ORIGINAL RESEARCH published: 25 July 2019 doi: 10.3389/fimmu.2019.01732 B-Lymphocyte Phenotype Determines T-Lymphocyte Subset Differentiation in Autoimmune Diabetes Edited by: Urs Christen, Leire Egia-Mendikute 1† , Berta Arpa 1† , Estela Rosell-Mases 1 , Marta Corral-Pujol 1 , Goethe University Frankfurt, Germany Jorge Carrascal 1 , Jorge Carrillo 1 , Conchi Mora 1 , Harold Chapman 2 , Anaïs Panosa 3 , Reviewed by: Marta Vives-Pi 4,5 , Thomas Stratmann 6 , David Serreze 2* and Joan Verdaguer 1,5* Ezio Bonifacio, Dresden University of 1 Immunology Unit, Department of Experimental Medicine, Faculty of Medicine, IRBLleida, University of Lleida, Lleida, Spain, Technology, Germany 2 The Jackson Laboratory, Bar Harbor, ME, United States, 3 Microscopy and Flow Cytometry Facility, IRBLleida, Universitat de Kathryn Haskins, Lleida, Lleida, Spain, 4 Immunology Section, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona, University of Colorado Denver, Barcelona, Spain, 5 CIBER of Diabetes and Associated Metabolic Diseases, Instituto de Salud Carlos III, Madrid, Spain, United States 6 Department of Cell Biology, Physiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain Thomas William Kay, St. Vincents Institute of Medical Research, Australia Previous studies indicate that B-lymphocytes play a key role activating diabetogenic *Correspondence: T-lymphocytes during the development of autoimmune diabetes. Recently, two David Serreze transgenic NOD mouse models were generated: the NOD-PerIg and the 116C-NOD dave.serreze@jax.org Joan Verdaguer mice. In NOD-PerIg mice, B-lymphocytes acquire an activated proliferative phenotype joan.verdaguer@mex.udl.cat and support accelerated autoimmune diabetes development. In contrast, in 116C-NOD † These authors share first authorship mice, B-lymphocytes display an anergic-like phenotype delaying autoimmune diabetes onset and decreasing disease incidence. The present study further evaluates the Specialty section: T- and B-lymphocyte phenotype in both models. In islet-infiltrating B-lymphocytes This article was submitted to Autoimmune and Autoinflammatory (IIBLs) from 116C-NOD mice, the expression of H2-Kd and H2-Ag7 is decreased, Disorders, whereas that of BAFF, BAFF-R, and TACI is increased. In contrast, IIBLs from a section of the journal NOD-PerIg show an increase in CD86 and FAS expression. In addition, islet-infiltrating Frontiers in Immunology T-lymphocytes (IITLs) from NOD-PerIg mice exhibit an increase in PD-1 expression. Received: 28 December 2018 Accepted: 09 July 2019 Moreover, proliferation assays indicate a high capacity of B-lymphocytes from NOD-PerIg Published: 25 July 2019 mice to secrete high amounts of cytokines and induce T-lymphocyte activation Citation: compared to 116C B-lymphocytes. This functional variability between 116C and PerIg Egia-Mendikute L, Arpa B, Rosell-Mases E, Corral-Pujol M, B-lymphocytes ultimately results in differences in the ability to shape T-lymphocyte Carrascal J, Carrillo J, Mora C, phenotype. These results support the role of B-lymphocytes as key regulators of Chapman H, Panosa A, Vives-Pi M, T-lymphocytes in autoimmune diabetes and provide essential information on the Stratmann T, Serreze D and Verdaguer J (2019) B-Lymphocyte phenotypic characteristics of the T- and B-lymphocytes involved in the autoimmune Phenotype Determines T-Lymphocyte response in autoimmune diabetes. Subset Differentiation in Autoimmune Diabetes. Front. Immunol. 10:1732. Keywords: type 1 diabetes, NOD mouse, transgenic mouse model, B-lymphocyte phernotype, T-lymphocyte doi: 10.3389/fimmu.2019.01732 phernotype Frontiers in Immunology | www.frontiersin.org 1 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes INTRODUCTION effect or functions, thus indicating why the development of the disease in both mice models is so different. CD4+ and CD8+ T-lymphocytes are considered to be the major effectors of ß-cell damage during the development of autoimmune diabetes in both NOD mice and humans. MATERIALS AND METHODS However, T-lymphocytes require the intervention of antigen Mice presenting cells (APCs), such as B-lymphocytes, which drive NOD, NOD-PerIg, and 116C-NOD mice were maintained by and modulate their response. The involvement of B-lymphocytes brother-sister mating under specific pathogen-free conditions in autoimmune diabetes development was proved when disease at the University of Lleida. This study was carried out in protection was observed in NOD mice after introducing accordance with the principles of the Basel Declaration and the Igµnull mutation or after anti-B lymphocyte antibody recommendations of the Catalan Government (Generalitat treatments (1–8). Further studies indicated that B-lymphocytes de Catalunya) concerning the protection of animals for promote autoimmune diabetes development through their experimentation. The protocol was approved by the Committee APC function activating ß-cell-reactive cytotoxic T-lymphocytes on the Ethics of Research in Animal Experimentation of the (CTLs) (9, 10). In parallel to this diabetogenic role, a protective University of Lleida, Spain. Protocol #: CEEA 02-04/16. effect of some B-lymphocytes on autoimmunity through a potential regulator/suppressor mechanism has been described. Diabetes Incidence Adoptive transfer of LPS-activated splenic B-lymphocytes Mice were checked weekly for glycosuria with Glucocard strips protects prediabetic NOD female mice from disease development (Meranini, Barcelona, Spain). After two positive readings (>3+) by triggering the apoptosis of ß-cell-reactive CTLs or/and and with a confirmation of glucose levels on blood (higher values inhibiting APC activity (11). than 250 mg/dL), mice were considered diabetic. Two transgenic NOD mouse models, the 116C-NOD and the NOD-PerIg mice (12, 13), have been recently generated Flow Cytometry to analyze the natural B-lymphocyte immune response during Splenocytes and islet-infiltrating leukocytes from 6-week- autoimmune diabetes development. On the one hand, the old female NOD, 116C-NOD and NOD-PerIg mice were 116C-NOD mouse model expresses the Ig genes from an obtained as described elsewhere (16) and then analyzed by islet-infiltrating B lymphocyte of a diabetes resistant but flow cytometry using FACS CANTO II instrumentation (BD, insulitis prone (8.3-NODxNOR)F1 mouse with pancreatic Biosciences, San Jose, CA) and Flowjo (version 8.7) software islet ß-cell specificity (14). Autoimmune diabetes incidence [see also Supplementary Figure 2 for histogram analysis for is decreased in 116C-NOD mice compared to NOD mice fluorescence minus one (FMO) control of each staining and (Supplementary Figure 1). B-lymphocytes from 116C-NOD for a representative analysis of transcription factor expression mice display an anergic-like phenotype but retain the ability in CD4+ T lymphocytes]. CD19-V450 (75-0193-U100) was to express some co-stimulatory molecules after activation and obtained from Tonbo Biosciences (San Diego, CA). CD3- induce a T cell shift toward a Th17 phenotype. On the other hand, FITC (561798), CD40-Biotin (553789), CD8-Biotin (553033), the NOD-PerIg mouse expresses a representative Ig of a large CD4-PerCp (553052), CD279-APC (562584), CD274-PacificBlue proportion of naturally occurring islet-infiltrating B-lymphocytes (564715), CD86-PE (561963), CD80-Biotin (09602D), I-Ak- in NOD mice recognizing the neuronal antigen peripherin Biotin (553539) which cross-reacts with I-Ag7, H-2Kd -Biotin (14, 15). In contrast to 116C-NOD, B-lymphocytes from NOD- (553564), Fc(CD16/CD32)-PeCy7 (560829), and streptavidin- PerIg mice acquire an activated proliferative phenotype and amCyan (563261) were purchased from BD Biosciences (San promote an accelerated autoimmune diabetes development Jose, CA). Fas-APC (152603) was supplied by Biolegend (San (Supplementary Figure 1). Despite the differences observed Diego, CA). TACI-Biotin (ABIN1169259) and BAFF-R-Biotin in autoimmune diabetes development, B-lymphocytes from (ABIN1169050) were purchased from Antibodies-online.com 116C-NOD and NOD-PerIg still share certain phenotypic and (USA). BAFF-APC (130-111-029) was obtained from Miltenyi functional similarities, such as an enlargement of the marginal Biotec (Madrid, Spain). For transcription factors and APRIL zone B-lymphocyte subset, and stimulation-induced cytokine intracellular staining, mononuclear cells were permeabilized production. Thus, the present study aims at studying in depth following the manufacturer’s instructions of the FoxP3 Stainning the functional and phenotypic differences between T- and B- Buffer Set (00-5523-00, ebioscience) and using the following lymphocytes in both mouse models, to better understand the set of MoAbs to T-bet-Pe/Cy7 (25-5825-82), RORgt-APC mechanisms underlying the function of both cell populations (53-9966-42), FoxP3-PacificBlue (48-5773-82), GATA3-FITC in inhibiting or accelerating autoimmune diabetes development. (53-9966-42), all from ebioscience (San Diego, CA), and APRIL- The results reveal variations in cytokine production and APC (130-105-388) from Miltenyi Biotec (Madrid, Spain). expression of MHC, as well as costimulatory and inhibitory molecules in T- and B-lymphocytes between both mouse models Proliferation Assays that consistently result in differences in their ability to modulate Splenocytes from 6-week-old female NOD, BDC2.5-NOD, T-lymphocyte responses. The results also highlight the capacity of 116C-NOD, and NOD-PerIg mice were obtained mechanically B-lymphocytes to shape T-lymphocyte phenotypes and show that disrupting their spleens, and red cells were lysed in 0.87% T- and B-lymphocytes of both transgenic mice exert disparate ammonium chloride solution. Spleens from BDC2.5-NOD mice Frontiers in Immunology | www.frontiersin.org 2 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes were donated by Dr. Pau Serra from his mouse colony at down-regulated in both IIBLs and splenic B cells from 116C- the Institut d’Investigacions Biomèdiques August Pi i Sunyer, NOD, whereas only MHC class II was down-regulated in Barcelona, Spain. T- and B-lymphocytes were purified by splenic B cells from NOD-PerIg mice compared to wild type negative selection using MACS T- and B-lymphocyte isolation NOD mice (Figure 1). In regard to costimulatory molecules, reagent (130-095-130 and 130-090-862 respectively, Myletnyi the percentages of IIBLs positive for CD86 and APRIL were Biotec, Madrid, Spain) following the manufacturer’s instructions. lower, but higher for BAFF, BAFF-R, and TACI in 116C- The purity of T- and B-lymphocytes was always above NOD when compared to wild type NOD and/or NOD-PerIg 90%. For B proliferation assays, previous CFSE (C34554, life mice (Figure 1). In fact, the number of IIBLs positive for technologies) labeled B-lymphocytes were incubated in 96-well BAFF-R and TACI was almost absent in NOD-PerIg. Since tissue culture plates (3 × 105 /well) at 37◦ C in 5% CO2 in TACI acts as an inhibitor for lymphocyte activation through complete culture medium using the following stimuli: 10 ug/ml BAFF/BAFF-R interaction, these results suggest that 116C- LPS (L3012-5MG, Sigma-Aldrich Inc., St. Louis, MO), or 10 NOD IIBLs cannot induce activation signals for T cells. In ug/mL anti-CD40 (553787, BD Biosciences, clone 3/23) plus 10 contrast, the high percentage of IIBLs with up-regulation of U/mL rIL-4 (404-ML-010, R&D Systems, Minneapolis, MN), costimulatory molecules suggests a high capacity of these cells or anti-IgM, affinity pure F(ab′ )2 (JAC-715-006-020, Jackson from NOD-PerIg mice to be potential T-lymphocyte activators. Immunoresearch, West Grove, PA). After 48 h of culture, the The level and number of Fas+ IIBLs was higher in NOD- phenotype of B-lymphocytes was analyzed by flow cytometry PerIg compared to NOD and 116C-NOD mice, thus suggesting and culture supernatants were stored at −80◦ C until use. that IIBLs from NOD-PerIg mice exhibit a high activation A minimum of four animals per group was analyzed. For status. An increased percentage of BAFF+ B-lymphocytes is T-lymphocyte proliferation assays, previous CFSE labeled T- observed in the spleen of NOD-PerIg when compared to 116C- lymphocytes from spleens of NOD or BDC2.5-NOD mice were NOD and wild type NOD mice. A number of other less co-cultured (3 × 105 cell/well) with purified B-lymphocytes pronounced differences in the percentage or the expression (3 × 105 cell/well) of NOD, NOD-PerIg or 116C-NOD of above mentioned molecules in splenic B-lymphocytes to check B-lymphocyte capacity to promote T-lymphocyte were also observed between mouse strains (Figure 1). The proliferation. 10 ug/mL of anti-CD3 MoAb (553058, BD), or expression of PD-1 and PD-L1 coinhibitory molecules was 20 ug/mL of 2.5HIP peptide (LQTLALWSRMD) synthesized then tested in IIBLs and splenic B-lymphocytes from 116C- by the proteomics and protein chemistry unit of Pompeu NOD and NOD-PerIg mice. No differences were observed in Fabra University (Barcelona), was also added to cultures the percentage of PD-1+ B-lymphocytes between mice. On or anti-CD3 coated plates to boost T-lymphocyte activation. the contrary, the percentage of PD-L1+ in IIBLs was lower Seventy-two hours later, cells were analyzed by flow cytometry in all three mouse models (Figure 1) suggesting low ability and culture supernatants stored at −80◦ C until use. All of B-lymphocytes to down regulate T-lymphocyte activity in B- and T-lymphocyte proliferation assays were performed islet pancreatic-infiltrate. in duplicate. In addition to the phenotypic changes, the ability to respond to different stimuli determines the functional capacity of B- Cytokine Profile Analysis lymphocytes. As previously demonstrated, 116C B-lymphocytes Culture supernatants from proliferation assays were collected have a decreased ability to proliferate but can still secrete some and IL-2, IL-4, IL-6, IFN-γ, IL-10, TNF-α, and IL-17 cytokines in response to LPS, anti-CD40 + IL-4, and anti- cytokine amounts analyzed by flow cytometry using CBA BCR (13). Proliferation and cytokine secretion analysis under kit (560485, BD). the same stimuli were carried out with splenic B-lymphocytes from NOD-PerIg, 116C-NOD, and wild type NOD mice to Statistics compare their activation capacity. Significant differences were PRISM Graphpad software was used for analysis. Statistics were observed in the proliferation assays (Figure 2A). Interestingly, performed using log-rank (Mantel-Cox) and Mann-Whitney compared to 116C-NOD and wild type NOD mice, NOD- U-tests. All mean values ± SD are shown. PerIg B-lymphocyte proliferation was higher at baseline and under anti-CD40 + IL-4 stimuli. Of note, under anti-BCR RESULTS stimulation, NOD-PerIg B-lymphocytes did not proliferate (Figure 2A), and an increased number of dead cells was Marked Phenotype Differences Between observed in the SSC-A/FSC-H flow cytometry analysis compared B-Lymphocytes From 116C-NOD and to baseline conditions (Supplementary Figure 3). Since NOD- NOD-PerIg Mice PerIg B-lymphocytes seem to be already highly activated just Since B-lymphocytes play an important role in presenting ß- after isolation (baseline condition), these results suggest that they cell autoantigens to autoreactive T-lymphocytes in autoimmune might have undergone activation-induced cell death after BCR diabetes, the present study first analyzed the expression of stimulation. Significant differences were also observed in the B- MHC and CD80, CD86, BAFF, BAFF-R, APRIL, and TACI lymphocyte cytokine production of the different mouse strains. costimulatory and coinhibitory molecules in spleen and islet B-lymphocyte secretion of TNF-α to all different stimuli, IL-6 infiltrating B-lymphocytes (IIBLs) from 116C-NOD and NOD- secretion after LPS and anti-CD40 + IL-4, and IL-10 secretion PerIg mice. MHC class I and class II expression were deeply after LPS stimulation was significantly higher in NOD-PerIg Frontiers in Immunology | www.frontiersin.org 3 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 1 | Expression of MHC, costimulatory and coinhibitory molecules in B-lymphocyte membrane. Median fluorescent intensity (MFI) of H2Kd , I-Ag7 , and FAS expression, and percentage values of BAFF, APRIL, BAFF-R, TACI, CD86, CD80, PD-1, PD-L1, and FAS positive islet-infiltrating or splenic B-lymphocytes from 6-week-old NOD, 116C-NOD and NOD-PerIg were analyzed (N = 5 per group, with two replicates for each experimental condition). Bars show the SD of the median of MFI or from the percentage values. Mann-Whitney U was used for analysis and statistically significant differences are shown as *P < 0.05 or **P < 0.01. compared to 116C-NOD and wild type NOD mice (Figure 2B). of spleen and islet-infiltrating T-lymphocytes were analyzed Interestingly, B-lymphocytes from NOD-PerIg mice secreted in NOD, 116C-NOD, and NOD-PerIg mice. As previously high amounts of IL-6 and TNF-α cytokines, even without reported (12), B-lymphocytes from 116C-NOD mice induced stimulation. Despite the external supply of IL-4, almost no IL- a shift toward a dominant T-lymphocyte Th17 differentiation, 4 was detected in the B-lymphocyte culture wells under the especially through double Th1/Th17 bias compared to NOD stimulus of anti-CD40 + IL-4, thus suggesting a high uptake of mice (Figure 3A). However, no significant differences were found this cytokine by NOD-PerIg B-lymphocytes during the culture regarding the T helper phenotype from CD4+ T-lymphocytes period compared to 116C-NOD and wild type NOD mice between NOD-PerIg and NOD mice either in the spleen or in the (Figure 2B). Altogether, these results suggest that NOD-PerIg B- pancreatic islet infiltrates. Only a significant increase in triple T- lymphocytes display an activated phenotype and, consequently, bet+RORγt+FoxP3+ CD4+ T-lymphocytes was detected in the might be able to activate autoreactive T-lymphocytes and induce spleen of NOD-PerIg compared to NOD mice. The expression autoimmune diabetes development more efficiently than 116C- of PD-1 and PD-L1 by splenic and islet infiltrating CD4+ and NOD B-lymphocytes. CD8+ T-lymphocytes from NOD, 116-NOD and NOD-PerIg mice was then analyzed (Figure 3B). A significant increase in Spleen and Islet Infiltrating T-Lymphocytes the expression of PD-1 was observed in CD4+ IITLs from From 116C-NOD and NOD-PerIg Mice Are NOD-PerIg compared to 116C-NOD, and in CD8+ IITLs from Phenotypically Different NOD-PerIg and 116C-NOD compared to NOD mice. These Since the functional status of B-lymphocytes can decisively data indicate that the CD8+ IITLs in both NOD-PerIg and impact T-lymphocyte behavior, the phenotypic characteristics 116C-NOD mice have a highly activated phenotype. However, Frontiers in Immunology | www.frontiersin.org 4 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 2 | B-lymphocyte proliferation assay. (A) CFSE analysis of proliferation responses of splenic B-lymphocytes from 6-week-old female NOD, 116C-NOD and NOD-PerIg mice (n = 4 per group, with two replicates for each experimental condition). Splenic B-lymphocytes were purified, labeled with CFSE and cultured in the presence (narrow line) of LPS, anti-CD40 + IL-4, anti-BCR stimuli or without stimulus (bold line) for 48 h. (B) IL-4, IL-6, TNF-α, and IL-10 cytokine concentrations were assessed in culture supernatants of purified B lymphocytes cultured in the same conditions as described above. (a) = Amount of IL-4 added to the culture media in absence of cells. Bars show the SD of the proliferation index or from the cytokine concentration values. Mann-Whitney U was used for analysis and statistically significant differences are shown as *P < 0.05. a high percentage of CD4+ IITLs express PD-1 only in NOD- differences between 116C-NOD and NOD-PerIg B-lymphocytes PerIg, suggesting that full activation of the IILTs occurs just in could explain their dual role in respectively inhibiting or these mice. promoting autoimmune diabetes development. Thus, the ability of B-lymphocytes from 116C-NOD and NOD-PerIg to induce NOD-PerIg B-Lymphocytes Induce High distinct NOD T-cell responses was then evaluated. The ability CD4+ and CD8+ T-Cell Activation of 116C-NOD and NOD-PerIg B-lymphocytes to induce T- A growing body of evidence indicates that B-lymphocytes lymphocyte proliferation was analyzed first. The results showed can contribute to autoimmune diabetes development by acting that NOD-PerIg B-lymphocytes had a higher capacity than those as APCs activating ß-cell autoreactive T-cells (1, 4, 10, 17–20). from 116C-NOD and NOD mice to induce CD4+ T-lymphocyte However, it has also been demonstrated that some B-lymphocytes proliferation (Figure 4A). Interestingly, the percentage of PD- can protect from disease development by inducing ß-cell 1 positive CD4+ and CD8+ T-lymphocytes was significantly autoreactive CTLs apoptosis or/and inhibiting APC activity higher when co-cultured with NOD-PerIg B-lymphocytes (11). Previous studies with 116C-NOD and NOD-PerIg mice compared to those co-cultured with B-lymphocytes from support the idea that B-lymphocytes can perform both actions NOD and/or 116C-NOD mice (Figure 4B), thus confirming depending on their functional status (12, 13). The present the T-lymphocyte activation status. Next, the expression of study indicates that the profound phenotypic and functional BAFF, BAFF-R, APRIL, and TACI costimulatory molecules, Frontiers in Immunology | www.frontiersin.org 5 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 3 | Phenotype of unstimulated splenic and islet infiltrating NOD, 116C-NOD, and NOD-PerIg T-lymphocytes. (A) T-bet, RORγt, GATA3, and FoxP3 expression was assessed by flow cytometry in splenic and islet-infiltrating T-lymphocytes from 6-week-old female NOD, 116C-NOD and NOD-PerIg mice (n = 4 per group, with two replicates for each experimental condition). “Total” and “SP” stand for the percentage of “whole positive” and “single positive” cells for the aforementioned marker, respectively. (B) Expression of PD-1 was analyzed by flow cytometry in the spleen and islet-infiltrating T-lymphocytes from 6-week-old female NOD, 116C-NOD and NOD-PerIg mice (n = 4 per group, with two replicates for each experimental condition). Bars show the SD of the percentage values. Mann-Whitney U, statistically significant differences: *P < 0.05 or **P < 0.01. and PD-1 and PD-L1 was also assessed in B-lymphocytes CD4 T helper responses was evaluated. As in previous assays, after co-culture with T-cells. The results indicated that a transcription factor expression and cytokine production by significant number of B-lymphocytes from NOD-PerIg, but T-lymphocytes from NOD mice after co-culture with B- not from NOD or 116-NOD mice, expressed PD-L1, thus lymphocytes from NOD-PerIg, 116C-NOD or wild type NOD being protected from the CTLs killing (Figure 4C). These mice was analyzed. Interestingly, the results indicated that B- results indicated that NOD-PerIg B-lymphocytes display a highly lymphocytes from NOD-PerIg mice led most NOD CD4+ T- activated phenotype that promoted both CD4+ and CD8+ T- lymphocytes toward a quadruple T-Bet+ GATA3+ RORγT+ lymphocyte activation. Foxp3+ phenotype (Figure 5A). Moreover, cytokine production analysis showed that B-lymphocytes from NOD-PerIg mice B-Lymphocytes From NOD-PerIg Mice prompted NOD T-lymphocytes to secrete significant amounts of cytokines from different T helper subsets, although with Enforce NOD T Cells Toward a Quadruple predominance of proinflammatory and Th1 cytokines, due to T-Bet+ GATA3+ RORγT+ Foxp3+ large amounts of IFN-γ secretion (Figure 5B). These results Phenotype indicate that B-lymphocytes from NOD-PerIg mice are highly As previously shown, B-lymphocytes from 116C-NOD mice active that mainly promote Th1 responses but not exclusively, induce a shift toward a dominant T-lymphocyte Th1/Th17 confirming that B-lymphocytes can enforce CD4 T-lymphocytes differentiation (12). In the present study, the capacity of B- toward different T helper functions depending on their lymphocytes from NOD-PerIg mice to modulate and conduct phenotype and behavior. Frontiers in Immunology | www.frontiersin.org 6 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 4 | Proliferation assay and molecular marker expression by NOD T-lymphocytes co-cultured with B-lymphocytes from NOD, 116C-NOD, and NOD-PerIg mice. (A) NOD T-lymphocyte proliferation assay. Purified splenic T-lymphocytes from NOD mice were CFSE-labeled and cultured in the presence (narrow line) of soluble anti-CD3, or plate-coated anti-CD3, and either purified NOD, 116C-NOD, or NOD-PerIg B-lymphocytes plus anti-CD3, or without stimulus (bold line) for 72 h. Ten NOD mice were used to obtain the T-lymphocyte pool and 4 mice of each group to obtain purified B-lymphocytes. Two replicates were carried out for each experimental condition. The proliferation of CD4+ or CD8+ T-lymphocytes was analyzed independently. (B) The expression of PD-1 and PD-L1 in CD4+ and CD8+ NOD T-lymphocytes was analyzed after co-culturing them as previously described. (C) The proportion of NOD, 116C-NOD, and NOD-PerIg B-lymphocytes expressing BAFF, APRIL, BAFF-R, TACI, PD-1, or PD-L1 was also assessed. All B-lymphocytes were incubated alone without stimulus or in the presence of NOD T-lymphocytes plus anti-CD3 for 72 h (n = 4 per group, with two replicates for each experimental condition). Bars show the SD of the percentage values. Mann-Whitney U, statistically significant differences: *P < 0.05. B-Lymphocytes From NOD-PerIg activation assays were performed. To this end, T-lymphocytes Presenting the 2.5HIP Epitope Promote from BDC2.5-NOD were incubated with their 2.5HIP cognate High BDC2.5-NOD T-Lymphocyte epitope in the presence of B-lymphocytes from NOD, 116C- NOD, and NOD-PerIg mice. Although the proliferation of Activation T-lymphocytes was not as high as expected, NOD-PerIg B- In the above experiments, the differences observed in T- lymphocytes were able to induce more efficiently CD4 T- lymphocyte activation could be due to the ability of anti-CD3 antibody to bind to B-lymphocytes of the different mouse lymphocyte proliferation than their counterparts (Figure 6B). strains. To shed light on this issue, Fc receptor (CD16/CD32) Although no differences in the transcription factor expression expression on NOD-PerIg, NOD, and 116C-NOD B-lymphocytes were detected (Figure 6C), BDC2.5-NOD T-lymphocytes co- was analyzed. Higher Fc expression was observed in NOD- cultured with NOD-PerIg produced larger amounts of cytokines PerIg compared to NOD and 116C-NOD B-lymphocytes than with NOD or 116C-NOD B-lymphocytes (Figure 6D). In (Figure 6A). Thus, in order to confirm that the ability of fact, BDC2.5-NOD T-lymphocytes produced larger amounts NOD-PerIg B-lymphocytes to activate T-lymphocytes was due of different cytokines distinctive of different T-lymphocyte to their high capacity to express co-stimulation molecules subsets. Surprisingly, BDC2.5-NOD T-lymphocytes incubated and cytokine secretion, rather than or only by the increase with 116C-NOD proliferate as efficiently as when they were in anti-CD3 binding capacity, BDC2.5-NOD T-lymphocyte in the presence of B-lymphocytes from NOD. These results Frontiers in Immunology | www.frontiersin.org 7 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 5 | Phenotype analysis of NOD T-lymphocytes after culture with B-lymphocytes from NOD, 116C-NOD, and NOD-PerIg mice. (A) Transcription factors expression analysis in splenic NOD T-lymphocytes cultured in the presence of soluble anti-CD3, or plate-coated anti-CD3, or either purified NOD, 116C-NOD, or NOD-PerIg B-lymphocytes plus anti-CD3, or without stimulus for 72 h (n = 6, with two replicates for each experimental condition). “Total” and “SP” stand for the percentage of “whole positive” and “single positive” cells for the aforementioned marker, respectively. (B) Cytokine concentration analysis in culture supernatants of NOD T-lymphocytes cultured in the same condition as described above. Bars show the SD of the percentage values or cytokine concentrations (n = 4, with two replicates for each experimental condition). Mann-Whitney U, statistically significant differences: *P < 0.05 or **P < 0.01. suggest the ability of 116C-NOD B-lymphocytes to overcome B-lymphocytes in NOD mice prevents disease development their “anergic” state in order to stimulate T-lymphocytes in the (1–8), whereas depending on their antigenic specificity the presence of an autoantigen. Nevertheless, their ability to induce transgenic expression of BCRs either induces, delays, or prevents cytokine production was not as good as the one of NOD or autoimmune diabetes (10, 12, 13, 21–24). The diabetogenic NOD-PerIg B-lymphocytes. capacity of B-lymphocytes in two BCR transgenic mice, the NOD-PerIg and the 116C-NOD strains (12, 13) was analyzed DISCUSSION in previous studies. In 116C-NOD mice, B-lymphocytes display an anergic-like phenotype delaying autoimmune diabetes onset It is currently well accepted that B-lymphocytes contribute and decreasing disease incidence, whereas in NOD-PerIg mice, to the development of autoimmune diabetes. The lack of B-lymphocytes acquire an activated proliferative phenotype that Frontiers in Immunology | www.frontiersin.org 8 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes FIGURE 6 | Analysis of BDC2.5-NOD T-lymphocyte activation after co-culture with B-lymphocytes from NOD, 116C-NOD, and NOD-PerIg mice. (A) The expression of Fc Receptors (CD16/CD32) from B-lymphocytes was analyzed by flow cytometry before culture (n = 4 per group). (B) BDC2.5-NOD T-lymphocyte proliferation assay. Purified splenic T-lymphocytes from BDC2.5-NOD mice were CFSE-labeled and cultured in the presence of 2.5HIP alone and with purified NOD, 116C-NOD, or NOD-PerIg B-lymphocytes, or without stimulus for 72 h. (C) Transcription factor expression was analyzed after culturing cells as mentioned above. “Total” and “SP” stand for the percentage of “whole positive” and “single positive” cells for the aforementioned marker, respectively. (D) Cytokine release was checked in supernatants after 72 h. Bars show the SD of the percentage values or cytokine concentrations. In all experiments N = 4. Mann-Whitney U, statistically significant differences: *P < 0.05. promotes disease development. Moreover, a T helper response also analyzed. With this approach, the effect was assessed imbalance toward a Th17 phenotype was observed in 116C- independently from their antigenic specificity of expressed NOD compared to NOD mice, thus suggesting a strong linkage accessory molecules and secreted cytokines by the B-lymphocytes between B-lymphocyte phenotype and T-lymphocyte response. in the activation of the T-lymphocytes. Finally, B lymphocyte By comparing the phenotype of the three mouse strains, capacity to act as APCs in autoimmune diabetes environment the NOD, the 116C-NOD, and the NOD-PerIg, the present was tested by analyzing the ability of these cells to present study sought to determine the most relevant B-lymphocyte a known diabetes-related autoantigen, known as 2.5HIP, a traits during autoimmune diabetes. To this end, the linkage Hybrid Insulin Peptide. In contrast to those from the NOD between phenotypic characteristics of B-lymphocytes and the and 116C-NOD strains, B-lymphocytes from NOD-PerIg mice ability of these cells to induce phenotypic and functional display an activated phenotype, secrete high amounts of changes in T-lymphocytes was further analyzed. To this end, proinflammatory cytokines (even without any B-lymphocyte the expression of accessory molecules and cytokine secretion stimulation), and induce significant T-lymphocyte activation. In in B-lymphocytes from NOD-PerIg, 116C-NOD, and NOD addition, B-lymphocytes from116C-NOD express significantly mice was evaluated. Then, through a nonspecific anti-CD3 lower levels of MHC molecules compared to those from stimulus that mimics a signal through the TCR, the ability NOD and NOD-PerIg mice, thus indicating their inability to of these B-lymphocytes to induce T-lymphocyte activation was perform a good antigen presentation. Although a previous study Frontiers in Immunology | www.frontiersin.org 9 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes showed that CD86 expression levels in B-lymphocytes from double, triple or even quadruple Th positive may also be found 116C-NOD mice were high (12) suggesting perhaps a greater (26). For instance, CD4+ T-lymphocytes that are at the same capacity for activation of T-lymphocytes, the percentage of B- time Th1 and Th17, or other multiple possible combinations, lymphocytes expressing CD86 is lower in 116C-NOD than even some that simultaneously display multiple Th type markers. in NOD and NOD-PerIg mice. Moreover, since BAFF-R and A cell population can also pass from one phenotype to another in TACI is higher in 116C-NOD than in NOD and NOD-PerIg the presence of certain cytokines. For example, a subpopulation mice and taking into account that TACI acts as an inhibitor of Th9 can be differentiated from Th2 in the presence of for lymphocyte activation through BAFF/BAFF-R interaction, TGF-β (27). these results suggest that 116C-NOD IIBLs cannot induce a In summary, this study highlights the existing strong T-lymphocyte activation. In fact, current and previous interdependence between B- and T-lymphocytes, which data (12) suggest that 116C-NOD B-lymphocytes would show a gives to the former populations the chance to set up T- greater tendency to fostering a response toward Th17 phenotype. lymphocyte phenotypes through the secretion of cytokines and In contrast to the 116C-NOD B-lymphocytes, the expression the expression of costimulatory and co-inhibitory molecules. levels of MHC molecules in B-lymphocytes from NOD-PerIg In terms of autoimmune diabetes, this study also confirms that mice are similar to those from NOD mice. In addition, the the most harmful response for disease development comes percentage of B-lymphocytes expressing high levels of the CD86 from a concomitant Th1/proinflammatory response, and molecule and low levels of TACI and BAFF-R is higher in that B-lymphocytes play the key role in the establishment of NOD-PerIg mice compared to NOD and 116C-NOD mice, this response. indicating the strong ability of B lymphocytes to activate T lymphocytes. Consistent with these results and those obtained in the analysis of the incidence of autoimmune diabetes, this ETHICS STATEMENT study also shows that the NOD-PerIg B-lymphocytes have the This study was carried out in accordance with the capacity to proliferate and secrete proinflammatory cytokines, recommendations of Catalan and Spanish Government even in the absence of any stimulus, and induce a strong T- guidelines for Animal Experimentation (Directiva 2010/63/UE; lymphocyte proliferation. Moreover, the results also indicate Real Decreto 53/2013; Llei 5/1995/GC; Orden 214/1997/GC). that NOD-PerIg B-lymphocytes induce CD4+ T-lymphocytes Ethical Committee of Animal Experimentation of the University to differentiate toward a quadruple Bet+ GATA3+ RORγT+ of Lleida. Experimental procedure # CEEA, 02-04/16. Foxp3+ phenotype and to secrete cytokines characteristic from most different T helper subsets, although with predominance of proinflammatory and Th1 cytokines, thus contributing to AUTHOR CONTRIBUTIONS autoimmune diabetes development (25). These findings were confirmed when we analyzed the ability of the different LE-M researched data and wrote the manuscript. BA, ER-M, B-lymphocytes to induce specific responses to the known MC-P, JCarrascal, JCarrillo, HC, and AP researched data. diabetes-related peptide 2.5HIP. Even without a polyclonal CM, MV-P, and TS contributed to discussion, and reviewed stimulus, NOD-PerIg B-lymphocytes were able to activate the manuscript. DS co-conceived the study, contributed to specific responses from BDC2.5-NOD T-lymphocytes, inducing discussion, and reviewed manuscript. JV conceived the study, a higher production of different cytokines when compared to evaluated data, wrote, reviewed and edited to the manuscript. JV the other two strains. On the contrary, no changes were found is the guarantor of this work and, as such, had full access to all the between strains in the phenotype of BDC2.5-NOD CD4+ T- data in the study and takes responsibility for the integrity of the lymphocytes after stimulation with the 2.5HIP cognate epitope. data and the accuracy of the data analysis. All authors revised the This could be due to their transgenic nature, being their prefixed work and gave final approval of the version to be published. phenotype strong enough to support any input, compared to NOD CD4+ T-lymphocytes. As observed in previous works ACKNOWLEDGMENTS carried out with the transgenic model 125tg-NOD (20), our results indicate that 116C-NOD B-lymphocytes can induce T- The research described in this publication was partially supported lymphocyte activation and cytokine production in very specific by the Plan Nacional de I+D+i of the Spanish Ministry of Science situations despite their “anergic phenotype.” and Innovation (SAF2016-77227-R), CIBER of Diabetes and Thus, this study also clearly shows that depending on Associated Metabolic Diseases (CIBERDEM) that is an initiative their phenotype and behavior (secreted cytokine and accessory from Instituto de Salud Carlos III (Spain) and the Juvenile molecule expression), B-lymphocytes from NOD, 116C-NOD, Diabetes Research Foundation 5-2005-1133 innovative research and NOD-PerIg mice can promote CD4+ T lymphocytes grant. LE-M, ER-M, and MC-P, were supported by pre-doctoral toward different T helper functions. The present findings also fellowships from the University of Lleida and the IRBLleida. illustrate that to face any insult, the immune system can bring JCarrascal was supported by a FPI pre-doctoral fellowship a combined T helper response exerted by several CD4+ T- (BES-2007-15221) from the Spanish Ministry of Science and lymphocyte populations if required. Moreover, since the immune Innovation. JV, MC-P, and TS are associate professors from system has an enormous plasticity and redundancy, other CD4+ the Serra-Hunter Program from the Catalan Government. DS T-lymphocytes populations of T helper that are phenotypically is supported by NIH grants DK46266 and DK95735 as well Frontiers in Immunology | www.frontiersin.org 10 July 2019 | Volume 10 | Article 1732 Egia-Mendikute et al. T- and B-Lymphocyte Phenotype in Autoimmune Diabetes as by grants from the Juvenile Diabetes Research Foundation, SUPPLEMENTARY MATERIAL the American Diabetes Association, and Helmsley Charitable Trust (#2014PG-T1D048). We thank Dr. Jeremy Racine and Mr. The Supplementary Material for this article can be found Qiming Wang for reviewing the manuscript. Special thanks to online at: https://www.frontiersin.org/articles/10.3389/fimmu. Ms. D. Cullell-Young for English grammar assistance. 2019.01732/full#supplementary-material REFERENCES by islet-infiltrating B lymphocytes. J Immunol. (2007) 178:6533–9. doi: 10.4049/jimmunol.178.10.6533 1. Serreze DV, Chapman HD, Varnum DS, Hanson MS, Reifsnyder PC, Richard 16. Puertas MC, Carrillo J, Pastor X, Ampudia RM, Alba A, Planas R, et al. SD, et al. 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Leeth CM, Racine J, Chapman HD, Arpa B, Carrillo J, Carrascal J, be construed as a potential conflict of interest. et al. B-lymphocytes expressing an immunoglobulin specificity recognizing the pancreatic ß-cell autoantigen peripherin are potent contributors to Copyright © 2019 Egia-Mendikute, Arpa, Rosell-Mases, Corral-Pujol, Carrascal, type 1 diabetes development in NOD mice. Diabetes. (2016) 65:1977–87. Carrillo, Mora, Chapman, Panosa, Vives-Pi, Stratmann, Serreze and Verdaguer. doi: 10.2337/db15-1606 This is an open-access article distributed under the terms of the Creative Commons 14. Carrillo J, Puertas MC, Alba A, Ampudia RM, Pastor X, Planas R, et al. Islet- Attribution License (CC BY). The use, distribution or reproduction in other forums infiltrating B-cells in nonobese diabetic mice predominantly target nervous is permitted, provided the original author(s) and the copyright owner(s) are credited system elements. Diabetes. (2005) 54:69–77. doi: 10.2337/diabetes.54.1.69 and that the original publication in this journal is cited, in accordance with accepted 15. Puertas MC, Carrillo J, Pastor X, Ampudia RM, Planas R, Alba A, academic practice. No use, distribution or reproduction is permitted which does not et al. Peripherin is a relevant neuroendocrine autoantigen recognized comply with these terms. Frontiers in Immunology | www.frontiersin.org 11 July 2019 | Volume 10 | Article 1732