Deficiency in B7-H1 (PD-L1)/PD-1 coinhibition triggers pancreatic beta-cell destruction by insulin-specific, murine CD8 T-cells

Abstract

Objective: RIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic beta-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic beta-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells.

Research design and methods: DNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1(-/-), PD-1(-/-) or bone marrow chimeric mice.

Results: Preproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1(-/-) or PD-1(-/-) mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic beta-cells or of PD-1 in autoreactive CD8 T-cells induced EAD.

Conclusions: An imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic beta-cells can trigger pancreatic beta-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the beta-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.

FIG. 1.

The RIP-B7.1 diabetes model. A: RIP-B7.1 or C57BL/6 (B6) mice were immunized with pCI/preproinsulin (■, n = 8) or the noncoding pCI (□, n = 8). At indicated times after immunization cumulative diabetes incidences (%) were determined. B: RIP-B7.1 mice were immunized with pCI/preproinsulin DNA. At 3 weeks after immunization, we selected three mice that had developed an early stage of EAD (with blood glucose levels between 300–440 mg/dl). These mice were injected twice (at days 21 and 23) with 200 μg mAb YTS-169 (anti-CD8). The glucose level (■; mg/dl) and CD8 T-cell numbers in the blood (□, CD8 T-cells number of nontreated mice were set for 100%) were determined at indicated time points after immunization. The injections of pCI/preproinsulin DNA and anti-CD8 mAb are indicated (arrows). C: RIP-B7.1 mice (n = 10) were immunized with pCI/preproinsulin DNA. Pancreatic CD8 T-cells derived from diabetic mice (blood glucose level >400 mg/dl) were pooled and restimulated ex vivo with a preproinsulin-specific peptide library (i.e., 10mers with two amino acids offset), and frequencies of IFNγ⁺ CD8 T-cells were determined by flow cytometry. The mean percentage of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown. D: Pancreatic cells were prepared from pCI/preproinsulin (group 1) or pCI/preproinsulin_N110A (group 2) immunized, diabetic RIP-B7.1 mice, or control pCI-immunized, healthy (group 3) RIP-B7.1 mice and restimulated ex vivo with the K^b/A_12–21, K^b/A_12-N21A or K^b/OVA_257–264 peptides, and specific IFNγ⁺ CD8 T-cell levels were determined by flow cytometry. The mean percentage of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (+ SD) of a representative experiment (n = three mice per group) are shown. pCI/ppins, pCI/preproinsulin.

FIG. 2.

Priming of preproinsulin-specific CD8 T-cells in B6 mice. A: C57BL/6 (B6) mice were immunized with pCI/preproinsulin_N110A or control pCI. Twenty-four days after immunization, histology was performed by analyzing pancreatic sections for insulin, CD8, and CD3 T-cells. B and C: B6 mice remained either untreated (□, group 1) or were immunized with pCI/preproinsulin_N110A (■, group 2). IFNγ-deficient mice were immunized with pCI/preproinsulin_N110A (○, group 3). B: Fourteen days after injection, CD8 T-cells were isolated from spleens. Next, 3 × 10⁶ splenic CD8 T-cells were transferred intravenously into sublethally (650rad) irradiated RIP-B7.1 hosts, and cumulative diabetes incidences were determined. C: Pancreatic CD8 T-cells were prepared from healthy hosts (transferred with nonimmune CD8 T-cells, group 1) or diabetic (transferred with immune CD8 T-cells, group 2) RIP-B7.1 hosts, and restimulated ex vivo with the K^b/A_12-N21A or control K^b/OVA_257–264 peptide. Specific IFNγ⁺ CD8 T-cell frequencies were determined by flow cytometry. The mean percentage of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (± SD) of a representative experiment (n = 3 mice per group) is shown. pCI/ppins_N110A, pCI/preproinsulin_N110A. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Blocking B7–H1 coinhibition induced EAD in pCI/preproinsulin_N110A-immunized B6 mice. B6 mice were immunized with pCI (A) or pCI/preproinsulin_N110A (B and C). At days 12 and 15 after immunization, mice were injected with blocking B7–H1 antibody (A and B) or control antibody (C), and blood glucose levels were followed up during the experiment. B,D, and E: Preproinsulin-immune and B7–H1 antibody-treated B6 mice developed either a severe hyperglycemia (with blood glucose levels >550 mg/dl, group 1) or a moderate hyperglycemia (with blood glucose levels of 250–360 mg/dl, group 2a) which subsequently returned to normoglycemia (with blood glucose levels of <200 mg/dl, group 2b). Representative mice in groups 1, 2a, and 2b were analyzed histologically for insulin expression and CD8 T-cell influx (D), or were analyzed for preproinsulin-specific CD8 T-cells (E). Pancreatic CD8 T-cells were restimulated ex vivo with the K^b/A_12-N21A or control K^b/OVA_257–264 peptide, and IFNγ⁺ CD8 T-cell levels were determined by flow cytometry (E). pCI/ppins_N110A, pCI/preproinsulin_N110A. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Preproinsulin-specific EAD development in B7–H1^−/− and PD-1^−/− mice. A: B7–H1^−/− mice were immunized with pCI/preproinsulin_N110A (●, n = 5), or the noncoding pCI (○, n = 8), and cumulative diabetes incidences (%) were determined. B: RIP-B7.1 (■) and B7–H1^−/− (●) mice were immunized with pCI/preproinsulin_N110A. Spleens were removed from early diabetic mice and 3 × 10⁶ CD8 T-cells were transferred intravenously into sublethally irradiated RIP-B7.1 hosts, and cumulative diabetes incidences (%) were determined. C: In addition, spleens were removed from early diabetic RIP-B7.1 mice, and 3 × 10⁶ CD8 T-cells were transferred intravenously into sublethally irradiated B7–H1^−/− (□, n = 8) or RIP-B7.1⁺/B7–H1^−/− (▵, n = 4) hosts. D: PD-1^−/− mice were immunized with pCI/preproinsulin_N110A (●, n = 12), or the noncoding pCI (○, n = 12), and cumulative diabetes incidences (%) were determined. pCI/ppins_N110A, pCI/preproinsulin_N110A.