NKRP1 receptors were discovered more than 20 years ago, but due to a lack of appropriate reagents, our understanding of them has remained limited. Using a novel panel of mAbs that specifically recognize mouse NKRP1A, D, and F molecules, we report here that NKRP1D expression is limited to a subpopulation of NK cells, but in contrast to Ly49 receptors appears to be expressed in a normal codominant manner. NKRP1D− and NKRP1D+ NK cells are functionally distinct, NKRP1D+ cells showing reduced expression of various Ly49 receptors, elevated expression of CD94/NKG2 receptors, and higher IFN-γ secretion and cytotoxicity than NKRP1D− cells. Furthermore, NKRP1D+ NK cells were unable to kill transfected cells expressing high levels of Clr-b molecules, but readily killed MHC class-I-deficient blast cells that express only low levels of Clr-b. NKRP1A and NKRP1F were expressed at low levels on all splenic and bone marrow NK cells, but mAb-induced cross-linking of NKRP1A and NKRP1F caused no significant enhancement or inhibition of NK cell cytotoxicity and no detectable production of IFN-γ. NKRP1A, D, and F expression could not be detected on NKT cells, all of which express NKRP1C, and although some activated T cells expressed NKRP1C and perhaps low levels of NKRP1A, no significant expression of NKRP1D or F could be detected. NKRP1 molecules expressed on NK cells or transfectants were down-regulated by cross-linking with mAbs or cell surface ligands, and using this phenomenon as a functional assay for NKRP1-ligand interaction revealed that NKRP1F can recognize CLR-x.

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3+ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3+ and NKR-P1B+ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D− and NKR-P1D+ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.

Intravenous injection of rabbit anti-asialo-GM1 serum, an antiserum previouslY shown to eliminate splenic natural killer (NK) activity in vitro, profoundly depressed NK activity in CBA, DBA/2 and BALB/c nu/nu mice. The effect on NK activity was selective, as treatment of mice with anti-asialo-GM1 serum did not affect the development of other cytotoxic cells including cytotoxic macrophages following injection of poly I:C, or cytotoxic T cells in response to allogeneic cells. The role of NK cells in controlling tumor cell growth was investigated using an NK-sensitive (cl 27v-1C2) and an NK-resistant (cl 27av) subline of the murine lymphoma L5178Y. Initial studies showed that cl 27v-1C2 cells were at least 100 times less tumorigenic than were cl 27av cells in both syngeneic DBA/2 mice and BALB/c nu/nu mice. In addition, treatment of DBA/2 mice with poly I:C, which boosted NK activity, markedly depressed the growth of cl 27v-1C2 cells, but not of cl 27av cells. On the other hand, treatment of DBA/2 mice and BALB/c nu/nu mice with anti-asialo-GM1 serum led to a marked increase in tumorigenicity of cl 27v 1C2 cells, but had no effect on the tumorigenicity of cl 27av cells. In addition, the protection against cl 27v-1C2 growth afforded by poly-I:C treatment was abrogated by injection oif anti-asialo-GM1 serum. The possibility that the effects observed were caused by binding of the injected antibodies to the tumor cells was minimized by: (1) using a clone of tumor cells (cl 27v-1C2) that lacks chemically detectable asialo-GM1, and (2) pretreating animals with anti-asialo-GM1 rather than administering antiserum and tumor cells concurrently. These studies provided compelling evidence that NK cells could play an active role in controlling tumor growth. Selective depletion of NK activity by injection of anti-asialo-GM1 serum is a method which would be generally applicable to studying the role of NK cells in disease processes.

Key Points NKR-P1B is involved in NK cell tolerance and MHC-I-independent missing-self recognition of Clr-b-deficient target cells. The NKR-P1B:Clr-b system plays a role in tumor surveillance and immune escape in the Eμ-myc transgenic mouse model of B-cell lymphoma.

Natural killer (NK) cells arise from immature progenitors present in fetal tissues and adult bone marrow, but the factors responsible for driving the proliferation and differentiation of these progenitors are poorly understood. Mouse NK cells had previously been thought not to express interleukin (IL)-2Ralpha chains, but we show here that immature and mature mouse NK cells express IL-2Ralpha chain mRNA and that low levels of IL-2Ralpha chains can be detected on the surface of immature and mature NK cells provided they are cultured in the absence of IL-2. Despite their potential expression of high-affinity IL-2 receptors, immature NK cells only proliferate if IL-2 is present at extremely high concentrations. Surprisingly, IL-15 can also only support the growth of immature NK cells at high, presumably nonphysiological concentrations. Although NK cells express mRNA for the high-affinity IL-15Ralpha chain, they also express a variety of alternately spliced transcripts whose protein products could potentially disrupt signaling through IL-15 receptors. The requirement for high concentrations of IL-2 and IL-15 suggests that if these cytokines play any role in the proliferative expansion of NK cells in vivo, they act indirectly via other cells or in cooperation with other factors. In support of the latter possibility, we report that the recently described cytokine IL-21 can markedly enhance the proliferation of immature (and mature) NK cells in the presence of doses of IL-2 and IL-15 that by themselves have little growth-promoting activity.