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. 2021 Aug 10:12:720133.
doi: 10.3389/fimmu.2021.720133. eCollection 2021.

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice

Deepika Watts  1   2   3 , Marthe Janßen  1   2   3 , Mangesh Jaykar  1 , Francesco Palmucci  1   2   3 , Marc Weigelt  4 , Cathleen Petzold  1 , Angela Hommel  4 , Tim Sparwasser  5 , Ezio Bonifacio  2   3   4 , Karsten Kretschmer  1   2   3
Affiliations

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice

Deepika Watts et al. Front Immunol. .

Abstract

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

Keywords: Foxp3; Treg cells; cell ablation; immune regulation; type 1 diabetes.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Rapid progression to overt diabetes after acute Treg cell ablation in adult NOD.DEREG females. Mice were either left untreated or injected with DT on two consecutive days, and subjected to further analysis on day 14, unless otherwise stated. (A) Efficiency of Treg cell ablation. Representative dot plots of CD4-gated cells from peripheral blood of 16-week-old NOD.DEREG females before (day 0) and after (day 3) injection with DT. Numbers in dots plots indicate mean percentage ± SD of 8 mice (unpaired t-test, p < 0.001) from a single experiment representative of 6 experiments performed (5-9 mice per experiment). (B–E) Pancreatic pathohistological changes. Histological sections were scored as described in Materials and Methods. (B) Insulitis scores of 4-5-week-old female (left, n = 10) and male (right, n = 9) NOD.DEREG mice. As all mice remained normoglycemic by day 7 after the first DT injection, two additional doses of DT were administered (day 7 and 8) prior to histology on day 14. (C) Representative histology and (D) insulitis score of 8-9-week-old NOD.DEREG females (8-10 mice per group). Untreated NOD.RAG (n = 4) and DT-injected DEREG– NOD (n = 5) females were included as controls. (E) Insulitis scores of 16-week-old DEREG females on different genetic backgrounds (4-7 mice per group). (F) Expression of mRNA encoding immune effector molecules with known function in autoimmune β cell destruction. Freshly isolated mRNA from pancreas and pLN (pooled from 4-5 mice per group) of 10-12-week-old NOD.DEREG females was subjected to real-time RT-PCR using β-Actin for normalization. Heat map (row normalized) shows mean values of triplicate samples from a single experiment representative of at least two independent experiments performed. Blue and red represent lowest and highest gene expression values, respectively. Pancreas, Unpaired t-test: p < 0.001: Gzma, Gzmb, Prf1, Tnf; p ≤ 0.01: Ifng. pLN, Unpaired t-test: p <0.001: Gzma, Gzmb; p ≤ 0.01: Prf1; not significant: Tnf, Ifng. (G, H) Manifestation of overt diabetes. (G) Cohorts of 16-week-old, initially non-diabetic NOD.DEREG mice were repeatedly injected with 2 doses of DT at 5-day-intervals, as indicated. Blood glucose levels (BGL) were determined at least three times per week. (H) Diabetes incidence of female NOD.DEREG mice. Mice were considered diabetic at blood glucose levels above 200 mg/dl on at least two consecutive measurements or with blood glucose levels once above 400 mg/dl. Data are from a single experiment (n = 10) representative of > 8 experiments performed. Note that the diabetes incidence of male NOD.DEREG mice is depicted in Supplementary Figure S3.
Figure 2
Figure 2
Transient nature of Treg cell ablation in NOD.DEREG mice. (A) Kinetics of DT-mediated ablation and subsequent recovery of the CD4+GFP+ Treg cell compartment in pancreas and indicated lymphoid organs of 10-12-week-old NOD.DEREG mice. Data are from a single experiment (2 mice/timepoint) representative of 3 experiments performed. Arrows indicate days of DT injection. (B, C) GFP+ Treg cell recovery in (B) pancreas and (C) pLN is preceded by the rapid accumulation of CD4+CD25+ cells lacking DTR/GFP expression (GFP–). Each symbol corresponds to an individual mouse (10-12-week-old). (D, E) Impact of repeated DT administration on glycemic state and Treg cell depletion. (D) Blood glucose concentrations of individual NOD.DEREG mice presented in Figure 1 (H). Arrows indicate days of DT injection. (E) Kinetics of CD25+GFP+ and CD25+GFP– cells among CD4-gated cells, as revealed by flow cytometry among peripheral blood-derived CD4+ T cells at different timepoints (Two-way ANOVA in combination with Bonferroni’s Multiple Comparison post-test: ***p < 0.001). (F) Acute Treg cell ablation in the absence of Treg cell rebound. NOD.RAG1–/– mice were repopulated with diabetogenic BDC2.5 T conventional cells, either alone (red circles) or with GFP+ (blue squares) or DTR/GFP– (green squares) Treg cells, and blood glucose levels of recipients were routinely assessed at least twice a week for up to 9 weeks. DT was injected at three consecutive days of week 6 (arrows). (Mann-Whitney-U test; two weeks: p = 0.0159, nine weeks: p = 0.0476). (G–I) Pancreatic NKT cell accumulation after Treg cell ablation. (G) Percentages of CD3+CD49b+ NKT cells among FSC/SSC-gated cells in pLNs (left) and pancreas (right) of 12-16-week-old NOD.DEREG females before (day 0) and at indicated days after DT administration. Symbols and horizontal lines indicate individual mice (4-8 mice per timepoint) and mean values, respectively. Unpaired t-test: ns, not significant; *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001). (H, I) Kinetics of pancreas-infiltrating CD3+CD49b+ NKT and CD3–CD49b+ NK cells (FSC/SSC-gated) in (H) female and (I) male NOD.DEREG mice (10-12-week-old). Shown are mean percentages ± SD (day 0: n = 7) or mean percentages and range of replicate mice (days 1-7, day 10: n = 2).
Figure 3
Figure 3
Anti-CD3 mAb therapy after Treg cell ablation ameliorates insulitis and prevents progression to hyperglycemia. (A) Kinetics of CD8+ T cells with a naïve (CD62LhighCD44low), central memory (CD62LintCD44int), and effector/memory (CD62LlowCD44high) phenotype in pancreas (top), pLN (middle), and scLN (bottom) of 10-12-week-old NOD.DEREG mice before (day 0) and at indicated days after DT administration. (B, C) Adoptive splenocyte transfer model. (B) Splenocytes from adult (16-18-week-old) but not young (4-5-week-old) DTR/GFP+ NOD.DEREG donors promote overt diabetes in NOD.RAG1–/– recipient mice after DT treatment. Black arrow indicates the day of splenocyte transfer (2 x 106 total cells), and red arrows indicate the days of DT administration. (Log-Rank (Mantel-Cox) test, p = 0.0097). (C) Proportions of CD8+ T cells with a CD62LlowCD44high effector/memory phenotype in the pLN of NOD.RAG1–/– recipients of splenocytes from young (red circles) or adult (blue squares) NOD.DEREG donors. Unpaired t-test: ***p < 0.001. (D–F) anti-CD3 mAb treatment of Treg cell-depleted NOD.DEREG mice. (D) Scheme of experimental design. Cohorts of adult NOD.DEREG females were injected with DT only (n = 13), or were additionally treated with anti-CD3 mAb (n = 13). Untreated DTR/GFP– NOD.DEREG mice were included for comparison (n = 4). Arrows: red, DT injection; blue, anti-CD3 injection; black, analysis by histology and flow cytometry. Blood glucose concentrations were assessed before (day 0) and every second day after the first injection with DT until the end of the observation period on day 14. (E) Histological insulitis score of indicated experimental groups, and (F) blood glucose levels of individual mice (one-way ANOVA: p ≤ 0.01). Each symbol in (A, C, F) corresponds to an individual mouse.
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