Tumor-associated macrophages impair NK cell IFN-γ production and contribute to tumor progression in clear cell renal cell carcinoma.

Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.

The MICA-NKG2D axis in clear cell renal cell carcinoma bolsters MICA as target in immuno-oncology.

NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.

Circulating and Tumor-Infiltrating NK Cells From Clear Cell Renal Cell Carcinoma Patients Exhibit a Predominantly Inhibitory Phenotype Characterized by Overexpression of CD85j, CD45, CD48 and PD-1.

Although natural killer (NK) cells infiltrate clear cell renal cell carcinomas (ccRCC), the most frequent malignancy of the kidney, tumor progression suggests that they become dysfunctional. As ccRCC-driven subversion of NK cell effector functions is usually accompanied by phenotypic changes, analysis of such alterations might lead to the identification of novel biomarkers and/or targets in immuno-oncology. Consequently, we performed a phenotypic analysis of peripheral blood NK cells (PBNK) and tumor-infiltrating NK cells (TINK) from ccRCC patients. Compared to HD, PBNK from ccRCC patients exhibited features of activated cells as shown by CD25, CD69 and CD62L expression. They also displayed increased expression of DNAM-1, CD48, CD45, MHC-I, reduced expression of NKG2D, and higher frequencies of CD85j+ and PD-1+ cells. In addition, compared to PBNK from ccRCC patients, TINK exhibited higher expression of activation markers, tissue residency features and decreased expression of the activating receptors DNAM-1, NKp30, NKp46, NKp80 and CD16, suggesting a more inhibitory phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that CD48, CD45, CD85j and PD-1 are significantly overexpressed in ccRCC and that their expression is associated with an NK cell infiltration signature. Calculation of z-scores revealed that their expression on PBNK, alone or combined, distinguished ccRCC patients from HD. Therefore, these molecules emerge as novel potential biomarkers and our results suggest that they might constitute possible targets for immunotherapy in ccRCC patients.

Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein.

BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells.

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation.

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca2+ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca 2+ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca 2+ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca 2+ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca 2+ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca 2+ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH 4 Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca 2+ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.