Human lung tissue-resident NK cells (trNK cells) are likely to play an important role in host responses towards viral infections, inflammatory conditions and cancer. However, detailed insights into these cells are still largely lacking. Here we show, using RNA sequencing and flow cytometry-based analyses, that subsets of human lung CD69 + CD16 − NK cells display hallmarks of tissue-residency, including high expression of CD49a, CD103, and ZNF683, and reduced expression of SELL, S1PR5, and KLF2/3. CD49a + CD16 − NK cells are functionally competent, and produce IFN-γ, TNF, MIP-1β, and GM-CSF. After stimulation with IL-15, they upregulate perforin, granzyme B, and Ki67 to a similar degree as CD49a − CD16 − NK cells. Comparing datasets from trNK cells in human lung and bone marrow with tissue-resident memory CD8 + T cells identifies core genes co-regulated either by tissue-residency, cell-type or location. Together, our data indicate that human lung trNK cells have distinct features, likely regulating their function in barrier immunity.

29The concept of adaptive-like "memory" NK cells has been extensively investigated in 30 recent years. In humans, NK cells with adaptive-like features have been identified in 31 peripheral blood and liver of cytomegalovirus (CMV)-seropositive individuals. The 32 human lung is a major target organ for infections and is also a significant reservoir for 33 CMV. However, it remains unknown whether adaptive-like NK cells can be found in 34 this organ. Using RNAseq and flow cytometry analysis, we here identify a novel 35 adaptive-like KIR + NKG2C + NK cell subset with a CD49a + CD56 bright CD16tissue-36 resident (tr)NK cell phenotype in human lung and in peripheral blood. Adaptive-like 37 lung trNK cells were found to be present independently of adaptive-like CD56 dim CD16 + 38 NK cells, to be hyperresponsive towards target cells, and to exhibit signs of metabolic 39 reprogramming. In conclusion, adaptive-like trNK cells constitute a novel subset of 40 human lung NK cells, likely contributing to unique defense mechanisms in context of 41 infection at this site. 42 4 Results 99 Adaptive-like NK cells can be identified in human lung 100We first set out to investigate whether expansions of KIR + NKG2C + adaptive-101 like NK cells could be identified in the human lung. The majority of NK cells in the 102 lung are phenotypically similar to NK cells found in peripheral blood (CD69 -103 CD56 dim CD16 + ), suggesting that these cells may recirculate between this organ and 104 peripheral blood 20 . Accordingly, KIR + NKG2C + CD56 dim CD16 + NK cells could be 105 identified not only in peripheral blood but also in the lung (Fig. 1a). Surprisingly, KIR 106 and NKG2C were also co-expressed on CD56 bright CD16lung NK cells, with varying 107 frequencies between donors (Fig. 1b, c) (see Supplementary Fig. 1a for the gating 108 strategy to identify KIR + NKG2C + CD56 bright CD16and CD56 dim CD16 + NK cells). In 109 several donors the frequencies of KIR + NKG2C + CD56 bright CD16 -NK cells in human 110 lung vastly exceeded those previously described in the liver 7 , with up to 98% of 111 CD56 bright CD16lung NK cells co-expressing KIR and NKG2C (Fig. 1 a, b). 112Next, we assessed phenotypic features of KIR + NKG2C + CD56 bright CD16lung 113 NK cells in an unbiased manner. Uniform manifold approximation and expression 114 (UMAP) analysis revealed a distinct subset of cells with an expression pattern 115 consistent with adaptive-like NK cells found in peripheral blood and liver, including 116 low expression of Siglec-7 and CD161, and high expression of NKG2C, KIRs, and 117 CD2 7,8,23,24 (Fig. 1d). Unlike KIR + NKG2C + CD56 dim CD16 + NK cells, 118 KIR + NKG2C + CD56 bright CD16 -NK cells expressed lower levels of CD45RA and 119 NKp80, and higher levels of CD8 (Fig. 1d). In addition to these expression patterns, 120 manual analysis of individual samples additionally confirmed low expression of ILT2 121 and FcεR1g, as compared to KIR + NKG2C + CD56 dim CD16 + lung NK cells (Fig. 1e, f). 122Furthermore, KIR + NKG2C + CD56 bright CD16lu...

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The developing fetal and neonatal immune system is known to be exceedingly prone to tolerance induction following exposure to foreign antigens. In mice this has been partially attributed to the absence of a functional adaptive immune system prior to birth. By contrast, the adaptive immune system is highly developed in human beings as early as the second trimester of fetal gestation. Nonetheless, evidence for intrauterine tolerance to non-inherited maternal alloantigens has been observed in clinical studies suggesting that the human fetal immune system is also prone to tolerance induction. In these studies we provide the first evidence for a mechanism underlying the development of immunological tolerance in human beings involving the generation of fetal CD4+CD25+ regulatory T cells (TReg) in the fetal peripheral lymphoid tissues. Additionally we provide evidence that the tolerogenic properties of the fetal adaptive immune system are in part attributed to the presence of a unique population of fetal T cells distinct from those found after birth. Our findings suggest that fetal hematopoietic stem cells (HSC) give rise to phenotypically and functionally distinct T cells than those derived from adult HSC. These findings have significant implications for many different clinical settings including intrauterine infections, transplantation, and autoimmunity.

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