Interplay of human adaptative NKG2C+ NK cells with specific antibodies and T cells in the response to cytomegalovirus infection

Human cytomegalovirus (HCMV) infection induces persistent changes in the host immune system, namely a sustained expansion of effector memory and cytotoxic specific T lymphocytes, the production of antibodies (Ab) and, in some individuals, associates with the differentiation of a functionally competent Natural Killer (NK) cell subset with adaptive features, identified by the elevated expression of the activating receptor CD94/NKG2C. NK cells are considered important players in the defence against HCMV, yet the exact mechanisms by which these cells contribute to primary infection and viral reactivation control remain partially understood. In the present study, we have characterized three distinct mechanisms by which NK cells could participate in the defence to HCMV infection. On one hand, we have shown that NK cell direct sensing of HCMV particles leads to the priming of their anti-viral effector functions by mechanisms involving TLR2 and type I IFN. We have also shown that NK cells cooperate with specific antibodies in the recognition of HCMV-infected cells, overriding viral immunoevasion strategies from early to late stages of the lytic cycle. Of note, adaptive NKG2C+ NK cells were highly responsive to Ab-driven activation, being particularly efficient in the production of antiviral cytokines. Finally, we have also shown that the increased expression of HLA class II molecules in circulating adaptive NKG2C+ NK cells endows them with the capacity to present HCMV-derived antigens to specific CD4+ T cells. Remarkably, CD4+ T cells activated in response to HLA-DR+ NK cells displayed an effector memory phenotype, lacked the co-stimulatory molecule CD28 and showed a cytotoxic and Th1 functional profile. Altogether, these results support that the interplay of human NK cells, particularly the adaptive NKG2C+ subset, with specific antibodies and T cells contributes to the defence against HCMV infection. La infección por el citomegalovirus humano (HCMV) induce cambios persistentes en el sistema inmunitario del huésped, consistentes en la expansión de linfocitos T citotóxicos específicos, la producción de anticuerpos y, en algunos individuos, la diferenciación y expansión adaptativa de una subpoblación de células Natural Killer (NK), identificada por la elevada expresión del receptor activador CD94/NKG2C. Se considera que las células NK juegan un papel importante en la defensa contra el HCMV, aunque el conocimiento de los mecanismos con los que contribuyen al control de la infección primaria y de la reactivación viral es parcial. En este estudio, hemos caracterizado tres mecanismos mediados por células NK que pueden contribuir a la defensa frente la infección por HCMV. Por un lado, mostramos que el reconocimiento directo de partículas de HCMV incrementa sus funciones anti-virales mediante mecanismos dependientes de TLR2 e IFN de tipo I. Por otra parte, demostramos in vitro la importancia de la citotoxicidad dependiente de anticuerpos mediada por las células NK como mecanismo de respuesta frente a las células infectadas, superando los mecanismos virales de inmunoevasión. En comparación con la subpoblación NK NKG2A+, las células adaptativas NKG2Cbright mostraron una producción de citocinas anti-virales superior en respuesta a la activación mediada por anticuerpos. Finalmente, describimos como la expresión constitutiva de HLA-DR en células NK NKG2C+ adaptativas circulantes les permite la presentación de antígenos del HCMV a linfocitos T CD4+ específicos con un fenotipo efector-memoria, carentes de la molécula co-estimuladora CD28 y con un perfil funcional Th1 y citotóxico. En conjunto, estos resultados apoyan que la interacción de las células NK, en especial la subpoblación NKG2C+ adaptativa, con anticuerpos y linfocitos T específicos contribuye a la defensa frente al HCMV

Human cytomegalovirus antigen presentation by HLA-DR+ NKG2C+ adaptive NK cells specifically activates polyfunctional effector memory CD4+ T lymphocytes

Natural killer (NK) cells play a dual role in the defense against viral pathogens by directly lysing infected cells as well as by regulating anti-viral T cell immunity. Infection by human cytomegalovirus (HCMV) promotes a persistent expansion of NKG2C+ adaptive NK cells which have been shown to display enhanced antibody-dependent responses against infected targets and associated to viral control in transplanted patients. Based on gene expression data showing increased transcription of CIITA and several genes related to the MHC class II pathway in adaptive NK cells, we explored their putative capacity for antigen presentation to CD4+ T cells. Phenotypic analysis confirmed a preferential steady-state expression of HLA-DR by circulating NKG2C+ adaptive NK cells in healthy individuals. Expression of HLA-DR in NKG2C+ adaptive NK cells was variable and unrelated to the expression of activation (i.e., CD69 and CD25) or differentiation (i.e., FcRγ chain, CD57) markers, remaining stable over time at the individual level. Incubation of purified NK cells with HCMV complexed with serum specific antibodies induced an up-regulation of surface HLA-DR concomitant to CD16 loss whereas no changes in CD80/CD86 co-stimulatory ligands were detected. In addition, surface CX3CR1 decreased upon antigen-loading while HLA-DR+ NK cells maintained a CCR7-, CXCR3low homing profile. Remarkably, HCMV-loaded purified NK cells activated autologous CD4+ T cells in an HLA-DR dependent manner. The fraction of T lymphocytes activated by antigen-loaded NK cells was smaller than that stimulated by monocyte-derived dendritic cells, corresponding to CD28-negative effector-memory CD4+ T cells with cytotoxic potential. Antigen presentation by NK cells activated a polyfunctional CD4+ T cell response characterized by degranulation (CD107a) and the secretion of Th1 cytokines (IFNγ and TNFα). Overall, our data discloses the capacity of NKG2C+ adaptive NK cells to process and present HCMV antigens to memory CD4+ cytotoxic T cells, directly regulating their response to the viral infection.The authors are supported by Plan Estatal I+D Retos (SAF2016-80363-C2-1-R/-2-R), Spanish Ministry of Economy and Competitiveness (MINECO, FEDER); EU FP7-MINECO Infect-ERA program (PCIN-2015-191-C02-01/02); Fundación Española contra el Cáncer (GCB15152947MELE); Proyecto Integrado de Excelencia ISCIII (PIE 2015/00008); and Worldwide Cancer Research Foundation (15–1146)

Adaptive features of natural killer cells in multiple sclerosis

Human cytomegalovirus (HCMV) has been recently related with a lower susceptibility to multiple sclerosis (MS). HCMV promotes an adaptive development of NK cells bearing the CD94/NKG2C receptor with a characteristic phenotypic and functional profile. NK cells are proposed to play an immunoregulatory role in MS, and expansion of the NKG2C(+) subset was recently associated with reduced disability progression. To further explore this issue, additional adaptive NK cell markers, i.e., downregulation of FcεRIγ chain (FcRγ) and PLZF transcription factor, as well as antibody-dependent NK cell activation were assessed in controls and MS patients considering HCMV serology and clinical features. In line with previous reports, increased proportions of NKG2C(+), FcRγ(-), and PLZF(-) CD56dim NK cells were found in HCMV(+) cases. However, PLZF(-) NK cells were detected uncoupled from other adaptive markers within the CD56bright subset from HCMV(+) cases and among CD56dim NK cells from HCMV(-) MS patients, suggesting an additional effect of HCMV-independent factors in PLZF downregulation. Interferon-β therapy was associated with lower proportions of FcRγ(-) CD56dim NK cells in HCMV(+) and increased PLZF(-) CD56bright NK cells in HCMV(-) patients, pointing out to an influence of the cytokine on the expression of adaptive NK cell-associated markers. In addition, proportions of NKG2C(+) and FcRγ(-) NK cells differed in progressive MS patients as compared to controls and other clinical forms. Remarkably, an adaptive NK cell phenotype did not directly correlate with enhanced antibody-triggered degranulation and TNFα production in MS in contrast to controls. Altogether, our results provide novel insights into the putative influence of HCMV and adaptive NK cells in MS.This work was supported by grants FIS/PI17/00254, SAF 2016-80363-C2-1-R (Spanish Ministry of Economy and Competitiveness and FEDER), the EU FP7-MINECO Infect-ERA Program (PCIN-2015-191-C02-01), and Red Española de Esclerosis Múltiple (REEM) from the Instituto de Salud Carlos III, the European Regional Development Fund (Grant RD16/0015/0011), and the Spanish Ministry of Economy and Competitiveness

Pretransplant adaptive NKG2C+ NK cells protect against cytomegalovirus infection in kidney transplant recipients

Cytomegalovirus (CMV) infection constitutes a complication for kidney transplant recipients (KTR) and CMV-specific T cells reduce the risk of viral replication in seropositive patients. CMV promotes the adaptive differentiation and expansion of an NK cell subset, hallmarked by expression of the CD94/NKG2C receptor with additional characteristic features. We previously reported an association of pretransplant NKG2C+ NK cells with a reduced incidence of CMV infection. We have strengthened the analysis in cryopreserved peripheral blood mononuclear cells from an enlarged KTR cohort (n = 145) with homogeneous immunosuppression, excluding cases at low risk of infection (ie, CMV D-R-) or receiving antiviral prophylaxis. Moreover, adaptive NKG2C+ NK cell-associated markers (ie, NKG2A, CD57, Immunoglobulin-like transcript 2 [LIR1 or LILRB1], FcεRI γ chain, and Prolymphocytic Leukemia Zinc Finger transcription factor) as well as T lymphocyte subsets were assessed by multicolor flow cytometry. The relation of NKG2C+ NK cells with T cells specific for CMV antigens was analyzed in pretransplant patients (n = 29) and healthy controls (n = 28). Multivariate Cox regression and Kaplan-Meier analyses supported that NKG2C+ NK cells bearing adaptive markers were specifically associated with a reduced incidence of posttransplant symptomatic CMV infection; no correlation between NKG2C+ NK cells and CMV-specific T cells was observed. These results support that adaptive NKG2C+ NK cells contribute to control CMV infection in KTR.We thank Sara Alvarez and Anna Faura in the Nephrology Research Support team for coordinating sample acquisition from KTR and ESRD patients on transplant waiting list. This study was supported by grants from: Fundació la Marató deTV3 (105/U/2018); Spanish Ministry of Economy and Competitiveness MINECO-FEDER (SAF2016-80363-C2-1-Rand-C2-2-R); Spanish Ministry of Health ISCIII FIS-FEDER (PI13/00598, PI16/00617) and ISCIII RedinRen-FEDER (RD16/0009/0013)

NK cell-triggered CCL5/IFNγ-CXCL9/10 axis underlies the clinical efficacy of neoadjuvant anti-HER2 antibodies in breast cancer

Background: The variability in responses to neoadjuvant treatment with anti-HER2 antibodies prompts to personalized clinical management and the development of innovative treatment strategies. Tumor-infiltrating Natural Killer (TI-NK) cells can predict the efficacy of HER2-targeted antibodies independently from clinicopathological factors in primary HER2-positive breast cancer patients. Understanding the mechanism/s underlying this association would contribute to optimizing patient stratification and provide the rationale for combinatorial approaches with immunotherapy. Methods: We sought to uncover processes enriched in NK cell-infiltrated tumors as compared to NK cell-desert tumors by microarray analysis. Findings were validated in clinical trial-derived transcriptomic data. In vitro and in vivo preclinical models were used for mechanistic studies. Findings were analysed in clinical samples (tumor and serum) from breast cancer patients. Results: NK cell-infiltrated tumors were enriched in CCL5/IFNG-CXCL9/10 transcripts. In multivariate logistic regression analysis, IFNG levels underlie the association between TI-NK cells and pathological complete response to neoadjuvant treatment with trastuzumab. Mechanistically, the production of IFN-ɣ by CD16+ NK cells triggered the secretion of CXCL9/10 from cancer cells. This effect was associated to tumor growth control and the conversion of CD16 into CD16-CD103+ NK cells in humanized in vivo models. In human breast tumors, the CD16 and CD103 markers identified lineage-related NK cell subpopulations capable of producing CCL5 and IFN-ɣ, which correlated with tissue-resident CD8+ T cells. Finally, an early increase in serum CCL5/CXCL9 levels identified patients with NK cell-rich tumors showing good responses to anti-HER2 antibody-based neoadjuvant treatment. Conclusions: This study identifies specialized NK cell subsets as the source of IFN-ɣ influencing the clinical efficacy of anti-HER2 antibodies. It also reveals the potential of serum CCL5/CXCL9 as biomarkers for identifying patients with NK cell-rich tumors and favorable responses to anti-HER2 antibody-based neoadjuvant treatment.AM is supported by ISCiii/FEDER (PI19/00328, PI22/00040 and CIBERONC). The authors are supported by coordinated research projects from Asociación Española contra el Cáncer (GCB15152947MELE). AM and MLB are supported by Generalitat de Catalunya (2017 SGR 888 and 2023 SGR 863). ARe is funded by EC Horizon 2020. Marie Sklodowska Curie-Innovative Training Network (No. 765104; 2018–2021). JA, FR and AR are supported by ISCiii/FEDER (PI18/00006; PI21/00002 and CIBERONC) and by Generalitat de Catalunya (2017 SGR 507; 2021 SGR00776)