ORIGINAL RESEARCH published: 20 January 2021 doi: 10.3389/fimmu.2020.599623 Vitamin D3-Induced Tolerogenic Dendritic Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a Functional Hyporesponsiveness Juan Navarro-Barriuso 1,2, Marı́a José Mansilla 1,2, Bibiana Quirant-Sánchez 1,2, Aina Teniente-Serra 1,2, Cristina Ramo-Tello 3 and Eva M. Martı́nez-Cáceres 1,2* Edited by: Fang Zhou, 1 Division of Immunology, LCMN, Germans Trias i Pujol University Hospital and Research Institute, Barcelona, Spain, CAS Lamvac Biotech Co., Ltd., China 2 Department of Cellular Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Multiple Sclerosis Unit, Department of Neurosciences, Germans Trias i Pujol University Hospital, Barcelona, Spain Reviewed by: Ali Akbar Pourfathollah, Tarbiat Modares University, Iran Paulina A. Garcı´a-González, The use of autologous tolerogenic dendritic cells (tolDC) has become a promising University of Chile, Chile alternative for the treatment of autoimmune diseases. Among the different strategies Willem Van Eden, available, the use of vitamin D3 for the generation of tolDC (vitD3-tolDC) constitutes one of Utrecht University, Netherlands Carolina Llanos, the most robust approaches due to their immune regulatory properties, which are Pontificia Universidad currently being tested in clinical trials. However, the mechanisms that vitD3-tolDC Católica de Chile, Chile Grant P. Parnell, trigger for the induction of tolerance remain elusive. For this reason, we performed a full Westmead Institute for Medical phenotypical, functional, and transcriptomic characterization of T cells upon their Research, Australia interaction with autologous, antigen-specific vitD3-tolDC. We observed a strong *Correspondence: antigen-specific reduction of T cell proliferation, combined with a decrease in the Eva M. Martı´nez-Cáceres emmartinez.germanstrias@gencat.cat relative prevalence of TH1 subpopulations and IFN-g production. The analysis of the transcriptomic profile of T CD4+ cells evidenced a significant down-modulation of genes Specialty section: involved in cell cycle and cell response to mainly pro-inflammatory immune-related stimuli, This article was submitted to Immunological Tolerance highlighting the role of JUNB gene as a potential biomarker of these processes. and Regulation, Consequently, our results show the induction of a strong antigen-specific a section of the journal Frontiers in Immunology hyporesponsiveness combined with a reduction on the TH1 immune profile of T cells Received: 27 August 2020 upon their interaction with vitD3-tolDC, which manifests the regulatory properties of these Accepted: 30 November 2020 cells and, therefore, their therapeutic potential in the clinic. Published: 20 January 2021 Keywords: tolerogenic dendritic cells, immune tolerance, T cells, antigen-specific response, transcriptomic study Citation: Navarro-Barriuso J, Mansilla MJ, Quirant-Sánchez B, Teniente-Serra A, Ramo-Tello C and INTRODUCTION Martı´nez-Cáceres EM (2021) Vitamin D3-Induced Tolerogenic Dendritic In the last years, tolerogenic dendritic cells (tolDC) have become one of the most promising alternatives Cells Modulate the Transcriptomic Profile of T CD4+ Cells Towards a for the treatment of autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis, or type Functional Hyporesponsiveness. 1 diabetes. In fact, several Phase I clinical trials have already finished or are currently ongoing, with Front. Immunol. 11:599623. positive results regarding the safety and the tolerability of this therapeutic cell-based approach (1). In doi: 10.3389/fimmu.2020.599623 general, tolDC are commonly defined as a stable and semi-mature subset of dendritic cells (DC), Frontiers in Immunology | www.frontiersin.org 1 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness between antigen-capturing immature DC (iDC) and immunogenic according to the institutional Standard Operating Procedures for mature DC (mDC)—characterized by their increased expression of blood donation, including a signed informed consent. In parallel, MHC class II and co-stimulatory molecules. But most importantly, whole blood samples from 12 different healthy donors were tolDC are presumably capable to induce immune tolerance collected by standard venipuncture in lithium heparin tubes for towards the peptides these cells are presenting, in an antigen- the allogeneic functional assays (see below). specific manner (2–5). TolDC can be generated in vitro from peripheral blood Monocyte Isolation monocytes. In the last years, a wide variety of protocols for their Healthy donor buffy coat samples were processed first depleting production have been reported, ranging from the use of different CD3 + cells using the RoseetteSep ® Human Monocyte drugs and chemical agents to genetic engineering techniques (6, 7). Enrichment Kit (StemCell Technologies, Vancouver, Canada) In this regard, the use of 1,25-dyhydroxyvitamin D3, the active form prior to a density gradient separation using ficoll-hypaque of vitamin D3, constitutes one of the most widely studied (Rafer, Zaragoza, Spain). Afterwards, CD14+ cells were isolated approaches for the differentiation of tolDC. Briefly, vitamin D3- using the EasySep ® Human CD14 Positive Selection Kit induced tolDC (vitD3-tolDC) are thought to develop their (StemCell), according to the manufacturer’s instructions. Cell regulatory properties through a semi-mature profile, their ability viability was determined using 7-amino-actinomycin D (7-AAD) to inhibit or reduce T cell responses, and a switch of the immune (BD Biosciences, Franklin Lakes, NK, USA) and phycoerythrin response towards a TH2 profile (8–18). Furthermore, vitD3-tolDC (PE)-conjugated annexin V (Immunotools, Friesoythe, are characterized by a reduced NF-kB-mediated activity and an Germany) staining for 20 min at 4°C, protected from light, and increase of mTOR-mediated glucose metabolism (10, 19). cell counts were quantified simultaneously using PerfectCount Even though tolDC—and vitD3-tolDC in particular—have beads (Cytognos, Salamanca, Spain). Samples were acquired on a been characterized with a developing knowledge over their FACSCanto II flow cytometer (BD Biosciences), and monocyte metabolism, molecular mechanisms, and functional pathways, purity was determined using forward and side scatter gating the specific effect of these cells over the rest of the immune- strategies on FACSDiva software (BD Biosciences). related components still remains elusive. It is known that tolDC can usually induce either anergy, hyporesponsiveness or depletion TT-Loaded DC Cultures over activated T cells, as well as regulatory T cell (Treg) The protocol for the generation of antigen-loaded tolDC was differentiation (20). However, to our knowledge, so far only one adapted from a previous study (25). Briefly, isolated monocytes study has focused its attention on the actual processes that were cultured for 6 days in 24-well plates at 37°C at a density autologous T cells might be undergoing upon tolDC interaction of 1 × 106 cells/ml in X-VIVO 15 medium, in the presence of —reporting an induction of hyporesponsiveness of CD4+ memory 400 U/ml granulocyte-macrophage colony-stimulating factor and naïve T cells towards antigen-specific stimulation mediated by (GM-CSF) and 500 U/ml IL-4 (both from Peprotech, London, dexamethasone-induced tolDC (21)—but neither at the UK). The whole volume of medium and cytokines was transcriptomic level nor with vitD3-tolDC in particular. replenished on day 4. If no further treatment was performed, In previous studies, our group has already extensively monocytes were differentiated into iDC. For the generation characterized vitD3-tolDC phenotypically, functionally, and of mDC, we further added a maturation cocktail, containing transcriptomically, evidencing the regulatory potential of these 1,000 U/ml IL-1b, 1,000 U/ml TNF-a (both from Peprotech) and cells both in vitro and in vivo in the animal model of MS, 1 µM prostaglandin E2 (PGE2) (Pfizer, New York, NY, USA) on experimental autoimmune encephalomyelitis (EAE) (13, 16, 22– day 4. Finally, in addition to the maturation cocktail, we added 24). Consequently, we wanted to take one step further for the 1 nM vitamin D3 (Calcijex, Abbott, Chicago, IL, USA) on days elucidation of the mechanisms of immune tolerance induction of 0 and 4 for the differentiation of vitD3-tolDC. For the generation vitD3-tolDC. With that aim, here we present a full phenotypical, of TT-loaded mDC (mDC-TT) and TT-loaded vitD3-tolDC functional, and transcriptomic characterization of T CD4+ cells after (vitD3-tolDC-TT) as antigen-specific experimental conditions, their interaction with autologous vitD3-tolDC loaded with tetanus 0.1 µg/ml of the whole TT protein (Sigma-Aldrich, St. Louis, toxin (TT), in order to study the antigen-specific effect mediated by MO, USA) were added to the mDC and vitD3-tolDC cultures on these cells compared to TT-loaded immunogenic mDC. The day 3, 18 h before the addition of the maturation stimulus, while purpose of this study is to identify one or several potential still in an immature status. On day 6, cells were harvested after an biomarkers of the immune modulation developed by vitD3-tolDC accutase (Invitrogen, Carlsbad, CA, USA) detaching treatment over T cells, which could constitute an interesting tool for the for 30 min, and washed twice. As shown above, cell counts and monitoring of patients treated with these cells in clinical trials, and viability were determined by flow cytometry. the understanding of the mechanisms of tolerance induction. Autologous PBMC Isolation, Co-Culture, and Sorting MATERIAL AND METHODS For the isolation of autologous PBMC, 3 ml of the buffy coat samples from each healthy donor was processed using a ficoll- Sample Collection hypaque density gradient separation and washed twice. Buffy coat samples from 16 randomized healthy controls were Afterwards, cells were counted by flow cytometry, as described obtained from the Banc de Sang i Teixits (Barcelona, Spain), above, and plated in round-bottom 96-well plates at a density of Frontiers in Immunology | www.frontiersin.org 2 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness 1 × 106 cells/ml in RPMI medium supplemented with 10% heat- Allogeneic and Autologous Cell inactivated fetal bovine serum, 2 mM L-glutamine (Lonza, Basel, Proliferation Assays Switzerland), 100 U/ml penicillin (Reig Jofre, Sant Joan Despı́, For the determination of the reactivity of PBMC from each Spain) and 100 µg/ml streptomycin (Normon, Tres Cantos, donor against TT, 2 × 105 PBMC were plated in 96-well round Spain). The plates were then incubated for 6 days at 37°C in a bottom plates at day 0 of each culture in supplemented RPMI 5% CO2 atmosphere. Afterwards, cells were harvested, and cell medium containing 0.1 µg/ml TT. As control conditions, the counts and viability were determined by flow cytometry. same number of cells was cultured with either supplemented Subsequently, an antigen-specific proliferation experimental RPMI medium only (negative control) or 50 ng/ml phorbol 12- setup was performed in 96-well round-bottom plates with co- myristate-13-acetate (PMA) and 500 ng/ml ionomycin (positive cultures of 105 autologous PBMC and 5,000 either mDC-TT or control). Ten replicates were performed for the negative control vitD3-tolDC-TT (1:20 ratio) in a final volume of 200 µl of and the condition of analysis, and six replicates for the positive supplemented RPMI medium. For each condition, 48 replicates control. Cells were then cultured for 5 days at 37°C in a 5% CO2 were performed. Cells were then incubated for 5 days at 37°C in a atmosphere. Afterwards, 1 µCi [3H]-thymidine (PerkinElmer, 5% CO2 atmosphere. Afterwards, cells were harvested and the Waltham, MA, USA) was added to each well, and the plate was whole volume of each cell suspension was incubated for 20 min, incubated for further 18 h under the same conditions. Cells were protected from light, with the adequate amounts of monoclonal then collected using a HARVESTER96 2M cell harvester antibodies anti-CD3 Violet 450 (V450) and anti-CD4 PerCP- (Tomtec Inc, Hamdem, CT, USA) and read on a 1450 Cyanine dye (Cy)5.5. Finally, cells were washed and the whole MicroBeta TriLux liquid scintillation counter (Wallac, Turku, CD3+CD4+ cell subpopulation was isolated and counted using a Finland). Donors were considered positive for TT reactivity FACSAria II cell sorter (BD Biosciences). Finally, dry pellets of when the counts per minute (cpm) of at least five replicates mDC-TT-conditioned CD3+CD4+ cells (mDC-Tcell) and vitD3- from the condition of analysis were over the mean plus two times tolDC-TT-conditioned CD3+CD4+ cells (vitD3-Tcell) were the standard deviation (SD) of the negative control. obtained by centrifugation and stored at −80°C. For the isolation of allogeneic PBMC, whole blood samples of different healthy donors were processed by ficoll-hypaque Phenotype Characterization of DC density gradient separation. Cells were washed twice, and and Autologous PBMC afterwards, their absolute number and viability was determined Surface expression of CD11c, CD14, CD83, CD86 and HLA-DR as shown above. Subsequently, 10 5 either allogeneic or in iDC, mDC, mDC-TT, vitD3-tolDC and vitD3-tolDC-TT was autologous viable PBMC were co-cultured with 5,000 either determined by flow cytometry. In each case, DC suspensions iDC, mDC, mDC-TT, vitD3-tolDC or vitD3-tolDC-TT (1:20 were incubated for 20 min, protected from light, with the ratio) in 96-well round bottom plates, in a total volume of 200 µl appropriate amounts of monoclonal antibodies anti-: CD11c of supplemented RPMI medium. Again, as negative and positive PE-Cy7, CD14 V450, CD83 allophycocyanin (APC), CD86 controls, either supplemented RPMI medium or a mix of 50 ng/ fluorescein isothiocyanate (FITC) and HLA-DR Violet 500 ml PMA and 500 ng/ml ionomycin was used, respectively. Six (V500) (all of them from BD Biosciences). Subsequently, at replicates of each condition were performed. Cells were then least 10,000 CD11c+ events of each condition were acquired plated for 4 days at 37°C in a 5% CO2 atmosphere, and using a FACSCanto II flow cytometer and analyzed using afterwards, 1 µCi [3H]-thymidine was added to each well, and FACSDiva software. the plates were incubated, harvested, and read as described above. For the phenotypical characterization of mDC-Tcell and vitD3- Tcell, cell suspensions of these conditions were incubated for 20 Cytokine and Soluble Protein Production min with the adequate amounts of monoclonal antibodies The production of granzyme B (GZMB), as well as of IL-1b, indicated below. Afterwards, samples were washed twice and IL-6, IL-10, IFN-g, IL-12p70 and TNF-a cytokines, was acquired on a LSRFortessa flow cytometer, setting the stopping quantified in the supernatants of mDC-TT and vitD3-tolDC- gate at 300,000 peripheral blood mononuclear cells. The definition TT with autologous PBMC co-cultures, using the Human of each peripheral blood mononuclear cell subpopulation was Soluble Protein CBA Flex Set (BD biosciences) according to determined as specified in Supplementary Table 1, using several manufacturer’s instructions. Samples were acquired on an LSR combinations of the following monoclonal antibodies anti-: Fortessa flow cytometer (BD Biosciences), and the results CXCR3 AlexaFluor (AF)488, CD4 PerCP-Cy5.5, CCR7 PE, were analyzed using FACSDiva software. The production of CD45RA PE-Cy7, CD38 APC, CD45 AF700, CD8 APC-H7, TGF-b was determined using the Human/Mouse TGF beta 1 CD3 V450, HLA-DR V500, CCR6 Brilliant Violet (BV) 605, Uncoated ELISA kit (Invitrogen) in 100 µl of the co-culture CD25 PE, CCR4 PE-Cy7, CD127 AF647, CD45RO APC-H7, supernatants after sample activation with HCl 1N, following the CD49b FITC and LAG-3 PE (BD Biosciences). Results were manufacturer’s instructions. The optical density of each well was analyzed with FACSDiva software (BD Biosciences). Forward measured at l = 450 nm, and the optical density at l = 570 nm and side scatter gating strategy was used in order to select the was then subtracted as background signal, using a Varioskan desired lymphocyte subpopulations, and their relative percentages Flash Multimode Reader (Thermo Fisher Scientific, Waltham, were analyzed for each cell subset. MA, USA). Frontiers in Immunology | www.frontiersin.org 3 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness RNA Extraction and RNA-seq Analysis (ranked according to p-value), the FDR q-value is the p-value Total RNA of autologous mDC-Tcell and vitD3-Tcell samples multiplied by the number of GO terms assessed and divided by i. was isolated using the automated Maxwell 16 LEV simplyRNA Purification Kit (Promega Biotech, Madison, WI, USA), Statistical Analysis including a DNAse I digestion step, according to manufacturer’s All the statistical analyses were performed with either parametric instructions. Samples were quantified using a Nanodrop ND-1000 or non-parametric tests depending on the normality of each spectrophotometer (Thermo Fisher Scientific), and subsequently compared data set, as determined by the D’Agostino & Pearson stored at −80°C in RNAse-free tubes. RNA integrity number test using Prism 6.0 software (GraphPad, La Jolla, CA, USA). For (RIN) was determined in an Agilent BioAnalyzer with the multiple comparisons, either the non-parametric Friedman test RNA6000 Pico assay (Agilent Technologies, Santa Clara, CA, with Dunn’s correction or the one-way ANOVA test with USA). Afterwards, the sequencing libraries were prepared using Geisser–Greenhouse correction were used, and analogously, the TruSeq Stranded Total RNA Sample Preparation kit (Illumina, either paired t tests or Wilcoxon tests for the comparisons San Diego, CA, USA) with 200 ng of total RNA per sample as between two groups if they were normally distributed or not, input. Paired-end sequencing (2 × 75 bp) was then performed on a respectively. Results were expressed as mean ± SD, unless noted HiSeq-2500 instrument (Illumina). Reads were quality trimmed otherwise, and they were considered statistically significant when and adapters removed using Trimmomatic V0.30. TopHat p < 0.05. software v2.1.0 was used to map RNA-seq reads to the human reference genome (Ensembl release 78) (26). FeatureCounts function was used to assign reads to genomic features focusing RESULTS on RNA biotypes. A matrix with summarized raw counts of reads assigned through mapping to high confidence protein coding Functional and Phenotypical genes only was generated (“golden” annotation label), Characteristics of TT-Loaded vitD3-tolDC corresponding to stable and unlikely to change transcripts from Monocytes from 16 healthy donor samples were isolated (94.4 ± the Consensus CDS (CCDS) Project. Data exploration results 2.8% purity) with viabilities of CD14+ cells above 95%. After from hierarchical clustering and principal component analysis their differentiation into DC, with or without exposition to TT, (PCA) in R software were used to exclude any outliers and assess cells were harvested and their purity, viability, and phenotype sample similarities based on global gene expression patterns, and were determined by flow cytometry, as shown previously (25). In to guide the modeling design to be used for subsequent analyses. all cases, purity was >90%, as determined by the percentage of CD11c+ cells, with a mean viability of 94.2 ± 3.3%, which was not Differential Gene Expression Analysis affected by the addition of TT (Supplementary Table 2). The Transcriptional changes at the gene level between mDC-Tcell study of the phenotype of vitD3-tolDC-TT showed significant and vitD3-Tcell were assessed using the Bioconductor DeSeq2 reductions in the surface expression of CD86 (77.2 ± 8.7%) and package in R (27). A paired sample comparison design, factoring HLA-DR (79.5 ± 7.7%) compared to mDC, but more in inter-individual differences, was applied. The results were importantly, evidenced that the exposure of DC to TT on day considered statistically significant with an adjusted p-value 3 of the culture did not have an effect per se over the expression of (padj) < 0.05. We set a 20% fold change (FC) cutoff as the these molecules, neither in vitD3-tolDC nor in mDC, since there threshold for relevant biological effects (|FC| > 1.2). were no relevant differences on the percentages of reduction (Supplementary Figure 1). The same could be observed Gene Ontology Enrichment Analysis regarding the functionality of these cells. On the one hand, as Unranked lists of the significant differentially upregulated (FC > also shown in Supplementary Figure 1, both vitD3-tolDC and 1.2 and padj < 0.05) and downregulated genes (FC < −1.2 and vitD3-tolDC-TT exhibited a similar and strongly reduced padj < 0.05) were tested for enrichment in Gene Ontology (GO) induction of allogeneic proliferation compared to mDC (vitD3- functional categories using the GOrilla web tool, applying the tolDC: 50.6 ± 30.7, p < 0.001; vitD3-tolDC-TT: 49.2 ± 36.7, p = default settings for comparison to the background list of genes 0.001). On the other hand, there were no statistically significant found in the dataset (28). We tested for enrichment in three types differences in the mean induction of allogeneic proliferation of GO categories: “biological process” (GOPROCESS), induced by mDC-TT compared to mDC (p = 0.916). “molecular function” (GOFUNCTION), and “cellular Altogether, our results evidence that vitD3-tolDC-TT show the component” (GOCOMPONENT). Enrichment score (ES) was same tolerogenic properties as vitD3-tolDC, thus demonstrating defined as ES = (b/n)/(B/N), where “N” is the total number of that loading these cells with TT does not affect their phenotype genes in the background list, “B” is the total number of genes in nor their functionality. N associated with a specific GO term, “n” is the number of differentially expressed genes being tested for enrichment and VitD3-tolDC-TT Induce “b” is the number of n intersecting with B. Enrichment p-value is an Antigen-Specific Response Over computed according to the hypergeometric (HG) model. False Autologous PBMC discovery rate (FDR) q-value is the Benjamini and Hochberg In order to test the antigen-specific functionality of vitD3-tolDC- multiple testing correction adjusted p-value. For the ith term TT in an autologous setup, we assessed the baseline reactivity of Frontiers in Immunology | www.frontiersin.org 4 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness each donor against the TT itself to measure their potential to VitD3-tolDC-TT Drive a Reduction of TH1 respond under these conditions. As shown in Figure 1A, we were CD4+ Cell Subpopulations able to assess the TT reactivity in all of our healthy donors, but Once determined that an antigen-specific modulation was only nine of them resulted positive, according to the criteria established by TT-loaded DC, we studied which changes were described in the Material and Methods section—when the mean being induced over the autologous T lymphocytes. Therefore, we proliferation of at least 5 out of 10 replicates was over the mean characterized the phenotype of CD3+CD4+ and CD3+CD8+ cells plus two times the SD of the control condition—and reaching using an exhaustive multiparametric flow cytometry panel, statistical significance (p < 0.05). described in previous studies (29). First, our results evidenced Subsequently, we analyzed the capability of our cells to induce a reduction in the prevalence of activated T CD4 + cells, proliferation over autologous PBMC. As shown in Figure 1B, a determined by HLA-DR and/or CD38 staining, in vitD3-Tcell significant proliferation was only induced by mDC-TT, as compared to mDC-Tcell (Activated CD4+ mDC-Tcell: 23.57 ± evidenced by the statistically significant differences observed 15.81 vs Activated CD4+ vitD3-Tcell: 18.52 ± 14.16; p = 0.002). with the remaining conditions. Specifically, reductions of a The same effect was observed over T CD8+ cells (Activated 38.4 ± 44.3% (p = 0.020), a 40.0 ± 21.0% (p < 0.001), a 56.9 ± CD8+ mDC-Tcell: 15.94 ± 12.48 vs Activated CD8+ vitD3-Tcell: 19.2% (p < 0.001) and a 37.3 ± 17.4% (p < 0.001) were observed 11.33 ± 9.81; p = 0.002). Furthermore, we found a reduction in in iDC, mDC, vitD3-tolDC and vitD3-tolDC-TT, respectively, the relative percentages of CD4+ TH1 Central Memory (CM) and compared to mDC-TT. Our results therefore evidence that Effector Memory (EM) subpopulations in vitD3-T cell (TH1 CM autologous proliferation is only primed if an antigenic peptide mDC-Tcell: 33.98 ± 6.44 vs TH1 CM vitD3-Tcell: 30.23 ± 7.48; p = is presented by an immunogenic DC condition, such as mDC- 0.013; TH1 EM mDC-Tcell: 44.46 ± 8.72 vs TH1 EM vitD3-Tcell: TT, confirming the antigen-specific modulation developed by 40.95 ± 8.08; p = 0.001). All these results are shown in Figures our cells. Furthermore, reduced autologous proliferation 2A, B. Thus, our data suggest that vitD3-tolDC-TT are driving mediated by vitD3-tolDC-TT was observed in all donors an antigen-specific switch towards a more anti-inflammatory—or (Figure 1C). less TH1-like—profile over T CD4+ lymphocytes. We could not A B C FIGURE 1 | PBMC reactivity and antigen-specific induction of autologous proliferation mediated by DC against tetanus toxin. (A) Induction of proliferation of PBMC without stimuli (C−) and against tetanus toxin (TT) after 5 days of culture (n = 16). Data presented as counts per minute (cpm), measured as tritiated thymidine incorporation after 18 h. Ten replicated measurements of each condition were performed. (B) Induction of antigen-specific autologous proliferation against TT mediated by immature DC (iDC), mature DC (mDC), TT-loaded mDC (mDC-TT), vitamin D3-induced tolerogenic DC (vitD3-tolDC) and TT-loaded vitD3-tolDC (vitD3- tolDC-TT), as well as a negative control (C−), without any stimuli (n = 16) and (C) comparison of autologous antigen-specific proliferation against TT mediated by mDC-TT and vitD3-tolDC-TT on each donor. Data presented as relative percentage of induced proliferation compared to mDC-TT, measured as tritiated thymidine incorporation after 18 h. Six replicated measurements of each condition were performed. Error bars corresponding to SEM. ns, not significant; *p < 0.05. One-way ANOVA test with Geisser–Greenhouse correction or paired t test. Frontiers in Immunology | www.frontiersin.org 5 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness A B C FIGURE 2 | Phenotype and cytokine production of DC-co-cultured autologous PBMC. (A) Gating strategy. (B) Relative percentages of CD4+CD38+HLA-DR+, CD8+CD38+HLA-DR+, TH1 Central Memory and TH1 Effector Memory subpopulations on PBMC co-cultured with either tetanus toxin (TT)-loaded mature DC (mDC- TT) or TT-loaded vitamin D3-induced tolerogenic DC (vitD3-tolDC-TT) after 5 days of culture (n = 10). Data presented as the relative percentage of each lymphocyte subpopulation within its respective parent subpopulation, measured by multiparametric flow cytometry, in a box and whiskers representation. Error bars corresponding to the maximum and minimum values of each condition. (C) Analysis of the secretion of granzyme B (GZMB) (n = 12), IFN-g (n = 12), IL-1b (n = 8), IL-6 (n = 8), IL-10 (n = 10), TGF-b (n = 7), and TNF-a (n = 8) in the supernatants of PBMC co-cultured with either mDC-TT or vitD3-tolDC-TT after 5 days of culture using either ELISA (TGF-b) or cytometric bead array (GZMB, IFN-g, IL-1b, IL-6, IL-10, and TNF-a) techniques. One ELISA experiment was performed for all samples, with duplicated measurements for each sample. One single cytometric bead array experiment was performed for the analysis of all the samples, with one single measurement for each sample. Sample sizes vary due to some measurements being negative or under the detection limit of the technique. Error bars corresponding to SEM. ns, not significant; * p < 0.05. Paired t test or Wilcoxon test. Frontiers in Immunology | www.frontiersin.org 6 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness detect any significant changes over any other T cell subpopulation, their RNA and selected 10 donors that showed sufficient nucleic nor Treg nor Tr1 subpopulations (data not shown). acid concentration and integrity for the RNA-seq analysis (RIN > 7) Next, we analyzed the cytokine secretion profile present in the in both mDC-Tcell and vitD3-Tcell conditions. Consequently, autologous co-cultures of mDC-TT and vitD3-tolDC-TT. Our donors HD4, HD5, HD9, HD10, HD11, and HD12 were results, as shown in Figure 2C, evidenced a statistically significant discarded from downstream studies. increased secretion of the cytokine IL-6 and lower levels of IFN-g in After processing the samples through the RNA-seq analysis, the co-culture of autologous PBMC with vitD3-tolDC-TT compared 39% of total reads could be assigned to different known RNA to mDC-TT (IL-6 mDC-TT: 61.4 ± 84.3 pg/ml vs IL-6 vitD3-tolDC-TT: classes (Supplementary Figure 2A), and out of them, around 77.7 ± 94.5 pg/ml; p = 0.039; and IFN-g mDC-TT: 3.3 ± 2.9 pg/ml vs 47% of these assigned reads could be related to protein coding IFN-g vitD3-tolDC-TT: 2.2 ± 2.8 pg/ml; p = 0.002). Therefore, the genes (Supplementary Figure 2B). Interestingly, the hierarchical reduction in the production of IFN-g, combined with the increase clustering analysis revealed that our samples tended to cluster by of IL-6, again suggest a reduction of the TH1-like cytokine profile, in individual rather than by treatment (Figure 3A), but also that line with the phenotype results. No statistically significant changes there is a consistent pattern by which vitD3-Tcell samples ranked could be found in the production of GZMB, IL-1b, IL-10, TGF-b nor higher on both axes from the PCA (Figure 3B). These results led TNF-a. to choose a paired comparative analysis approach for the differential expression analysis. VitD3-tolDC Induce a General After the subsequent filtering process described in the Material Transcriptomic Repression Over and Methods section, a total of 16,333 protein coding genes with T CD4+ Cells detectable reads were tested for differential expression. Among all For all the 16 donors, at least 700,000 CD3+CD4+ cells in both of them, 546 genes showed a statistically significant change in their conditions (mDC-Tcell and vitD3-Tcell) were successfully expression (adjusted p < 0.05) in vitD3-Tcell compared to mDC- isolated by flow cytometry cell sorting. The gating strategy is Tcell, and only 373 also presented an absolute value of FC superior shown in Supplementary Figure 1. Afterwards, we extracted to 1.20 (|FC vitD3-Tcell vs mDC-Tcell| > 1.20). While only 29 of these A B C FIGURE 3 | Exploratory analysis of the RNA-seq study of T CD4+ cells co-cultured with autologous antigen-specific DC. (A) Hierarchical clustering analysis by gene expression of the 20 samples of CD4+ T cells of the RNA-seq study. (B) Representation of the first two principal component analysis (PCA) components at the gene level. Each color corresponds to a different sample of CD4+ T cells, as depicted in the legend, and the co-culture condition of each sample is indicated with a prefix, either “m” for tetanus toxin (TT)-loaded mature DC or “v” for TT-loaded vitamin D3-induced tolerogenic DC. (C) Volcano plot showing the significant differentially expressed genes. Axis is the log2 fold change. Color code: green, significantly regulated genes (padj < 0.05; |FC| > 1.2) considered in the Gene Ontology enrichment analysis; orange, genes with |FC| > 1.2 below the significance threshold; red, genes with padj < 0.05 below the relevant fold change cutoff. Frontiers in Immunology | www.frontiersin.org 7 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness genes were up-modulated in vitD3-Tcell compared to mDC-Tcell, many more results. Within the 50 most down-regulated results the majority of them, 344 genes, were down-modulated, indicating (FC vitD3-Tcell vs mDC-Tcell < 1.78) we found several genes encoding a strong transcriptomic repression induced by vitD3-tolDC-TT proteins involved in the immune response (CCL17, CCL22, EBI3, over these cells (Figure 3C). IL13 and LIF), antigen presentation (HLA-DQA1, HLA-DQA2 and HLA-DRB5) and microtubule binding (KIF4A, KIF15, KIF18B T CD4+ Cells Selectively Undergo a Strong and KIFC1), among others (Table 2). Furthermore, when we Functional and Immune-Related analyzed the whole list, we could find not only several more genes Transcriptomic Down-Modulation Upon included in these categories, but also many other genes encoding Interaction With vitD3-tolDC proteins related to cytoskeleton and cell adhesion (ARPC1B, When we studied those differentially expressed genes that CAPG, CTNNA1, LGALS1, MYL6B, LGALS9 or SDC4), actin appeared up-modulated (FC vitD3-Tcell vs mDC-Tcell > 1.20) in our related functionalities (ACTB, ACTG1, PARVB or TPM4), G- analysis (Table 1), we did not find many relevant or immune- proteins and modulators (GBP2, GBP4, GNA15, GNG4, related genes. Specifically, 18 of these 29 genes did not have any IQGAP3, MYO1G, MYO1E or SRGAP3), nucleic acid binding GO annotation, and among the rest, we could only find the genes (ASF1B, DEPDC1, EXO1 or FEN1), histones (HIST1H2BL, encoding the JUNB and SCML1 transcription factors and several HIST2H2BF or, HIST1H4H), the pro-inflammatory transcription other genes encoding different molecule transporters (ABCC2 factor STAT1 and other pro-inflammatory mediators (TNFSF4), and SLC10A1), G-protein modulators (GRTP1 and RASA4) and different kinase activators and modulators, proteases and protease kinases (AK5 and CKMT2). inhibitors, oxydases, oxygenases, transferases and many other However, as mentioned above, the study of the down- metabolic mediators. The whole list is shown in Supplementary modulated genes (FC vitD3-Tcell vs mDC-Tcell < 1.20) yielded Table 3. Altogether, these results indicate that vitD3-tolDC-TT mediate a strong down-modulation of metabolic and immune- related functions over vitD3-Tcell. TABLE 1 | Up-modulated genes in vitD3-Tcell compared to mDC–Tcell. VitD3-Tcell Present Decreased Cell Cycle Gene GO annotation FC vs Adj. Symbol mDC–Tcell p-value and Mitotic Activity In this regard, the GO enrichment analysis further supported the TMIE NA 1.62 0.01199 results observed in the differential gene expression (DGE) study. PRH2 NA 1.61 0.04136 Thus, first, the enrichment analysis produced a total of 482 ARHGEF26 NA 1.53 0.01404 GRTP1 G-Protein Modulator; Cysteine Protease 1.50 0.03547 protein sets and pathways with p < 0.001, four of them up- AKAP6 NA 1.45 0.01310 modulated—although none of them showed an FDR value below CKMT2 Amino Acid Kinase 1.43 0.03068 0.25—and the remaining 478 down-modulated (Supplementary SLC10A1 Cation Transporter 1.40 0.00264 Table 4). We further filtered the results to analyze only those GO C17orf107 NA 1.40 0.01419 SULT1B1 NA 1.38 0.00117 terms that presented a much more significant enrichment (p < TTC16 NA 1.37 0.03271 10-9). This process left us with a total of 66 down-modulated GO TEC NA 1.35 0.02676 terms, but none up-modulated. These 66 elements, ordered by KRT72 NA 1.31 0.00543 decreasing ES, are shown in Table 3. Interestingly, among the ABCC2 ATP-Binding Cassette (ABC) Transporter 1.30 0.00598 most significantly enriched down-modulated pathways, we KRT73 NA 1.28 0.02747 SORBS3 NA 1.26 0.00279 found several GO annotations referring to immune-related AK5 Nucleotide Kinase 1.24 0.02996 functionality (for instance Interferon-Gamma-Mediated EDAR NA 1.24 0.00041 Signaling Pathway, ES: 10.65; Cytokine-Mediated Signaling ADAM23 Metalloprotease 1.24 0.01495 Pathway, ES: 5.09; or Immune Response, ES: 3.03), class II- RALGPS2 Guanyl-Nucleotide Exchange Factor 1.23 0.02922 related antigen presentation (like MHC Class II Protein ALS2CL NA 1.22 0.04815 KBTBD11 NA 1.22 0.00543 Complex, ES: 30.23; Antigen Processing And Presentation Of JUNB Basic Leucine Zipper Transcription Factor; 1.22 0.00360 Exogenous Peptide Antigen Via MHC Class II, ES: 8.08; or Nucleic Acid Binding Antigen Processing And Presentation Of Exogenous Peptide RASA4 G-Protein Modulator 1.22 0.01328 Antigen, ES: 6.74), cell response to different stimuli (Cell C9orf72 NA 1.22 0.00818 Surface Receptor Signaling Pathway, ES: 2.21; Response To ZC4H2 NA 1.21 0.04572 ADPRM NA 1.21 0.01438 Stress, ES: 1.93; or Cellular Response To Stimulus, ES: 1.80) SCML1 Chromatin/Chromatin-Binding Protein; 1.21 0.03955 and, specially, to cell cycle and mitotic division (for instance Transcription Factor Condensed Chromosome Outer Kinetochore, ES: 21.98; Mitotic MEGF6 Extracellular Matrix Protein 1.21 0.00102 Spindle Organization, ES: 8.79; Microtubule Cytoskeleton LMTK3 NA 1.21 0.03156 Organization Involved In Mitosis, ES: 7.48; or Cell Cycle Gene expression values from an RNA-seq analysis with healthy donors (n = 10). Data Checkpoint, ES: 6.05). Our results, in line with the DGE presented as the mean fold change (FC) of expression in vitD3-Tcell compared to mDC- analysis, would suggest that vitD3-Tcell are undergoing a Tcell. GO, Gene Ontology; mDC-Tcell, mature dendritic cell-conditioned CD3+CD4+ cells; NA, not available; padj, adjusted p-value; vitD3-Tcell, vitamin D3-induced tolerogenic process of transcriptomic down-modulation leading to reduced dendritic cell-conditioned CD3+CD4+ cells. immune-related, metabolic and proliferative functionalities. Frontiers in Immunology | www.frontiersin.org 8 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness TABLE 2 | Top 50 down-modulated genes in vitD3-Tcell compared to mDC-Tcell. GeneSymbol GO annotation FC vs mDC-Tcell padj IL13 Cytokine −2.59 0.00082 C1orf106 NA −2.43 0.00486 RYR2 Ligand-Gated Ion Channel −2.35 0.00802 HLA-DQA2 Major Histocompatibility Complex Antigen −2.25 0.03271 UBE2C NA −2.23 0.00000 DEPDC1 Nucleic Acid Binding −2.23 0.00367 NEK2 Protein Kinase −2.20 0.00206 EBI3 Cytokine; Defense/Immunity Protein −2.18 0.00009 CCL17 Chemokine −2.18 0.01852 AURKB Non-Receptor Serine/Threonine Protein Kinase −2.14 0.00006 CYP1B1 Oxygenase −2.12 0.02334 SPC25 Enzyme Modulator −2.11 0.01556 KIF4A Microtubule Binding Motor Protein −2.09 0.00001 KIF18B Microtubule Binding Motor Protein −2.07 0.00001 CCNB2 Kinase Activator −2.06 0.00000 HLA-DRB5 Major Histocompatibility Complex Antigen −2.02 0.00970 MCM10 NA −2.01 0.00003 HIST1H3C Histone −2.01 0.00000 KIF15 Microtubule Binding Motor Protein −1.97 0.00000 BIRC5 Protease Inhibitor −1.96 0.00002 CHST3 NA −1.95 0.01782 FAM111B NA −1.95 0.00000 MYBL2 NA −1.92 0.00000 ATP8B4 Cation Transporter. Hydrolase −1.92 0.00000 SKA1 NA −1.91 0.00898 KIAA0101 NA −1.91 0.00292 TK1 Nucleotide Kinase −1.90 0.00001 HIST1H3J Nucleic Acid Binding; Transcription Factor −1.89 0.03504 E2F8 Nucleic Acid Binding; Transcription Factor −1.89 0.00209 HLA-DQA1 Major Histocompatibility Complex Antigen −1.88 0.00011 HIST1H3F Reductase −1.88 0.00005 RRM2 Reductase −1.88 0.00337 GNG4 Heterotrimeric G-Protein −1.87 0.01479 PRR11 NA −1.86 0.00041 CEP55 NA −1.85 0.00008 CKAP2L NA −1.84 0.00151 CDCA8 NA −1.84 0.00015 HIST1H3G Histone −1.83 0.00000 CDK1 Non-Receptor Serine/Threonine Protein Kinase; Non-Receptor Tyrosine Protein Kinase −1.83 0.00283 HMMR NA −1.83 0.00012 PKMYT1 Non-Receptor Serine/Threonine Protein Kinase −1.83 0.00825 CCL22 Chemokine −1.82 0.02508 CREB3L3 NA −1.81 0.02749 CDC25A Protein Phosphatase −1.81 0.02454 DTL NA −1.81 0.00008 RAD51AP1 NA −1.80 0.00912 ESCO2 NA −1.79 0.01020 LIF Cytokine −1.78 0.00000 KIFC1 Microtubule Binding Motor Protein −1.78 0.00000 ASB2 NA −1.78 0.00000 Gene expression values from an RNA-seq analysis with healthy donors (n = 10). Data presented as the mean fold change (FC) of expression in vitD3-Tcell compared to mDC-Tcell. GO, Gene Ontology; mDC-Tcell, mature dendritic cell-conditioned CD3+CD4+ cells; NA, not available; padj, adjusted p-value; vitD3-Tcell, vitamin D3-induced tolerogenic dendritic cell- conditioned CD3+CD4+ cells. DISCUSSION induced on T cells due to a lack of one or more of the three immunogenic activation signals. This causes T cells to become In this study we analyzed the specific effect of vitD3-tolDC over hyporesponsive or to die (20, 30). However, in the case of autologous CD4+ T cells. Thus, we switched the attention from autoimmunity, where T cells are already activated and the study of tolDC themselves—widely studied so far—to focus developing an immunogenic response, an antigen-specific on the study of the functional effect that these cells develop over process of tolerance induction is required. In this regard, T cells upon their interaction. In homeostatic conditions, either previous in vivo studies with vitD3-tolDC in the EAE model depletion, inactivation and/or induction of anergy is often showed that an antigen-specific setup—and therefore an active Frontiers in Immunology | www.frontiersin.org 9 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness TABLE 3 | Most significantly down-regulated Gene Ontology terms in vitD3-Tcell compared to mDC-Tcell. GO category GO term ES p-value FDR GOCOMPONENT MHC Class II Protein Complex 30.23 5.24E-16 2.39E-13 GOCOMPONENT Condensed Chromosome Outer Kinetochore 21.98 3.96E-10 4.81E-08 GOCOMPONENT MHC Protein Complex 18.71 1.94E-12 5.88E-10 GOCOMPONENT Clathrin-Coated Endocytic Vesicle Membrane 16.92 6.86E-10 7.81E-08 GOFUNCTION Peptide Antigen Binding 16.92 6.86E-10 7.06E-07 GOPROCESS Interferon-Gamma-Mediated Signaling Pathway 10.65 1.35E-13 1.44E-10 GOCOMPONENT DNA Packaging Complex 10.26 5.61E-19 5.11E-16 GOPROCESS Nuclear Chromosome Segregation 10.21 9.93E-10 2.98E-07 GOCOMPONENT Nucleosome 9.82 2.16E-16 1.31E-13 GOPROCESS Mitotic Spindle Organization 8.79 1.87E-11 8.07E-09 GOPROCESS Antigen Processing And Presentation Of Exogenous Peptide Antigen Via MHC Class II 8.08 1.20E-12 8.70E-10 GOPROCESS Regulation Of Chromosome Segregation 7.98 9.85E-14 1.24E-10 GOPROCESS Antigen Processing And Presentation Of Peptide Or Polysaccharide Antigen Via MHC Class II 7.98 1.50E-12 9.88E-10 GOPROCESS Antigen Processing And Presentation Of Peptide Antigen Via MHC Class II 7.98 1.50E-12 9.43E-10 GOPROCESS Microtubule Cytoskeleton Organization Involved In Mitosis 7.48 1.93E-11 8.07E-09 GOPROCESS Chromosome Segregation 7.21 8.19E-13 7.07E-10 GOPROCESS Mitotic Cell Cycle 7.20 1.49E-15 2.29E-12 GOPROCESS Nucleosome Assembly 7.08 9.78E-14 1.35E-10 GOPROCESS Antigen Processing And Presentation Of Exogenous Peptide Antigen 6.74 1.09E-11 5.59E-09 GOPROCESS Antigen Processing And Presentation Of Exogenous Antigen 6.62 1.57E-11 6.98E-09 GOCOMPONENT Protein-DNA Complex 6.57 5.01E-14 1.82E-11 GOPROCESS Antigen Processing And Presentation Of Peptide Antigen 6.32 3.73E-11 1.47E-08 GOPROCESS Spindle Organization 6.11 2.12E-10 7.32E-08 GOPROCESS Cell Cycle Checkpoint 6.05 2.87E-11 1.16E-08 GOPROCESS Antigen Processing And Presentation 5.91 1.55E-11 7.15E-09 GOCOMPONENT Midbody 5.75 9.66E-12 1.95E-09 GOPROCESS Nucleosome Organization 5.71 1.12E-11 5.52E-09 GOPROCESS Regulation Of Mitotic Nuclear Division 5.46 7.83E-11 2.92E-08 GOPROCESS Regulation Of Nuclear Division 5.39 1.37E-11 6.52E-09 GOPROCESS Cytokine-Mediated Signaling Pathway 5.09 3.61E-27 2.49E-23 GOCOMPONENT Spindle 5.07 1.32E-10 1.85E-08 GOCOMPONENT Chromosome 4.67 9.10E-12 2.07E-09 GOPROCESS Mitotic Cell Cycle Process 4.64 3.48E-28 4.81E-24 GOPROCESS Positive Regulation Of Cell Cycle Process 4.55 4.10E-11 1.57E-08 GOPROCESS Protein-DNA Complex Assembly 4.47 7.59E-10 2.33E-07 GOPROCESS Cell Division 4.25 5.31E-16 9.17E-13 GOPROCESS Negative Regulation Of Cell Cycle Process 4.04 6.43E-10 2.02E-07 GOPROCESS Regulation Of Mitotic Cell Cycle Phase Transition 3.99 1.04E-12 8.42E-10 GOPROCESS Chromosome Organization 3.94 1.50E-12 1.04E-09 GOPROCESS Regulation Of Cell Cycle Phase Transition 3.82 1.92E-12 1.15E-09 GOPROCESS Cell Cycle Process 3.63 2.27E-25 1.05E-21 GOPROCESS Cell Cycle 3.56 1.11E-12 8.53E-10 GOPROCESS Regulation Of Cell Cycle Process 3.47 7.37E-17 1.69E-13 GOFUNCTION Protein Heterodimerization Activity 3.44 5.36E-10 7.36E-07 GOCOMPONENT Chromosomal Part 3.27 8.53E-20 1.55E-16 GOCOMPONENT Nuclear Chromosome Part 3.14 3.70E-11 6.75E-09 GOPROCESS Regulation Of Mitotic Cell Cycle 3.12 2.72E-12 1.44E-09 GOPROCESS Negative Regulation Of Cell Cycle 3.12 2.28E-10 7.66E-08 GOPROCESS Immune Response 3.03 2.51E-12 1.45E-09 GOPROCESS Regulation Of Cell Cycle 2.82 6.87E-17 1.90E-13 GOPROCESS Positive Regulation Of Immune System Process 2.68 5.07E-10 1.67E-07 GOFUNCTION Protein Dimerization Activity 2.58 1.83E-12 3.76E-09 GOPROCESS Immune System Process 2.36 3.18E-16 6.27E-13 GOPROCESS Regulation Of Immune System Process 2.31 1.35E-10 4.91E-08 GOCOMPONENT Cytoskeletal Part 2.26 7.15E-12 1.86E-09 GOPROCESS Cell Surface Receptor Signaling Pathway 2.21 2.80E-13 2.76E-10 GOPROCESS Response To Stress 1.93 6.04E-13 5.56E-10 GOCOMPONENT Extracellular Region Part 1.83 1.79E-10 2.33E-08 GOCOMPONENT Non-Membrane-Bounded Organelle 1.82 1.01E-10 1.67E-08 GOCOMPONENT Intracellular Non-Membrane-Bounded Organelle 1.82 1.01E-10 1.53E-08 GOPROCESS Cellular Response To Stimulus 1.80 6.19E-10 1.99E-07 GOPROCESS Response To Stimulus 1.78 6.82E-17 2.35E-13 GOPROCESS Signal Transduction 1.76 9.95E-14 1.14E-10 (Continued) Frontiers in Immunology | www.frontiersin.org 10 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness TABLE 3 | Continued GO category GO term ES p-value FDR GOPROCESS Regulation Of Cellular Process 1.28 1.91E-10 6.76E-08 GOFUNCTION Protein Binding 1.26 4.87E-15 2.01E-11 GOPROCESS Cellular Process 1.15 2.64E-12 1.46E-09 Results presented as Enrichment Score (ES) values of different Gene Ontology (GO) terms in vitD3-Tcell compared to mDC-Tcell using data from an RNA-seq analysis with healthy donors (n = 10). FDR, False Discovery Rate; GOCOMPONENT, GO cellular component; GOFUNCTION, GO molecular function; GOPROCESS, GO biological process; mDC-Tcell, mature dendritic cell-conditioned CD3+CD4+ cells; NA, not available; padj, adjusted p-value; vitD3-Tcell, vitamin D3-induced tolerogenic dendritic cell-conditioned CD3+CD4+ cells. process—is required, provided that a beneficial effect of this mediated signaling pathway was observed, which on the other therapy was only observed when vitD3-tolDC were pulsed with hand supported the robustness of our RNA-seq study. Beyond the adequate immunogenic peptide (23, 24). Consequently, we this, the results pointed towards a generalized down-modulation developed an experimental model for the generation of of the transcriptomic profile of vitD3-Tcell that could either autologous antigen-specific vitD3-tolDC and T cells from respond to an induction of T CD4+ cell hyporesponsiveness or healthy donors, using an immunogenic peptide presented via even to a process of clonal deletion. On the one hand, the down- class II MHC with the aim to reproduce antigen presentation in modulation of genes and pathways involved in crucial cellular the context of CD4+-mediated autoimmune diseases. In this mechanisms—in particular those related to cell proliferation, regard, we selected TT for its compliance with this feature— mitosis, cell cycle and response to immune stimuli, some of them since the vaccination against TT is included in European health never reported before—could be explained by both of these systems—which also allowed us to use healthy donors instead of processes. However, on the other hand, the lack of induction patients of a determined autoimmune disease, thus eliminating of cell death and apoptotic-related pathways makes clonal disease-conditioned immune variations. deletion very unlikely to be happening. Therefore, our results All in all, our approach aimed to be as versatile as possible, suggest that the antigen-specific interaction of vitD3-tolDC with and to serve as a preliminary study for future research, with the autologous T CD4+ cells is mediating, in fact, an induction of idea that the immunogenic peptide/s might be replaced hyporesponsiveness over these cells. Furthermore, previous depending on the disease of interest, as well as, of course, studies from our group already pointed in this direction (16), a using patient cells instead of healthy donors. On the one hand, biological situation that, potentially, might lead to the abrogation for autoimmune diseases with identified autoantigens, this of an autoimmune immunogenic response in patients. Moreover, decision would be trivial. On the other hand, for those our current results provide evidence that these modulations are conditions in which autoimmune antigens are yet to be taking place at the transcriptomic level in T CD4+ cells, identified, the experimental design should be considered case indicating that the antigen-specific modulation induced by by case. For instance, using autologous synovial fluid as a source vitD3-tolDC is deeper than expected and, in consequence, for autoantigens to load tolDC has provided promising results in probably also long-lasting. rheumatoid arthritis (31), so analog workaround solutions could Unfortunately, the lack of strongly up-regulated genes among be taken into account. the protein-coding RNA transcripts did not allow us to point After validating our experimental setup—meaning that towards many clear candidate biomarkers that might become vitD3-tolDC-TT were able to induce an antigen-specific indicators of the response of T cells upon their interaction with response—we focused on the study of the actual phenotypic, vitD3-tolDC to monitor patients in clinical trials. One of the functional and transcriptomic modulations induced by vitD3- most relevant exceptions came given by JUNB gene, encoding a tolDC. First, the analysis of the phenotype of T CD4+ cells member of the AP-1 family of transcription factors. In evidenced that their interaction with vitD3-tolDC-TT caused a experimental models, this gene has been reported to be crucial relative reduction in the activation of these cells. More in maintaining Treg suppressive function (32), although it is also importantly, a switch in the immune response of these cells apparently involved in the induction and maintenance of IL-23- towards a more immunoregulatory profile was induced, with a related pathogenicity of TH17 cells (33, 34). However, neither of reduction in the prevalence of TH1 memory subpopulations. these functionalities seems to fit in our model based on our These results were further supported by the decrease of IFN-g results, since neither TH17 nor Treg induction was evidenced. production in the autologous co-culture supernatants, Consequently, it would be interesting to elucidate the specific consequently supporting that vitD3-tolDC were inducing a role of JunB in our experimental setting, and to what extent small switch towards a more anti-inflammatory immune profile, in changes in its expression can actually be sufficient or not for the line with previous findings in the literature (16–18). induction and maintenance of immune tolerance. Furthermore, When we deepened into the analysis of the vitD3-tolDC- if this hypothesis proves to be valid, either JUNB and/or other mediated transcriptomic profile of T cells, we observed several related genes might also constitute potential biomarkers of genes and GO terms regulated in line with the abovementioned response to vitD3-tolDC treatment in the clinic. It is also phenotypical and functional switch towards a less activated an worth noting that we may have overlooked other potential more immunoregulatory profile; for instance, a down- biomarkers that might be found among the non-protein- modulation of STAT1 gene and the interferon-gamma- coding and alternate splicing RNA transcripts. Although this Frontiers in Immunology | www.frontiersin.org 11 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness possibility, if true, would have a limited functional value in our identified several specific genes and pathways selectively down- experimental model, it could be addressed in future studies. modulated, as well as the induction of JUNB, which might In addition, our results did not allow us to reach any constitute a putative biomarker of the modulation mediated by conclusion regarding a potential induction of anergy, and, as vitD3-tolDC over CD4+ T cells and, consequently, a potential discussed above, they also rule out any kind of Treg or Tr1 biomarker to monitor the effect of vitD3-tolDC after their response. Even though our previous in vivo experiments with the administration to patients. Therefore, the results presented in murine EAE model pointed towards an induction of Treg this article allowed us to better understand the process of T cell mediated by vitD3-tolDC (23, 24), we have observed that, at hyporesponsiveness at the molecular level and, more importantly, least in this experimental setting, this is not the case with human to set the path for future studies to fully elucidate the specific cells. These results are in line with what previous studies from processes that are taking place in one of the most important both our group and other authors have already reported (13, 16, mechanisms that the promising tolDC-based therapies can 35), although there seems to be some controversy (36, 37). trigger in order to restore tolerance in autoimmune diseases. However, it is also worth mentioning that in these reports, Treg induction was only observed after two rounds of stimulation of T cells, which might explain why we have not detected it. This is definitely something to be taken into account, DATA AVAILABILITY STATEMENT since Treg induction is, undoubtedly, one of the main The datasets presented in this study can be found in online mechanisms for the induction of immune tolerance of tolDC repositories. The names of the repository/repositories and and other antigen presenting cell approaches (38, 39). Indeed, accession number(s) can be found below: https://www.ncbi. Treg themselves, when expanded in vitro, present a huge nlm.nih.gov/geo/, GSE128816. therapeutic potential as a cell therapy for autoimmune diseases in humans (40). Consequently, the transcriptomic study of vitD3-tolDC-induced Treg should probably be addressed separately in future studies, since two rounds of T cell ETHICS STATEMENT stimulation might have masked some of the results that we This study was reviewed and approved by the Germans Trias i have reported here. Pujol Hospital Ethical Committee. The participants provided Our current study presents some limitations. First, since we their written informed consent to participate in this study. focused on the study of CD4+ T cells alone, we were naturally omitting the potential modulation that vitD3-tolDC might be mediating through other subpopulations, such as regulatory B cells or regulatory NK cells. Furthermore, the election of the AUTHOR CONTRIBUTIONS timepoint for the RNA-seq analysis intrinsically establishes another limitation, which is the status of the transcriptomic EM-C, JN-B and MM conceived the experiments. JN-B and MM profile at different timepoints of the co-culture. However, our performed the cell cultures and the cell sortings, and prepared selection came based on the phenotypical and functional results the samples for the RNA-seq analysis. JN-B and MM analyzed shown in the study, which evidence that, by day 5 of the co- the results. EM-C, JN-B and MM interpreted the results. JN-B culture, there is a significant and differential modulation wrote the manuscript. AT-S, BQ-S, CR-T, EM-C, JN-B, and MM mediated by vitD3-tolDC over T CD4+ cells. Consequently, reviewed the manuscript. All authors contributed to the article even though it is true that other timepoints might provide and approved the submitted version. valuable additional information, we think that our election provided the best compromise, and a full time-course characterization of the antigen-specific transcriptomic changes FUNDING induced by vitD3-tolDC will be addressed in future studies. On the other hand, we cannot fully discard the presence of non- This work was partially supported by projects PI14/01175, PI16/ antigen-specific CD4+ cells by the time the cell sorting was 01737 and PI17/01521, integrated in the Plan Nacional de I+D+I performed. However, even with a residual amount of non- and co-supported by the ISCIII-Subdirección General de antigen-specific T cells, the obtained results were consistent Evaluación and the Fondo Europeo de Desarrollo Regional not only within the different techniques, but also with the (FEDER), and by grant 140191 from IWT-TBM (Belgium). literature, supporting our findings. JN-B was beneficiary of a FI-DGR PhD contract from AGAUR In conclusion, our results evidence that vitD3-tolDC are (Agència de Gestió d’Ajuts Universitaris i de Recerca), supported inducing a strong antigen-specific transcriptomic down- by the Government of Catalonia (02/2015-01/2018). The modulation over autologous T CD4+ cells, with a reduced ability contracts of JN-B and MM have been partially supported by to respond to immune- and non-immune-related stimuli. grant 140191 from IWT-TBM. The authors are members of a Consequently, it constitutes one of the first attempts to consolidated group recognized by AGAUR (2017 SGR 103), understand the changes that T cells are undergoing at the supported by the Government of Catalonia. This work has transcriptomic level upon an antigen-specific interaction with a been supported by positive discussion through A FACTT tolerogenic cell product, such as vitD3-tolDC. In that regard, we network (COST Action BM1305: www.afactt.eu) and project Frontiers in Immunology | www.frontiersin.org 12 January 2021 | Volume 11 | Article 599623 Navarro-Barriuso et al. VitD3-tolDC Induce T-Cell Transcriptomic Hyporresponsiveness 779316–ReSToRe (www.h2020restore.eu). COST and ReSToRe SUPPLEMENTARY FIGURE 1 | Phenotype and functionality of DC. (A) Gating strategy. (B) Surface expression of CD86 (FITC) and HLA-DR (V500) on immature are supported by the EU Framework Programme Horizon 2020. DC (iDC), mature DC (mDC), tetanus toxin (TT)-loaded mDC (mDC-TT), vitamin D3- induced tolerogenic DC (vitD3-tolDC) and TT-loaded vitD3-tolDC (vitD3-tolDC-TT) (n = 8). Data presented as relative percentage of median fluorescence intensity (MFI) normalized versus mDC, measured by flow cytometry. In all cases, DC populations ACKNOWLEDGMENTS were CD11c+ (C) Allogeneic proliferation of PBMC from healthy donors co-cultured with either iDC, mDC, mDC-TT, vitD3-tolDC or vitD3-tolDC-TT (n = 14) in a DC : We want to thank Marco A. Ferná ndez-Sanmartı́n and Gerard PBMC 1:20 ratio. Data presented as relative percentage of induced proliferation Requena, from the IGTP Flow Cytometry Platform, Edurne compared to mDC, measured as tritiated thymidine incorporation after 18 h. Six Pedrosa, Veró nica Guirao and Ingrid Liarte, from the IGTP replicated measurements of each condition were performed. Error bars corresponding to SEM. ns = not significant; * p < 0.05. Friedman test with Dunn’s Biobank, Lauro Sumoy and Raquel Pluvinet, from the IGTP correction. (D) Gating strategy for the sorting of autologous T CD4+ cells. High Content Genomics and Bioinformatics Unit, and, specifically, Jorge Carrascal and all the members of the IGTP SUPPLEMENTARY FIGURE 2 | Assignment and functional classification of the Immunology Division for their support and technical assistance. total reads from the RNA-seq study of T CD4+ cells co-cultured with autologous antigen-specific DC. (A) Assignment of the total reads from the RNA-seq analysis to known RNA classes. (B) Classification of the assigned reads into known RNA functionalities. SUPPLEMENTARY MATERIAL SUPPLEMENTARY TABLE 3 | Up and down-modulated genes in vitD3-Tcell compared to mDC-Tcell. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.599623/ SUPPLEMENTARY TABLE 4 | Differentially enriched Gene Ontology terms in full#supplementary-material vitD3-Tcell compared to mDC-Tcell. changes leading to a tolerogenic dendritic cell phenotype. J Immunol Baltim REFERENCES Md 1950 (2009) 182:2074–83. doi: 10.4049/jimmunol.0803345 13. Naranjo-Gó mez M, Raïch-Regué D, Oñate C, Grau-Ló pez L, Ramo-Tello C, 1. Ten Brinke A, Hilkens CMU, Cools N, Geissler EK, Hutchinson JA, Lombardi G, Pujol-Borrell R, et al. Comparative study of clinical grade human tolerogenic et al. Clinical Use of Tolerogenic Dendritic Cells-Harmonization Approach in dendritic cells. J Transl Med (2011) 9:89. doi: 10.1186/1479-5876-9-89 European Collaborative Effort. Mediators Inflammation (2015) 2015:471719. 14. 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Available at: doi: 10.1016/j.clim.2014.03.016 https://www.ncbi.nlm.nih.gov/books/NBK26921/ (Accessed February 21, 2019). Conflict of Interest: The authors declare that the research was conducted in the 31. Bell GM, Anderson AE, Diboll J, Reece R, Eltherington O, Harry RA, et al. absence of any commercial or financial relationships that could be construed as a Autologous tolerogenic dendritic cells for rheumatoid and inflammatory potential conflict of interest. arthritis. Ann Rheum Dis (2016) 76:227–34. doi: 10.1136/annrheumdis- 2015-208456 Copyright © 2021 Navarro-Barriuso, Mansilla, Quirant-Sánchez, Teniente-Serra, 32. Koizumi S, Sasaki D, Hsieh T-H, Taira N, Arakaki N, Yamasaki S, et al. JunB Ramo-Tello and Martı ́nez-Cáceres. This is an open-access article distributed under regulates homeostasis and suppressive functions of effector regulatory T cells. the terms of the Creative Commons Attribution License (CC BY). The use, distribution Nat Commun (2018) 9:5344. doi: 10.1038/s41467-018-07735-4 or reproduction in other forums is permitted, provided the original author(s) and the 33. Hasan Z, Koizumi S, Sasaki D, Yamada H, Arakaki N, Fujihara Y, et al. JunB is copyright owner(s) are credited and that the original publication in this journal is essential for IL-23-dependent pathogenicity of Th17 cells. Nat Commun cited, in accordance with accepted academic practice. No use, distribution or (2017) 8:15628. doi: 10.1038/ncomms15628 reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 14 January 2021 | Volume 11 | Article 599623