Natural killer (NK) cells are innate immune cells with the inherent ability to directly kill tumor and virus infected cells. Due to their ability to kill cancer cells without any prior priming, and their role in preventing metastasis NK cells have since long been the choice for autologous adoptive cell transfer therapy in cancer. However, poor expansion potential of PBMC derived NK cells in vitro is a major roadblock preventing the widespread use of NK cells in immunotherapy. We found that human NK cells isolated from lymph nodes (LNs) express higher levels of genes encoding for stem-like transcription factors (TCF7, LEF1, MYC) compared to NK cells from blood, spleen, bone marrow (BM) and lung. Therefore, we hypothesized that NK cells isolated from LNs will show superior expansion potential in vitro. Flow cytometric analysis shows that LN derived NK cells express high levels of TCF1 protein ex vivo, and show greater proliferation compared to NK cells isolated from blood, spleen and BM following stimulation with IL-2 and IL-15 in vitro. We also observed that a significant frequency of LN NK cells expressed TCF1 even after expansion, suggesting preserved proliferation potential of these cells. Additionally, the expanded NK cells from LN acquired properties of mature, highly functional NK cells such as increased expression of CD16, CD57 and higher Granzyme B expression. Lastly, LN derived NK cells also demonstrate enhanced cytolytic activity in vitro after expansion. Taken together our results suggest that in vitro expanded NK cells from LNs are potentially efficacious anti-tumor agents and could be leveraged for the development of future generation of NK cell directed immunotherapies.
The majority of T cells throughout the human body persist as non-circulating tissue-resident memory T cells (TRM) in lymphoid and mucosal sites, and are vital for orchestrating protective immune responses. However, given the difficulty of sampling healthy human tissues, a full understanding of TRM identity and function across tissue sites remains lacking. Here, we utilize a unique human tissue resource where tissues are obtained from organ donors to dissect CD4+ and CD8+ TRM heterogeneity using high-dimensional flow cytometry and single cell RNA-sequencing (scRNA-seq). We identify remarkable heterogeneity in expression of key surface markers that define TRM, namely CD103, CD101, CD49a, PD-1 and CXCR6, particularly in the lung and the intestines. These phenotypic groups of TRM denote different functional subsets after activation: CD103+ and CD101+ TRM preferentially produce IL-17A, CD49a+ and CXCR6+ TRM show enhanced IFNγ production, and PD-1+ TRM produce granzyme-B. To investigate the transcriptional programming underlying TRM, we profile ~20,000 resting and activated T cells from the lungs and intestines of human organ donors with scRNA-seq and identify TRM based on our previously defined tissue signature. In both tissues, we detect multiple clusters of TRM enriched for expression of individual or a combination of TRM marker genes. Importantly, these clusters show variable expression of genes coding for cytokines (IFNG, IL23A, IL26), transcription factors (TBX21, GATA3, BATF), and cytotoxic molecules (GNLY, GZMs), suggesting differentially regulated TRM subsets. Our results therefore highlight the heterogeneity of TRM across human tissue sites and define the transcriptional programming underlying TRM function.
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