Intact B7-H3 signaling promotes allograft prolongation through preferential suppression of Th1 effector responses

Abstract

Ligands of the B7 family provide both positive and negative costimulatory signals to the CD28 family of receptors on T lymphocytes, the balance of which determines the immune response. B7-H3 is a member of the B7 family whose function in T-cell activation has been the subject of some controversy: in autoimmunity and tumor immunity, it has been described as both costimulatory and coinhibitory, while in transplantation, B7-H3 signaling is thought to contribute to graft rejection. However, we now demonstrate results to the contrary. Signaling through a putative B7-H3 receptor prolonged allograft survival in a fully MHC-mismatched cardiac model and promoted a shift toward a Th2 milieu; conversely, B7-H3 blockade, achieved by use of a blocking antibody, resulted in accelerated rejection, an effect associated with enhanced IFN-γ production. Finally, graft prolongation achieved by CTLA4 Ig was shortened both by B7-H3 blockade and the absence of recipient B7-H3. These findings suggest a coinhibitory role for B7-H3. However, experience with other CD28/B7 family members suggests that immune redundancy plays a crucial role in determining the functions of various pathways. Given the abundance of conflicting data, it is plausible that, under differing conditions, B7-H3 may have both positive and negative costimulatory functions.

Figure 1. Signaling through the putative B7-H3 receptor leads to prolonged allograft survival

(A, B) Treatment with B7-H3 Ig significantly prolonged allograft survival in wild-type (MST =16 vs 7d, n=7 treated/5 control mice, p<0.01, Log-rank test) and CD28KO recipients (MST =22 vs 12.5d, n=6 mice per group, p<0.01, Log-rank test). (C) B7-H3KO recipients rejected their grafts at a comparable rate to wild-type recipients. However, treatment of B7-H3KO recipients with B7-H3 Ig still led to graft prolongation (MST=19 vs 7d, n=5 treated KO mice/5 control KO mice, p<0.01, Log-rank test). (D) B7-H3 blockade does not affect survival of BALB/c allografts in WT B6 recipients (MST=8d vs 7d, n=6 treated/5 control mice, p=ns, Log-rank test). (E, F) Blockade of B7-H3 with anti-B7-H3 mAb precipitates rejection of BALB/c allografts in CD28KO (MST=8d vs 12.5d, n=6 mice per group, p<0.001, Log-rank test) and B7DKO recipients (MST=34d vs >100d, n=7 treated/5 control mice, p<0.001, Log-rank test) respectively. Data shown in panels A–F are pooled from 2 experiments. (G) Histopathological staining of hearts in B7DKO recipients of fully-mismatched allografts after B7-H3 blockade. Compared with hearts from control mice (top), hearts from anti-B7-H3-treated mice (bottom) showed more severe cellular rejection, with a dense chronic inflammatory infiltrate and extensive myocyte damage (day 40). 100× original magnification; H&E staining.

Figure 2. B7-H3 signaling promotes a protective Th2 milieu

(A) Frequency of cytokine-producing T cells (as determined by flow cytometric analysis of intracellular cytokine staining) in B6 WT recipients treated with B7-H3 Ig, anti-B7-H3, or control. (B) The number of cytokine-producing CD4⁺ or CD8⁺ T cells from recipient mice upon allo-antigen-specific restimulation was determined by ELISpot. (A, B) Data are shown as mean + SEM of at least n=3 mice per group, representative of three independent experiments, all performed at day 5 post-transplant. *p<0.05, **p<0.01, one-way ANOVA. (C) B7-H3 signaling promotes graft survival in STAT4KO recipients (MST=14.5d vs 9.5d, n=5 treated/6 control mice, p<0.05, Log-rank test), but not STAT6KO recipients (MST=10d vs 9d, n=5 treated/6 control mice). Data shown are pooled from 2 experiments.

Figure 3. Kinetics of B7-H3 mediated cytokine expression following transplantation

The frequency of IFN-γ (A) and IL-4 (B) producing T cells in the draining LNs and spleens of B6 WT recipients treated with anti-B7-H3 or rIgG control was determined by flow cytometry on day 2 or 4 post-transplantation. Data are shown as mean + SEM of at least n=3 mice per group and are representative of 2–3 experiments per time point. *p<0.05, Student’s t test.

Figure 4. B7-H3 signaling promotes a Th2 milieu through the selective inhibition of Th1 responses

(A) Frequency of cytokine-producing CD4⁺ T cells following 2 or 4 days in vitro culture under Th0 conditions, in the presence of B7-H3 Ig or control. Representative flow dot plots of cytokine-producing CD4⁺ T cells following 4 days of culture are shown. (B) Frequency of cytokine-producing CD4⁺ T cells in suboptimal Th2-polarizing conditions. (C) Frequency of cytokine production in Th1-primed CD4⁺ cells upon secondary restimulation in the presence of B7-H3 Ig or control. (D) Percentage of Annexin V⁻ 7AAD⁻ live cells in restimulated Th1- or Th2-primed CD4⁺ cells. All data are shown as mean + SEM of n=3–4 samples/replicates and are representative of 2–3 experiments. *p<0.05, **p<0.01, Student’s t-test.

Figure 5. Intact B7-H3 signaling is required for induction of acquired tolerance by CTLA4 Ig

(A) B7-H3 Ig acts in concert with CTLA4 Ig to promote allograft survival (MST=48 in the combination therapy vs MST=16 in B7-H3Ig alone vs MST=29.5 in CTLA4 Ig alone, n=5 combination treatment/5 B7-H3 Ig-treated/7 CTLA4 Ig-treated mice, p<0.05, Log-rank test) (B) Blockade of B7-H3 signaling with anti-B7-H3 mAb abrogates graft-prolonging effects of CTLA4 Ig (MST=20d vs 29.5d, n=7 mice per group, p<0.05, Log-rank test). (C) CTLA4 Ig-treated B7-H3KO recipients reject BALB/c grafts earlier than CTLA4 Ig-treated WT recipients (MST=16d vs 29.5d, n=6 treated/7 control mice, p<0.001, Log-rank test). This was associated with a more severe lymphocellular infiltration (day 20). 100× original magnification; H&E staining.