The human tissue-resident T cell pool consists of a diverse array of conventional adaptive as well as unconventional invariant T cells that populate barrier sites and carefully balance immune defense in the context of microbial exposure. Here, we investigated the tissue-resident CD4 T cell compartment across donor-matched lymphoid and nonlymphoid tissues and uncovered a population enriched primarily in the ileum and liver displaying a rich polyfunctional profile with diverse T helper 1/17/22 characteristics.These CD4 T cells were highly responsive, susceptible to innate cytokine-driven activation, and identified by CD161 and CD56 coexpression. Their transcriptional and protein expression profile included innate-like effector features including NKp80, NKG2D, NKG7, as well as granzyme K, and exhibited characteristics of tissue residence, persistence, and barrier repair, and they were depleted in the colon of patients with inflammatory bowel disease. Notably, the CD161(+) CD56(+) CD4 T cell pool was expanded in cytomegalovirus (CMV)-seropositive individuals and enriched in CMV-specific T cell receptors and responses. Together, we identify a potent effector-memory CD4 T cell subset resident in the gastrointestinal tract and liver, defined by enhanced innateness and enriched in CMV-specificity.

CXCR5+ CD8+ T cells emerged as key mediators of antiviral immunity in the context of chronic infection. However, their functional attributes and tissue distribution remain incompletely defined, especially in relation to antigen specificity. Here, we investigated the anatomical localization and antiviral properties of CXCR5+ CD8+ T cells across multiple sites throughout the human body, with an emphasis on oropharyngeal lymphoid tissues. Tonsils harbored the highest frequencies of CXCR5+ CD8+ T cells compared to other tissues, many of which expressed Granzyme K and concurrently displayed tissue residency features, as demonstrated by single cell profiling. Irrespective of clonal identity and virus specificity, CD8+ T cells expressed CXCR5 more commonly in tonsils compared to vascular circulation. CXCR5 expression was particularly prominent among tonsil-localized CD8+ T cells targeting Epstein-Barr virus (EBV) latent antigens and associated with a PD-1+ resident stem-like phenotype. These data identify a CXCR5+ tissue-resident memory CD8+ T cell subset in human tonsils with a potential role in immune surveillance of EBV.

Killer cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands regulate the functions of natural killer (NK) cells. However, extensive sequence homology within the KIR family limits the ability of monoclonal antibodies to selectively recognise individual receptors, and no KIR2DS1-specific reagent is available to date. Here, we comprehensively delineate the binding profile of the monoclonal antibody S22019F. Using Ba/F3 cells, NK cell clones, and primary NK cells, we demonstrate that S22019F selectively binds KIR2DS1. Moreover, functional assays reveal that binding of S22019F to KIR2DS1 is preserved upon activation by its HLA-C ligand. We further confirm that S22019F does not cross-react to KIR2DL3*005 allotypes and we extend its application to the analysis of KIR2DS1+ T cells. Collectively, these findings underscore the value of S22019F as a reagent that selectively recognises KIR2DS1, enabling improved analyses of KIR2DS1-bearing lymphocytes in fundamental and clinical research.

BACKGROUND:Flavivirus infections pose a significant global health burden, highlighting the need for safe and effective vaccination strategies. Co-administration of different vaccines, including licensed flavivirus vaccines, is commonly practiced providing protection against multiple pathogens while also saving time and reducing visits to healthcare units. However, how co-administration of different flavivirus vaccines de facto affects immunogenicity, particularly with respect to T cell responses, is only partially understood. METHODS AND FINDINGS:Antigen-specific T cell responses were assessed in study participants enrolled in a previously conducted open-label, non-randomized clinical trial. In the trial, vaccines against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), or yellow fever virus (YFV) were administered either individually or concomitantly in different combinations in healthy study participants. Peripheral blood samples were collected before vaccination and at multiple time points afterward. To analyze antigen-specific CD4+ and CD8+ T cell responses, PBMCs were stimulated with overlapping peptide pools from TBEV, JEV, YFV, and Zika virus (ZIKV) envelope (E), capsid (C), and non-structural protein 5 (NS5) viral antigens. A flow cytometry-based activation-induced marker (AIM) assay was used to quantify antigen-specific T cell responses. The results revealed remarkably similar frequencies of CD4+ and CD8+ T cell responses, regardless of whether vaccines were administered individually or concomitantly. In addition, administering the vaccines in the same or different upper arms did not markedly affect T cell responses. Finally, limited cross-reactivity was observed between the TBEV, JEV, and YFV vaccines, and related ZIKV-specific antigens. CONCLUSIONS:TBEV or JEV vaccines can be co-administered with the live attenuated YFV vaccine without any markedly altered antigen-specific CD4+ and CD8+ T cell responses to the respective flaviviruses. Additionally, the vaccines can be delivered in the same or different upper arms without any significant altered influence on the T cell response. From a broader perspective, these results provide valuable insights into the outcome of immune responses following simultaneous administration of different vaccines for different but related pathogens.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that mediate rapid antimicrobial immune responses to antigens derived from microbial riboflavin pathway metabolites presented by the evolutionarily conserved MR1 molecules. MAIT cells represent a large pre-expanded T cell subset in humans and are involved in both protective immunity and inflammatory immunopathology. However, what controls the functional heterogeneity of human MAIT cell responses is still largely unclear. Here, combining functional and transcriptomic analyses, we investigate how MAIT cell response programs are influenced by the cytokine milieu at the time of antigen recognition. Activation by MR1-presented antigen together with IL-12 induces intermediate levels of IFNγ and TNF, as well as a regulatory profile with substantial IL-10 production and elevated expression of TIM-3, LAG-3, and PD-1. Activation by the combination of antigen and IL-12 induces a c-MAF-dependent program required for IL-10 production. The MAIT cell-derived IL-10 mediates both autocrine and paracrine immune regulation. In contrast, coactivation of MAIT cells with IL-18 induces IL-17, GM-CSF, IFNγ, and TNF, without IL-10. Notably, IL-18 dominantly counteracts IL-10 expression. The activation states biased toward IL-10 or IL-17 production are reversible and do not represent stable subsets. Finally, MR1-restricted TCR-mediated activation without cytokine coactivation drives primarily granzyme B cytolytic arming. Altogether, these findings demonstrate that human MAIT cells adapt their functional effector response during antigen recognition to cytokine cues in the microenvironment, and identify programs biased toward either regulatory c-MAF-dependent IL-10 expression, or an inflammatory IL-17 and GM-CSF profile.

Tissues and organs develop from single founder cells, which give rise to distinct cell lineages that contribute to regeneration and maintenance of homeostasis in the adult. Tracing the genealogical relationships between individual cells and their gene expression signatures is an important step towards understanding how these processes are regulated in human health and disease. Here, we present mt-SCITE, a computational method for inferring the evolutionary history of dividing cells based on mitochondrial mutations detected in single cells. We show that mt-SCITE outperforms existing methods in accurately estimating the correct mitochondrial mutation tree from simulated data. To validate our method, we applied mt-SCITE on in vitro expanded T-cells profiled with bulk ATAC sequencing, where the clonal relationships were determined independently from T cell receptor sequences. We used our method to reconstruct the division histories and transcriptional heterogeneity of clonally related CD8+ T cells from single-cell RNA sequencing data obtained from a healthy human donor. This allowed us to track the in vivo development of CD8+ T cell clones post vaccination and resolve subclonal lineage relationships without the need for genetic barcoding. Our analysis revealed that T cell clones can adopt predominantly memory-like or effector-like states, while some exhibit mixed identities. Taken together, our method represents a novel approach to study cell lineage development in humans. ### Competing Interest Statement The authors have declared no competing interest.

CD8 + T cells are classically defined by cytotoxic activity, but it has remained unclear whether cytotoxic programs are compartmentalized across tissues and memory subsets. Here, we established a human organ donor cohort and found that expression of conventional cytotoxic molecules—granulysin, perforin, and granzyme B—was most prominent among circulating memory CD8 + T cells and decreased progressively with tissue residency, inversely mirroring the expression of CD69 and CD103. Other cytotoxic molecules, including granzymes A, H, K, and M, were variably expressed across tissues, and memory CD8 + T cells targeting persistent viruses expressed multiple granzymes coordinately. In an in vitro tonsil system, transforming growth factor–β induced discordant regulation of cytotoxic molecules and CD103. Combined with interleukin-15, this circuitry modulated proliferation and the acquisition of redirected killing activity via perforin and granzyme B. Our findings suggest that human memory CD8 + T cell cytotoxicity is intricately regulated by environmental cues reflecting tissue location and antigen specificity.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.

Although human twin studies have revealed the combined contribution of heritable and environmental factors in shaping immune system variability in blood, the contribution of these factors to immune system variability in tissues remains unexplored. The human uterus undergoes constant regeneration and is exposed to distinct environmental factors. To assess uterine immune system variation, we performed a system-level analysis of endometrial and peripheral blood immune cells in monozygotic twins. Although most immune cell phenotypes in peripheral blood showed high genetic heritability, more variation was found in endometrial immune cells, indicating a stronger influence by environmental factors. Cytomegalovirus infection was identified to influence peripheral blood immune cell variability but had limited effect on endometrial immune cells. Instead, hormonal contraception shaped the local endometrial milieu and immune cell composition with minor influence on the systemic immune system. These results highlight that the magnitude of human immune system variation and factors influencing it can be tissue specific.

The human fetal immune system starts to develop in the first trimester and likely plays a crucial role in fetal development and maternal-fetal tolerance. Innate lymphoid cells (ILCs) are the earliest lymphoid cells to arise in the human fetus. ILCs consist of natural killer (NK) cells, ILC1s, ILC2s, and ILC3s that all share a common lymphoid origin. Here, we studied fetal ILC subsets, mainly NK cells and ILC3s and their potential progenitors, across human fetal tissues. Our results show that fetal ILC subsets have distinct distribution, developmental kinetics, and gene expression profiles across human fetal tissues. Furthermore, we identify CD34+RORγt+Eomes- and CD34+RORγt+Eomes+ cells in the fetal intestine, indicating that tissue-specific ILC progenitors exist already during fetal development.

This is the processed data underlying the paper "Clonally heritable gene expression imparts a layer of diversity within cell types" by Mold, Weissman, et al. Data has been gone through preprocessing steps, using the Python Notebooks found at https://github.com/MartyWeissman/ClonalOmics/tree/main/Data. Smaller files are provided in .csv (comma-separated-value) format and larger files such as expression matrices are provided in .loom format (using the AnnData package).

The functional diversity of natural killer (NK) cell repertoires stems from differentiation, homeostatic receptor-ligand interactions, and adaptive-like responses to viral infections. Here, we generated a single-cell transcriptional reference map of healthy human blood and tissue-derived NK cells, with temporal resolution and fate-specific expression of gene regulator networks defining NK cell differentiation. Using transfer learning, transcriptomes of tumor-infiltrating NK cells from seven solid tumor types (427 patients), combined from 39 datasets, were incorporated into the reference map and interrogated for tumor microenvironment (TME)-induced perturbations. We identified six functionally distinct NK cellular states in healthy and malignant tissues, two of which were commonly enriched for across tumor types: a dysfunctional ‘stressed’ CD56 bright state susceptible to TME-induced immunosuppression and a cytotoxic TME-resistant ‘effector’ CD56 dim state. The ratio of ‘stressed’ CD56 bright and ‘effector’ CD56 dim was predictive of patient outcome in malignant melanoma and osteosarcoma. This resource may inform the design of novel NK cell therapies and can be extended endlessly through transfer learning to interrogate new datasets from experimental perturbations or disease conditions.

Lung cancer is a leading cause of cancer-related death worldwide. Despite recent advances in tissue immunology, little is known about the spatial distribution of tissue-resident lymphocyte subsets in lung tumors. Using high-parameter flow cytometry, we identified an accumulation of tissue-resident lymphocytes including tissue-resident NK (trNK) cells and CD8+ tissue-resident memory T (TRM) cells toward the center of human non-small cell lung carcinomas (NSCLC). Chemokine receptor expression patterns indicated different modes of tumor-infiltration and/or residency between trNK cells and CD8+ TRM cells. In contrast to CD8+ TRM cells, trNK cells and ILCs generally expressed low levels of immune checkpoint receptors independent of location in the tumor. Additionally, granzyme expression in trNK cells and CD8+ TRM cells was highest in the tumor center, and intratumoral CD49a+CD16- NK cells were functional and responded stronger to target cell stimulation than their CD49a- counterparts, indicating functional relevance of trNK cells in lung tumors. In summary, the present spatial mapping of lymphocyte subsets in human NSCLC provides novel insights into the composition and functionality of tissue-resident immune cells, suggesting a role for trNK cells and CD8+ TRM cells in lung tumors and their potential relevance for future therapeutic approaches.

Background COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. Methods We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. Results We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. Conclusions This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

Long-read sequencing has dramatically increased our understanding of human genome variation. Here, we demonstrate that long-read technology can give new insights into the genomic architecture of individual cells. Clonally expanded CD8+ T-cells from a human donor were subjected to droplet-based multiple displacement amplification (dMDA) to generate long molecules with reduced bias. PacBio sequencing generated up to 40% genome coverage per single-cell, enabling detection of single nucleotide variants (SNVs), structural variants (SVs), and tandem repeats, also in regions inaccessible by short reads. 28 somatic SNVs were detected, including one case of mitochondrial heteroplasmy. 5473 high-confidence SVs/cell were discovered, a sixteen-fold increase compared to Illumina-based results from clonally related cells. Single-cell de novo assembly generated a genome size of up to 598 Mb and 1762 (12.8%) complete gene models. In summary, our work shows the promise of long-read sequencing toward characterization of the full spectrum of genetic variation in single cells.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy.