*Purpose: Unconventional T-cells (e.g. Tregs and γδT-cells) are increasingly recognized for their roles in maintaining homeostasis and protecting non-lymphoid tissues against injury/stress. Double negative (DN) αβT-cells represent a significant component of T-cells in human and mouse kidneys, actively dividing in steady state and involved in protecting against kidney ischemia reperfusion injury (IRI). However, little is known about the regulation of kidney DN T-cells.

*Methods: C57BL/6J (WT), B6.129P2-B2mtm1Unc/J (β2m KO), B6.129S2-H2dlAb1-Ea/J (MHC II KO), B6.129P2-H2-K1tm1Bpe H2-D1tm1Bpe/DcrJ (KD KO) and Nu/J mice were used for this study, n=3-7/group. Human kidney samples were from the normal section of nephrectomies for renal cancers. Kidney lymphocytes were isolated and analyzed by flow cytometry.

*Results: Our data demonstrates that kidney DN T-cells are thymus-derived as lack of thymus significantly reduced the absolute count of DN T-cells in kidney (WT, 2.2×10 vs. Nu/J, 0.24×10 , P<0.05) and also comprised of two (PD-1 and NK1.1 ) subsets. Deficiency of β2m-dependent MHC class I, but not MHC class II molecules led to significant reduction in frequency, absolute numbers of kidney DN T-cells due to impaired activation (>60%, P<0.001), proliferation (>40%, P<0.05), increased apoptosis, and loss of a NK1.1 subset. Development of the NK1.1 subset is restricted by β2m-dependent nonclassical MHC molecules as the frequency of NK1.1 DN T-cells was significantly reduced in β2m KO (10.3±2) but not in KD KO or WT mice (23.1±3, P<0.001). In contrast, homeostasis but not development of the PD-1 subset is regulated by β2m-dependent non-classical MHC molecules as the frequency of PD-1 DN T-cells was significantly higher in β2m KO (70.2±5) then WT mice (44.2±3 P<0.001). DN T-cells retained in kidneys of β2m KO mice are mostly PD-1 and remain functionally responsive to external stimuli as indicated by their rapid activation and expansion in response to kidney IRI. Both normal human kidney and cancer samples express the PD-1 (% normal, 39.5±8 vs. tumor, 47.9±7.8) and NK1.1 subsets (% normal, 26.2±8.4 vs. tumor, 23.0±8.3).

*Conclusions: These findings demonstrate that kidney DN T-cells, particularly PD-1+ DN T-cells, are early responders of the renal immune surveillance system utilizing recognition/activation/ regulation mechanisms that differ from conventional T-cells.

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