LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

Natural killer cell responses to human oncogenic γ-herpesvirus infections.

The two γ-herpesviruses Epstein Barr virus (EBV) and Kaposi sarcoma associated herpesvirus (KSHV) are each associated with more than 1% of all tumors in humans. While EBV establishes persistent infection in nearly all adult individuals, KSHV benefits from this widespread EBV prevalence for its own persistence. Interestingly, EBV infection expands early differentiated NKG2A+KIR- NK cells that protect against lytic EBV infection, while KSHV co-infection drives accumulation of poorly functional CD56-CD16+ NK cells. Thus persistent γ-herpesvirus infections are sculptors of human NK cell repertoires and the respectively stimulated NK cell subsets should be considered for immunotherapies of EBV and KSHV associated malignancies.

Phenotypic and functional exhaustion of circulating CD3+ CD56+ NKT-like cells in colorectal cancer patients.

CD3+ CD56+ NKT-like cells are crucial to antitumor immune surveillance and defense. However, research on circulating NKT-like cells in colorectal cancer (CRC) patients is limited. This investigation selected 113 patients diagnosed with primary CRC for preoperative peripheral blood collection. The blood from 106 healthy donors at the physical examination center was acquired as a healthy control (HC). The distribution of lymphocyte subsets, immunophenotype, and functional characteristics of NKT-like cells was comprehensively evaluated. Compared to HC, primary CRC patients had considerably fewer peripheral NKT-like cells in frequency and absolute quantity, and the fraction of NKT-like cells was further reduced in patients with vascular invasion compared to those without. The NKT-like cells in CRC patients had a reduced fraction of the activating receptor CD16, up-regulated expression of inhibitory receptors LAG-3 and NKG2A, impaired production of TNF-α and IFN-γ, as well as degranulation capacity. Moreover, the increased frequency of NKG2A+ NKT-like cells and the decreased expression of activation-related molecules were significantly correlated with tumor progression. In detail, NKG2A+ NKT-like cells indicated increased PD-1 and Tim-3 and reduced TNF-α than NKG2A- subgroup. Blocking NKG2A in vitro restored cytokine secretion capacity in NKT-like cells from CRC patients. Altogether, this research revealed that circulating NKT-like cells in CRC patients exhibited suppressive phenotype and functional impairment, which was more pronounced in NKG2A+ NKT-like cells. These findings suggest that NKG2A blockade may restore anti-tumor effector function in NKT-like cells, which provides a potential target for immunotherapy in CRC patients.

NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody.

Natural killer (NK) cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9 ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared with anti-NKG2A antibody blockade, NKG2AKO NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2AKO NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences in molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

Long-term dynamics of natural killer cells in response to SARS-CoV-2 vaccination: Persistently enhanced activity postvaccination.

Natural Killer (NK) cells play a significant role in the early defense against virus infections and cancer. Recent studies have demonstrated the involvement of NK cells in both the induction and effector phases of vaccine-induced immunity in various contexts. However, their role in shaping immune responses following SARS-CoV-2 vaccination remains poorly understood. To address this matter, we conducted a comprehensive analysis of NK cell phenotype and function in SARS-CoV-2 unexposed individuals who received the BNT162b2 vaccine. We employed a longitudinal study design and utilized a panel of 53 15-mer overlapping peptides covering the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein to assess NK cell function at 0 and 20 days following the first vaccine, and 30 and 240 days following booster. Additionally, we evaluated the levels of total IgG anti-Spike antibodies and their potential neutralizing ability. Our findings revealed an increased NK cell activity upon re-exposure to RBD when combined with IL12 and IL18 several months after booster. Concurrently, we observed that the frequencies of NKG2A + NK cells declined over the course of the follow-up period, while NKG2C increased only in CMV positive subjects. The finding that NK cell functions are inducible 9 months after vaccination upon re-exposure to RBD and cytokines, sheds light on the role of NK cells in contributing to SARS-CoV-2 vaccine-induced immune protection and pave the way to further studies in the field.

The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8⁺ T Cells in Response to a Lethal Poxvirus Infection.

CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.

PACIFIC-9: Phase III trial of durvalumab + oleclumab or monalizumab in unresectable stage III non-small-cell lung cancer.

Evidence from the Phase III PACIFIC trial established durvalumab, a monoclonal antibody (mAb) targeting PD-L1, following concurrent chemoradiotherapy (cCRT) as a global standard of care for patients with unresectable, stage III non-small-cell lung cancer (NSCLC). There remains an unmet need to improve upon the outcomes achieved with the PACIFIC regimen. Combining durvalumab with other immunotherapies may improve outcomes further. Two such immunotherapies include oleclumab, an mAb targeting CD73, and monalizumab, an mAb targeting NKG2A. Both agents demonstrated antitumor activity in early-phase trials. PACIFIC-9 (NCT05221840) is an international, double-blind, randomized, placebo-controlled, Phase III trial comparing durvalumab plus either oleclumab or monalizumab with durvalumab plus placebo in patients with unresectable, stage III NSCLC and no disease progression following cCRT.Clinical Trial Registration: NCT05221840 (ClinicalTrials.gov).

Durvalumab is a treatment that helps the body's immune system to identify and attack cancer cells by binding to a protein called PD-L1. Studies show that durvalumab lowers the risk of cancer growing or spreading, and prolongs survival, when administered after chemotherapy and radiation therapy ('chemoradiotherapy') in patients with a type of lung cancer called stage III non-small-cell lung cancer (NSCLC) for whom surgery is not an option.Two antibody treatments have been developed that may help a patient's immune system to identify and attack cancer cells. Oleclumab binds to a protein on cancer cells called CD73, which prevents the production of adenosine, a chemical that obstructs the immune system from attacking the cancer. Monalizumab binds to NKG2A, a protein on immune cells that inhibits their ability to destroy cancer cells. Early studies suggest that combining either of these treatments with durvalumab may be better than durvalumab alone for slowing the growth and spread of cancer in patients with NSCLC.PACIFIC-9 is a study that aims to recruit approximately 999 patients with stage III NSCLC for whom surgery is not an option and who have completed chemoradiotherapy without the cancer growing or spreading. Patients will be randomly assigned in equal numbers to receive up to a year of treatment with durvalumab plus oleclumab, durvalumab plus monalizumab or durvalumab plus placebo. The primary measure of efficacy is the length of time that patients remain alive without the cancer growing or spreading for each combination versus durvalumab plus placebo.

[Effect of Spatholobi Caulis extract from ethyl acetate on immune killing function of NK cells].

This study aims to investigate the regulatory effect of the Spatholobi Caulis extract from ethyl acetate(SEA) on natural killer(NK) cells under physiological conditions and elucidate the underlying mechanism. The C57BL/6 mice were randomized into NC and SEA groups, and NK-92 cells were respectively treated with 0, 25, 50, and 100 µg·mL~(-1) SEA. The body weight and immune organ index of the mice were compared between groups. The lactate dehydrogenase(LDH) assay was employed to examine the cytotoxicity of NK-92 cells treated with SEA and the killing activity of mouse NK cells against YAC-1 cells. The cell-counting kit-8(CCK-8) was used to examine the impact of SEA on the proliferation of NK-92 cells. Flow cytometry was employed to measure the number of NK cells in the peripheral blood as well as the expression levels of natural killer group 2 member A(NKG2A) and natural killer group 2 member D(NKG2D). The enzyme-linked immunosorbent assay(ELISA) was performed to determine the interferon(IFN)-γ secretion in the serum. Semi-quantitative PCR was conducted to determine the mRNA levels of NKG2A, NKG2D, and IFN-γ in spleen cells. Western blot was employed to investigate the involvement of phosphoinositide 3-kinase(PI3K)/extracellular regulated protein kinase 1(ERK1) signaling pathway. The results showed that SEA exhibited no adverse effects on the body, while significantly enhance the number of NK cells and augment the cytotoxicity of NK-92 cells against YAC-1 cells. Moreover, it suppressed the expression of NKG2A, enhanced the expression of NKG2D, promoted IFN-γ secretion, and upregulated the protein levels of PI3K and ERK. The findings suggest that SEA has the potential to enhance the immune recognition and effector function of NK cells by increasing the cell number, modulating the expression of functional receptors, and promoting IFN-γ secretion via the PI3K/ERK signaling pathway.

Inhibitory receptors for HLA class I as immune checkpoints for natural killer cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.

Natural killer (NK) cells mediate potent anti-tumor responses, which makes them attractive targets for immunotherapy. The anti-tumor response of endogenous- or allogeneic NK cells can be enhanced through clinically available monoclonal antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is regulated by interaction of inhibitory receptors with classical- and non-classical human leukocyte antigens (HLA) class I molecules. Inhibitory receptors of the killer immunoglobulin-like receptor (KIR) family interact with HLA-A, -B or -C epitopes, while NKG2A interacts with the non-classical HLA-E molecule. Both types of inhibitory interactions may influence the strength of the ADCC response. In the present review, we provide an overview of the effect of inhibitory KIRs and NKG2A on NK cell-mediated ADCC, which highlights the rationale for combination strategies with ADCC triggering antibodies and interference with the NK cell relevant inhibitory immune checkpoints, such as KIR and NKG2A.

Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure.

BACKGROUND: Combination antiretroviral therapy (cART) has dramatically extended the life expectancy of people living with HIV-1 and improved their quality of life. There is nevertheless no cure for HIV-1 infection since HIV-1 persists in viral reservoirs of latently infected CD4+ T cells. cART does not eradicate HIV-1 reservoirs or restore cytotoxic natural killer (NK) cells which are dramatically reduced by HIV-1 infection, and express the checkpoint inhibitors NKG2A or KIR2DL upregulated after HIV-1 infection. Cytotoxic NK cells expressing the homing receptor CXCR5 were recently described as key subsets controlling viral replication. METHODS: We designed and evaluated the potency of "Natural killer activating Multimeric immunotherapeutic compleXes", called as NaMiX, combining multimers of the IL-15/IL-15Rα complex with an anti-NKG2A or an anti-KIR single-chain fragment variable (scFv) to kill HIV-1 infected CD4+ T cells. The oligomerization domain of the C4 binding protein was used to associate the IL-15/IL-15Rα complex to the scFv of each checkpoint inhibitor as well as to multimerize each entity into a heptamer (α form) or a dimer (ß form). Each α or ß form was compared in different in vitro models using one-way ANOVA and post-hoc Tukey's tests before evaluation in humanized NSG tg-huIL-15 mice having functional NK cells. RESULTS: All NaMiX significantly enhanced the cytolytic activity of NK and CD8+ T cells against Raji tumour cells and HIV-1+ ACH-2 cells by increasing degranulation, release of granzyme B, perforin and IFN-γ. Targeting NKG2A had a stronger effect than targeting KIR2DL due to higher expression of NKG2A on NK cells. In viral inhibition assays, NaMiX initially increased viral replication of CD4+ T cells which was subsequently inhibited by cytotoxic NK cells. Importantly, anti-NKG2A NaMiX enhanced activation, cytotoxicity, IFN-γ production and CXCR5 expression of NK cells from HIV-1 positive individuals. In humanized NSG tg-huIL-15 mice, we confirmed enhanced activation, degranulation, cytotoxicity of NK cells, and killing of HIV-1 infected cells from mice injected with the anti-NKG2A.α NaMiX, as compared to control mice, as well as decreased total HIV-1 DNA in the lung. CONCLUSIONS: NK cell-mediated killing of HIV-1 infected cells by NaMiX represents a promising approach to support HIV-1 cure strategies.

Association between human genetic variants affecting the host NK cell response and the development of herpes simplex virus type 1 encephalitis.

Herpes simplex virus encephalitis (HSE) is a rare complication of herpes simplex virus type 1 (HSV-1) infection or reactivation. It is so far unclear why only few patients develop HSE. As natural killer (NK) cells provide an important defense against HSV-1, we investigated whether there is an association between distinct human genetic variants associated with the host NK cell response and HSE. Forty-nine adult patients with confirmed HSE and 247 matched controls were analyzed for the distribution of the following genotypes: CD16A (FcγRIIIA) V/F and IGHG1 G1m3/17, both influencing antibody-dependent cellular cytotoxicity; HLA-E*0101/*0103, associated with NK cell activation; and SLFN13 rs9916629C/T  associated with NK cell response. Homozygous HLA-E*0101:0101 and HLA-E*0103:0103 variants as well as the rs9916629CC genotype were overrepresented in HSE patients compared to controls (p ≤ 0.001). Notably, cooccurrence of the homozygous HLA-E*0101 and rs9916629CC genotypes was present in 19% of patients but totally absent in controls (p ≤ 0.0001). Distribution of CD16A and IGHG1 variants did not differ between patients and controls. Our data show that the rare combination of HLA-E*0101:0101 and rs9916629CC is significantly associated with HSE. Possibly, these genetic variations could be useful as clinical markers predicting HSE prognosis and helping to adapt the treatment of HSE in the individual patient.

The imbalance between NKG2A and NKG2D expression is involved in NK cell immunosuppression and tumor progression of patients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Immunosuppression in a tumor microenvironment is associated with enhanced tumor progression. Natural killer group 2 (NKG2) family proteins, including inhibitory receptors and activators, can be used as attractive targets for immunotherapy of immune checkpoint inhibition. We further explore the expression level prognostic value of NKG2A and NKG2D in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). METHODS: This study was a prospective study involving 92 patients with HBV-HCC, 16 patients with HBV-related liver cirrhosis, 18 patients with CHB, and 38 healthy donors. We analyzed the expression and related functions of NKG2A, NKG2D, and the NKG2A/NKG2D ratio in the peripheral blood of patients with HBV-HCC and analyzed tumor progression. The tissue samples from patients with HBV-HCC were further used for multiple immunofluorescence and immunohistochemistry. RESULTS: In patients with HBV-HCC with tumor progression, the ratio of NKG2A/NKG2D is higher in NK cells and T cells. The Kaplan-Meier survival curve showed that the NKG2A/NKG2D ratio on NK cells could predict tumor progression in patients with HBV-HCC, and that an increase in this ratio was associated with inhibition of NK cell function. The Cancer Genome Atlas (TCGA) database was further used to verify that the higher the NKG2A/NKG2D ratio, the shorter the progression-free survival of patients with HCC, and the more likely the immune function was suppressed. CONCLUSIONS: The imbalance between NKG2A and NKG2D of NK cells is involved in NK cell immunosuppression, and the increase of the NKG2A/NKG2D ratio is related to the tumor progression of HBV-HCC.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

NKG2A-checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA-E.

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non-classical MHC class I molecule HLA-E. HLA-E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA-A, HLA-B and HLA-C) and the non-classical class I paralogue HLA-G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA-E is peptide-sensitive. Here, we have evaluated peptide dependence of NKG2A-mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell-expressed HLA-E molecules stably presenting disulfate-trapped peptide ligands. We show that different HLA-class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA-leader peptide presented by HLA-E. Monalizumab was less effective in augmenting NK cell-mediated killing of target cells displaying HLA-G peptide on HLA-E, than cells expressing HLA-E complexed with HLA-A, HLA-B and HLA-C peptides. Our results indicate that peptides displayed by HLA-E molecules on tumour cells might influence the effectivity of NKG2A-ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA-G levels.

NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-ß in vitro, although TGF-ß signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

Increased Peripheral NKG2A-NKG2D+CD3-CD16+CD56dim NK Cell Subset Was Positively Correlated with Antiphospholipid Antibodies in Patients of Obstetric Antiphospholipid Syndrome.

Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disorder with severe life-threatening complications shown during pregnancy. It has been reported that the increase in CD16+CD56dim natural killer (NK) cells in peripheral blood are risk factors for recurrent miscarriages, but this expression of CD16+CD56dim NK cells in OAPS patients has not been reported, and the mechanism is not clearly illustrated. In this study, we compared the distributional profiles of different NK cell subsets and the expressions of NK cell-activating receptors in peripheral blood of patients with OAPS and healthy women. Our results showed significantly increased NKG2A-NKG2D+ subset and decreased NKG2A+NKG2D- subset in CD3- CD16+CD56dim NK cells, CD3-CD16-CD56bright NK cells and CD56+T cells in OAPS patients compared with those in healthy control women. The CD27-CD11b+ subset significantly increased in CD3-CD16+CD56dim NK cells in OAPS patients compared with those in healthy control women. In addition, the NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK subset in triple positivity was higher than single positivity OAPS patients. At the optimal diagnostic threshold established by ROC analysis, using the cut-off of NKG2A-NKG2D+ and CD27-CD11b+ subset in CD3-CD16+CD56dim NK cells is 10.10% and 92.75%, the sensitivity of NKG2A-NKG2D+ and CD27-CD11b+ to detect patients with OAPS compared with healthy control results was 94.1% and 60.8%, and specificity was 84.2% and 89.5%, respectively, with an area under the curve (AUC) of 0.903 and 0.829, respectively. The NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK cells was positively correlated with the antiphospholipid antibodies lg anti-aCL IgG, lg anti-aCL IgM, lg anti-aCL IgA, lg anti-ß2GP1 IgM and Complement 4(C4), while the CD27+CD11b+ subset in CD3-CD16+CD56dim NK cells was correlated with lg anti-ß2GP1 IgG and lg anti-ß2GP1 IgA. These results suggested that the NK cytotoxic function enhanced in OAPS patients and unbalanced of NK activating receptors and inhibiting receptors may contribute to the immune pathogenesis of OAPS.