Abstract: Natural killer (NK) cells are innate immune cells with the inherent ability to kill tumor and virus infected cells without any prior priming. Recent studies have demonstrated the ability of NK cells to develop adaptive memory and tissue residence. However, our understanding of human NK cells in tissues and their site-specific properties is limited. Here we use our unique tissue resource established in collaboration with LiveOnNY to elucidate the diversity of human NK cell phenotypic subsets, functional profile and transcriptional signatures in non-mucosal and mucosal tissue sites. We identified tissue-specific patterns of NK cell maturation and show that NK cell subset (immature and mature) and memory NK cell distribution is a function of tissue site which is not affected by age. Our results also show that while NK cells from different tissue sites express similar levels of granzyme B, NK cell antibody-dependent cellular cytotoxicity response and cytokine driven activation may be influenced by tissue site. Whole transcriptome profiling of NK cell subsets revealed that mature NK cells from all the sites (blood, bone marrow, spleen, lung and LN) have very similar transcriptional profiles. Interestingly, immature NK cells from all tissue sites have very different transcriptional profiles compared to their blood counterpart and are enriched for tissue resident memory T cell transcriptional signature. Furthermore, we show that immature NK cells show site-specific expression patterns of tissue residence surface markers. Overall, our data demonstrates the effects of tissue microenvironment on the developmental and function potential of tissue NK cells and provides insight into the development of future NK cell mediated immunotherapies.

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.

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 αβ 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.