HLA-G/LILRBs: A Cancer Immunotherapy Challenge.

Despite some success, many patients do not benefit from immunotherapy. New strategies to improve clinical efficacy include identification of novel immune-checkpoint (IC) targets or a combination of immunotherapy with antiangiogenic treatments. Here, we propose the therapeutic use of IC, HLA-G/LILRB, and explore its enhanced synergistic antitumor activity when combined with antiangiogenic therapies.

Antagonistic anti-LILRB1 monoclonal antibody regulates antitumor functions of natural killer cells.

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 vitro and in vivo xenograft model using non-obese diabetic (NOD)-SCID interleukin-2Rγ-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 vitro and in 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.

Cytometry-based analysis of HLA-G functions according to ILT2 expression.

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8+ T cell cytotoxic functions. Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8+ T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition. This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2+ CD8+ T cell and ILT2+ NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation.

CD8+PD-1-ILT2+ T Cells Are an Intratumoral Cytotoxic Population Selectively Inhibited by the Immune-Checkpoint HLA-G.

Only some cancer patients respond to the immune-checkpoint inhibitors being used in the clinic, and other therapeutic targets are sought. Here, we investigated the HLA-G/ILT2 checkpoint in clear-cell renal-cell carcinoma (ccRCC) patients and focused on tumor-infiltrating CD8+ T lymphocytes (TIL) expressing the HLA-G receptor ILT2. Using transcriptomics and flow cytometry, we characterized both peripheral blood and tumor-infiltrating CD8+ILT2+ T cells from cancer patients as late-differentiated CD27-CD28-CD57+ cytotoxic effectors. We observed a clear dichotomy between CD8+ILT2+ and CD8+PD-1+ TIL subsets. These subsets, which were sometimes present at comparable frequencies in TIL populations, barely overlapped phenotypically and were distinguished by expression of exclusive sets of surface molecules that included checkpoint molecules and activating and inhibitory receptors. CD8+ILT2+ TILs displayed a more mature phenotype and higher expression of cytotoxic molecules. In ex vivo functional experiments with both peripheral blood T cells and TILs, CD8+ILT2+ T cells displayed significantly higher cytotoxicity and IFNγ production than their ILT2- (peripheral blood mononuclear cells, PBMC) and PD-1+ (TILs) counterparts. HLA-G expression by target cells specifically inhibited CD8+ILT2+ T-cell cytotoxicity, but not that of their CD8+ILT2- (PBMC) or CD8+PD-1+ (TIL) counterparts, an effect counteracted by blocking the HLA-G/ILT2 interaction. CD8+ILT2+ TILs may therefore constitute an untapped reservoir of fully differentiated cytotoxic T cells within the tumor microenvironment, independent of the PD1+ TILs targeted by immune therapies, and specifically inhibited by HLA-G. These results emphasize the potential of therapeutically targeting the HLA-G/ILT2 checkpoint in HLA-G+ tumors, either concomitantly with anti-PD-1/PD-L1 or in cases of nonresponsiveness to anti-PD-1/PD-L1.

LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia.

One of the cardinal features of chronic lymphocytic leukemia (CLL) is its association with a profound immunosuppression. NK cell function is markedly impaired in CLL patients, who show a significant dysregulation of the expression of activating and inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2, also termed LIR-1, LILRB1) in the regulation of NK cells in CLL. Our results show that ILT2 expression was significantly decreased on leukemic cells and increased on NK cells of CLL patients, particularly in those with advanced disease and with bad prognostic features, such as those carrying chromosome del(11q). The immunomodulatory drug lenalidomide may regulate the expression of ILT2 and its ligands in CLL since it significantly increased the expression of ILT2 and partially reestablished the expression of its ligands on leukemic cells. Furthermore, lenalidomide significantly increased the activation and proliferation of NK cells, which was strongly enhanced by ILT2 blockade. Combining ILT2 blockade and lenalidomide activated NK cell cytotoxicity resulting in increased elimination of leukemic cells from CLL patients. Overall, we describe herein the role of an inhibitory receptor involved in the suppression of NK cell activity in CLL, which is restored by ILT2 blockade in combination with lenalidomide, suggesting that it may be an interesting therapeutic strategy to be explored in this disease.