ORIGINAL RESEARCH published: 25 January 2021 doi: 10.3389/fimmu.2020.611522 NK Cell Subsets Changes in Partial Remission and Early Stages of Pediatric Type 1 Diabetes Laia Gomez-Muñoz 1, David Perna-Barrull 1, Adrian Villalba 1, Silvia Rodriguez-Fernandez 1, Rosa-Maria Ampudia 1, Aina Teniente-Serra 1, Federico Vazquez 2, Marta Murillo 3, Edited by: Jacobo Perez 4, Raquel Corripio 4, Joan Bel 3 and Marta Vives-Pi 1* Alessandra Fierabracci, 1Immunology Service, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona, Badalona, Spain, Bambino Gesù 2Endocrinology Service, Germans Trias i Pujol University Hospital, Badalona, Spain, 3 Pediatrics Service, Germans Trias i Children Hospital (IRCCS), Italy Pujol University Hospital, Badalona, Spain, 4 Department of Pediatric Endocrine, Parc Tauli Hospital Universitari, Institut Reviewed by: d’Investigació i Innovació Parc Taulı´ I3PT, Universitat Autonoma de Barcelona, Sabadell, Spain Matthias G. Von Herrath, La Jolla Institute for Immunology (LJI), United States Type 1 diabetes (T1D) is a chronic metabolic disease characterized by the autoimmune Antonino Bruno, MultiMedica (IRCCS), Italy destruction of b-cells in the pancreatic islets. T1D is preceded by islet-specific Mariella Della Chiesa, inflammation led by several immune cells. Among them, natural killer (NK) cells are University of Genoa, Italy emerging as important players in T1D development. Human NK cells are characterized Eddie A. James, Benaroya Research Institute, by CD56 and CD16 expression, which allows classifying NK cells into four subsets: 1) United States CD56dimCD16+ or effector NK cells (NKeff); 2) CD56brightCD16− or regulatory NK cells *Correspondence: (NKreg); 3) intermediate CD56brightCD16+ NK cells; and 4) CD56dimCD16− NK cells, whose Marta Vives-Pi mvives@igtp.cat function is not well determined. Since many studies have shown that T1D progression is associated with changes in various immune cell types, we hypothesize that the kinetics of Specialty section: NK cell subsets in the blood could correlate with different stages of T1D. To that aim, This article was submitted to NK and Innate pediatric patients newly diagnosed with T1D were recruited, and peripheral NK cell Lymphoid Cell Biology, subsets were analyzed by flow cytometry at several disease checkpoints: disease onset, a section of the journal partial remission (PR), 8 months (for non-remitters), and 12 months of progression. Our Frontiers in Immunology results showed that total NK cells and their four subsets are altered at the early stages of Received: 29 September 2020 Accepted: 04 December 2020 T1D. A decrease in the counts and percentage of total NK cells and NKeff cells at the Published: 25 January 2021 different disease stages was found when compared to controls. These results suggest the Citation: extravasation of these cells into the islets at disease onset, which is maintained throughout Gomez-Muñoz L, Perna-Barrull D, Villalba A, Rodriguez-Fernandez S, the follow-up. By contrast, NKreg cells increased during the early stages after T1D onset, Ampudia R-M, Teniente-Serra A, and both intermediate NK cells and CD56dimCD16- NK cells diminished at the PR stage, Vazquez F, Murillo M, Perez J, which might reflect the immunoregulatory attempts and could be candidate biomarkers Corripio R, Bel J and Vives-Pi M (2021) NK Cell Subsets Changes in Partial for this stage. Also, CD56dimCD16- NK cells increased during T1D progression. Finally, Remission and Early Stages of changes in CD16 expression were identified in the different T1D stages, highlighting a Pediatric Type 1 Diabetes. Front. Immunol. 11:611522. CD16 expression reduction in total NK cells and NKeff cells 1 year after diagnosis. That doi: 10.3389/fimmu.2020.611522 may reflect a state of exhaustion after multiple cell-to-cell interactions. Altogether, our Frontiers in Immunology | www.frontiersin.org 1 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes preliminary data provide a longitudinal picture of peripheral NK cell subpopulations during the different T1D stages, which could be potential candidate biomarkers indicators of disease progression. Keywords: NK cell subpopulations, type 1 diabetes, partial remission, pediatrics, autoimmunity INTRODUCTION functional intermediate stage of maturation between regulatory and effector NK cells (17), and CD56dimCD16− NK cells, a Type 1 diabetes (T1D) is an autoimmune disease caused by the unique non-classical subset that is not fully characterized. destruction of the insulin-producing b-cells. T1D most often Abnormalities in peripheral blood NK cell frequency and activity arises in children, and after diagnosis, up to 80% of pediatric have been described in patients with T1D (18, 19). The reduced patients experience a spontaneous and transient partial frequencies of NK cells found in recently diagnosed patients with remission (PR) phase, also known as honeymoon, which can T1D (20–22) probably correlate with an increase of these cells in the last up to 2 years (1). This interesting period is defined by low insulitis and pancreatic lymph nodes, thus reflecting the chronic requirements of exogenous insulin and diminished glycated aggression. However, the analysis of different NK cell subsets or hemoglobin (HbA1c) (2, 3), and could be the result of their activation status could contribute to the elucidation of the metabolic and immunological interactions that allow the exact role of NK cells in different stages of T1D, including the PR recovery of b-cell function. Because early stages of T1D are phase. In this regard, CD16 is a molecule found on the membrane of often chosen for immune interventions (4), to characterize new NK cells, especially in all CD56dim NK cells, which is involved in biomarkers and cell subset changes at this time-point is essential. antibody-dependent cell cytotoxicity (ADCC), triggering NK Autoreactive T cells are key mediators of b-cell destruction, but degranulation and the killing of target cells (23). Activated NK other leukocytes—macrophages, dendritic cells, natural killer (NK) cells lose CD16 expression on the membrane, probably to modulate cells, and B lymphocytes—are determinant in the NK cell function (24). immunopathogenesis of the disease, especially at the initial stages Since many studies have shown that the progression of T1D is of the autoimmune attack. Among the innate immune cells, NK associated with changes in various immune cell types and little is cells can participate in the initiation and maintenance of known about the contribution of the NK cell subsets in the autoimmune responses (5–7). These cells modulate several innate different T1D stages, this pilot study is aimed at analyzing the and adaptive components by their cytotoxic activity, which is frequency and absolute counts of these cells in peripheral blood regulated through a balance of activator and inhibitory receptors, at key time-points and to assess their CD16 expression in order and by the production of cytokines and chemokines (8). In T1D, to characterize their effector cytolytic activity. NK cells infiltrate the islets, being one of the first immune cells to extravasate into the pancreas, and can be cytolytic for b-cells. Studies in experimental models support an active role of NK cells in T1D pathogenesis (9–11), and most data point to NK MATERIALS AND METHODS contribution to the aggressivity of insulitis and the acceleration of the disease (12). However, their role in human T1D is controversial, Study Population with both protective and destructive functions (13, 14). This fact Pediatric patients with T1D at disease onset (n = 17) and age- could be explained by the different functions of NK cell subsets. and sex-matched non-diabetic control subjects (n = 17) were In human peripheral blood, NK cells are defined by the lack of included in this study. All patients fulfilled the American CD3 and the expression of the cell adhesion molecule CD56. Diabetes Association classification criteria for T1D (25). Moreover, based on the cell surface density of CD56 and the Inclusion criteria were 4–18 years of age and normal body expression of CD16 (also known as FcgRIII), NK cells can be mass index (BMI) according to the Spanish BMI pediatric subdivided into four different subsets (15, 16). Comprising cohort growth chart (26). Exclusion criteria were being under around 90% of circulating NK cells, CD56dimCD16+ or effector immunosuppressive or anti-inflammatory treatment, the NK (NKeff) cells possess a potent cytotoxic activity, while the presence of other autoimmune diseases, type 2 diabetes, remaining 10% characterized by CD56brightCD16- or regulatory pregnancy, compromised kidney function, or liver diseases. All NK (NKreg) cells, mainly produce and secrete diverse cytokines— the experiments were carried out in strict accordance with the including the immunoregulatory ones. The other two subsets are principles outlined in the Declaration of Helsinki for human CD56briCD16+ or intermediate NK cells, which represent a research and after the approval of the Committee on the Ethics of Research of the Germans Trias i Pujol Hospital and Hospital Parc Taulı́. Legal representatives of all participants signed Abbreviations: ADCC, antibody-dependent cell cytotoxicity; BMI, Body Mass informed consent forms at the beginning of the study. Index; HbA1c, Glycated Hemoglobin; IDAA1c, Insulin Dose-Adjusted HbA1c; M, Months; MFI, Median Fluorescence Intensity; NK, Natural Killer; NKeff, Effector Natural Killer; NKG2D, Natural Killer Group 2 member D; NKreg, Regulatory Study Design Natural Killer; NOD, Non-obese Diabetic; PBMCs, Peripheral Blood Blood samples were obtained at three different time-points Mononuclear Cells; PR, Partial Remission; T1D, Type 1 Diabetes. through T1D progression: at disease onset (n = 17), at PR (n = Frontiers in Immunology | www.frontiersin.org 2 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes 11) or 8 months (8M) for non-remitter patients (n = 6), and 12 LSR Fortessa Flow Cytometer (BD Biosciences). The gating months (12M) after disease onset (n = 6). At disease onset, strategy to analyze specific NK cell subsets was based on samples were collected between 1 and 14 days after diagnosis. At international consensus (28). Data were analyzed using this stage, all patients with T1D were tested for autoantibodies to FACSDiva software (BD Biosciences). The CD56 briCD16- GAD65, IA-2, and ZnT8. Patients were considered to be in PR population was used as an internal control population in the when they fulfilled the accepted criteria of ≤9 insulin dose- analysis of the MFI of CD16. adjusted HbA1c (IDAA1c), an index that is calculated as HbA1c (%) + [4 × insulin dose (U/kg/day)] (3). Longitudinal data Statistical Analysis collection occurred over 1 year. So far, only six patients have Cell concentration, percentage, and MFI data were imported into completed the year of follow-up since diagnosis and were R with the pzfx R package. Data reformatting, filtering, and included in this study. exploration were assessed with tidyverse and ggplot2 suites of packages. A generalized linear mixed model was fit with Sample Collection condition, time or time × remission as fixed effects and sample Blood samples of 6 ml from T1D patients were collected at three identifier and individual as random effect covariates using the longitudinal time-points in EDTA tubes (BD Biosciences, San glmer function in the lme4 R package (29), and the proportion of Jose, CA, USA) and processed within 6 h. Control samples from cell counts in the cluster of interest relative to total input cell non-diabetic subjects (sex and age-related) were obtained population as response variable following a binomial distribution simultaneously to the diagnosis of children with T1D. as implemented in the CyTOF workflow (30). Sex and age were excluded from the final model due to a lack of significant effects. Metabolic Parameters Contrast p-values were generated with the NK fraction. Values HbA1c was determined by high-performance liquid were analyzed referred to either total NK cell counts or PBMC chromatography (ADAMS A1c HA-8180V, Arkray, MN, USA) input cell counts. Differences in ratios between CD56dimCD16− in all patients at each time-point. Basal non-fasting C-peptide and NKreg relative to NKeff or total NK were tested with a linear was determined by ELISA (Architect i2000, Abbott, IL, USA) in mixed model with the lmer function in the lme4 package with a both controls and patients at each time-point. logarithmic transformation to approximate normality by Shapiro testing. CD16 MFI was normalized applying an arcsinh HLA Class II Typing (hyperbolic inverse sine) transformation with 150 cofactor; HLA typing of DRB1 alleles was determined in all patients at the samples with missing data were removed. Both cellular count Immunology Service by hybridization with sequence-specific ratios and CD16 MFI were analyzed using the same Gaussian oligonucleotide (SSO) probes (LABType SSO, One Lambda, CA, model with condition as the fixed effect and individual as a USA), following the manufacturer’s instructions. According to random effect, as previously described (30). To find statistically HLA-DRB1 allele combinations, three groups of HLA-DRB1 significant correlations between parameters, Spearman’s test was genotypes were formed: high-risk (positive for both HLA- used. A p-value of ≤0.05 was considered significant. DRB1*03 and HLA-DRB1*04 or homozygous for these alleles), moderate risk (positive for HLA-DRB1*03 or HLA-DRB1*04), and neutral risk (negative for HLA-DRB1*03 and HLA-DRB1*04). RESULTS Flow Cytometry Phenotypic analysis and the median fluorescence intensity (MFI) Clinical Features of Pediatric Patients With of CD16 of NK cell subpopulations in fresh peripheral blood T1D Throughout the Study were performed through multicolor flow cytometry with a four- Clinical data from control subjects and patients with T1D at each laser LSR Fortessa cytometer (BD Biosciences). For flow time-point are summarized in Table 1. All patients were positive cytometry assays, quality control procedures were implemented for autoantibodies to GAD65, IA-2, and/or ZnT8. No statistically weekly. Briefly, 1–2 ml of peripheral whole blood were washed significant differences were found in age and BMI when with 15 ml of FACSFlow Sheath Fluid (ThermoFisher Scientific), compared between control subjects and patients at disease and 100 ml were incubated with the corresponding monoclonal onset; as expected, they were found in terms of plasma C- antibodies. For NK phenotyping, the panel of antibodies was peptide concentration. Differences at PR stage were found in built as follows (27): CD45 AF700 (clone HI30, BioLegend), CD3 BMI, HbA1c, insulin dose, C-peptide, and IDAA1c values when APCH7 (clone SK7, BD Biosciences), CD19 APCH7 (clone compared to recently diagnosed patients. These differences were HIB19, BD Pharmingen), CD14 V450 (clone MjP9, BD not found 12M after the onset, time at which the plasma C- Horizon), CD16 APC (clone B73.1, BD Pharmingen), and peptide concentration reached the minimum values. Non- CD56 PE (clone MY31, BD Biosciences). After incubating the remitter patients (8M) presented higher HbA1c and IDAA1c cells for 20 min, erythrocytes were lysed for 7 min (Lysing Buffer, values than remitter patients, although they were still lower than BD Biosciences). Absolute counts (cells/ml) were analyzed for all those of newly diagnosed patients. Moreover, non-fasting C- subsets using Perfect Count Microspheres of known peptide remained as low as at the onset of the disease. concentration (Cytognos SL, Salamanca, Spain). At least 10.000 HLA typing of DRB1 alleles was determined in patients with leukocyte events per sample and 5.000 beads were acquired using T1D to determine genetic predisposition for the disease. As Frontiers in Immunology | www.frontiersin.org 3 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes expected, 50% of the patients presented a high-risk HLA-DRB1 subjects (Figure 2B). By contrast, NKreg cells tended to increase genotype and 44% of them were DRB1*03 or *04 positive, having in percentage in patients with T1D at all the time-points when a moderate risk for T1D. Only one patient was negative for compared to control subjects, although no statistically significant DRB1*03 and *04 (neutral risk genotype) (Table 1). differences were observed. When compared to the disease onset, these cells also tended to present higher levels at the PR phase NK Cells and Their Effector, Regulatory, and 8M of disease progression (for non-remitter patients), and Intermediate NK Subsets Are Altered reaching the highest level at 12M. Concerning absolute counts, at the Early Stages of T1D patients at disease onset showed decreased levels when compared To analyze the frequency and absolute counts of the four NK cell to control subjects (p ≤ 0.05), and NKreg cells recovered at PR subsets in T1D patients at disease onset, PR, no PR (8M), and and 8M of disease progression, reaching in the last case a after 12M of progression, NK cells were detected as scatter-gated significant difference when compared to patients at T1D onset peripheral blood mononuclear cells (PBMCs), CD3-CD19-, (p ≤ 0.01) (Figure 2C). Furthermore, we found that remitter CD56+, CD14− and CD16+ or − (Supplementary Figure 1). patients and patients at 12M tended to diminish the percentage Representative plots for CD56 and CD14 on CD3−CD19− cells of intermediate NK cells, while non-remitter patients maintained and for CD56 and CD16 on total NK cells of the different groups or even increased their levels in comparison to the control are depicted in Figure 1. subjects or patients at disease onset. Concerning the absolute Figure 2 shows quantitative alterations of NK cell subsets in counts, intermediate NK cells significantly decreased at the PR peripheral blood at all the described groups and time-points, as stage when compared to control subjects (p ≤ 0.05). At 12M, well as changes per patient during the year follow-up. As for total lower counts similar to those of the PR can be observed NK cells, percentages referring to total PBMCs were decreased in (Figure 2D). patients at disease onset (p ≤ 0.001), PR (p ≤ 0.01), 8M for non- Although when looking at the data individually, heterogeneity remitter patients (p = 0.07), and 12M (p ≤ 0.01) when compared can be found between patients in all the different NK cell subsets to control subjects. However, non-remitter patients showed a analyzed, the majority of them tended to increase the percentage higher percentage of NK cells in comparison to patients at onset and concentration of NKreg at PR, 8M, and 12M when compared and patients undergoing PR, although non-significant. Their to T1D onset. In fact, the ratios between NKreg cells and NKeff absolute counts also followed the same tendency, with lower cells and between NKreg cells and total NK cells were both counts of NK cells at disease onset (p ≤ 0.001), PR (p ≤ 0.01), and significantly higher at the PR phase in comparison to the ratios 12M (p ≤ 0.01) when compared to control subjects (Figure 2A). of control subjects (p ≤ 0.01) and disease onset (p ≤ 0.05). Also, Referring to total NK cells, we also found diminished percentages the 12M time-point showed both higher ratios than control of NKeff cells in patients with T1D at all the different time-points subjects (p ≤ 0.05) and T1D onset (p ≤ 0.05) (Figure 3). when compared to control subjects, reaching a statistically When referring NK cell subsets to total PBMCs, percentages significant decrease at 12M (p ≤ 0.05). Moreover, their of NKeff cells were significantly decreased in patients at disease absolute counts also decreased at disease onset (p ≤ 0.001), PR onset (p ≤ 0.001), PR (p ≤ 0.01), and 12M (p ≤ 0.01) when (p ≤ 0.01), and 12M (p ≤ 0.01) when compared to control compared to control subjects (Supplementary Figure 2A). Like TABLE 1 | Clinical data for pediatric control subjects and patients with T1D at disease onset, PR, 8M (no PR), and 12M of progression. Control subjects Patients at Disease Patients at PR (n = 11) Patients at 8M (no PR) (n = 6) Patients at 12M (n = 6) (n = 17) onset (n = 17) Age (years) 8.8 ± 3.3 8.7 ± 3.6 9 ± 4.1 8.5 ± 3 9.3 ± 3.6 Sex (M/F) 7/10 7/10 5/6 2/4 2/4 BMI (kg/m2) 18.3 ± 4.3 16.8 ± 2.5 17.7 ± 3.0* 17.2 ± 2.1 17 ± 1.7 HbA1c (%) ND 11.4 ± 2.4 6.8 ± 0.6** 8.1 ± 0.7*/xx 7.6 ± 1 HbA1c (mmol/mol) ND 101.3 ± 26.2 51.5 ± 6.4** 64.5 ± 7.4*/xx 59.2 ± 10.8 Insulin dose (U/Kg/day) ND 0.7 ± 0.2 0.4 ± 0.1** 0.85 ± 0.1*** 0.7 ± 0.1 C-peptide (ng/ml) 1.3 ± 0.4 0.3 ± 0.2++++ 0.7 ± 0.5++ 0.25 ± 0.1+++ 0.1 ± 0.1+++ IDAA1c (%) ND 14.3 ± 3 8.4 ± 0.5** 11.5 ± 1*/xxx 10.2 ± 1.4 Autoantibodies IA-2A (+/−) ND 13/2 ND ND ND Autoantibodies GAD65A (+/−) ND 13/2 ND ND ND Autoantibodies ZnT8A (+/−) ND 11/6 ND ND ND High-risk HLA-DRB1 genotype ND 8/16 ND ND ND Moderate risk HLA-DRB1 ND 7/16 ND ND ND genotype Neutral HLA-DRB1 genotype ND 1/16 ND ND ND Data are presented as mean ± SD; p-value calculated from Mann-Whitney test when comparing controls subjects and pediatric patients at the different time-points ( p ≤ 0.01, +++p ≤ ++ 0.001, and ++++p ≤ 0.0001) or from Wilcoxon test when comparing between patients at the different time-points (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for comparison with disease onset and xxp ≤ 0.01 and xxxp ≤ 0.001 for comparison with PR). PR, partial remission; M, male; F, female; BMI, body mass index; HbA1c, glycated hemoglobin; IDAA1c, insulin dose-adjusted HbA1c; IA-2A, anti-insulinoma antigen-2 autoantibody; GAD65A, anti-glutamic acid decarboxylase 65 autoantibody; ZnT8A, anti-zinc transporter 8 autoantibody; HLA, human leukocyte antigen; ND, not determined. Frontiers in Immunology | www.frontiersin.org 4 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes FIGURE 1 | Representative plots of NK subsets changes at the initial stages of T1D. Representative staining for CD56 and CD14 on CD3−CD19− cells and for CD56 and CD16 on total NK cells of control subjects (first row) and patients at disease onset (second row), at partial remission (PR, third row), 8 months without partial remission (8M no PR, fourth row) and at 12 months after onset (12M, fifth row). Frontiers in Immunology | www.frontiersin.org 5 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes A B C D FIGURE 2 | Peripheral blood NK subsets are quantitatively altered at the initial stages of T1D. Concentration (cells/µl, left panel) and percentages (%, right panel) of (A) total NK cells, (B) effector NK (NKeff) cells, (C) regulatory NK (NKreg) cells, and (D) intermediate NK cells. Dots represent control subjects, and patients are represented by squares at disease onset, triangles at partial remission (PR), rhombuses at 8 months without partial remission (8M no PR), and asterisks at 12 months after onset (12M). Lines link the same patient throughout the different time-points. Boxes indicate the median, first and third quartile range, and whiskers indicate the overall range without outliers of n ≥ 5 individuals per group. (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, generalized linear mixed model). Frontiers in Immunology | www.frontiersin.org 6 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes FIGURE 3 | Changes in regulatory NK cells at initial stages of T1D. NKreg cells/Total NK cells and NKreg cells/NKeff cells ratios were measured for each control subject (dots) and patient with T1DM at different time-points: disease onset (squares), partial remission (PR, triangles), 8 months without partial remission (8M no PR, rhombuses), and 12 months after onset (12M, asterisks). Lines link the same patient throughout the different time-points. Boxes indicate the median, first and third quartile range, and whiskers indicate the overall range without outliers of n ≥ 5 individuals per group. (*p ≤ 0.05 and **p ≤ 0.01, linear mixed model). total NK cells, these diminished percentages were not observed in regarding these cells, it is interesting to observe how they clearly non-remitter patients, who showed a higher percentage of NKeff increased in number at 12M in those patients that undergo the PR cells in comparison to the disease onset and patients undergoing phase. By analyzing the ratios of CD56dimCD16− NK cells/Total NK PR, although non-significant. Regarding NKreg cells, their cells and CD56dimCD16− NK cells/NKeff cells, we obtained that percentages in peripheral blood significantly decreased at T1D patients at the PR phase presented both lower ratios than control onset in comparison to control subjects (p ≤ 0.05) and tended to subjects (p ≤ 0.01) and in the case of the CD56dimCD16− NK cells/ recover again at the PR phase. In non-remitter patients, a NKeff cells ratio, than patients at disease onset (p ≤ 0.05). We also significant recovery can be observed at 8M of disease obtained higher ratios at 12M of disease progression than those of progression when compared to the disease onset (p ≤ 0.01). the other conditions, although not significant (Figure 4B). Nevertheless, this increase might not be maintained over the Altogether, these results indicate alterations in both concentration months (Supplementary Figure 2B). Furthermore, we found and percentage of the CD56dimCD16− NK cell subset during the that the percentage of intermediate NK cells tended to decrease at early progression of pediatric T1D. disease onset and at the PR phase, reaching in this last case a When referring CD56dimCD16− NK cells to total PBMCs, statistically significant reduction when compared to control percentages of these cells were significantly increased at 12M of subjects (p ≤ 0.05), but not at 8M of disease progression in the T1D progression when compared to the onset (p ≤ 0.01). Also, case of non-remitter patients (Supplementary Figure 2C). At non-remitter patients showed a higher percentage of this subset 12M it can also be observed lower percentages when compared to in comparison to the remitter ones (p ≤ 0.05) (Supplementary control subjects. Figure 2D). CD56dimCD16- NK Cell Subset Is Altered NK Cells and Their Subsets Do Not at 12 Months of T1D Progression Correlate With Clinical Features at T1D CD56dimCD16− NK cells are a unique and not fully characterized Onset subset of non-classical NK cells. First, albeit no differences in In the control subjects group, a positive correlation between the percentages were found between the control group and T1D percentage of NKeff cells and age (Spearman’s r = 0.542, p = onset, a trend to increase CD56dimCD16− NK cells can be noted. 0.0301) and a negative correlation between the percentage of While the percentages of these cells did not differ between patients NKreg cells and age (Spearman’s r = −0.6144, p = 0.0113) were at onset and remitters or non-remitters, a clear increase can be found (Figure 5A). However, these correlations were lost in T1D observed at 12M of disease progression when compared to the other patients. Also, no significant correlations were found between time-points, and especially when compared to the control subjects total NK cells or their subsets and BMI, HbA1c, and C-peptide at (p ≤ 0.01). Regarding their concentration, CD56dimCD16- NK cells T1D onset, neither in absolute counts nor in percentages. significantly increased at 12M in comparison to patients at disease However, C-peptide tended to positively correlate with the onset (p ≤ 0.01). Interestingly, a trend to expand this subset in percentage of total NK cells (% of PBMCs) (Spearman’s r = peripheral blood with disease progression can be noted, and at 8M, 0.44, p = 0.0771) (Figure 5B). At PR, absolute counts of both non-remitter patients presented higher counts than remitter total NK cells and NKeff cells tended to positively correlate with patients (p ≤ 0.05) (Figure 4A). Although when looking at the BMI values (respectively, Spearman’s r = 0.6061, p = 0.0633 and data individually heterogeneity can be found between patients Spearman’s r = 0.6214, p = 0.0552) (Figure 5C). Frontiers in Immunology | www.frontiersin.org 7 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes A B FIGURE 4 | CD56dimCD16- NK cell subset is altered along with the progression of T1D. (A) Concentration (cells/µl, left panel) and percentages (%, right panel) of total CD56dimCD16− NK cells. (B) Ratios between CD56dimCD16− NK cells and total NK cells (left panel) and between CD56dimCD16− NK cells and effector NK (NKeff) cells (right panel). Dots represent control subjects, and patients are represented by squares at disease onset, triangles at partial remission (PR), rhombuses at 8 months without partial remission (8M no PR), and asterisks at 12 months after onset (12M). Lines link the same patient throughout the different time-points. Boxes indicate the median, first and third quartile range, and whiskers indicate the overall range without outliers of n ≥ 5 individuals per group. (*p ≤ 0.05 and **p ≤ 0.01, generalized linear mixed model and linear mixed model). Modulation of CD16 Expression at Early progression expressed lower CD16 MFI levels than NKeff cells of Stages of T1D control subjects (p ≤ 0.01) and patients at disease onset (p ≤ To determine ADCC potential, CD16 expression levels in the NK 0.001), and these cells of non-remitter patients also reduced the cell membrane were assessed by flow cytometry. A tendency to a expression of CD16 in comparison to the remitter ones (p ≤ 0.01) lower CD16 expression over time in patients with T1D can be (Figure 6, middle graph). Finally, in the intermediate NK cells, observed on both total NK cells and NKeff cells. Total NK cells of CD16 membrane expression tended to be higher at disease onset patients at 12M of disease progression expressed lower CD16 in comparison to control subjects. Following the same tendency MFI levels than NK cells of control subjects (p ≤ 0.01) and as the NKeff cells, CD16 also tended to be reduced at the PR patients at T1D onset (p ≤ 0.01). Furthermore, these cells of non- phase; however, intermediate NK cells of non-remitter patients remitter patients exhibited a lower CD16 expression in are marked by a reduction in the membrane expression of CD16 comparison to those of remitter patients (p ≤ 0.01) (Figure 6, in comparison to all the other groups, especially to both patients upper graph). CD16 expression was determined in the two at disease onset (p ≤ 0.01) and patients at 12M (p ≤ 0.05) (Figure positive subsets, NKeff cells, and intermediate NK cells. The 6, lower graph). Although heterogeneity can be found between MFI of CD16 in NKeff cells was similar to that of total NK remitter and non-remitter patients, we can observe that the cells. In the same way, NKeff cells of patients at 12M of disease majority of both of them tended to decrease CD16 expression Frontiers in Immunology | www.frontiersin.org 8 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes A B C FIGURE 5 | Correlations between NK cells and age, C-peptide, and BMI. (A) NKeff cells and NKreg cells percentages of control subjects are shown in correlation to age (years) (respectively, Spearman’s r = 0.542, p = 0.0301 and Spearman’s r = −0.6144, p = 0.0113). (B) NK cell percentages of patients at disease onset are shown in correlation to basal C-peptide (ng/ml) (Spearman’s r = 0.44, p = 0.0771). (C) NK cells and NKeff cells absolute counts of patients at the PR phase are shown in correlation to BMI (kg/m2) (respectively, Spearman’s r = 0.6061, p = 0.0633 and Spearman’s r = 0.6214, p = 0.0552). (r and p-values by Spearman’s correlation analysis, *p ≤0.05). at 12M of disease progression on total NK and NKeff cells, while relied on a simple and fast multicolor flow cytometry assay to its expression is decreased on intermediate NK cells both at PR study four NK cell subsets through the first year of T1D and 8M (more remarkably) in comparison to the disease onset. progression in pediatric patients. We report here that NK cells are diminished in patients with T1D during this follow-up. Moreover, we have observed crucial DISCUSSION changes in the four NK cell subsets at the different studied time- points, which could reflect the immunoregulatory attempts that Despite different strategies capable of stopping the autoimmune patients undergo during the PR phase. Also, membrane process and promote b-cell recovery have been developed in the expression of CD16 in NK cells differed at the different disease last years, none of them has achieved total remission in humans. stages, specifically between remitters and non-remitters and 1 This failure could be partially explained by the lack of biomarkers year after diagnosis. that allow the proper stratification of patients, the identification The variations found in NK cell subsets both in concentration of patients with a better glycemic prognosis, and the discovery of and percentage reflect the dynamic changes of the innate optimal checkpoints for immune interventions, e.g., the immune system at the early stages of T1D, as recently spontaneous PR phase. In this sense, alterations of immune described (20). Following previous studies, our results cell subsets in peripheral blood, including NK cells, could be showed a decrease in the number and percentage of total NK candidate biomarkers for disease staging and for understanding cells and NKeff cells at disease onset when compared to control the immunoregulatory changes that take place at the early stages subjects (18, 20, 21), while the absolute counts of PBMCs are of the disease. Specifically, NK cells have been detected in the similar between groups (data not shown). This reduction is pancreas of both experimental models and patients with T1D, sustained throughout the study follow-up, both at the PR and specific subsets of these cells are related to an increased risk phase and one year after T1D onset. The presence of NK cells of developing the disease (31). The functional differences of NK in healthy human pancreatic islets evidences the innate capacity cell subpopulations could explain their also reported dual of these cells to migrate to the pancreas (36) as well as in behavior, one that promotes b-cell destruction and another response to inflammatory mediators (37). In this sense, the that protects against the autoimmune attack (14, 15, 32). immunophenotyping of the insulitis in T1D patients revealed Nowadays, recent advances in single-cell RNA sequencing the presence of NK cells (38, 39). The reduction in peripheral NK analysis has revealed novel NK cell subsets in human peripheral cells at T1D onset is often correlated to an activated phenotype, blood, allowing their deep characterization and a better and these alterations are no longer observed in individuals with stratification of these cells than the classical cytometry-based long-standing disease (40). Because NK cells are among the first approach (33, 34). Also, this technique provides new insights ones to migrate to the pancreas upon cytokine activation, we into NK cell biology (35). However, biomarkers for clinical use propose that the reduction in NKeff cells we observed in the must be quickly and easily obtained. In the present study, we periphery could be explained by an active migration of these cells Frontiers in Immunology | www.frontiersin.org 9 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes FIGURE 6 | CD16 membrane expression is modulated depending on the phase of the disease. Median of fluorescence intensity (MFI) of CD16 surface expression on NK cells (upper graph), NK effector cells (NKeff cells, middle graph), and intermediate NK cells (lower graph). Dots represent control subjects, and patients are represented by squares at disease onset, triangles at partial remission (PR), rhombuses at 8 months without partial remission (8M no PR), and asterisks at 12 months after onset (12M). Lines link the same patient throughout the different time-points. Boxes indicate the median, first and third quartile range, and whiskers indicate the overall range without outliers of n ≥ 5 individuals per group. (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, linear mixed model). into the pancreas and draining nodes. Taking into account the NK data from experimental models (10, 11, 32, 41, 42), these results point to an active role of these cells in the disease pathogenesis: in the islets, they will exert a cytotoxic function against b-cells, thus contributing to disease development. In fact, NK cells are responsible for accelerated T1D in non-obese diabetic (NOD) mice (12). Additionally, in this model, pancreatic NK cells were found to exhibit higher levels of activation markers, a more marked mature phenotype and greater proliferation rates in comparison to other mouse strains, which supports a role for these cells in promoting b- cell destruction (32). However, controversial results have been reported in humans. One study described in subjects from 6 months to 2 years after disease onset a decreased responsiveness of NKeff cells to IL-2 and IL-15 stimulation and aberrant signaling through the activating natural killer group 2 member D (NKG2D) receptor (19), which could also explain the reduction in peripheral NK cell frequency. Given that the chronic exposure to ligands recognizing their activating receptors can result in NK cell hyporesponsiveness (43–45), we propose that the observed NK cell reduction after 1 year of follow-up could be explained by their loss of functionality. Indeed, another study demonstrated that the reduction in NK cell activation (including NKeff and NKreg cells) in patients with T1D occurs approximately 1 year after the onset of the disease and that the lower expression of NKG2D in these cells was not a genetic defect, suggesting an indirect modulatory effect (40). Alternatively, NK cells regulate adaptive immunity by suppressing autoreactive T cell responses and proliferation and by killing over-stimulated lymphocytes, macrophages, or dendritic cells (46, 47). In this sense, the here reported follow- up of pediatric patients with T1D showed a tendency to increase NKreg cells in blood during the first stages of T1D, especially the first months after the disease onset, when the PR occurs. That might reflect an attempt at immunoregulation during the chronic autoimmune attack at secondary lymphoid organs, as NKreg cells can mold the autoreactive response through anti- inflammatory cytokines such as IL-10 (48). Actually, CD56bright NK cells are the predominant subset in secondary lymphoid organs since they express CCR7 and CD62L (49). In other autoimmune diseases, such as multiple sclerosis, these FIGURE 6 | Continued CD56bright NK cells exercise a regulatory role by suppressing Frontiers in Immunology | www.frontiersin.org 10 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes self-reactive T cell responses and they expand after a successful period, thus increasing in blood and contributing less to the response to immunotherapies (50). In NOD mice treated with autoimmune attack. Interestingly, it has been shown that patients Complete Freund’s Adjuvant, NK cells were the ones to suppress with diabetic ketoacidosis at diagnosis display a lower frequency of b-cell-specific T cells and to prevent diabetes (51). Nevertheless, NKreg cells (21). Rodacki et al. found a tendency of NK cells to the exhaustion of these cells over time might result in sustained negatively correlate with HbA1c in recently diagnosed but not in macrophage and b-cell-specific T cell activation and enhanced long-standing patients (40), suggesting that NK cell reduction is not proinflammatory responses, contributing in this way to the end solely caused by the loss of glucose control. Nevertheless, further of the PR stage. research is needed to confirm the impact of glucose metabolism on Regarding intermediate NK cells, a transitional form between NK cell subsets. NKreg and NKeff cells with cytotoxic capacity, the observed reduction Further in this study, we determined the expression of CD16 at the PR phase could be due to the immunomodulation of early because it can hint at the NK cell functional state as this receptor stages. It has been described that T-cell derived cytokines and is involved in ADCC. As mentioned before, CD16 on NK cells cell-cell contacts are needed for the acquisition of a functional suffers a shedding process after cell activation and its expression CD16 and the transition of NKreg cells into the intermediate NK is reduced after multiple cell-to-cell interactions, leading NK cells subset (17, 49). Therefore, the immunoregulatory attempts to a state of exhaustion (24, 56). This effect could be the reason during the PR phase may be dampening this conversion. for the observed reduction in CD16 expression on total NK cells Finally, CD56dimCD16− NK cell subset increased through and NKeff cells in patients with T1D after diagnosis. Despite it is disease progression and non-remitter patients showed higher difficult to establish if this reduction is caused by NK cell numbers of these cells than the remitter ones. In pediatric degranulation and CD16 shedding or by the downregulation of healthy subjects, this subset is more abundant in bone marrow CD16 membrane expression, these results could support the than in peripheral blood (52), but it is not fully characterized in progression to a state of hyporesponsiveness after 1 year of terms of function and ontogeny. However, antitumor cytotoxic disease evolution. Concerning intermediate NK cells, this subset activity and effector functions stronger than other NK subsets increases the expression of CD16 during the differentiation have been demonstrated (53, 54). Therefore, the increase in these process from NKreg to NKeff cells (57). In our study, these cells during T1D progression could reflect their higher migration cells showed a tendency to increase CD16 membrane expression from bone marrow towards peripheral blood and possibly the at disease onset in comparison to controls. This might reflect a pancreas, where they could exert their cytotoxic functions against strong cytotoxic profile and the ongoing differentiation into b-cells. On the contrary, this phenomenon could be less frequent NKeff cells. Although non-remitter patients at 8M had similar during the PR phase due to the lower peripheral counts found intermediate NK cell percentages as the disease onset, we found a in this stage, contributing in this way to the immunoregulation. remarkable decrease in CD16 membrane expression when The source of this subset is controversial, and they can arise compared both to the T1D onset and 12M. That could be from either precursor NK cells (CD56−CD16−) that start to explained by a higher number of NKreg cells differentiating into express the CD56 marker or NKeff cells that lose membrane intermediate NK cells in non-remitter patients (which would still CD16 after they have been activated, thus becoming express lower levels of CD16) since no immunomodulatory effect is CD56dimCD16− cells (24). Both mechanisms could contribute stopping this process. This transition could also be prompted by the to the here reported progressive increase of these cells during the necessity to resupply the exhausted NKeff cell pool. At 12 months, early stages of T1D. the recovery of CD16 membrane expression could reflect an In order to assess whether metabolism affects NK cell variations, enhanced cytotoxic activity of these cells and their conversion several correlations were determined. No statistically significant into the NKeff subset (17). correlations were observed between age, BMI, C-peptide, or We are well aware of the limitations of the present study. The HbA1c, and the different NK cell subsets of T1D patients in this main ones are the lack of functional experiments that makes it study. As previously described (55), we also have detected purely descriptive and the small sample size that lowers the statistically significant correlations in control subjects between the statistical power. Also, another major weak point of our study age and the percentage of NKeff (positive correlation) and NKreg population is the wide range of ages (from 4 to 18) and the high cells (negative correlation). Although our pediatric cohort presents a variability that this entails regarding the physiological changes wide range of ages, having found no correlations between this that the subjects undergo (e.g., puberty, among others). Despite parameter and the different NK cell subsets in T1D reflects the this, longitudinal data were analyzed with a generalized linear immunological imbalance present in patients regardless of the age. mixed model, and age was excluded from the final model due to a While other studies showed a negative correlation between absolute lack of significant effects. Moreover, the p-value of the NK counts of NK cells and the concentration of C-peptide (20), we subsets determines statistical differences and the obtained observed that C-peptide tended to positively correlate with the results are in accordance with those of previous studies (20– percentage of total NK cells at disease onset. Moreover, during the 22). As mentioned before, functional studies are required in PR phase, absolute counts of both total NK cells and NKeff cells order to elucidate potential mechanisms of action of NK cell tended to positively correlate with higher BMI values. These results subsets and to deepen more in the characterization of reliable might reflect that better glycemic control and the removal of glucose and reproducible biomarkers for the PR phase. The higher levels toxicity could impact the migration of NK cells during the remission of NKreg cells found during the first months after the disease Frontiers in Immunology | www.frontiersin.org 11 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes onset or the diminished concentration of CD56dimCD16− and AUTHOR CONTRIBUTIONS intermediate NK cells in remitter patients could be potential candidate biomarkers reflecting the immunoregulatory attempts LG-M, DP-B, AV, SR-F, AT-S, and MV-P designed the at the early stages of T1D. However, this work should be experiments. FV, MM, JP, RC, and JB selected the patients and considered as a pilot study, and further examination in larger obtained the samples. LG-M, DP-B, AV, R-MA, and SR-F cohorts is needed to validate our observations. Furthermore, the performed the experiments; LG-M, DP-B, AV, SR-F, and analysis of the targeted organ and other cell subsets would be MV-P analyzed the data and wrote the manuscript. All authors beneficial to clarify the exact role of each of the NK subsets and to contributed to the article and approved the submitted version. link peripheral NK cell variations with the pancreatic events. Nevertheless, the strength of this study is the reported longitudinal picture of NK cell subsets during T1D FUNDING progression, including the analysis at the PR phase, which enables us to explore the underlying pathophysiology and This work has been funded by the Spanish Government (FIS understand the immunoregulation during the early stages. PI18/00436) co-financed with the European Regional Overall, the here reported results show clear alterations of Development funds (FEDER), and by DiabetesCero Foundation. peripheral NK cell subsets during the progression of T1D, which can reflect both their involvement in the autoimmune attack and their contribution to the immunoregulatory attempts during the ACKNOWLEDGMENTS PR stage. We are grateful to N. Real from the Pediatrics Service of the Hospital Universitari Germans Trias i Pujol (HUGTiP) for DATA AVAILABILITY STATEMENT sample obtaining. We really appreciate the help of M. Martı́nez, B. Quirant, and Dr. E. Martı́nez-Cáceres from Immunology Service The original contributions presented in the study are included in (HUGTiP) and of Prof L. Castaño and Dr. S. Rivero from Molecular the article/Supplementary Material. Further inquiries can be Genetics Lab (Hospital Universitario Cruces, Barakaldo) for directed to the corresponding author. determining autoantibodies. Special thanks to Mr. M Fernandez and G. Requena from Flow Cytometry IGTP Platform for their support, and Dr. L. Sumoy from Bioinformatics Facility (IGTP) for helping with the statistical analysis. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Committee on the Ethics of Research of the SUPPLEMENTARY MATERIAL Germans Trias i Pujol Research Institute and Hospital (HUGTiP) and Hospital Parc Taulı́. Written informed consent The Supplementary Material for this article can be found online to participate in this study was provided by the participants’ legal at: https://www.frontiersin.org/articles/10.3389/fimmu.2020. guardian/next of kin. 611522/full#supplementary-material REFERENCES 7. Popko K, Gó rska E. The role of natural killer cells in pathogenesis of autoimmune diseases. Cent Eur J Immunol (2015) 40(4):470–6. 1. Fonolleda M, Murillo M, Vá zquez F, Bel J, Vives-Pi M. 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Leukemia (2005) 19(5):835–40. doi: 10.1038/sj.leu.2403704 Frontiers in Immunology | www.frontiersin.org 13 January 2021 | Volume 11 | Article 611522 Gomez-Muñoz et al. NK and Pediatric Autoimmune Diabetes 54. Stabile H, Nisti P, Peruzzi G, Fionda C, Pagliara D, Brescia PL, et al. Conflict of Interest: MV-P holds a patent that relates to immunotherapy for T1D Reconstitution of multifunctional CD56lowCD16low natural killer cell and is co-founder of Ahead Therapeutics S.L. SR-F is part-time employed at subset in children with acute leukemia given a/b T cell-depleted HLA- Ahead Therapeutics S.L. haploidentical haematopoietic stem cell transplantation. Oncoimmunology The remaining authors declare that the research was conducted in the absence of (2017) 6(9):e1342024. doi: 10.1080/2162402X.2017.1342024 any commercial or financial relationships that could be construed as a potential 55. Mahapatra S, Mace EM, Minard CG, Forbes LR, Vargas-Hernandez A, conflict of interest. Duryea TK, et al. High-resolution phenotyping identifies NK cell subsets that distinguish healthy children from adults. PLoS One (2017) 12(8): e0181134. doi: 10.1371/journal.pone.0181134 Copyright © 2021 Gomez-Muñoz, Perna-Barrull, Villalba, Rodriguez-Fernandez, 56. Srpan K, Ambrose A, Karampatzakis A, Saeed M, Cartwright ANR, Guldevall K, Ampudia, Teniente-Serra, Vazquez, Murillo, Perez, Corripio, Bel and Vives-Pi. This is et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts an open-access article distributed under the terms of the Creative Commons serial engagement of target cells. J Cell Biol (2018) 217(9):3267–83. doi: 10.1083/ Attribution License (CC BY). The use, distribution or reproduction in other forums jcb.201712085 is permitted, provided the original author(s) and the copyright owner(s) are credited 57. Abel AM, Yang C, Thakar MS, Malarkannan S. Natural Killer Cells: and that the original publication in this journal is cited, in accordance with accepted Development, Maturation, and Clinical Utilization. Front Immunol (2018) academic practice. No use, distribution or reproduction is permitted which does not 9:1869. doi: 10.3389/fimmu.2018.01869 comply with these terms. Frontiers in Immunology | www.frontiersin.org 14 January 2021 | Volume 11 | Article 611522