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.

Adaptive NK cell response to human cytomegalovirus: Facts and open issues.

Human cytomegalovirus (HCMV) infection exerts broad effects on the immune system. These include the differentiation and persistent expansion of a mature NK cell subset which displays a characteristic phenotypic and functional profile hallmarked by expression of the HLA-E-specific CD94/NKG2C activating receptor. Based on our experience and recent advances in the field, we overview the adaptive features of the NKG2C+ NK cell response, discussing observations and open questions on: (a) the mechanisms and influence of viral and host factors; (b) the existence of other NKG2C- NK cell subsets sharing adaptive features; (c) the development and role of adaptive NKG2C+ NK cells in the response to HCMV in hematopoietic and solid organ transplant patients; (d) their relation with other viral infections, mainly HIV-1; and (e) current perspectives for their use in adoptive immunotherapy of cancer.

NK Cells from Human Cytomegalovirus-Seropositive Individuals Have a Distinct Metabolic Profile That Correlates with Elevated mTOR Signaling.

CMV can elicit adaptive immune features in both mouse and human NK cells. Mouse Ly49H+ NK cells expand 100- to 1000-fold in response to mouse CMV infection and persist for months after exposure. Human NKG2C+ NK cells also expand after human CMV (HCMV) infection and persist for months. The clonal expansion of adaptive NK cells is likely an energy-intensive process, and the metabolic requirements that support adaptive NK cell expansion and persistence remain largely uncharacterized. We previously reported that NK cells from HCMV-seropositive donors had increased maximum capacity for both glycolysis and mitochondrial oxidative phosphorylation relative to NK cells from HCMV-seronegative donors. In this article, we report an extension of this work in which we analyzed the metabolomes of NK cells from HCMV-seropositive donors with NKG2C+ expansions and NK cells from HCMV seronegative donors without such expansions. NK cells from HCMV+ donors exhibited striking elevations in purine and pyrimidine deoxyribonucleotides, along with moderate increases in plasma membrane components. Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that, as a part of mTOR complex 1 (mTORC1), bridges nutrient signaling to metabolic processes necessary for cell growth. Signaling through mTORC1 induces both nucleotide and lipid synthesis. We observed elevated mTORC1 signaling on activation in both NKG2C- and NKG2C+ NK cells from HCMV+ donors relative to those from HCMV- donors, demonstrating a correlation between higher mTORC1 activity and synthesis of key metabolites for cell growth and division.

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.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

The nonclassical MHC class I Qa-1 expressed in layer 6 neurons regulates activity-dependent plasticity via microglial CD94/NKG2 in the cortex.

During developmental critical periods, circuits are sculpted by a process of activity-dependent competition. The molecular machinery involved in regulating the complex process of responding to different levels of activity is now beginning to be identified. Here, we show that the nonclassical major histocompatibility class I (MHCI) molecule Qa-1 is expressed in the healthy brain in layer 6 corticothalamic neurons. In the visual cortex, Qa-1 expression begins during the critical period for ocular dominance (OD) plasticity and is regulated by neuronal activity, suggesting a role in regulating activity-dependent competition. Indeed, in mice lacking Qa-1, OD plasticity is perturbed. Moreover, signaling through CD94/NKG2, a known cognate Qa-1 heterodimeric receptor in the immune system, is implicated: selectively targeting this interaction phenocopies the plasticity perturbation observed in Qa-1 knockouts. In the cortex, CD94/NKG2 is expressed by microglial cells, which undergo activity-dependent changes in their morphology in a Qa-1­dependent manner. Our study thus reveals a neuron­microglial interaction dependent upon a nonclassical MHCI molecule expressed in L6 neurons, which regulates plasticity in the visual cortex. These results also point to an unexpected function for the Qa-1/HLA-E (ligand) and CD94/NKG2 (receptor) interaction in the nervous system, in addition to that described in the immune system.

NKG2C+CD57+ natural killer cells with senescent features are induced during cutaneous leishmaniasis and accumulate in patients with lesional healing impairment.

Natural killer (NK) cells include different subsets with diverse effector capacities that are poorly understood in the context of parasitic diseases. Here, we investigated inhibitory and activating receptor expression on NK cells in patients with cutaneous leishmaniasis (CL) and explored their phenotypic and functional heterogeneity based on CD57 and NKG2C expression. The expression of CD57 identified NK cells that accumulated in CL patients and exhibited features of senescence. The CD57+ cells exhibited heightened levels of the activating receptor NKG2C and diminished expression of the inhibitory receptor NKG2A. RNA sequencing analyses based on NKG2C transcriptome have revealed two distinct profiles among CL patients associated with cytotoxic and functional genes. The CD57+NKG2C+ subset accumulated in the blood of patients and presented conspicuous features of senescence, including the expression of markers such as p16, yH2ax, and p38, as well as reduced proliferative capacity. In addition, they positively correlated with the number of days until lesion resolution. This study provides a broad understanding of the NK cell biology during Leishmania infection and reinforces the role of senescent cells in the adverse clinical outcomes of CL.

Mutual exclusivity and co-occurrence patterns of immune checkpoints indicate NKG2A relates to anti-PD-1 resistance in gastric cancer.

BACKGROUND: An increasing number of clinical studies have begun to explore combination strategies with immune checkpoint inhibitors, aiming to present new opportunities for overcoming anti-PD-1 treatment resistance in gastric cancer. Unfortunately, the exploration of certain immune checkpoint inhibitor combination strategies has yielded suboptimal results. Therefore, it is necessary to comprehensively analyze the expression patterns of immune checkpoints and identify optimal combination regimens of anti-PD-1 inhibitors with other immune checkpoint inhibitors. METHODS: Leveraging single-cell RNA sequencing (scRNA-seq) and multivariate linear regression interaction models, we dissected the immune checkpoint expression characteristics of CD8+ T cells in gastric cancer and the immune checkpoint expression pattern (ICEP) mediating anti-PD-1 treatment resistance. Furthermore, we employed transcription factor analysis and CellOracle to explore the transcriptional regulatory mechanisms governing CD8+ T cell differentiation fates. Finally, we utilized Nichenet and spatial transcriptomic analysis to investigate the spatial expression patterns of immune checkpoints. RESULTS: Interaction analysis indicated that, among the known immune checkpoints, co-expression of NKG2A and PD-1 might exert a more profound inhibitory effect on the proliferative capacity of CD8+ T cells. The co-expression analysis revealed differential co-expression pattern of PD-1 and NKG2A, defined as ICEP1 (CD8+ T cells co-expressing PD-1, CTLA-4, TIGIT, LAG-3 or CD38) and ICEP2 (CD8+ T cells solely expressing NKG2A or co-expressing with other immune checkpoints), reflecting the co-occurrence pattern of PD-1 and the mutual exclusivity of NKG2A. Further, these two ICEP CD8+ T cell subsets represented distinct CD8+ T cell differentiation fates governed by MSC and RUNX3. Notably, ICEP2 CD8+ T cells were associated with anti-PD-1 therapy resistance in gastric cancer. This phenomenon may be attributed to the recruitment of LGMN+ macrophages mediated by the CXCL16-CXCR6 signaling pathway. CONCLUSION: This study unveiled two distinct ICEPs and the mutually exclusivity and co-occurrence characteristics of CD8+ T cells in gastric cancer. The ICEP2 CD8+ T cell subset, highly expressed in gastric cancer patients resistant to anti-PD-1 therapy, may be recruited by LGMN+ macrophages through CXCL16-CXCR6 axis. These findings provide evidence for NKG2A as a novel immunotherapeutic target in gastric cancer and offer new insights into combination strategies for immune checkpoint inhibitors in gastric cancer.

A Single-Domain TCR-like Antibody Selective for the Qa-1b/Qdm Peptide Complex Enhances Tumoricidal Activity of NK Cells via Blocking the NKG2A Immune Checkpoint.

The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1b, the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1b/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1b/Qdm peptide complex and not to Qa-1b alone or Qa-1b loaded with control peptides. Conversely, currently available Abs to Qa-1b bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1b/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-γ; no binding was observed following genetic knockdown of Qa-1b or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1b/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1b/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.

Combination of NKG2A and PD-1 Blockade Improves Radiotherapy Response in Radioresistant Tumors.

Radiotherapy (RT) is commonly employed to treat solid tumors. Immune checkpoint blockade of programmed cell death protein 1 (PD-1) and CTLA-4 improves survival in RT patients, yet many fail to respond to combination therapy. Natural killer group 2 (NKG2) family receptors, particularly inhibitory NKG2A and activating NKG2D, have emerged as promising therapeutic targets to improve antitumor T cell responses; thus, we examined how these receptors and their ligands (Qa-1b and retinoic acid early inducible 1 [Rae-1], respectively) regulate the RT response in C57BL/6 mice bearing syngeneic B16F10 melanoma and MC38 colorectal adenocarcinoma tumors. RT (15 Gy) transiently reduced B16F10 tumor burden, whereas MC38 tumors exhibited durable response to RT. Intratumoral NK and CD8 T cells expressed NKG2A and NKG2D in both models, which was unaltered by RT. In vitro/in vivo RT increased tumor/stromal cell Qa-1b and Rae-1 expression in both models, especially B16F10 tumors, but IFN-γ stimulation induced both Qa-1b and Rae-1 only in B16F10 tumors. NKG2A/Qa-1b inhibition alone did not improve RT response in either model, but combined RT and NKG2A/PD-1 blockade improved survival in the B16F10 model. Depletion experiments indicate that the triple therapy efficacy is CD8 T cell-dependent with negligible NK cell contribution. RNA sequencing of CD8 T cells from triple therapy-treated B16F10 tumors showed increased proliferative capacity compared with RT and PD-1 blockade alone. Our work demonstrates that RT modulates NKG2A ligand expression, which inhibits RT-induced T cell responses in tumors that fail to respond to combined RT and PD-1 blockade. These results provide a rationale for combining NKG2A blockade with immune checkpoint blockade therapies and RT to improve clinical response.

A New Look at Immunogenetics of Pregnancy: Maternal Major Histocompatibility Complex Class I Educates Uterine Natural Killer Cells.

Our incomplete knowledge of maternal-fetal interface (MFI) physiology impedes a better understanding of the pathological mechanisms leading to pregnancy complications, such as pre-eclampsia and fetal growth restriction. At the MFI, uterine natural killer (uNK) cells do not attack fetal cells but engage in crosstalk with both fetal and maternal cells to support feto-placental development. However, mother and fetus are genetically half-mismatched and certain combinations of variable immune genes-human leukocyte antigens (HLAs) and killer-cell immunoglobulin-like receptor (KIR), indeed, the most variable gene sets in the genome-associate with pregnancy outcomes, suggesting that these interactions regulate uNK cell function. How do these interactions influence the physiology and pathology at the MFI? Uterine NK cell function is regulated by both maternal and fetal Major Histocompatibility Complex (MHC); however, evidence for fetal cells educating uNK cells is lacking, and new evidence shows that maternal rather than fetal MHC class I molecules educate uNK cells. Furthermore, uNK cell education works through self-recognition by the ancient and conserved NKG2A receptor. Pregnant mice lacking this receptor produce normal litter sizes, but a significant portion of the offspring have low birthweight and abnormal brain development. Evidence from a genome-wide association study of over 150,000 human pregnancies validates the finding because women whose NKG2A receptor is genetically determined to engage their own MHC class I molecules are exposed to lower risk of developing pre-eclampsia, suggesting that maternal uNK cell education is a pre-requisite for a healthy pregnancy and, likely, for healthy offspring too.

Phenotypic and functional analysis in HER2+ targeted therapy of human NK cell subpopulation according to the expression of FcεRIγ and NKG2C in breast cancer patients.

Adaptive NK cells constitute an NK cell subpopulation, which expands after human cytomegalovirus (HCMV) infection. This subpopulation has stronger production of cytokines after CD16 stimulation, longer life and persistence than conventional NK cells and are, therefore, interesting tools for cancer immunotherapy. Since there is limited information on adaptive NK cells in cancer patients, we described this population phenotypically and functionally, by flow cytometry, in the context of HER2 + breast cancer (BC) directed therapy. We assessed HCMV status in 78 patients with BC. We found that, similarly to healthy donors (HD), a high proportion of BC patients were HCMV-positive, and nearly 72% of them had an adaptive NK cell subpopulation characterized by the loss of FcεRIγ intracellular adaptor protein or the presence of NKG2C receptor. However, in BC patients, FcεRIγ- and NKG2C + NK cell populations overlapped to a lesser extent than in HD. Otherwise, no profound phenotypic differences were found between BC patients and HD. Although FcεRIγ- or NKG2C + NK cell subsets from BC patients produced more IFN-γ than their FcεRIγ + or NKG2C- NK cell counterparts, IFN-γ production increased only when NK cells simultaneously expressed FcεRIγ- and NKG2C + , whereas in HD the presence of NKG2C marker was sufficient to display greater functionality. Furthermore, in a group of patients treated with chemotherapy and Trastuzumab plus Pertuzumab, FcεRIγ-NKG2C + and FcεRIγ-NKG2C- NK cells retained greater functionality after treatment than FcεRIγ + NKG2C- NK cells. These results suggest that the presence or magnitude of adaptive NK cell subsets might serve as a key determinant for therapeutic approaches based on antibodies directed against tumor antigens.

All-Atom Simulations Reveal the Intricacies of Signal Transduction upon Binding of the HLA-E Ligand to the Transmembrane Inhibitory CD94/NKG2A Receptor.

Natural killer (NK) cells play an important role in the innate immune response against tumors and various pathogens such as viruses and bacteria. Their function is controlled by a wide array of activating and inhibitory receptors, which are expressed on their cell surface. Among them is a dimeric NKG2A/CD94 inhibitory transmembrane (TM) receptor which specifically binds to the non-classical MHC I molecule HLA-E, which is often overexpressed on the surface of senescent and tumor cells. Using the Alphafold 2 artificial intelligence system, we constructed the missing segments of the NKG2A/CD94 receptor and generated its complete 3D structure comprising extracellular (EC), TM, and intracellular regions, which served as a starting point for the multi-microsecond all-atom molecular dynamics simulations of the receptor with and without the bound HLA-E ligand and its nonameric peptide. The simulated models revealed that an intricate interplay of events is taking place between the EC and TM regions ultimately affecting the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) regions that host the point at which the signal is transmitted further down the inhibitory signaling cascade. Signal transduction through the lipid bilayer was also coupled with the changes in the relative orientation of the NKG2A/CD94 TM helices in response to linker reorganization, mediated by fine-tuned interactions in the EC region of the receptor, taking place after HLA-E binding. This research provides atomistic details of the cells' protection mechanism against NK cells and broadens the knowledge regarding the TM signaling of ITIM-bearing receptors.

Long-Term Evolution of the Adaptive NKG2C+ NK Cell Response to Cytomegalovirus Infection in Kidney Transplantation: An Insight on the Diversity of Host-Pathogen Interaction.

Human CMV infection is frequent in kidney transplant recipients (KTR). Pretransplant Ag-specific T cells and adaptive NKG2C+ NK cells associate with reduced incidence of infection in CMV+ KTR. Expansions of adaptive NKG2C+ NK cells were reported in posttransplant CMV-infected KTR. To further explore this issue, NKG2C+ NK, CD8+, and TcRγδ T cells were analyzed pretransplant and at different time points posttransplant for ≥24 mo in a cohort of CMV+ KTR (n = 112), stratified according to CMV viremia detection. In cryopreserved samples from a subgroup (n = 49), adaptive NKG2C+ NK cell markers and T cell subsets were compared after a longer follow-up (median, 56 mo), assessing the frequencies of CMV-specific T cells and viremia at the last time point. Increased proportions of NKG2C+ NK, CD8+, and TcRγδ T cells were detected along posttransplant evolution in viremia(+) KTR. However, the individual magnitude and kinetics of the NKG2C+ NK response was variable and only exceptionally detected among viremia(-) KTR, presumably reflecting subclinical viral replication events. NKG2C+ expansions were independent of KLRC2 zygosity and associated with higher viral loads at diagnosis; no relation with other clinical parameters was perceived. Increased proportions of adaptive NKG2C+ NK cells (CD57+, ILT2+, FcεRIγ-) were observed after resolution of viremia long-term posttransplant, coinciding with increased CD8+ and Vδ2- γδ T cells; at that stage CMV-specific T cells were comparable to viremia(-) cases. These data suggest that adaptive NKG2C+ NK cells participate with T cells to restore CMV replication control, although their relative contribution cannot be discerned.

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.

NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals.

Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer.Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021-000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1 .

Influence of NKG2C Genotypes on HIV Susceptibility and Viral Load Set Point.

NKG2C is an activating NK cell receptor encoded by a gene having an unexpressed deletion variant. Cytomegalovirus (CMV) infection expands a population of NKG2C+ NK cells with adaptive-like properties. Previous reports found that carriage of the deleted NKG2C- variant was more frequent in people living with HIV (PLWH) than in HIV- controls unexposed to HIV. The frequency of NKG2C+ NK cells positively correlated with HIV viral load (VL) in some studies and negatively correlated with VL in others. Here, we investigated the link between NKG2C genotype and HIV susceptibility and VL set point in PLWH. NKG2C genotyping was performed on 434 PLWH and 157 HIV-exposed seronegative (HESN) subjects. Comparison of the distributions of the three possible NKG2C genotypes in these populations revealed that the frequencies of NKG2C+/+ and NKG2C+/- carriers did not differ significantly between PLWH and HESN subjects, while that of NKG2C-/- carriers was higher in PLWH than in HESN subjects, in which none were found (P = 0.03, χ2 test). We were unable to replicate that carriage of at least 1 NKG2C- allele was more frequent in PLWH. Information on the pretreatment VL set point was available for 160 NKG2C+/+, 83 NKG2C+/-, and 6 NKG2C-/- PLWH. HIV VL set points were similar between NKG2C genotypes. The frequency of NKG2C+ CD3- CD14- CD19- CD56dim NK cells and the mean fluorescence intensity (MFI) of NKG2C expression on NK cells were higher on cells from CMV+ PLWH who carried 2, versus 1, NKG2C+ alleles. We observed no correlations between VL set point and either the frequency or the MFI of NKG2C expression. IMPORTANCE We compared NKG2C allele and genotype distributions in subjects who remained HIV uninfected despite multiple HIV exposures (HESN subjects) with those in the group PLWH. This allowed us to determine whether NKG2C genotype influenced susceptibility to HIV infection. The absence of the NKG2C-/- genotype among HESN subjects but not PLWH suggested that carriage of this genotype was associated with HIV susceptibility. We calculated the VL set point in a subset of 252 NKG2C-genotyped PLWH. We observed no between-group differences in the VL set point in carriers of the three possible NKG2C genotypes. No significant correlations were seen between the frequency or MFI of NKG2C expression on NK cells and VL set point in cytomegalovirus-coinfected PLWH. These findings suggested that adaptive NK cells played no role in establishing the in VL set point, a parameter that is a predictor of the rate of treatment-naive HIV disease progression.

NKG2Cpos NK Cells Regulate the Expansion of Cytomegalovirus-Specific CD8 T Cells.

Infection with the human CMV associates with phenotypic alterations in lymphocyte subsets. A highly reproducible finding in CMV-seropositive individuals is an expansion of NKG2Cpos NK cells. In this study, we analyzed if the altered NK cell compartment in CMV-seropositive human donors may affect CMV-specific CD8 T cells. Resting CMV-specific CD8 T cells were terminally differentiated and expressed high levels of the NKG2C ligand HLA-E. Activation of CMV-specific CD8 T cells with the cognate Ag further increased HLA-E expression. In line with a negative regulatory effect of NKG2Cpos NK cells on HLA-Ehigh CD8 T cells, depletion of NKG2Cpos NK cells enhanced Ag-specific expansion of CMV-specific CD8 T cells in vitro. In turn, the activation of NK cells in coculture with CMV-specific CD8 T cells promoted a selective loss of HLA-Ehigh CD8 T cells. To test if NKG2Cpos NK cells can target HLA-Ehigh CD8 T cells, Jurkat T cells with and without stabilized HLA-E on the surface were used. NKG2Cpos NK cells stimulated with HLA-Ehigh Jurkat cells released higher levels of Granzyme B compared with NKG2Cneg NK cells and NKG2Cpos NK cells stimulated with HLA-Elow Jurkat cells. Moreover, intracellular levels of caspase 3/7 were increased in HLA-Ehigh Jurkat cells compared with HLA-Elow Jurkat cells, consistent with higher rates of apoptosis in HLA-Ehigh T cells in the presence of NKG2Cpos NK cells. Our data show that NKG2Cpos NK cells interact with HLA-Ehigh CD8 T cells, which may negatively regulate the expansion of CMV-specific CD8 T cells upon activation.

HCMV-controlling NKG2C+ NK cells originate from novel circulating inflammatory precursors.

BACKGROUND: There is limited knowledge on the origin and development from CD34+ precursors of the ample spectrum of human natural killer (NK) cells, particularly of specialized NK subsets. OBJECTIVE: This study sought to characterize the NK-cell progeny of CD34+DNAM-1brightCXCR4+ and of other precursors circulating in the peripheral blood of patients with chronic viral infections (eg, HIV, hepatitis C virus, cytomegalovirus reactivation). METHODS: Highly purified precursors were obtained by flow cytometric sorting and cultured in standard NK-cell differentiation media (ie, SCF, FLT3, IL-7, IL-15). Phenotypic and functional analyses on progenies were performed by multiparametric cytofluorimetric assays. Transcriptional signatures of NK-cell progenies were studied by microarray analysis. Inhibition of cytomegalovirus replication was studied by PCR. RESULTS: Unlike conventional CD34+ precursors, Lin-CD34+DNAM-1brightCXCR4+ precursors from patients with chronic infection, rapidly differentiate into cytotoxic, IFN-γ-secreting CD94/NKG2C+KIR+CD57+ NK-cell progenies. An additional novel subset of common lymphocyte precursors was identified among Lin-CD34-CD56-CD16+ cells and characterized by expression of CXCR4 and lack of perforin and CD94. Lin-CD34-CD56-CD16+Perf-CD94-CXCR4+ precursors are also endowed with generation potential toward memory-like NKG2C+NK cells. Maturing NK-cell progenies mediated strong human cytomegalovirus-inhibiting activity. Microarray analysis confirmed a transcriptional signature compatible with NK-cell progenies and with maturing adaptive NK cells. CONCLUSIONS: During viral infections, precursors of adaptive NK cells are released and circulate in the peripheral blood.

Age-related changes in PD-1 expression coincide with increased cytotoxic potential in Vδ2 T cells during infancy.

Human Vγ9Vδ2 T cells respond to several diverse pathogens by sensing microbial cholesterol intermediates. Unlike CD4 T cells, they are poised for rapid Th1-like responses even before birth, which allows them to play a key role in the first line of defense against pathogens in early life. However, their regulation and functional maturation during infancy (in particular the acquisition of cytotoxic potential) remain understudied. We thus characterized their responses to cholesterol intermediates and Bacille Calmette-Guérin in a cohort of African neonates and 12-month-old infants. Infant Vδ2 lymphocytes exhibited intermediate or adult-like expression of markers associated with differentiation or function, intermediate proliferative responses, and adult-like cytotoxic potential. The enhancement of Vδ2 cell cytotoxic potential coincided with decreasing PD-1 and increasing NKG2A expression. Our results are consistent with the hypothesis that switching from a PD-1+ to a NKG2A+ phenotype during infancy indicates a shift in mechanisms regulating Vδ2 T cell function.