Site-specific development and progressive maturation of human tissue-resident memory T cells over infancy and childhood.

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated T cells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0-10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory T cells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory T cells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased T cell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory T cells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children.

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.

Infant T cells are developmentally adapted for robust lung immune responses through enhanced T cell receptor signaling.

Infants require coordinated immune responses to prevent succumbing to multiple infectious challenges during early life, particularly in the respiratory tract. The mechanisms by which infant T cells are functionally adapted for these responses are not well understood. Here, we demonstrated using an in vivo mouse cotransfer model that infant T cells generated greater numbers of lung-homing effector cells in response to influenza infection compared with adult T cells in the same host, due to augmented T cell receptor (TCR)­mediated signaling. Mouse infant T cells showed increased sensitivity to low antigen doses, originating at the interface between T cells and antigen-bearing accessory cells­through actin-mediated mobilization of signaling molecules to the immune synapse. This enhanced signaling was also observed in human infant versus adult T cells. Our findings provide a mechanism for how infants control pathogen load and dissemination, which is important for designing developmentally targeted strategies for promoting immune responses at this vulnerable life stage.

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.

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.

The Interaction between NKAP and HDAC3 Is Critical for T Cell Maturation.

NKAP and HDAC3 are critical for T cell maturation. NKAP and HDAC3 physically associate, and a point mutation in NKAP, NKAP(Y352A), abrogates this interaction. To evaluate the significance of NKAP and HDAC3 association in T cell maturation, transgenic mice were engineered for cre-mediated endogenous NKAP gene deletion coupled to induction of NKAP(Y352A) or a wild type (WT) control transgene, NKAP(WT), in double positive thymocytes or regulatory T cells (Tregs). T cell maturation was normal in mice with endogenous NKAP deletion coupled to NKAP(WT) induction. However, severe defects occurred in T cell and Treg maturation and in iNKT cell development when NKAP(Y352A) was induced, recapitulating NKAP deficiency. Conventional T cells expressing NKAP(Y352A) failed to enter the long-term T cell pool, did not produce cytokines, and remained complement susceptible, whereas Tregs expressing NKAP(Y352A) were eliminated as recent thymic emigrants leading to lethal autoimmunity. Overall, these results demonstrate the significance of NKAP-HDAC3 association in T cells.

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.

Factors Affecting Enrolment in the Community Based Health Insurance Scheme of Chandranigahapur Hospital of Rautahat District.

BACKGROUND: Low income countries face considerable challenges in financing health care for their populations. As its consequences, poor people don't have access to desired health services, drugs and medicine.To address the financial barriers to health services, Government of Nepal introduced Community Based Health Insurance scheme at selected health facilities. However, enrolment in the schemeis very low. This study aims to identify the associated factors affecting enrolment in the insurance scheme. METHODS: A community based case-control study was conducted within the coverage area of CBHI scheme of Chandranigahapur Hospital. CBHI Scheme of Chandranigahapur Hospital was selected purposively. Altogether 416 households were interviewed using a structured questionnaire. The required number of sample size from the enrolled households as cases and equal number of non-enrolled households as controls were selected randomly in 1:1 ratio. RESULTS: The odds of enrolment in the CBHI scheme among male-headed households were found lower than female-headed households (AOR 0.251, 95% CI 0.097 to 0.652). Similarly household head belonging to upper caste/ethnic groups (AOR 3.981, 95% CI 2.027 to 7.816) as well aseducated household head(AOR 6.184, 95% CI 3.137 to 12.188)were more likely to enrol in the CBHI scheme. Households having >60 years elderly were found significantly associated with enrolment in CBHI scheme(AOR 3.996, 95% CI 2.130 to 7.497). Time to reach health facility as well as affordability of premium of the insurance scheme was also found significantly associated with enrolment in the CBHI scheme. CONCLUSIONS: The enrolment in the CBHI scheme is determined by combination of householdhead, household and health service related factors.These determinants should be addressed to enhance the enrolment in the insurance scheme.

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.

Prevalence of cardiovascular disease risk factors: A community-based cross-sectional study in a peri-urban community of Kathmandu, Nepal.

BACKGROUND: As a low-income country, Nepal is experiencing cardiovascular diseases as an emerging health problem. However, studies are lacking on the risk factors of cardiovascular diseases in peri-urban communities; where the socio-demographical transition is in progress. Therefore, this study aimed to identify the prevalence and socio-demographic distribution of cardiovascular disease risk factors in one of the peri-urban communities in Kathmandu, Nepal. METHODS: We conducted a cross-sectional study in Sitapaila Village Development Committee, Kathmandu from February 2014 to February 2015. Altogether, 347 adults from 18 to 70 years of age were selected randomly. Data were collected through modified WHO STEPS questionnaire for non-communicable disease (NCD) risk factors survey and analyzed in SPSS V.16.0 software. RESULTS: Mean age of the participant was 42.5 ±â€¯13.2 years. Majority of them were female (n = 206; 59.4%), one-third (34%) represented Brahman and Chetri, and over a quarter (29.1%) did not attend school. Cardiovascular disease risk factors included smoking (17.6%), alcohol consumption (29.4%), insufficient fruit and vegetables intake (98%), insufficient physical activity (21.0%), obesity (15.3%), hypertension (34.4%), diabetes (10.5%), and high triglyceride levels (10.8%). They were significantly associated with different socio-demographic characteristics: smoking with gender, age groups and education level; alcohol consumption was with gender, age groups, ethnicity and occupation; insufficient physical activity with gender, age groups and occupation; hypertension with gender, age groups, ethnicity, education level and occupation. CONCLUSION: A high prevalence of cardiovascular disease risk factors and their disproportional distribution among the study population indicated an inevitable risk of cardiovascular events in near future.

The differentiation of ROR-γt expressing iNKT17 cells is orchestrated by Runx1.

iNKT cells are a unique lineage of T cells that recognize glycolipid presented by CD1d. In the thymus, they differentiate into iNKT1, iNKT2 and iNKT17 effector subsets, characterized by preferential expression of Tbet, Gata3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. We demonstrate that the transcriptional regulator Runx1 is essential for the generation of ROR-γt expressing iNKT17 cells. PLZF-cre Runx1 cKO mice lack iNKT17 cells in the thymus, spleen and liver. Runx1-deficient iNKT cells have altered expression of several genes important for iNKT17 differentiation, including decreased expression of IL-7Rα, BATF and c-Maf and increased expression of Bcl11b and Lef1. However, reduction of Lef1 expression or introduction of an IL-7Rα transgene is not sufficient to correct the defect in iNKT17 differentiation, demonstrating that Runx1 is a key regulator of several genes required for iNKT17 differentiation. Loss of Runx1 leads to a severe decrease in iNKT cell numbers in the thymus, spleen and liver. The decrease in cell number is due to a combined decrease in proliferation at Stage 1 during thymic development and increased apoptosis. Thus, we describe a novel role of Runx1 in iNKT cell development and differentiation, particularly in orchestrating iNKT17 differentiation.

Histone deacetylase 3 is required for iNKT cell development.

NKT cells are a distinct subset that have developmental requirements that often differ from conventional T cells. Here, we show that NKT-specific deletion of Hdac3 results in a severe reduction in the number of iNKT cells, particularly of NKT1 cells. In addition, there is decreased cytokine production by Hdac3-deficient NKT2 and NKT17 cells. Hdac3-deficient iNKT cells have increased cell death that is not rescued by transgenic expression of Bcl-2 or Bcl-xL. Hdac3-deficient iNKT cells have less Cyto-ID staining and lower LC3A/B expression, indicative of reduced autophagy. Interestingly, Hdac3-deficient iNKT cells also have lower expression of the nutrient receptors GLUT1, CD71 and CD98, which would increase the need for autophagy when nutrients are limiting. Therefore, Hdac3 is required for iNKT cell development and differentiation.

NKAP Regulates Invariant NKT Cell Proliferation and Differentiation into ROR-γt-Expressing NKT17 Cells.

Invariant NKT (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and they recognize glycolipids presented by an MHC class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2, and NKT17 functional subsets that preferentially produce cytokines IFN-γ, IL-4, and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and it is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of promyelocytic leukemia zinc finger; however, the promyelocytic leukemia zinc finger transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP conditional knockout mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation.

Coactosin-like 1 antagonizes cofilin to promote lamellipodial protrusion at the immune synapse.

Actin depolymerizing factor-homology (ADF-H) family proteins regulate actin filament dynamics at multiple cellular locations. Herein, we have investigated the function of the ADF-H family member coactosin-like 1 (COTL1) in the regulation of actin dynamics at the T cell immune synapse (IS). We initially identified COTL1 in a genetic screen to identify novel regulators of T cell activation, and subsequently found that it associates with F-actin and localizes at the IS in response to TCR+CD28 stimulation. Live cell microscopy showed that depletion of COTL1 protein impaired T cell spreading in response to TCR ligation and abrogated lamellipodial protrusion at the T cell - B cell contact site, producing only a band of F-actin. Significantly, re-expression of wild type COTL1, but not a mutant deficient in F-actin binding could rescue these defects. In addition, COTL1 depletion reduced T cell migration. In vitro studies showed that COTL1 and cofilin compete with each other for binding to F-actin, and COTL1 protects F-actin from cofilin-mediated depolymerization. While depletion of cofilin enhanced F-actin assembly and lamellipodial protrusion at the IS, concurrent depletion of both COTL1 and cofilin restored lamellipodia formation. Taken together, our results suggest that COTL1 regulates lamellipodia dynamics in part by protecting F-actin from cofilin-mediated disassembly.

Separation of plasmacytoid dendritic cells from B-cell-biased lymphoid progenitor (BLP) and Pre-pro B cells using PDCA-1.

B-cell-biased lymphoid progenitors (BLPs) and Pre-pro B cells lie at a critical juncture between B cell specification and commitment. However, both of these populations are heterogenous, which hampers investigation into the molecular changes that occur as lymphoid progenitors commit to the B cell lineage. Here, we demonstrate that there are PDCA-1(+)Siglec H(+) plasmacytoid dendritic cells (pDCs) that co-purify with BLPs and Pre-pro B cells, which express little or no CD11c or Ly6C. Removal of PDCA-1(+) pDCs separates B cell progenitors that express high levels of a Rag1-GFP reporter from Rag1-GFP(low/neg) pDCs within the BLP and Pre-pro B populations. Analysis of Flt3-ligand knockout and IL-7Rα knockout mice revealed that there is a block in B cell development at the all-lymphoid progenitor (ALP) stage, as the majority of cells within the BLP or Pre-pro B gates were PDCA-1(+) pDCs. Thus, removal of PDCA-1(+) pDCs is critical for analysis of BLP and Pre-pro B cell populations. Analysis of B cell potential within the B220(+)CD19(-) fraction demonstrated that AA4.1(+)Ly6D(+)PDCA-1(-) Pre-pro B cells gave rise to CD19(+) B cells at high frequency, while PDCA-1(+) pDCs in this fraction did not. Interestingly, the presence of PDCA-1(+) pDCs within CLPs may help to explain the conflicting results regarding the origin of these cells.

The transcriptional repressor NKAP is required for the development of iNKT cells.

Invariant natural killer T cells have a distinct developmental pathway from conventional αß T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.

Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX.

The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.