Anti-LILRB1 monoclonal antibody, clone 4F9

Is the natural killer (NK) cell receptor repertoire of endometrial NK (eNK) cells tissue-specific? The NK cell receptor (NKR) expression profile in pre-pregnancy endometrium appears to have a unique tissue-specific phenotype, different from that found in NK cells in peripheral blood, suggesting that these cells are finely tuned towards the reception of an allogeneic fetus. NK cells are important for successful pregnancy. After implantation, NK cells encounter extravillous trophoblast cells and regulate trophoblast invasion. NK cell activity is amongst others regulated by C-type lectin heterodimer (CD94/NKG2) and killer cell immunoglobulin-like (KIR) receptors. KIR expression on decidual NK cells is affected by the presence of maternal HLA-C and biased towards KIR2D expression. However, little is known about NKR expression on eNK cells prior to pregnancy. In this study, matched peripheral and menstrual blood (a source of endometrial cells) was obtained from 25 healthy females with regular menstrual cycles. Menstrual blood was collected during the first 36 h of menstruation using a menstrual cup, a non-invasive technique to obtain endometrial cells. KIR and NKG2 receptor expression on eNK cells was characterized by 10-color flow cytometry, and compared to matched pbNK cells of the same female. KIR and HLA-C genotypes were determined by PCR-SSOP techniques. Anti-CMV IgG antibodies in plasma were measured by chemiluminescence immunoassay. KIR expression patterns of eNK cells collected from the same female do not differ over consecutive menstrual cycles. The percentage of NK cells expressing KIR2DL2/L3/S2, KIR2DL3, KIR2DL1, LILRB1 and/or NKG2A was significantly higher in eNK cells compared to pbNK cells, while no significant difference was observed for NKG2C, KIR2DL1/S1, and KIR3DL1. The NKR repertoire of eNK cells was clearly different from pbNK cells, with eNK cells co-expressing more than three NKR simultaneously. In addition, outlier analysis revealed 8 and 15 NKR subpopulation expansions in eNK and pbNK cells, respectively. In contrast to the pbNK cell population, the expansions present in the eNK cell population were independent of CMV status and HLA-C genotype. Moreover, the typical NKG2C imprint induced by CMV infection on pbNK cells was not observed on eNK cells from the same female, suggesting a rapid local turnover of eNK cells and/or a distinct licensing process. Based on our previous work and the parameters studied here, menstrual blood-derived eNK cells closely resemble biopsy-derived eNK cells. However, sampling is not done at the exact same time during the menstrual cycle, and therefore we cannot exclude some, as yet undetected, differences. Our data reveals that NK cells in the pre-implantation endometrium appear to have a dedicated tissue-specific phenotype, different from NK cells in peripheral blood. This may indicate that eNK cells are finely tuned to receive an allogeneic fetus. Studying the endometrial NKR repertoire of women with pregnancy related problems could provide clues to understand the pathogenesis of pregnancy complications.

Background Current immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, checkpoint blockade monotherapies have not been successful against most hematological malignancies including multiple myeloma and leukemia. There is an urgent need to identify new targets for development of cancer immunotherapy. LILRB1, an immunoreceptor tyrosine-based inhibitory motif-containing receptor, is widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells and subsets of natural killer (NK) cells and T cells. The ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules, activate LILRB1 and transduce a suppressive signal, which inhibits the immune responses. However, it is not clear whether LILRB1 blockade can be effectively used for cancer treatment. Methods First, we measured the LILRB1 expression on NK cells from cancer patients to determine whether LILRB1 upregulated on NK cells from patients with cancer, compared with NK cells from healthy donors. Then, we developed specific antagonistic anti-LILRB1 monoclonal antibodies and studied the effects of LILRB1 blockade on the antitumor immune function of NK cells, especially in multiple myeloma models,in vitroandin vivoxenograft model using non-obese diabetic (NOD)-SCID interleukin-2R gamma-null mice. Results We demonstrate that percentage of LILRB1(+)NK cells is significantly higher in patients with persistent multiple myeloma after treatment than that in healthy donors. Further, the percentage of LILRB1(+)NK cells is also significantly higher in patients with late-stage prostate cancer than that in healthy donors. Significantly, we showed that LILRB1 blockade by our antagonistic LILRB1 antibody increased the tumoricidal activity of NK cells against several types of cancer cells, including multiple myeloma, leukemia, lymphoma and solid tumors,in vitroandin vivo. Conclusions Our results indicate that blocking LILRB1 signaling on immune effector cells such as NK cells may represent a novel strategy for the development of anticancer immunotherapy.