ORIGINAL RESEARCH published: 15 October 2019 doi: 10.3389/fimmu.2019.02403 Adaptive Features of Natural Killer Cells in Multiple Sclerosis Antía Moreira 1,2,3 , Elisenda Alari-Pahissa 4 , Elvira Munteis 1 , Andrea Vera 1 , Ana Zabalza 1 , Mireia Llop 1 , Noelia Villarrubia 5 , Marcel Costa-García 4 , Roberto Álvarez-Lafuente 6 , Luisa María Villar 5 , Miguel López-Botet 4,7* and Jose E. Martínez-Rodríguez 1* 1 Neurology Department, Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain, 2 Neurology Department, Althaia, Xarxa Assistencial i Universitària de Manresa, Manresa, Spain, 3 Departament de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 University Pompeu Fabra, Barcelona, Spain, 5 Immunology Department, Hospital Universitario Ramón y Cajal, Madrid, Spain, 6 Neurology Service, Instituto de Investigación Sanitaria del Hospital Clínico de San Carlos, Madrid, Spain, 7 Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain 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 Edited by: Zsolt Illes, CD94/NKG2C receptor with a characteristic phenotypic and functional profile. NK cells University of Southern are proposed to play an immunoregulatory role in MS, and expansion of the NKG2C(+) Denmark, Denmark subset was recently associated with reduced disability progression. To further explore Reviewed by: Catharina C. Gross, this issue, additional adaptive NK cell markers, i.e., downregulation of FcεRIγ chain University of Münster, Germany (FcRγ) and PLZF transcription factor, as well as antibody-dependent NK cell activation Anna Fogdell-Hahn, were assessed in controls and MS patients considering HCMV serology and clinical Karolinska Institute (KI), Sweden Bruno Gran, features. In line with previous reports, increased proportions of NKG2C(+), FcRγ(–), and Nottingham University Hospitals NHS PLZF(–) CD56dim NK cells were found in HCMV(+) cases. However, PLZF(–) NK cells Trust, United Kingdom were detected uncoupled from other adaptive markers within the CD56bright subset from *Correspondence: Miguel López-Botet HCMV(+) cases and among CD56dim NK cells from HCMV(–) MS patients, suggesting miguel.lopez-botet@upf.edu an additional effect of HCMV-independent factors in PLZF downregulation. Interferon-β Jose E. Martínez-Rodríguez therapy was associated with lower proportions of FcRγ(–) CD56dim NK cells in HCMV(+) jmartinezr@hospitaldelmar.cat and increased PLZF(–) CD56bright NK cells in HCMV(–) patients, pointing out to an Specialty section: influence of the cytokine on the expression of adaptive NK cell-associated markers. This article was submitted to In addition, proportions of NKG2C(+) and FcRγ(–) NK cells differed in progressive MS Multiple Sclerosis and Neuroimmunology, patients as compared to controls and other clinical forms. Remarkably, an adaptive NK a section of the journal cell phenotype did not directly correlate with enhanced antibody-triggered degranulation Frontiers in Immunology and TNFα production in MS in contrast to controls. Altogether, our results provide novel Received: 11 June 2019 insights into the putative influence of HCMV and adaptive NK cells in MS. Accepted: 25 September 2019 Published: 15 October 2019 Keywords: multiple sclerosis, natural killer cells, cytomegalovirus, NKG2C, FcεRIγ, PLZF Citation: Moreira A, Alari-Pahissa E, Munteis E, Vera A, Zabalza A, Llop M, INTRODUCTION Villarrubia N, Costa-García M, Álvarez-Lafuente R, Villar LM, Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized López-Botet M and by a highly variable clinical course. Both genetic and environmental factors are involved in MS, Martínez-Rodríguez JE (2019) Adaptive Features of Natural Killer with an influence of herpesvirus infections supported by seroepidemiological studies. Whereas Cells in Multiple Sclerosis. Epstein–Barr virus (EBV) is consistently associated with MS and might play a pathogenic role Front. Immunol. 10:2403. as an environmental trigger (1, 2), human cytomegalovirus (HCMV) was recently related to doi: 10.3389/fimmu.2019.02403 lower MS susceptibility (3–6); however, the basis for these observations remains uncertain. Frontiers in Immunology | www.frontiersin.org 1 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS HCMV establishes a lifelong latent infection whose prevalence lymphocytes on the clinical course (31). In the present study, appears inversely related with socioeconomical development and, we aimed to further explore whether HCMV and adaptive in line with the “hygiene hypothesis,” the reported associations NK cells play a protective role in MS. Adaptive NK cell with MS might be indirectly linked to the exposure to other immunophenotype and antibody-triggered NK cell activation environmental factors. On the other hand, a putative influence against EBV-transformed B cell lines were evaluated in MS of HCMV in MS immunopathology is conceivable (7), as this patients considering their HCMV serostatus and clinical profile, herpesvirus may exert a profound impact on the immune providing further insights into the involvement of NK cells in system (8), and could potentially modulate the response to other this disease. pathogens (i.e., heterologous immunity) (9, 10). Nevertheless, whether HCMV may exert a beneficial or detrimental effect on MATERIALS AND METHODS MS is currently uncertain (11). T lymphocytes and natural killer (NK) cells play Study Design and Population complementary key roles in controlling HCMV, which, A case–control study of MS patients was designed to analyze reciprocally, has developed a variety of immune evasion adaptive NK cell markers and antibody-dependent NK cell mechanisms (12). HCMV has been reported to induce a activation in accordance to HCMV serostatus and clinical persistent reconfiguration of NK cell compartment, both in characteristics. MS patients diagnosed based on McDonald healthy individuals and under pathological conditions. The criteria 2010 from Hospital del Mar, IMIM (Barcelona) and magnitude of this effect is variable and hallmarked by the Hospital Ramón y Cajal (Madrid) (REEM, Spanish Network differentiation and expansion of functionally mature NK for MS research) were recruited from routine clinical visits. cells displaying high levels of the CD94/NKG2C activating In addition, a subgroup of MS patients was prospectively receptor encompassed by additional phenotypic and functional evaluated after onset of interferon-beta (IFNβ) therapy to changes (13–16). Human adaptive NKG2C(+) NK cells assess the influence of this treatment on adaptive NK cells. are contained within the major circulating CD56dim subset, We excluded patients on disease-modifying therapies known which mediates cytotoxicity and cytokine production upon to deplete peripheral lymphocytes or alter their trafficking interaction with target cells, either directly or through IgG (e.g., natalizumab, fingolimod, dimethyl fumarate), as well as engagement of FcγRIIIA (CD16A). As compared to other those undergoing corticosteroid treatment in the last 30 days, subsets, adaptive NKG2C(+) NK cells effectively mediate pregnancy, or any severe concomitant disease. The following antibody-dependent effector functions, particularly cytokine demographic and clinical characteristics were evaluated in every production (17, 18). Additional associated-adaptive NK cell case: age, sex, disease duration, MS form (RRMS: relapsing– markers include epigenetic silencing of the promyelocytic remitting MS, SPMS: secondary progressive MS, PPMS: primary leukemia zinc finger (PLZF) transcription factor, with unknown progressive MS), disability scores (EDSS: Expanded Disability functional implications in NK cells (17, 19–21), and loss of the Status Scale, MSSS: multiple sclerosis severity score) and FcεRI gamma chain (FcRγ) adaptor coupled to CD16, whose treatment. Healthy controls matched for age and sex were replacement by the ζ chain may enhance antibody-dependent NK recruited from the same geographical area. cell activation (17, 19, 22, 23). Some adaptive NK cell-associated The study was approved by the local Ethics Committee, features have also been detected in NKG2C(–) NK cell subsets, including participants after written informed consent. Blood suggesting that NK cell differentiation is independent of NKG2C samples obtained from venous puncture were obtained expression and may be induced by stimuli other than HCMV from controls and MS patients, performing standard (17, 18, 24). Moreover, adaptive NK cell differentiation may clinical diagnostics test to evaluate EBV-(LIASON R ) and not be a coordinated process, as supported by the delayed FcRγ HCMV-specific circulating antibodies (Roche R , Cobas602). loss observed in adaptive NK cells following HCMV infection in hematopoietic stem cell transplantation recipients (25). The Analysis of NK Cell Immunophenotype cellular and molecular mechanisms underlying the development Peripheral blood mononuclear cells (PBMCs) from 47 healthy of adaptive NK cells and their implications in human health controls and 151 MS patients were isolated from blood samples remain partially defined. collected in EDTA tubes using Ficoll–Hypaque density gradient NK cells have been previously studied in the context of centrifugation and cryopreserved in fetal calf serum with 10% MS (26, 27), and changes associated with the response to dimethyl sulfoxide until analysis. Sample staining for flow some immunomodulatory therapies have been reported (28, cytometry analysis was performed using anti-NKG2A (clone 29), proposing an immunoregulatory role played by the minor Z199) indirectly labeled with a secondary goat anti-mouse PE- circulating CD56bright NK cell subset (30). These cells display Cy7 (Biolegend) and the following fluorochrome-conjugated a CD16(–) NKG2A(+) phenotype and have a limited cytotoxic antibodies: anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC capacity but efficiently produce cytokines, constituting the (Biolegend), antiCD16-eFluor450 (eBioscience), and NKG2C- predominant subset in peripheral lymphoid tissues. However, PE (R&D Systems). For the analysis of FcRγ (MS, n = 139; the role of NK cells in MS immunology is only partially controls n = 47) and PLZF expression (MS, n = 86; controls understood. Recently, we described an association of HCMV- n = 26), cells were treated with a fixation/permeabilization kit induced expansion of NKG2C(+) NK cells with a lower risk of (BD Biosciences) followed by incubation with anti-FcRγ-FITC disability progression in MS, suggesting an influence of these (Millipore) and anti-PLZF-PE CF594 (BD Biosciences). Samples Frontiers in Immunology | www.frontiersin.org 2 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS were acquired in LSRFortessa (BD Biosciences) and data were in NK cells. Multivariate lineal regression analysis determined analyzed using FlowJo software (Tree Star, Oregon, USA), using predictors for adaptive NK cell markers adjusting the models the gating strategy shown in Figure 1. for HCMV serostatus, clinical variables, and IFNβ treatment. Results were considered significant at the two-sided level of 0.05. Functional Assessment of Analyses were performed using SPSS v.23 software. Antibody-Dependent NK Cell Activation PBMCs from 42 MS patients (22 RRMS, 8 SPMS and 12 PPMS) and 17 controls matched for HCMV serostatus were RESULTS incubated overnight at 37◦ C with recombinant IL-2 (200 HCMV-Related Expansion of Adaptive NK U/ml). The response of NK cells to the HLA class I- Cells in MS Patients defective 721.221 B-lymphoblastoid cell line with or without We comparatively studied in controls and MS patients the rituximab (50 ng/ml) was assessed following a 4-h incubation influence of HCMV on the expression of adaptive NK cell (E/T = 1/1). A complementary approach was performed using differentiation markers (NKG2C, FcRγ, and PLZF) based on EBV(+) AKBM cells as targets following induction of the the gating strategy for flow cytometry analysis illustrated in lytic cycle in the presence of EBV(+) or EBV(–) sera, as Figure 1. Demographic and clinical characteristics of MS patients previously described (32, 33). Surface expression of CD107 as a and controls are shown in Table 1. In our study, HCMV marker of degranulation and intracellular TNFα production was seroprevalence was comparable in both groups (Table 1), and no analyzed by flow cytometry as previously reported (34), using clinical differences were observed in MS patients associated with the anti-CD107-APC (BD Pharmigen) monoclonal antibody HCMV serostatus (data not shown). during incubation together with monensin (GolgiStop R ; BD) HCMV seropositivity was associated with greater proportions and brefeldin (GolgiPlug R ; BD). Cultures were then stained of NKG2C(+) NK cells, with no significant differences between with anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC-Cy7 MS patients and controls (Figure 2A). The expansion of (Biolegend), and anti-NKG2C-PE (R&D System), permeabilized, NKG2C(+) cells was confined to the CD56dim NK cell fixed and stained intracellularly with anti-TNFα-CFBlue (labeled subset, whereas detection of CD56bright NKG2C(+) NK cells by Immunostep), anti-FcRγ-FITC (Millipore), and anti-PLZF-PE appeared unrelated to HCMV serology (Figure 2A). Similarly, CF594 (BD Biosciences). Data acquisition was performed with an the frequency of CD56dim FcRγ(–) NK cells was also increased LSRFortessa cytometer (BD Biosciences). in HCMV(+) patients and controls as compared to HCMV(–) Multidimensional Flow Cytometry Analysis individuals (Figure 2B). By contrast, HCMV(+) controls and MS patients showed increased proportions of PLZF(–) cells not only Using Barnes-Hut t-SNE in CD56dim NK cells but, remarkably, also among the CD56bright A multidimensional flow cytometry analysis was performed as subset (Figure 2C); these PLZF(–) NK cells were confirmed previously described (35), compensating raw flow cytometry to display the characteristic CD56bright NKG2A(+) CD16(–) data using FlowJo software (Tree Star, Oregon, USA) and phenotype (Supplementary Figure 1). Of note, an increased later imported into R using flowCore and openCyto packages. frequency of PLZF(–) cells was also detected among CD56dim NK Lymphocytes were gated on forward scatter and side scatter cells from HCMV(–) MS patients as compared to seronegative characteristics and then on CD56dim NK cells. FITC channel controls (Figure 2C). Overall, these results support previous was normalized using flowStats R package in order to reduce studies describing the HCMV-related expansion of adaptive NK experimental variability on fluorescence intensity. Subsequently, cells displaying the NKG2C(+), FcRγ(–), and PLZF(–) markers randomly selected data from 500 CD56dim NK cells per sample in CD56dim NK cells; however, a lack of PLZF expression was concatenated. The most positive and negative one per associated to HCMV seropositivity was also perceived in the mille values for each parameter were reduced to their less CD56bright NK cell subset, in both controls and MS patients, in extreme border. Next, Barnes-Hut t-SNE was conducted using addition to an influence of HCMV-independent factors in PLZF the Rtsne package. Graphics were produced using ggplot2 and downregulation in CD56dim NK cells found in MS cases. RcolorBrewer R packages. Statistical Analysis Relationship Between Adaptive NK Cell Normal distribution was assessed using Kolmogorov–Smirnov Markers in MS Patients and Controls test. Continuous variables were expressed as mean ± standard We next evaluated the relation of adaptive NK cell markers deviation (SD) or median (first–third quartile) for parametric in CD56dim NK cells from controls and MS patients. and non-parametric variables, respectively. Relationship between Three-dimensional Venn diagrams illustrated that NK cells continuous and dichotomous variables was assessed by Student’s co-expressing adaptive NK cell markers were increased in t-test or Mann–Whitney U-test, respectively. Pearson or HCMV(+) compared to HCMV(–) MS patients (Figures 3A,B). Spearman correlation coefficients were calculated for pairwise The presence in MS patients of increased proportions of PLZF(–) comparison of continuous variables. Wilcoxon test for paired CD56dim NK cells without other adaptive markers was also samples was used to compare matched prospectively followed- confirmed (Figure 3A). up samples before and after IFNβ therapy. Three-dimensional t-SNE multidimensional analysis of adaptive markers in Venn analysis evaluated the co-expression of adaptive markers CD56dim NK cells (Figure 3C) revealed an HCMV-related Frontiers in Immunology | www.frontiersin.org 3 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS FIGURE 1 | Gating strategy for adaptive NK cells. Lymphocytes were identified based on their forward scatter (FCS) and side scatter (SSC) characteristics, defining NK cells as CD3(–) CD56(+) lymphocytes. Representative examples were selected based on the expression of adaptive NK cell markers, showing a case with a low expression of the three adaptive markers (MS.01), a case with low NKG2C(+), FcRγ(–), and PLZF(–) expression in CD56dim NK cells but with a higher proportions of PLZF(–) CD56bright NK cells (HC.01), and a case with higher proportions of NKG2C(+), FcRγ(–), and PLZF(–) CD56dim NK cells. expression of NKG2C, more pronounced in controls than in MS NKG2C(+) CD56dim NK cells (Figure 4A). However, lower patients. By contrast, NKG2C(–) FcRγ(–) PLZF(–) NK cells were proportions of FcRγ(–) CD56dim NK cells were found in more evident in MS patients than in controls, with a less marked IFNβ-treated patients as compared to untreated cases (9.6% relation to HCMV serology (Figure 3C). These results further ± 8.2 vs. 19.3% ± 19.7, p < 0.05), a finding only perceived point out an uncoupled expression of adaptive markers in NK in CD56dim NK cells from HCMV(+) but not in HCMV(–) cells from MS patients as compared to controls, suggesting the MS cases, who displayed low proportions of FcRγ(–) NK influence of factors other than HCMV. cells independently of treatment (Figure 4B). Interestingly, PLZF(–) CD56bright NK cells tended to increase in IFNβ- Influence of IFNβ Therapy on Adaptive NK treated HCMV(–) patients (Figure 4C), vanishing the differences Cells in RRMS related to HCMV serostatus noticed in both CD56dim and We next evaluated the impact of IFNβ therapy on adaptive CD56bright NK cell subsets (Figure 2C). These results suggest NK cell markers in RRMS (mean time of treatment, 7.0 ± that HCMV-driven adaptive NK cell development in RRMS 4.2 years). As previously reported (36), IFNβ-treated patients might be modulated by IFNβ therapy. However, the above- displayed increased proportions of CD56bright NK cells as mentioned IFNβ-related modifications in adaptive markers were compared to untreated cases (14.8% ± 11.6 vs. 8.3% ± not perceived in a subgroup of MS patients prospectively 7.1, p < 0.05), which appeared unrelated to any of the followed after 1 year of treatment (Supplementary Figure 2), adaptive markers analyzed (data not shown). IFNβ therapy indicating that the cytokine does not directly modify the adaptive did not alter the effect of HCMV on the proportions of NK cell immunophenotype. Frontiers in Immunology | www.frontiersin.org 4 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS TABLE 1 | Demographic and clinical characteristics of MS patients and controls. Controls MS patients P-value* RRMS SPMS PPMS P-value# n = 47 n = 151 n = 88 n = 44 n = 19 Age (years) 46.6 ± 13.3 50.1 ± 11.4 0.08 45.6 ± 10.0 55.0 ± 8.8 59.4 ± 13.0 <0.001 HCMV seroprevalence, n (%) 37 (78.7) 103 (68.7) 0.126 59 (67.8) 33 (75) 11 (57.9) 0.217 EBV seroprevalence, n (%) 40 (87) 147 (98.7) <0.01 86 (98.9) 43 (97.7) 18 (100) 0.152 Sex (female), n (%) 30 (63.8) 101 (66.9) 0.413 59 (67.0) 28 (63.6) 14 (73.7) 0.861 MS duration (years) – 15.1 ± 10.0 11.6 ± 9.1 21.5 ± 8.7 16.2 ± 9.8 <0.001 DMT, n (%) – 50 (33.1) 42 (47.7) 7 (15.9) 1 (5.3) <0.001 DMT-naive, n (%) – 47 (34.1) 22 (28.9) 9 (20.9) 16 (84.2) <0.001 EDSS – 3.5 (2.0–6.0) 2.0 (1.0–3.0) 6.5 (5.5–7.5) 6.0 (4.5–7.0) <0.001 MSSS – 4.23 ± 2.92 2.60 ± 2.20 6.58 ± 2.25 6.56 ± 1.91 <0.001 2y-RR – 0.19 ± 0.41 0.34 ± 0.52 0.01 ± 0.08 0.0 ± 0.0 <0.001 ARR – 0.37 ± 0.41 0.42 ± 0.42 0.52 ± 0.39 0.01 ± 0.02 <0.001 Values are expressed as mean ± SD or median (1st−3rd quartile). HCMV, human cytomegalovirus; EBV, Epstein–Barr virus; DMT, disease-modifying therapy (interferon-beta, n = 45; glatiramer acetate, n = 5), EDSS, Expanded Disability Status Scale; MSSS, multiple sclerosis severity score; 2y-RR, relapsing rate in the previous 2 years; ARR, annualized relapsing rate; RRMS, relapsing–remitting MS; SPMS, secondary progressive MS; PPMS, primary progressive MS. *P-values comparing controls and MS patients using t test or Mann–Whitney U-test. # P-values comparing RRMS, SPMS, and PPMS patients using one-way ANOVA. FIGURE 2 | Expression of adaptive markers in NK cells from controls and MS patients according to HCMV serostatus. (A) Proportions of NKG2C(+), (B) FcRγ(–), and (C) PLZF(–) in CD56dim and CD56bright NK cells from healthy controls (HC) and MS patients according to HCMV serostatus. Higher proportions of NKG2C(+), FcRγ(–), and PLZF(–) in CD56dim NK cells were found in HCMV(+) as compared to HCMV(–) cases (A–C), in addition to higher proportion of PLZF(–) NK cells perceived in the CD56bright subset (C). Increased proportions of PLZF(–) CD56dim NK cells were also found in HCMV(–) MS patients as compared to HCMV(–) controls (C). NKG2C, MS = 151, HC 139; FcRγ, MS = 139, HC = 47; PLZF, MS = 86, PLZF = 26. Individuals with an NKG2C del/del genotype (HC, n = 1; MS, n = 6) were excluded from the analysis of NKG2C expression. P values (Mann–Whitney U test): * <0.05, ** <0.01, *** <0.001, **** <0.0001. Frontiers in Immunology | www.frontiersin.org 5 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS FIGURE 3 | Interrelationship of adaptive NK cell markers in MS. Venn diagrams depicting the proportions of NKG2C(+), FcRγ(–), and PLZF(–) CD56dim NK cells in healthy controls (HC) (n = 26) and MS patients (n = 86) (A), and stratifying results according to HCMV serostatus (B). Values expressed the mean proportion for each group. P-values (Mann–Whitney U-test): ** <0.01, comparing controls vs. MS patients; ##<0.01, comparing HCMV(–) vs. HCMV(+). (C) t-SNE plots of NKG2C, NKG2A, FcRγ, and PLZF expression in CD56dim NK cells evaluated in HC and MS patients according to HCMV serostatus. Distribution of Adaptive NK Cells in MS FcRγ(–) NK cells appeared greater in SPMS patients as Patients According to Clinical Forms compared to RRMS, in both CD56dim and CD56bright Considering MS form, NKG2C(+) CD56dim NK cell proportions NK cells (Supplementary Figure 3B). No differences were were lower in PPMS patients as compared to SPMS patients observed for PLZF expression according to MS form (Supplementary Figure 3A). By contrast, proportions of (Supplementary Figure 3C). After multivariate regression Frontiers in Immunology | www.frontiersin.org 6 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS FIGURE 4 | Influence of IFNβ therapy in the expression of adaptive markers in NK cells from RRMS patients. Proportions of NKG2C(+) (A), FcRγ(–) (B), and PLZF(–) (C) in CD56dim and CD56bright NK cells according to the presence of IFNβ therapy and HCMV serostatus. The influence of IFNβ was mainly perceived in the expression of FcRγ in CD56dim NK cells in HCMV(+) cases, and in PLZF expression, which vanished differences related to HCMV serostatus in both PLZF(–) CD56dim and CD56bright NK cells. NKG2C(+): IFNβ-treated, n = 33, and untreated patients, n = 44; FcRγ: IFNβ-treated patients, n = 37, untreated patients, n = 33; PLZF, IFNβ-treated patients, n = 20, untreated patients, n = 24. Individuals with an NKG2C del/del genotype (RRMS, n = 5) were excluded from the analysis of NKG2C expression. P values (Mann–Whitney U-test): * <0.05, ** <0.01. analysis, the association of MS form with adaptive markers patients (Figure 5B). Comparable results were observed in appeared independent of HCMV and IFNβ-therapy only for the experiments using the EBV-infected AKBM cell line as target proportions of NKG2C(+) CD56dim and FcRγ(–) CD56bright NK (Supplementary Figure 4A). cells (Supplementary Table 1). No additional correlations were We subsequently evaluated antibody-dependent NK cell observed between adaptive NK cell markers and age, disease functions according to MS form (Figure 5C). RRMS patients duration, disability scores, or disease activity (data not shown). displayed increased proportions of degranulating CD56dim NK These results suggest that the expression of some adaptive NK cells as compared with controls and PPMS; moreover, the cell markers may vary in progressive MS forms independently of proportions of TNFα-secreting cells were lower in PPMS as the influence of HCMV and IFNβ therapy. compared to controls and other MS forms (Figure 5C). Similar results for TNFα production were obtained using the AKBM cell Adaptive NK Cell Markers and line (Supplementary Figure 4B). IFNβ therapy in MS patients Antibody-Dependent Activation in MS did not influence the results obtained in these experiments (data In order to evaluate antibody-dependent NK cell functions, not shown). degranulation and TNFα production were measured in Next, we evaluated the relationship of adaptive markers in NK cells from 42 MS patients and 17 controls following CD56dim NK cells and antibody-triggered responses. In controls, stimulation with the 721.221 cell line in the absence or the proportions of NKG2C(+), FcRγ(–), and PLZF(–) CD56dim presence of rituximab (Figure 5A). As expected, CD56dim NK cells appeared unrelated to degranulation, whereas a direct NK cells in controls and MS patients showed a greater correlation with TNFα production was observed (Figure 6). By degranulation and TNFα production in the presence of contrast, the proportions of adaptive NK cells in MS patients rituximab (Figure 5B). In addition, an increased degranulation did not correlate with TNFα secretion and, moreover, an inverse of CD56bright NK cells in the direct response to the relation between degranulation and the proportions of FcRγ(–) 721.221 cell line without rituximab was perceived in MS and PLZF(–) CD56dim NK cells was detected (Figure 6); similar Frontiers in Immunology | www.frontiersin.org 7 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS FIGURE 5 | Antibody-dependent NK cell activation in MS patients and controls. (A) Experimental design evaluating NK cell degranulation (CD107) and intracellular TNFα production in functional experiments using 721.221 lymphoblastoid cell lines as NK cell targets. (B) CD107 expression and TNFα production by CD56dim and CD56bright NK cells in controls and MS patients. (C) CD107 expression and TNFα production by CD56dim NK cells in controls and MS patients according to clinical form. PBMC: peripheral blood mononuclear cells. Controls, n = 17; MS patients, n = 42 (RRMS, 22; SPMS, 8, PPMS 12). P-values (Mann–Whitney U-test for CD107 and Student’s t-test for TNFα expression): * <0.05, ** <0.01, *** <0.001, **** <0.0001. observations were found in CD56bright NK cells from MS patients that antibody-triggered responses were greater in NKG2C(+) (data not shown). An analysis considering the co-expression CD56dim NK cells independently of FcRγ or PLZF expression of adaptive markers in controls and MS patients showed (Supplementary Figure 5). Frontiers in Immunology | www.frontiersin.org 8 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS FIGURE 6 | Correlations between adaptive NK markers and antibody-dependent CD56dim NK cell activation. Spearman rank correlations (R) between proportions of NKG2C(+), FcRγ(–), and PLZF(–) CD56dim NK cells and intracellular TNFα production (A), and CD107 expression (B) in functional experiments using rituximab and 721.221 lymphoblastoid cell lines as NK cell targets. HC, healthy controls; MS, multiple sclerosis. Controls, n = 17; MS patients, n = 42 (RRMS, 22; SPMS, 8, PPMS 12). Overall, these results suggest that adaptive NK cell function (10, 38–40). In line with reports relating HCMV with a reduced may differ in MS according to clinical form, being greater MS susceptibility (3–5), an association of HCMV-driven adaptive in RRMS as compared to progressive MS forms; however, in NKG2C(+) NK cell expansion with a lower risk of clinical contrast to healthy individuals, an adaptive NK cell profile in progression was previously described (31). Yet, it remains MS patients did not correlate with proficient antibody-triggered uncertain whether and how adaptive NK cells might influence the effector functions. development and clinical course of MS. In agreement with extensive observations in healthy blood DISCUSSION donors and different clinical settings, our results pointed out that HCMV infection is the leading factor that promotes the Considering the high prevalence of lifelong HCMV infection development of conventional adaptive NKG2C(+), FcRγ(–), and its broad impact on the immune system (8), the possibility and PLZF(–) CD56dim NK cells in MS patients (17–19, 22, that this herpesvirus might somehow impact the development 23). In previous reports, an uncoupled distribution of the of autoimmune disorders is plausible (37). A lower HCMV expression of some adaptive NK cell-associated markers, with seroprevalence has been reported in studies of pediatric and potential functional implications, was noticed (17, 18, 25). In adult MS populations (3–7, 11), suggesting that this infection this regard, we observed increased proportions of PLZF(–) NK might have a protective effect. Among different hypotheses to cells, but not of NKG2C(+) or FcRγ(–) NK cells, among the explain how HCMV might influence MS, we considered the CD56bright NK cell subset from HCMV(+) individuals. Such involvement of NK cells, which have been previously related phenotype may result from an influence of HCMV at early with the disease (26–30). The virus promotes to a variable stages of adaptive NK cell development, according to the linear extent an adaptive differentiation of NK cells (13–15, 17, model of differentiation from CD56bright NK cells (41), but 19), which effectively mediate antibody-triggered cytotoxicity also of HCMV-unrelated factors (20, 42), as suggested by the and cytokine production, thus potentially contributing to the detection of increased PLZF(–) CD56dim NK cells in HCMV(–) immune response against HCMV and other microbial pathogens MS patients. Frontiers in Immunology | www.frontiersin.org 9 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS No significant differences in NKG2C expression were be involved in MS, as supported by their participation in perceived between controls and progressive forms; yet, the marked antibody-dependent response against EBV-infected lower proportions of NKG2C(+) NK cells in PPMS as cells (33). In this regard, the adaptive NK cell phenotype compared to SPMS were detected, in line with the lower in MS patients was not associated with proficient antibody- NKG2C pattern of expression in MS patients observed in dependent effector functions, in contrast to the pattern of the multidimensional analysis as well as in previous studies response observed in healthy donors. Third, PLZF(–) CD56bright with a larger number of cases (31). An additional factor NK cells were detected in HCMV(+) controls and MS patients related with changes in the expression of adaptive NK cell and, moreover, IFNβ therapy was associated with increased markers in MS was IFNβ therapy, which was associated PLZF(–) CD56bright NK cells in HCMV(–) RRMS. This finding with reduced proportions of CD56dim FcRγ(–) NK cells deserves further attention in the context of the regulatory role in HCMV(+) RRMS patients. Moreover, HCMV-related of CD56bright NK cells and their contribution to control EBV differences in the proportions of PLZF(–) CD56dim and infection (45, 46). CD56bright NK cell subsets vanished in IFNβ-treated cases, an In conclusion, our results provide novel insights into effect related with an increase of PLZF(–) cells in HCMV(–) the putative influence of HCMV in MS involving the NK patients. However, no phenotypic changes in adaptive NK cell compartment. The study shows that the distribution cells were perceived in a subset of RRMS cases prospectively of adaptive NK cell markers in MS may vary not only followed up for 1 year after initiating IFNβ therapy, pointing depending on HCMV seropositivity but also on IFNβ therapy out that this effect likely results from complex mechanisms and disease form, revealing differences in their antibody- eventually requiring a longer exposure to be perceived. dependent activation as compared to healthy individuals. Further studies would be required to assess whether PLZF Further studies are required to explore the involvement of downregulation might be related to clinical response to HCMV in the immune response to EBV in MS patients, IFNβ therapy. and the putative influence of HCMV-unrelated factors in Adaptive NK cells have been reported to effectively mediate PLZF downregulation. antibody-triggered effector functions, particularly cytokine production (22, 23, 34). NK cell activation upon incubation DATA AVAILABILITY STATEMENT with rituximab-coated 721.221 cells was mainly exerted by CD56dim NK cells, without significant differences between The datasets generated for this study will not be made publicly controls and MS patients, as previously reported (43). Other available. The work is based on flow cytometry analysis of clinical variables (i.e., age and IFNβ-therapy) appeared as peripheral blood mononuclear cells. Data will be available well unrelated with antibody-triggered responses mediated by under request. CD56dim NK cells (data not shown). Notwithstanding, NK cell activation differed in MS according to the clinical form, being lower in PPMS as compared to RRMS patients. Similar ETHICS STATEMENT results were obtained using as a target the AKBM cell line, The studies involving human participants were reviewed suggesting a comparable pattern of NK cell response against EBV- and approved by Comité Etic d’Investigacions Cliniques infected targets. In accordance to previous studies addressing (CEIC), Hospital del Mar Medical Research Institute. The the relation of adaptive markers with NK cell antibody- patients/participants provided their written informed consent to triggered function (18, 25, 44), a proficient antibody-dependent participate in this study. activity was related to NKG2C expression in CD56dim NK cells rather than to other adaptive-associated markers. This finding, in conjunction with the low NKG2C expression found AUTHOR CONTRIBUTIONS in progressive MS (31), deserves attention, suggesting that an adaptive NK cell dysfunction may be associated with a risk of AM, EA-P, AV, and JM-R designed the study, executed disability progression. experiments, and performed the statistical analysis. AM, EA-P, Based on our observations, some considerations need to be ML-B, and JM-R contributed to interpretation of the results made regarding the putative connection of HCMV infection and and wrote the final draft that was revised for all authors. AZ, adaptive NK cells with MS immunopathology. First, the ability EM, and ML contributed to data interpretation and critically of NK cells to eliminate activated T lymphocytes suggested that reviewed the manuscript. MC-G performed and contributed they may play a relevant immunoregulatory role in the context to the interpretation of the t-SNE analysis. NV, LV, and RÁ-L of MS (27, 28, 30). Yet, adaptive NK cells do not effectively provided samples, performed serological analysis, and critically exert this function (17), possibly reflecting their expression reviewed the manuscript. of KIRs and ILT2 HLA class I-specific inhibitory receptors. Second, given their CD16-triggered antibody-dependent effector FUNDING functions and proliferation, adaptive NK cells likely participate in the immune response to different microbial pathogens (17, This work was supported by grants FIS/PI17/00254, SAF 19, 22, 40). Thus, HCMV-induced adaptive NK cells might 2016-80363-C2-1-R (Spanish Ministry of Economy and contribute to the control of other herpesviruses proposed to Competitiveness and FEDER), the EU FP7-MINECO Frontiers in Immunology | www.frontiersin.org 10 October 2019 | Volume 10 | Article 2403 Moreira et al. Adaptive NK Cells in MS Infect-ERA Program (PCIN-2015-191-C02-01), and Red SUPPLEMENTARY MATERIAL Española de Esclerosis Múltiple (REEM) from the Instituto de Salud Carlos III, the European Regional Development Fund The Supplementary Material for this article can be found (Grant RD16/0015/0011), and the Spanish Ministry of Economy online at: https://www.frontiersin.org/articles/10.3389/fimmu. and Competitiveness. 2019.02403/full#supplementary-material REFERENCES cells with altered signaling and effector function. Immunity. (2015) 42:443–56. doi: 10.1016/j.immuni.2015.02.008 1. Ascherio A, Munger KL, Lennette ET, Spiegelman D, Hernán MA, Olek MJ, 18. Muntasell A, Pupuleku A, Cisneros E, Vera A, Moraru M, Vilches C, et al. Epstein-Barr virus antibodies and risk of multiple sclerosis: a prospective et al. Relationship of NKG2C copy number with the distribution of study. JAMA. (2001) 286:3083–8. doi: 10.1001/jama.286.24.3083 distinct cytomegalovirus-induced adaptive NK cell subsets. 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Novartis, Almirall, Bayer, and Genzyme. EM has received personal fees for Neurol Sci. (2007) 28:121–6. doi: 10.1007/s10072-007-0803-3 consulting services and lectures from Merck-Serono, Biogen Idec, Teva, Genzyme, 37. Halenius A, Hengel H. Human cytomegalovirus and autoimmune disease. Novartis, Bayer, and Almirall. AZ has received travel funding from Biogen Biomed Res Int. (2014) 2014:1–15. doi: 10.1155/2014/472978 Idec, Novartis, and Genzyme. RÁ-L has received honoraria for lectures from 38. Kheav VD, Busson M, Scieux C, Peffault de Latour R, Maki G, Haas Merck-Serono, Novartis, and Biogen Idec, and grants from Merck-Serono, Teva, P, et al. Favorable impact of natural killer cell reconstitution on chronic Sanofi-Avertis, Biogen Idec, and Bayer. LV reports personal fees for consulting graft-versus-host disease and cytomegalovirus reactivation after allogeneic services from Biogen Idec, and grants from Merck-Serono, Biogen Idec, Teva, hematopoietic stem cell transplantation. Haematologica. (2014) 99:1860–7. and Genzyme. JM-R has participated as principal investigator in pharmaceutical doi: 10.3324/haematol.2014.108407 company-sponsored clinical trials including Novartis, Roche, Merck-Serono, 39. Redondo-Pachón D, Crespo M, Yélamos J, Muntasell A, Pérez-Sáez MJ, Pérez- Actelion, and Celgene, and personal fees for consulting services and lectures from Fernández S, et al. Adaptive NKG2C + NK cell response and the risk of Novartis, Biogen Idec and Merck-Serono. cytomegalovirus infection in kidney transplant recipients. J Immunol. (2017) 198:94–101. doi: 10.4049/jimmunol.1601236 The remaining authors declare that the research was conducted in the absence of 40. Hart GT, Tran TM, Theorell J, Schlums H, Arora G, Rajagopalan S, any commercial or financial relationships that could be construed as a potential et al. Adaptive NK cells in people exposed to Plasmodium falciparum conflict of interest. correlate with protection from malaria. J Exp Med. (2019) 216:1280–90. doi: 10.1084/jem.20181681 Copyright © 2019 Moreira, Alari-Pahissa, Munteis, Vera, Zabalza, Llop, Villarrubia, 41. Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, Costa-García, Álvarez-Lafuente, Villar, López-Botet and Martínez-Rodríguez. This et al. Expression patterns of NKG2A, KIR, and CD57 define a process of is an open-access article distributed under the terms of the Creative Commons CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood. Attribution License (CC BY). The use, distribution or reproduction in other forums (2010) 116:3853–64. doi: 10.1182/blood-2010-04-281675 is permitted, provided the original author(s) and the copyright owner(s) are credited 42. Tesi B, Schlums H, Cichocki F, Bryceson YT. Epigenetic regulation and that the original publication in this journal is cited, in accordance with accepted of adaptive NK cell diversification. Trends Immunol. (2016) 37:451–61. academic practice. No use, distribution or reproduction is permitted which does not doi: 10.1016/j.it.2016.04.006 comply with these terms. Frontiers in Immunology | www.frontiersin.org 12 October 2019 | Volume 10 | Article 2403