HLA-G/LILRBs: A Cancer Immunotherapy Challenge

Abstract

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. 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. Throughout tumor progression, several mechanisms to escape immune destruction are induced. Nascent transformed cells lose their antigenicity and/or immunogenicity. They then acquire the expression of inhibitory molecules regulating T cell activation, termed ICs, and secrete suppressive cytokines, which promote an immunosuppressive microenvironment. Overall, these mechanisms lead to tumor escape via T cell exhaustion and suppressed cell induction. A breakthrough in cancer therapy to control tumor progression and to re-establish immune function has been the development of immunotherapies, mainly based on antibodies directed at ICs or their ligands. Administration of these antibodies is clinically successful in restoring T cell co-stimulatory signals and reactivating antitumor T cell responses [1.Wei S.C. et al.Fundamental mechanisms of immune checkpoint blockade therapy.Cancer Discov. 2018; 8: 1069-1086Crossref PubMed Scopus (953) Google Scholar]. Despite the success of these therapies, a considerable proportion of patients remains unresponsive. To overcome this challenge, it is necessary to identify new IC targets or synergistic combinations that might restore an immune-supportive microenvironment, reactivate antitumor immunity, and improve the success of cancer immunotherapy. HLA-G, a nonclassical MHC class I molecule that plays a crucial role in fetal–maternal tolerance, is an IC molecule [2.Carosella E.D. et al.HLA-G: an immune checkpoint molecule.Adv. Immunol. 2015; 127: 33-144Crossref PubMed Scopus (208) Google Scholar]. HLA-G via interaction of the LILRB1 (ILT2) and LILRB2 (ILT4) receptors (Figure 1 and Table 1) inhibits cytotoxic T cells, natural killer (NK) cells, and B cells, induces T cell anergy, modulates myeloid cells, and promotes T regulatory cells (Tregs). Moreover, HLA-G expressed on antigen-presenting cells (APCs), such as myeloid-derived suppressor cells (MDSCs) or tolerogenic dendritic cells (DCs), promotes T cell hyporesponsiveness and induces Treg differentiation [2.Carosella E.D. et al.HLA-G: an immune checkpoint molecule.Adv. Immunol. 2015; 127: 33-144Crossref PubMed Scopus (208) Google Scholar]. LILRB1 is expressed on different leukemia and solid tumors. While LILRB1 prevents primary cutaneous T cell lymphoma cell death and enhances gastric tumor growth, HLA-G/LILRB1 interaction inhibits neoplastic B cell proliferation. Blocking LILRB1 on myeloma or lymphoblastic cells does not prevent NK-mediated lysis [3.Villa-Alvarez M. et al.Ig-like transcript 2 (ILT2) blockade and lenalidomide restore NK cell function in chronic lymphocytic leukemia.Front. Immunol. 2018; 9: 2917Crossref PubMed Scopus (12) Google Scholar]. LILRB2 is expressed in various solid tumors such as clear cell renal carcinoma (ccRCC) and stroma cells within the tumor microenvironment [4.Li Q. et al.Overexpressed immunoglobulin-like transcript (ILT) 4 in lung adenocarcinoma is correlated with immunosuppressive T cell subset infiltration and poor patient outcomes.Biomark. Res. 2020; 8: 11Crossref PubMed Scopus (4) Google Scholar]. LILRB2 expression correlates with poor cell differentiation and advanced metastasis. One underlying mechanism is the upregulation of vascular endothelial growth factor (VEGF)-C [5.Garcia M. et al.The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma.BMC Cancer. 2020; 20: 624Crossref PubMed Scopus (7) Google Scholar], a well-characterized and efficient growth factor involved in lymphangiogenesis and lymphatic metastases.Table 1HLA-G-Mediating Activities Following Interaction with Its Inhibitory Receptors LILRB1/LILRB2Cell TypeLILRBsEffectSolid tumor cellsLILRB1/LILRB2ProliferationCell migration, thus cancer disseminationSecretion of VEGF by endothelial cells, favoring cancer disseminationEndothelial cellsLILRB2Proliferation, leading to neoangiogenesisPromote VEGF secretion and consequently vascular permeabilityT cellsLILRB1Inhibition of proliferation and cytotoxicityInduction of TregsPrevent migrationNK cellsLILRB1Inhibition of interferon-γ production and cytotoxicityPrevent migrationMacrophagesLILRB1/LILRB2Polarization towards M2 phenotypeNeutrophilsLILRB2Inhibition of reactive oxygen speciesReduced phagocytosisMDSCsLILRB1/LILRB2ExpansionInduction of TregsDCsLILRB1/LILRB2Conversion into tolerogenic cellsPrevention of maturationModulation of T cell responsesInduction of TregsDC-10sLILRB2Production of IL-10Inhibition of T cell responsesInduction of Tr1 cells Open table in a new tab In immunocompetent mice, HLA-G-expressing tumor cells proliferate, while in immunodeficient mice, administration of HLA-G-expressing tumor cells results in widespread metastasis, suggesting that HLA-G plays a role in immune escape in tumors. Moreover, de novo expression of HLA-G is found in most human tumors analyzed, but not in surrounding healthy tissues [2.Carosella E.D. et al.HLA-G: an immune checkpoint molecule.Adv. Immunol. 2015; 127: 33-144Crossref PubMed Scopus (208) Google Scholar]. These findings are consistent with the involvement of the checkpoint HLA-G in tumor progression and invasiveness. Moreover, HLA-G/LILRBs affect a wider spectrum of immune cells compared to those modulated by cytotoxic T lymphocyte-associated antigen (CTLA)-4/B7 and programmed death (PD)-1/PD ligand-1; known targets of current IC blockade therapies. Therefore, we propose that the checkpoint HLA-G/LILRB should be explored as an additional target for cancer immunotherapy. Tregs, NK cells, tolerogenic DCs, macrophages, and MDSCs are consistently found within tumors, where they co-operate in promoting tumor escape. HLA-G/LILRB pathways modulate these cell types by inhibiting DC maturation and differentiation, promoting macrophage differentiation into M2-like macrophages, and allowing MDSC expansion. There is also an increased frequency of HLA-G-expressing DC-10s [6.Gregori S. et al.Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway.Blood. 2010; 116: 935-944Crossref PubMed Scopus (372) Google Scholar] in peripheral blood of patients with high grade gastric cancer [7.Xu D.P. et al.Elevation of HLA-G-expressing DC-10 cells in patients with gastric cancer.Hum. Immunol. 2016; 77: 800-804Crossref PubMed Scopus (9) Google Scholar]. Since HLA-G expression in DC-10s is induced by interleukin (IL)-6 it can be assumed that the presence of IL-10 in the tumor microenvironment might promote the differentiation of T regulatory type 1 (Tr1) cells, an inducible subset of IL-10-producing Tregs characterized by coexpression of CD49b and lymphocyte-activation gene 3 protein (LAG-3) [8.Roncarolo M.G. et al.The biology of T regulatory type 1 cells and their therapeutic application in immune-mediated diseases.Immunity. 2018; 49: 1004-1019Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar], which have been frequently associated with tumors [7.Xu D.P. et al.Elevation of HLA-G-expressing DC-10 cells in patients with gastric cancer.Hum. Immunol. 2016; 77: 800-804Crossref PubMed Scopus (9) Google Scholar], indicating that these cells may be involved in tumor escape. During tumor development, antigen-specific T cells become dysfunctional under prolonged antigen stimulation, which leads to the induction of exhausted T cells. T cell exhaustion is characterized by poor effector functions and expression of PD-1, Tim-3, LAG-3, CTLA-4, and TIGIT [9.Blank C.U. et al.Defining ‘T cell exhaustion.Nat. Rev. Immunol. 2019; 19: 665-674Crossref PubMed Scopus (212) Google Scholar]. Targeting CTLA-4 or PD-1 on T cells can be effective in several cancer settings. However, cancer-infiltrating T cells can become refractory to checkpoint-blockade-mediated reactivation. Recently, a population of tumor-infiltrating CD8+ T cells expressing LILRB1, distinct from CD8+PD1+ T cells, was characterized in ccRCC patients [10.Dumont C. et al.CD8(+)PD-1(-)ILT2(+) T cells are an intratumoral cytotoxic population selectively inhibited by the immune-checkpoint HLA-G.Cancer Immunol. Res. 2019; 7: 1619-1632Crossref PubMed Scopus (21) Google Scholar]. The CD8+LILRB1+-mediated cytotoxicity is prevented by HLA-G expression and neutralized by anti-HLA-G antibodies, supporting the notion that targeting HLA-G can rejuvenate cancer-infiltrating exhausted T cells. Two HLA-G-targeting approaches are under development for cancer immunotherapy: generation of anti-HLA-G neutralizing antibodies and chimeric antigen receptor (CAR) T cells specific for HLA-G. Better insight into the transcriptional regulation of HLA-G gene and the availability of appropriate anti-HLA-G antibodies is needed to implement these approaches. Alternative splicing of the primary HLA-G transcript might generate various isoforms, which lack the α1, α2, or α3 domains [11.Tronik-Le Roux D. et al.Novel landscape of HLA-G isoforms expressed in clear cell renal cell carcinoma patients.Mol. Oncol. 2017; 11: 1561-1578Crossref PubMed Scopus (33) Google Scholar]. Whether these isoforms are differentially regulated or have different functions is not yet known. Currently available antibodies recognize only the α1 domain of the protein, preventing the detection of HLA-G expression in some cancer patients. Therefore, producing HLA-G antibodies that specifically recognize other domains, in particular an antibody directed to the HLA-G α3 domain, will be essential for developing future therapeutic approaches. The α3 domain is found in most HLA-G isoforms expressed in tumors and contains the epitope recognized by LILRB1 and LILRB2. Therefore, an α3 domain directed antibody will specifically block the interaction of HLA-G with LILRB1 and LILRB2 simultaneously. The use of such an antibody will also have a great advantage over using antibodies directed exclusively against one of the known HLA-G receptors LILRB1 or LILRB2, independently. Since LILRB1 and LILRB2 have different roles in immunity and are found in most immune cells, blocking these receptors might eliminate the complete immune response. In addition, HLA-G might act through other yet undescribed receptors besides LILRB1 or LILRB2, therefore, blocking LILRB1 or LILRB2 will not prevent the action of HLA-G that signals through this receptor. Tumor vasculature and lymphatic endothelial cells have important roles in immune cell trafficking and constitute key elements that influence tumor behavior and treatment responses. When tumors cannot be sufficiently oxygenated, hypoxia occurs and triggers the upregulation of several genes, including VEGF and a variety of ICs, overall driving a shift towards an immunosuppressive environment. The persistent hypersecretion of proangiogenic factors within the tumor leads to the disorganization of nascent vessels, preventing T cell infiltration. Consequently, the tumor vasculature, mainly targeted by antiangiogenic drugs, has been considered a strategic challenge for antitumor treatment [12.Liu Z. et al.Tumor vasculatures: a new target for cancer immunotherapy.Trends Pharmacol. Sci. 2019; 40: 613-623Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. Although this approach would promote tumor vessel normalization, favor T cell infiltration, and enhance drug delivery to the tumor, antiangiogenic drugs offer only a modest survival benefit, suggesting that multiple nonredundant immunosuppressive mechanisms coexist within tumors. The relationship of HLA-G and vascular remodeling has been shown in decidua, where embryo-derived HLA-G mediates tolerance by interacting with LILRB1 on maternal decidual (d)NK cells and promotes VEGF expression [13.Gregori S. et al.HLA-G orchestrates the early interaction of human trophoblasts with the maternal niche.Front. Immunol. 2015; 6: 128Crossref PubMed Scopus (44) Google Scholar]. In the tumor, NK cells might promote tumor angiogenesis with a similar mechanism: transforming-growth-factor-β-mediated activation of tumor cells promotes VEGF secretion by NK cells supporting neoangiogenesis. The rationale underpinning the use of IC immunotherapy to enhance the efficacy of cancer antiangiogenic treatments comes from several studies. HLA-G/LILRB2 transcriptionally modifies the expression of VEGF-A/C in renal carcinoma cells derived from a highly vascularized tumor with immunogenic properties [5.Garcia M. et al.The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma.BMC Cancer. 2020; 20: 624Crossref PubMed Scopus (7) Google Scholar]. LILRB2 also induces the upregulation of VEGF-C in non-small cell lung cancer, promoting tumor progression [14.Gao A. et al.ILT4 functions as a potential checkpoint molecule for tumor immunotherapy.Biochim. Biophys. Acta Rev. Cancer. 2018; 1869: 278-285Crossref PubMed Scopus (16) Google Scholar]. In addition, pharmacological inhibition of VEGF-A leads to increased intratumoral CD8+ T cells and decreased Treg recruitment, resulting in reduced tumor growth. Moreover, VEGF promotes the upregulation of PD-1 on T cells leading to T cell exhaustion [15.Rahma O.E. Hodi F.S. The intersection between tumor angiogenesis and immune suppression.Clin. Cancer Res. 2019; 25: 5449-5457Crossref PubMed Scopus (73) Google Scholar]. Further studies that identify the molecular mechanisms underlying this two-way regulation would be valuable to understand the biology of the tumor and develop innovative therapeutic approaches. Immunotherapy protocols based on HLA-G blockade with novel HLA-G antibodies, alone or in combination with antiangiogenesis protocols might constitute a promising strategy for breaking down tolerance in tumors and promote rejuvenation of exhausted tumor-infiltrating CD8+ T cells. In addition, minor adverse effects could be produced by targeting HLA-G, since its expression under physiological conditions is highly restricted. In the future, an extended understanding of the crosstalk between tumor cells, endothelial cells, and immune cells could improve the effectiveness of protocols that may relieve endothelial anergy and enhance the abundance of activated T cells in tumors, ultimately improving outcomes for cancer patients. The authors declare no potential conflicts of interest.

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