 2 cytokines and while significant advances have been made in understanding the cytokine milieu that promotes ILC2 responses4–9, there are fundamental gaps in knowledge regarding how ILC2 responses are regulated by other stimuli. In this report, we demonstrate that ILC2s in the gastrointestinal tract co-localize with cholinergic neurons that express the neuropeptide neuromedin U (NMU)10,11. In contrast to other hematopoietic cells, ILC2s selectively express the NMU receptor 1 (NMUR1). In vitro stimulation of ILC2s with NMU induced rapid cell activation, proliferation and secretion of type 2 cytokines IL-5, IL-9 and IL-13 that was dependent on cell-intrinsic expression of NMUR1 and Gαq protein. In vivo administration of NMU triggered potent type 2 cytokine responses characterized by ILC2 activation, proliferation and eosinophil recruitment that was associated with accelerated expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis or induction of lung inflammation. Conversely, worm burden was higher in Nmur1−/− mice compared to control mice. Further, use of gene-deficient mice and adoptive cell transfer experiments revealed that ILC2s were necessary and sufficient to mount NMU-elicited type 2 cytokine responses. Together, these data indicate that the NMU-NMUR1 neuronal signaling circuit provides a selective and previously unrecognized mechanism through which the enteric nervous system and innate immune system integrate to promote rapid type 2 cytokine responses that can induce anti-microbial, inflammatory and tissue-protective type 2 responses at mucosal sites.

Graphical Abstract Highlights d High-resolution map of human NK cells shows tissue-driven distribution across ages d Differentiated NK cells predominate in blood, bone marrow, spleen, and lungs d Tissue-resident NK cells exhibit specific adaptations in mucosal and lymphoid sites d Lymph nodes and intestines are reservoirs for precursor and immature NK cells

SUMMARY 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 aged <1–93years. The distribution of cDC1 (CD141hiCD13hi) and cDC2 (Sirp-α+CD1c+) subsets was a function of tissue site and 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 resident memory T cells (TRM) maintain immunity in diverse sites as determined in mouse models, while their establishment and role in human tissues has been difficult to assess. Here, we investigated human lung TRM generation, maintenance and function in airway samples obtained longitudinally from HLA-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of TRM signature markers including CD69, CD103, and CD49a, while lung-infiltrating recipient T cells gradually acquire TRM phenotypes over months in vivo. Single cell transcriptome profiling of airway T cells reveals that donor T cells comprise two TRM-like subsets with varying levels of expression of TRM-associated genes while recipient T cells comprised non-TRM and similar TRM-like subpopulations, suggesting de novo TRM generation. Transplant recipients exhibiting higher frequencies of persisting donor TRM experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared to recipients with low donor TRM persistence, suggesting that monitoring TRM dynamics could be clinically informative. Together, our results provide novel spatial and temporal insights into how human TRM develop, function, persist, and impact tissue integrity within the complexities of lung transplantation.

The proportion of deceased donor kidneys procured for transplant but subsequently discarded has been growing steadily in the United States, but factors contributing to the rising discard rate remain unclear. To assess the reasons for and probability of organ discard we assembled a cohort of 212,305 deceased donor kidneys recovered for transplant from 2000-2015 in the SRTR registry that included 36,700 kidneys that were discarded. 'Biopsy Findings' (38.2%) was the most commonly reported reason for discard. The median Kidney Donor Risk Index of discarded kidneys was significantly higher than transplanted organs (1.78 vs 1.12), but a large overlap in the quality of discarded and transplanted kidneys was observed. Kidneys of donors who were older, female, Black, obese, diabetic, hypertensive or HCV-positive experienced a significantly increased odds of discard. Kidneys from donors with multiple unfavorable characteristics were more likely to be discarded, whereas unilaterally discarded kidneys had the most desirable donor characteristics and the recipients of their partner kidneys experienced a one-year death-censored graft survival rate over 90%. There was considerable geographic variation in the odds of discard across the United States, which further supports the notion that factors beyond organ quality contributed to kidney discard. Thus, while the discard of a small fraction of organs procured from donors may be inevitable, the discard of potentially transplantable kidneys needs to be avoided. This will require a better understanding of the factors contributing to organ discard in order to remove the disincentives to utilize less-than-ideal organs for transplantation.

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 8 different anatomic compartments in 6 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.

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.