Genetic Investigation of MHC-Independent Missing-Self Recognition by Mouse NK Cells Using an In Vivo Bone Marrow Transplantation Model

Abstract

MHC-I–specific receptors play a vital role in NK cell–mediated “missing-self” recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b−/− bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2Db−/− MHC-I–deficient BM cells. Selective rejection of Clr-b−/− BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b−/− BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1Bhi NK cells, leaving the remaining NKR-P1Blo NK subset and MHC-I–dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b−/− hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I–deficient NK cells, Clr-b−/− NK cells were hyporesponsive to both NK1.1 (NKR-P1C)–stimulated and IL-12/18 cytokine–primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

The mouse NKR-P1B:Clr-b recognition system is a negative regulator of innate immune responses

“…17,27,50 Clr-b recognition by NKR-P1B provides an MHC-I-independent missing-self recognition mechanism. Although Clr-b-deficient splenocytes are acutely rejected in WT mice treated with TLR agonists, they are not rejected in NKR-P1B-deficient mice in vivo, providing a genetic proof-of-principle for MHC-independent missing-self recognition by NK cells (this study and Chen et al 41 ). This complementation resembles the inability of Ly49-deficient mice to reject MHC-I-deficient cells.…”

“…However, in our study, IFN-g production and degranulation in response to general activation stimuli appear to be normal in NKR-P1B-deficient NK cells. In contrast, NK cells from Clr-b-deficient mice, like full MHC-I-deficient mice, appear to be hyporesponsive to activating receptor cross-linking and cytokine stimulation, 41 despite a complementarity of deficiencies in either the Clr-b ligand or the NKR-P1B receptor. We have encountered a similar situation in Ly49-deficient mice, in which NK cell responses to various general activating stimuli were preserved.…”

“…on May 12, 2018. by guest www.bloodjournal.org From Rejection of Clr-b 2/2 splenocytes by WT mice is mediated by NK cells, because NK cell depletion abrogated this response ( Figure 5B). 41 We next performed in vivo rejection assays using MHC-I-deficient cells. Strikingly, MHC-I-deficient (B2m 2/2 ) splenocytes were more efficiently rejected by NKR-P1B-deficient mice compared with WT mice ( Figure 5C, left).…”

“…32 Similarly, Clr-bdeficient mice are unable to reject Clr-b-deficient splenocytes due to self-tolerance mechanisms influencing NKR-P1B:Clr-b-dependent NK cell education. 41 Note that MHC-dependent NK cell education and missing-self responses are intact in NKR-P1B-deficient mice, as demonstrated by enhanced rejection of MHC-I-deficient cells. Thus, NKR-P1B:Clr-b-dependent missing-self recognition appears to function independently and in parallel with missing-self recognition of MHC-I ligands by Ly49 receptors.…”

Expansion and Protection by a Virus-Specific NK Cell Subset Lacking Expression of the Inhibitory NKR-P1B Receptor during Murine Cytomegalovirus Infection

“…This could also explain why a genetic deficiency of NKR-P1B confers only a modest protective advantage against MCMV infection. In contrast, Clr-b-deficient mice have normal NKR-P1B expression and function (38,68), yet they are slightly more susceptible to MCMV infection than WT and NKR-P1B-deficient mice. Clr-b-deficient NK cells have been shown to be functionally hyporesponsive (68), which may contribute to their higher susceptibility to MCMV infection.…”