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The expression of classical human leukocyte antigen class I antigens (HLA‐I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA‐I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA‐I and non–classical HLA‐I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA‐I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune‐related genes to characterize PDAC tumor microenvironments. Lower expression of HLA‐I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA‐E and HLA‐G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA‐I antigens was significantly correlated with shorter survival. Higher HLA‐I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD‐L1 and PD‐L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA‐I in three PDAC cell lines. It also upregulated surface expression of HLA‐E, HLA‐G and immune checkpoint molecules, including PD‐L1 and PD‐L2. These results suggest that the higher expression of HLA‐I, HLA‐E and HLA‐G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA‐I expression on PDAC cells. human leukocyte antigen class I antigens (HLA‐I) are needed for T cells to recognize target cells. Here, we showed that higher HLA‐I expression on pancreatic cancer cells is associated with poor prognosis, where formation of the tolerant microenvironment may be involved in IFNγ.

The implementation of immune checkpoint inhibitors to the oncology clinic signified a new era in cancer treatment. After the first indication of melanoma, an increasing list of additional cancer types are now treated with immune system targeting antibodies to PD-1, PD-L1 and CTLA-4, alleviating inhibition signals on T cells. Recently, we published proof-of-concept results on a novel checkpoint inhibitor, NKG2A. This receptor is expressed on cytotoxic lymphocytes, including NK cells and subsets of activated CD8+ T cells. Blocking antibodies to NKG2A unleashed the reactivity of these effector cells resulting in tumor control in multiple mouse models and an early clinical trial. Monalizumab is inhibiting this checkpoint in human beings and future clinical trials will have to reveal its potency in combination with other cancer treatment options.

The major challenge for universal chimeric antigen receptor T cell (UCAR-T) therapy is the inability to persist for a long time in patients leading to inferior efficacy clinically. The objective of this study was to design a novel UCAR-T cell that could avoid the occurrence of allo-rejection and provide effective resistance to allogeneic Natural Killer (NK) cell rejection, together with the validation of its safety and efficacy and . We prepared T-cell receptor (TCR), Human leukocyte antigen (HLA)-I/II triple-edited (TUCAR-T) cells and evaluated the anti-tumor efficacy ex vivo and in vivo. We measured the resistance of exogenous HLA-E expressing TUCAR-T (ETUCAR-T) to NK rejection by using an enhanced NK. Furthermore, we established the safety and efficacy of this regimen by treating Nalm6 tumor-bearing mice with a repeated high-dose infusion of ETUCAR-T. Moreover, we analyzed the effects of individual gene deficiency CAR-T on treated mice and the changes in the transcriptional profiles of different gene-edited T cells RNA-Seq. Data showed that HLA-II editing didn't impair the anti-tumor efficacy of TUCAR-T ex vivo and in vivo and we found for the first time that HLA-II deficiency could facilitate the persistence of CAR-T. Contrastively, as the most commonly eliminated target in UCAR-T, TCR deficiency was found to be a key disadvantageous factor for the shorter-term anti-tumor efficacy . Our study demonstrated ETUCAR-T could effectively resist allogeneic NK rejection and . Our research provided a potential and effective strategy for promoting the persistence of UCAR-T cells in clinical application. And it reveals the potential key factors of the poor persistence of UCAR-T along with new insights for future development.

NK‐cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non‐classical MHC‐I molecules HLA‐E in humans or Qa‐1 b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co‐injected with anti‐NKG2A and anti‐Qa‐1 b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type‐1 conventional dendritic cells (cDC1). Moreover, the anti‐tumor effects were enhanced upon combination with systemic anti‐PD‐1 mAb treatment and achieved partial abscopal efficacy against distant non‐injected tumors. In xenografted mice bearing HLA‐E‐expressing human cancer cells, intratumoral co‐injection of activated allogeneic human NK cells and clinical‐grade anti‐NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell‐based immunotherapies that exert their anti‐tumor efficacy as a result of eliciting endogenous T‐cell responses. Synopsis An approach that involves intratumoral injections of NK cells together with neutralizing antibodies against the NKG2A inhibitory receptor was developed, aiming at extending the efficacy of NK cell‐based immunotherapies to solid malignances. Intratumoral co‐injection of pre‐activated NK cells and NKG2A neutralizing antibodies achieves synergistic anti‐tumor activity against solid mouse tumor models. CD8 T cell‐ and cDC1 responses mediate the anti‐tumor efficacy of intratumoral NK cells and anti‐NKG2A/Qa‐1b mAb administrations. Systemic anti‐PD‐1 mAb injections synergistically enhance the efficacy of intratumoral NK cells + anti‐NKG2A/Qa‐1 b mAbs. Intratumoral delivery of human NK cells and monalizumab (anti‐NKG2A) to xenografted mouse tumors exerts synergistic effects. Graphical Abstract An approach that involves intratumoral injections of NK cells together with neutralizing antibodies against the NKG2A inhibitory receptor was developed, aiming at extending the efficacy of NK cell‐based immunotherapies to solid malignances.

Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. To restore NK cell cytotoxicity against HLA-E tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated gene editing. knockout resulted in a reduction of 81% of NKG2A cell frequency in expanded human NK cells post-cell sorting. , the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with -values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E solid tumor cell lines, with -values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in NK cells and contributed to the improved NK cell cytotoxicity against HLA-E tumors. , the adoptive transfer of human NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E metastatic breast cancer, as compared to WT NK cells ( = 0.0015). Our results demonstrate that knockout is an effective strategy to improve NK cell antitumor activity against HLA-E tumors and could be applied in the development of NK cell therapy for solid tumors.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes ( GGTA1 , CMAH , β4galNT2 , SLA-I α chain , and β-2 microglobulin ) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3 - CD56 + population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Senescent cells accumulate in human tissues during ageing and contribute to age-related pathologies. The mechanisms responsible for their accumulation are unclear. Here we show that senescent dermal fibroblasts express the non-classical MHC molecule HLA-E, which interacts with the inhibitory receptor NKG2A expressed by NK and highly differentiated CD8 + T cells to inhibit immune responses against senescent cells. HLA-E expression is induced by senescence-associated secretary phenotype-related pro-inflammatory cytokines, and is regulated by p38 MAP kinase signalling in vitro. Consistently, HLA-E expression is increased on senescent cells in human skin sections from old individuals, when compared with those from young, and in human melanocytic nevi relative to normal skin. Lastly, blocking the interaction between HLA-E and NKG2A boosts immune responses against senescent cells in vitro. We thus propose that increased HLA-E expression contributes to persistence of senescent cells in tissues, thereby suggesting a new strategy for eliminating senescent cells during ageing. Senescent cells increase with ageing and may cause inflammatory conditions, but how this accumulation is mediated is still unclear. Here the authors show that senescent cells express HLA-E to suppress NKG2A-mediated natural killer and CD8 T cell activation to avoid targeted elimination, while blocking NKG2A helps promote immunity against senescent cells.

Background: Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumour development in colon cancer. The goal of this study was to establish a tumour profile based on biomarkers that reflect a tumour’s immune susceptibility status and to determine their relation to patient outcome. Methods: The study population consisted of 285 stage I-IV colon cancer patients of which a tissue micro array (TMA) was available. Sections were immunohistochemically stained for the presence of Foxp3+ cells and tumour expression of HLA Class I (HLA-A, -B, -C) and non-classical HLA-E and HLA-G. All markers were combined for further analyses, resulting in three tumour immune phenotypes: strong immune system tumour recognition, intermediate immune system tumour recognition and poor immune system tumour recognition. Results: Loss of HLA class I expression was significantly related to a better OS ( P -value 0.005) and DFS ( P -value 0.008). Patients with tumours who showed neither HLA class I nor HLA-E or -G expression (phenotype a) had a significant better OS and DFS ( P -value <0.001 and 0.001, respectively) compared with phenotype b (OS HR: 4.7, 95% CI: 1.2–19.0, P =0.001) or c (OS HR: 8.2, 95% CI: 2.0–34.2, P =0.0001). Further, the tumour immune phenotype was an independent predictor for OS and DFS ( P -value 0.009 and 0.013, respectively). Conclusion: Tumours showing absence of HLA class I, HLA-E and HLA-G expressions were related to a better OS and DFS. By combining the expression status of several immune-related biomarkers, three tumour immune phenotypes were created that related to patient outcome. These immune phenotypes represented significant, independent, clinical prognostic profiles in colon cancer.

The contribution of the HLA-E/NKG2X axis in NK-mediated control of HIV infection remains unclear. We have studied the relationship between HLA-E expression and phenotypical as well as functional characteristics of NK cells, in the context of chronic HIV infection and in an in vitro model of acute infection. High viremia in HIV+ individuals was related to increased HLA-E expression, and changes in NK subpopulations, especially a reduction of the CD56 bright as well as an increase in adaptive NK subpopulation. Uncontrolled HIV infection was also characterized by a reversion of the NKG2A/NKG2C expression ratio and a loss of positive and negative regulation of NK mediated by HLA-E. This was reflected in a lower cytotoxic, degranulation and cytokine production capacity, especially in CD56 bright and adaptive NK. In line with these results, HLA-E expression showed a positive correlation with viral growth inhibition in an in vitro model of acute infection at day 7, which was lost after 14 days of culture. Using HLA-E expressing K562 cells, we determined that only one out of 11 described HIV-derived HLA-E epitopes increased HLA-E surface stability. In spite of that, eight of the 11 epitopes were capable of increasing degranulation and three drove differences in NK-cell mediated cell lysis or cytokine secretion. In conclusion, our results indicate that HLA-E molecules presenting HIV-derived epitopes may sensitize target cells for NK lysis in early HIV infection. However, prolonged exposure to elevated HLA-E expression levels in vivo may lead to NK cell dysfunction and reduced viral control In chronic infection.

A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression, without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were significantly more powerful than NKG2A+ NK cells against HLA-E-expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune-checkpoint, and increases the anti-tumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.