Regulatory T cells control diabetes without compromising acute anti-viral defense

Abstract

While previous reports have demonstrated the efficacy of regulatory T cell therapy in the prevention of diabetes, systemic immunocompromise and Treg instability remain key safety concerns. Here we examined the influence of induced Treg (iTreg) cell therapy on anti-viral host defense and autoimmune T cell responses during acute viral infection in a murine model of autoimmune diabetes. Protective transfers of iTregs maintained IL-10 expression, expanded in vivo and controlled diabetes, despite losing FoxP3 expression. Adoptive transfer of iTregs affected neither the primary anti-viral CD8 T cell response nor viral clearance, although a significant and sustained suppression of CD4 T cell responses was observed. Following acute viral clearance, iTregs transferred early suppressed both CD4 and CD8 T cell responses, which resulted in the reversion of diabetes. These observations indicate that iTregs suppress local autoimmune processes while preserving the immunocompetent host's ability to combat acute viral infection.

Keywords: Diabetes;; Regulatory T cells;; Safety;; Stability;; Therapy; Viral infection;.

Figure 1

Generation and characterization of induced FoxP3+ regulatory T cells. Negatively selected CD4⁺CD25⁻T cells were isolated from the spleens of LCMV-GP_61–80 specific (Smarta) FoxP3-GFP reporter mice and cultured with α-CD3 and α-CD28 antibodies in the presence of rhIL-2 and TGFβ. (A) Overview of model system employed. (B) GFP reporter and surface marker expression of iTregs pre-sort (top) versus naïve CD4⁺ splenocytes (bottom). (C) iTreg culture supernatants analyzed by BioPlex. (D) Histograms demonstrate the surface expression phenotype of iTregs (black histogram) versus CD4⁺ splenocyte controls (grey filled histograms).

Figure 2

Early iTreg transfer confers protection from diabetes in the RIP-GP model. Female RIP-GP mice were infected with LCMV Armstrong either 4 (A) or 9 days prior (B) to Treg transfer (LATE Treg transfer or on the same day as Treg transfer (EARLY Treg transfer) and monitored for blood glucose values (BGVs) on a weekly basis. Mice with BGVs in excess of 300 mg/dL were considered diabetic.

Figure 3

Antigen specific FoxP3⁺ iTregs expand in vivo in response to LCMV Arm and transiently lose FoxP3 expression. (A) C57BL/6 iTreg expansion (left panel) in C57BL/6 mice is shown as the fold change in the percent of CD4T that are positive for the CD45.1 congenic marker. Loss of FoxP3 expression during LCMV infection is shown as the percent of transferred (CD45.1⁺) cells that are FoxP3⁺. Solid triangles indicate kinetics in LCMV infected C57BL/6. Open squares indicate kinetics in uninfected C57BL/6 mice. (B) iTreg expansion (upper three panels) and FoxP3 reporter expression (lower three panels) in transferred cells were monitored 8 days post transfer in female RIP-GP mice receiving iTregs LATE (9dpi) and EARLY (0dpi) versus mice that were not infected. iTregs were tracked by congenic marker, CD45.1, expression. Data are from 3 independent experiments.

Figure 4

FoxP3 iTreg transfer does not significantly affect the acute anti-viral CD8 T cell response. (A) Viral titers in the kidney, liver and spleen were monitored at day 6 and day 8 (data not shown) post early iTreg transfer/LCMV infection. No significant difference was observed between iTreg transferred mice and those not receiving iTregs. By day 8 post-infection/iTreg transfer virus was undetectable regardless of iTreg transfer (data not shown). (B) Anti-LCMV CD8 T cell responses were monitored 8 and 15 days post-iTreg transfer in RIP-GP mice. Cells recovered from the spleen were restimulated in vitro with GP_33–41 peptide and assessed by ICCS for TNFα and IFNγ expression.

Figure 5

Early FoxP3 iTreg transfer diminishes the activity of CD8 T at d15pi and CD4 T at both d8 and d15pi. Anti-LCMV CD4 T cell responses were monitored at days 8 (left) and 15 (right) post iTreg transfer in RIP-GP mice. Cells recovered from the spleen were restimulated in vitro with GP33 peptide and assessed by ICCS for TNFα and IFNγ expression. Data shown is from the spleen. Similar data was observed in the PLN and blood.

Figure 6

Early iTreg transfer results in an increase in the frequency of IL10⁺IFNγ⁺ iTregs. Transferred iTreg CD4 T cell responses were monitored 8 days post iTreg transfer (left) and 15 days post transfer (right). Cells recovered from the spleen were restimulated in vitro with GP_61–80 peptide and assessed by ICCS for IL-10 and IFN-γ expression. Similar data was observed in the PDLN and blood. (A) Summarized data is shown. (B) Representative dot plots shown.