Coinhibitory receptor PD-1H preferentially suppresses CD4⁺ T cell-mediated immunity

Abstract

T cell activation is regulated by the interactions of surface receptors with stimulatory and inhibitory ligands. Programmed death-1 homolog (PD-1H, also called VISTA) is a member of the CD28 family of proteins and has been shown to act as a coinhibitory ligand on APCs that suppress T cell responses. Here, we determined that PD-1H functions as a coinhibitory receptor for CD4⁺ T cells. CD4⁺ T cells in mice lacking PD-1H exhibited a dramatically increased response to antigen stimulation. Furthermore, delivery of a PD-1H-specific agonist mAb directly inhibited CD4⁺ T cell activation both in vitro and in vivo, validating a coinhibitory function of PD-1H. In a murine model of acute hepatitis, administration of a PD-1H agonist mAb suppressed CD4⁺ T cell-mediated acute inflammation. PD-1H-deficient animals were highly resistant to tumor induction in a murine brain glioma model, and depletion of CD4⁺ T cells, but not CD8⁺ T cells, promoted tumor formation. Together, our findings suggest that PD-1H has potential as a target of immune modulation in the treatment of human inflammation and malignancies.

Figure 1. Analysis of CD4⁺ T cells in PD-1H–deficient mice.

(A) Total cell numbers calculated in the spleen and liver of 4-, 8-, 16-, and 36-week-old PD-1H–KO mice and WT littermates. (B) Absolute CD4⁺ cell numbers were determined by total cell count multiplied by percentage of total cells/100 as determined by flow cytometry. (C) CD44⁺CD62L^– and CD44⁺CD62L⁺ populations were determined as a percentage of CD4⁺ T cells at 4 and 36 weeks in both the spleen and liver. 5 to 6 mice were analyzed per group at each time point. PD-1H–deficient CD4⁺ T cell proliferation and cytokine production are enhanced in vitro. 96-well plates were coated overnight with anti-CD3 mAb, and purified CD4⁺ T cells were added to the wells. Plates were pulsed overnight with 3-HTdR thymidine. (D) Proliferation of PD-1H–KO or WT CD4⁺ T cells at serial dilutions of anti-CD3 was determined at 96 hours. Other time points were similar. (E) Proliferation at an anti-CD3 mAb of 0.63 μg/ml is shown at 24, 48, 72, and 96 hours. (F and G) Supernatants were harvested from 96-well proliferation plates at 24, 48, 72, and 96 hours and analyzed for IFN-γ, IL-17A, and TNF-α. In F, cytokine concentrations at 96 hours are shown at serial dilutions of anti-CD3 mAb. In E, cytokine concentrations are shown at 24, 48, 72 and 96 hours at a single anti-CD3 concentration. All experiments were repeated at least 3 times in triplicate. *P < 0.05.

Figure 2. PD-1H inhibits antigen-specific CD4⁺ T cell responses in vitro.

OT-II^+/+ and OT-II^–/– T cells were evaluated for response to antigen. (A) Expression of PD-1H on naive OT-II^+/+ T cells (white) and OT-II^–/– T cells (gray). (B–F) Purified PD-1H^–/– or PD-1H^+/+ OT-II T cells were added to 96-well plates with irradiated PD-1H–KO splenocytes. Proliferation was assessed at specific peptide concentration (B) and at specific time points (C). Supernatants from proliferation assays were assessed for cytokines at specific peptide concentrations (D) and at time points using 0.5 μg/ml OVA peptide (E). (F) Proliferation was assessed in the presence of 2 μg/ml of PD-1H mAb or mouse Ig. (G) Irradiated WT APCs or PD1H KO APCs were cultured with either OT-II^+/+ or OT-II^–/– T cells and analyzed for proliferation. (H and I) Naive purified OT-II^+/+ T cells were adoptively transferred (i.v.) on day –1 to PD-1H–KO recipient mice. OVA and polyI:C were injected on day 0 with either mouse Ig or anti–PD-1H. OT-II T cell numbers (H) and activation (I) as determined by CD44 expression were assessed on day 3. (J and K) Naive OT-II^+/+ or OT-II^–/– T cells were purified and identical numbers were transferred (i.v.) on day –1 to PD-1H–KO mice. OVA and polyI:C were injected on day 0. Mice bled at indicated time points were analyzed for percentage of OT-II T cells of total CD4⁺ T cells (J) and PD-1H expression (K). All experiments were repeated a minimum of 3 times. *P < 0.05.

Figure 3. PD-1H–deficient mice are more susceptible to acute Con A–induced hepatitis.

(A) PD-1H–KO or WT C57BL/6 littermates received i.v. injections of Con A at 30 mg/kg and were monitored for survival. (B) PD-1H–KO or WT C57BL/6 littermates received sublethal i.v. injections of Con A at 15 mg/kg,and serum was isolated at the indicated times to analyze cytokine levels. (C) As in B, serum at 24 hours was analyzed for ALT concentration. (D) The livers of PD-1H–KO or littermates receiving 15 mg/kg Con A were isolated at 24 hours, and the cells were stained with anti-CD4 and anti-NK1.1 mAbs for calculation of absolute cell numbers. (E) C57BL/6 mice were pretreated with i.p. injections of control mouse Ig or anti–PD-1H (mam82) 3 hours before i.v. injections of Con A at 30 mg/kg and monitored for survival. (F) As in E, mice were bled at 20 hours, and the serum was analyzed for ALT concentration. The experiments were repeated 3 to 5 times with 5 mice/group. *P < 0.05.

Figure 4. PD-1H expression on hematopoietic cells regulates the severity of acute Con A–induced hepatitis.

(A) Depiction of BM chimeric models for the Con A hepatitis experiments and analysis of chimerism in blood for CD4⁺ and CD8⁺ T cell reconstitution at 3 weeks. (B) Chimeric mice were bled 3 weeks after reconstitution, and CD4⁺ and CD8⁺ percentages were determined. (C) Rag-1–KO mice adoptively transferred with either PD-1H–KO or WT TCD-BM and injected with 15 mg/kg Con A were bled at 3 hours for analysis of serum cytokine levels as indicated. (D) As in C, liver lymphocytes were stained with anti-CD4 and anti-NK1.1 to determine absolute cell numbers in liver. (E) PD-1H–KO mice reconstituted with PD-1H–KO or WT TCD-BM were injected (i.v.) with 15 mg/kg of Con A and bled at 2, 5, and 24 hours for analysis of serum ALT concentrations. (F) As in C, serum at the 2- hour time point was analyzed for cytokines. *P < 0.05.

Figure 5. PD-1H–KO mice are resistant to the growth of GL261 glioma.

3 × 10⁵ GL261-luc cells were injected into the left hemisphere of the brains of PD-1H–KO or WT mice. Mice were subsequently treated with or without 4 Gy radiotherapy of the whole brain on day 5 after tumor inoculation. (A) Whole-brain tissues were isolated on day 15 for H&E staining. The brain from a WT mouse without tumor inoculation was used as a control. T, tumor area. Scale bars: 600 μm. (B) Survival of WT or PD-1H–KO mice with or without radiotherapy was monitored daily (n = 8). (C) Growth of GL261 tumors in individual mice was measured using a Lumina XR imaging system every 5 days and presented as bioluminescence of tumor in radiance (n = 8). **P < 0.01.

Figure 6. Resistance to GL261 tumor growth in PD-1H–KO mice is mediated by CD4⁺ T cell immunity.

3 × 10⁵ GL261-luc tumor cells were injected into the left hemisphere of the brains of PD-1H–KO or WT mice. Mice were treated with a total dose of 4 Gy radiotherapy of the whole brain on day 5 after tumor inoculation. (A) Ten days after radiotherapy, splenocytes and brain lymphocytes were isolated and restimulated with irradiated GL261 cells for 5 days. Percentages of IFN-γ–producing CD4⁺ T cells of total CD4⁺ T cells (left panels) or IFN-γ–producing CD8⁺ T cells of total CD8⁺ T cells (middle panels) were detected by intracellular staining. IFN-γ levels in culture supernatant were detected by CBA (right panels). One point indicates the result from 1 mouse (n = 6). Naive mice (n = 2) without tumor inoculation were used as negative control. (B) CD4⁺ T cells or CD8⁺ T cells were depleted by i.p. injection of anti-CD4 (GK1.5) or anti-CD8a (53-6.72) antibody at 500 μg every 5 to 7 days from 5 days before tumor inoculation. Survival of mice was monitored daily up to 50 days (n = 5). *P < 0.05; **P < 0.01.