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.

Liver biopsy in assessment of extended criteria donors.

The safety and liver utilization with prerecovery liver biopsy (PLB) in extended criteria liver donors are unclear. We conducted a retrospective cohort study in 1323 brain death donors (PLB = 496) from 3 organ procurement organizations (OPOs). Outcomes were complications, preempted liver recovery (PLR), and liver transplantation (LT). Additional analyses included liver-only and propensity score-matched multiorgan donor subgroups. PLB donors were older (57 versus 53 years; P < 0.001). Hepatitis C antibody positivity (14.3% versus 9.6%, P = 0.01) and liver-only donors (42.6% versus 17.5%; P < 0.001) were more prevalent. The PLB cohort had fewer complications (31.9% versus 42.3%; P < 0.001). In the PLB cohort, PLR was significantly higher (odds ratio [OR], 3.45; 95% confidence interval [CI], 2.42-4.92) and LT lower (OR, 0.69; 95% CI, 0.52-0.91). In liver-only and propensity score-matched multiorgan donor subgroups, PLR was significantly higher (OR, 1.76; 95% CI, 1.06-2.94 and OR, 2.29; 95% CI, 1.37-3.82, respectively) without a decrease in LT (OR, 0.71; 95% CI, 0.43-1.18 and OR, 0.91; 95% CI, 0.63-1.33, respectively) in PLB subgroups. In conclusion, in extended criteria liver donors, PLB is safe and decreases futile liver recovery without decreasing LT. Increased use of PLB, especially in liver-only donors, is likely to save costs to OPOs and transplant centers and improve efficiencies in organ allocation. Liver Transplantation 24 182-191 2018 AASLD.

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.

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.

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.

Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues.

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life.

Lesson From the New York City Out-of-Hospital Uncontrolled Donation After Circulatory Determination of Death Program.

STUDY OBJECTIVE: In 2006, the Institute of Medicine emphasized substantial potential to expand organ donation opportunities through uncontrolled donation after circulatory determination of death (uDCDD). We pilot an out-of-hospital uDCDD kidney program for New York City in partnership with communities that it was intended to benefit. We evaluate protocol process and outcomes while identifying barriers to success and means for improvement. METHODS: We conducted a prospective, participatory action research study in Manhattan from December 2010 to May 2011. Daily from 4 to 12 pm, our organ preservation unit monitored emergency medical services (EMS) frequencies for cardiac arrests occurring in private locations. After EMS providers independently ordered termination of resuscitation, organ preservation unit staff determined clinical eligibility and donor status. Authorized parties, persons authorized to make organ donation decisions, were approached about in vivo preservation. The study population included organ preservation unit staff, authorized parties, passersby, and other New York City agency personnel. Organ preservation unit staff independently documented shift activities with daily operations notes and teleconference summaries that we analyzed with mixed qualitative and quantitative methods. RESULTS: The organ preservation unit entered 9 private locations; all the deceased lacked previous registration, although 4 met clinical screening eligibility. No kidneys were recovered. We collected 837 notes from 35 organ preservation unit staff. Despite frequently recounting protocol breaches, most responses from passersby including New York City agencies were favorable. No authorized parties were offended by preservation requests, yielding a Bayesian posterior median 98% (95% credible interval 76% to 100%). CONCLUSION: In summary, the New York City out-of-hospital uDCDD program was not feasible. There were frequent protocol breaches and confusion in determining clinical eligibility. In the small sample of authorized persons we encountered during the immediate grieving period, negative reactions were infrequent.

Longterm maintenance of human naive T cells through in situ homeostasis in lymphoid tissue sites.

Naïve T cells develop in the thymus and coordinate immune responses to new antigens; however, mechanisms for their long-term persistence over the human lifespan remain undefined. Here, we investigated human naïve T cell development and maintenance in primary and secondary lymphoid tissues obtained from individual organ donors aged 3 months-73 years. In the thymus, the frequency of double-positive thymocytes declined sharply in donors over age 40 coincident with reduced recent thymic emigrants (RTE) in lymphoid tissues, while naïve T cells were functionally maintained predominantly in lymph nodes (LN). Analysis of TCR clonal distribution by CDR3 sequencing of naïve CD4+ and CD8+ T cells in spleen and LNs reveal site-specific clonal expansions of naïve T cells from individuals >40 years of age with minimal clonal overlap between lymphoid tissues. We also identified biased naïve T cell clonal distribution within specific lymph nodes based on VJ usage. Together these results suggest prolonged maintenance of naïve T cells through in situ homeostasis and retention in lymphoid tissue.

Spatial map of human T cell compartmentalization and maintenance over decades of life.

Mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid, and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naive, central, and effector memory, and terminal effector subsets is contingent on both their differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is different in CD4+ or CD8+ T cell lineages, varies with their differentiation stage and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting distinct pathways for human T cell fate determination and homeostasis.

A pilot program to evaluate deceased donor disease transmission risk: the New York Organ Donor Network Infectious Disease Working Group.

BACKGROUND: Recent cases of donor-derived infections raise the question of how best to screen donors without excessive restriction of the donor pool. METHODS: The New York Organ Donor Network (NYODN) established an Infectious Diseases Working Group (IDWG) in 2008, which established an on-call schedule of voluntary transplant infectious disease physicians to provide remote evaluations for donors at increased risk for disease transmission. RESULTS: Data were reviewed from 40 available IDWG evaluations from 2008 to 2011. Eighteen cases (45%) were considered to be at unacceptable risk for infection transmission. Sixteen of these cases were excluded from donation secondary to IDWG recommendation; there was limited recipient center interest in the remaining two cases. Approximately 22 (55%) cases were categorized by the IDWG as acceptable, with 14 proceeding to recovery of 49 organs. IDWG physician recommendations were conveyed to recipient centers, and screening guidelines for donors were revised based on the IDWG experiences. CONCLUSION: Establishment of a donation service area disease transmission evaluation service is a valuable program for donor screening and may promote dissemination of more detailed donor information to recipient centers.

Hemoglobin A1c testing is associated with improved pancreas utilization for transplant.

CONTEXT: Aging, higher prevalence of diabetes, worsening obesity, and hyperglycemia among potential donors increase the likelihood that pancreata will be declined by transplant centers. Hemoglobin A1c testing, also known as glycated hemoglobin testing, identifies a donor's average blood glucose concentration for the preceding 2 to 3 months and is the standard test for identifying prolonged periods of hyperglycemia. OBJECTIVE: To compare pancreas utilization rates before and after implementation of hemoglobin A1c testing. DESIGN: A retrospective study of data from the New York Organ Donor Network was conducted. Potential donors were defined as standard criteria donors who had no history of diabetes and were not seropositive for hepatitis B or C. Criteria for "ideal" potential pancreas donors were based on age, body mass index, lipase level, and terminal creatinine level. Potential donors who did not meet the criteria for ideal donors were considered "expanded" potential pancreas donors. Pancreas utilization rate was defined as the number of pancreata transplanted divided by the number of potential pancreas donors. RESULTS: Of 779 standard criteria donors, 691 (89%) were potential pancreas donors: 251 ideal (36%) and 440 expanded (64%) donors. In 2005 and 2006, before hemoglobin A1c testing, pancreas utilization rates were 21% and 18%, respectively. In 2008, 2009, and 2010, after hemoglobin A1c testing was incorporated, utilization rates were 27%, 28%, and 32%, respectively. Utilization of ideal donors increased from 33% to 51% (P= .003), and utilization of expanded donors increased from 11% to 17% (P= .05). Pancreas utilization increased 51.0%, and pancreas discards decreased 50.8% with the implementation of hemoglobin A1c testing. CONCLUSION: Hemoglobin A1c testing may increase utilization of ideal and expanded criteria pancreata.

Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets.

Knowledge of human T cells derives chiefly from studies of peripheral blood, whereas their distribution and function in tissues remains largely unknown. Here, we present a unique analysis of human T cells in lymphoid and mucosal tissues obtained from individual organ donors, revealing tissue-intrinsic compartmentalization of naive, effector, and memory subsets conserved between diverse individuals. Effector memory CD4(+) T cells producing IL-2 predominated in mucosal tissues and accumulated as central memory subsets in lymphoid tissue, whereas CD8(+) T cells were maintained as naive subsets in lymphoid tissues and IFN-γ-producing effector memory CD8(+) T cells in mucosal sites. The T cell activation marker CD69 was constitutively expressed by memory T cells in all tissues, distinguishing them from circulating subsets, with mucosal memory T cells exhibiting additional distinct phenotypic and functional properties. Our results provide an assessment of human T cell compartmentalization as a new baseline for understanding human adaptive immunity.