Blocking properties of mAbs against PD-1, PD-L1, and PD-L2. (A) Anti–PD-1 (J43) inhibits binding of both PD-L1-Ig and PD-L2-Ig (bold lines) to PD-1 transfectants. Dashed lines indicate staining with control human IgG. (B) Anti–PD-L1 (MIH6) inhibits PD-L1-Ig binding and anti–PD-L2 (TY25) inhibits PD-L2-Ig binding to PD-1 transfectants (bold lines). Dashed lines indicate staining with control human IgG.

PD-1–PD-L1 and CTLA-4 blockade accelerates insulitis in prediabetic female NOD mice. (A) Islets from control IgG–treated mouse at 5 wk of age. (B–D) Inflamed islets from anti–CTLA-4, anti–PD-1, or anti–PD-L1 mAb-treated mice, respectively, at 5 wk of age (all stained with hematoxylin and eosin; ×400).

(A) PD-L1 expression in inflamed islets of NOD mice and PD-1 expression on infiltrating cells. Inflamed islets from 10-wk-old unmanipulated prediabetic female NOD mice showing negative staining with isotype control Ig, positive PD-1 staining of infiltrating cells (arrows), positive PD-L1 staining of islets, and negative PD-L2 staining (×400). (B) Injected anti–PD-L1 mAb binds to islets. Inflamed islet, from a 10-wk-old NOD mouse that developed diabetes after injection of anti–PD-L1, showing positive direct staining with anti–rat antibody (×400).

PD-1–PD-L1 blockade accelerates autoimmune diabetes in NOD mice. All p-values are for comparisons with respective controls. (A) Diabetes-free survival after PD-1–PD-L1 blockade in 10-wk-old female NOD mice: anti–PD-1 (•; n = 17; P < 0.0001), anti–PD-L1 (▪; n = 17; P < 0.0001), anti–PD-L2 (▾; n = 7; P = 0.1904), anti–CTLA-4 (▴; n = 6; P = NS), and control antibody (♦; n = 12) treatment. (B) Diabetes-free survival after PD-1–PD-L1 blockade in 4-wk-old female NOD mice: anti–PD-1 (•; n = 15; P < 0.0184), anti–PD-L1 (▪; n = 18; P < 0.0001), anti–PD-L2 (▾; n = 7; P = NS), anti–CTLA-4 (▴; n = 10; P = NS), and control antibody (♦; n = 12) treatment. (C) Diabetes-free survival after PD-1–PD-L1 blockade in 1-wk-old female NOD mice: anti–PD-1 (•; n = 18; P = 0.0071), anti–PD-L1 (▪; n = 18; P = 0.0018), anti–PD-L2 (▾; n = 11; P = NS), anti–CTLA-4 (▴; n = 11; P = 0.0016), and control antibody (♦; n = 10) treatment. (D) Diabetes-free survival after PD-1–PD-L1 blockade in 10-wk-old male NOD mice: anti–PD-1 (•; n = 5; P = 0.0126), anti–PD-L1 (▪; n = 5; P = 0.0126), and control antibody (♦; n = 5) treatment. In 10-wk-old female NOR mice: anti–PD-1 (▾; n = 5; P = NS) and anti–PD-L1 (▴; n = 5; P = NS) treatment. (E) Diabetes-free survival after PD-1–PD-L1 blockade in 4-wk-old male NOD mice: anti–PD-1 (•; n = 5; P = NS), anti–PD-L1 (▪; n = 6; P = 0.038), control Ig (♦; n = 5) treatment. In 4-wk-old female NOR mice: anti–PD-1 (▾; n = 5; P = NS) and anti–PD-L1 (▴; n = 6; P = NS) treatment. (F) Insulitis scores in 4–5-wk-old female NOD mice treated with anti–PD-1 (▪; mean score 1.249 ± 0.2349; P < 0.001), anti–PD-L1 (•; mean score 2.275 ± 0.1987; P < 0.001), and control IgG (♦; mean score 0.03634 ± 0.01987).

Increased frequency of IFN-γ–producing GAD-specific splenocytes in NOD mice treated with anti–PD- or anti–PD-L1 mAb. ELISPOT responses are expressed as number of IFN-γ spots per million splenocytes from anti–PD-1 mAb, anti–PD-L1 mAb, and control IgG–treated 4-wk-old female NOD mice.

PD-L1 expression is down-regulated on stimulated splenocytes of older NOD mice. Expression of PD-L1 on CD3⁺ (A and B) or CD3⁻ (C and D) splenocytes of female NOD (A and C) or BALB/c (B and D) mice of various ages before and after stimulation with immobilized anti-CD3 mAb for 72 h. *, P < 0.01 and **, P < 0.05 versus stimulated CD3⁺ cells from 9-wk-old female NOD mice.