CD11b is a novel alternate receptor for CD154 during alloimmunity

Abstract

Antagonism of the CD154/CD40 pathway is a highly effective means of inducing long-term graft survival in preclinical models. Using a fully allogeneic murine transplant model, we found that CD154 blockade was more effective in prolonging graft survival than was CD40 blockade, raising the possibility that CD154 binds a second receptor. To test this, we queried the impact of CD154 antagonism in the absence of CD40. Data indicated that anti-CD154 functioned to reduce graft-infiltrating CD8⁺ T cells in both WT and CD40^-/- hosts. Because it has recently been reported that CD154 can ligate CD11b, we addressed the impact of blocking CD154-CD11b interactions during transplantation. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti-CD40 in prolonging allograft survival as compared to anti-CD40 plus control peptide. Mechanistically, CD154:CD11b antagonism functioned to reduce the frequency of graft-infiltrating CD8⁺ T cells and innate immune cells. These data therefore demonstrate that blocking CD154 interactions with both CD40 and CD11b is required for optimal inhibition of alloimmunity and provide an explanation for why CD40 blockers may be less efficacious than anti-CD154 reagents for the inhibition of allograft rejection.

Keywords: T cell biology; basic (laboratory) research/science; cellular biology; costimulation; immunobiology; immunosuppressant-fusion proteins and monoclonal antibodies: costimulation molecule specific.

Figure 1.. CD154 blockade inhibits CD8⁺ T cell infiltration into allografts in CD40^−/− mice.

A, WT B6 recipients received BALB/c skin grafts and were treated with 1×10⁷ BALB/c splenocytes (donor-specific transfusion, DST), and either no further treatment, anti-CD154 (clone MR-1) or anti-CD40 (clone 7E1-G2b) on days 0, 2, 4, 6. p<0.0001 by log-rank test; n=9–12/group. B-D, WT B6 or CD40^−/− recipients received BALB/c skin grafts and either no further treatment or anti-CD154 on days 0, 2, 4,6. Animals were sacrificed on day 10 and draining lymph nodes (DLN) and skin graft (SG) were analyzed. C, Representative flow cytometry plots and summary data of frequencies of IFN-γ-producing cells in CD8⁺ cell following ex vivo re-stimulation with BALB/c stimulators. D. Representative flow cytometry plots and summary data of frequencies of infiltrating H-2K^b+ CD3⁺ CD8⁺ T cells isolated from skin allografts. Data are representative of 3 independent experiments with a total 10–15 mice/group. *p<0.05, **p<0.005 by one way ANOVA with Kruskal-Wallis post-test. ns, not significant.

Figure 2.. Blockade of CD154-CD11b interaction synergizes with anti-CD40 mAb to prolong graft survival.

A, MACS-enriched CD11b⁺ cells (purity 60%) from CD40^−/− mouse splenocytes were incubated with 20 μg /ml of mouse soluble CD154 or media alone as a control. Subsequently, cells were stained with anti-CD154, and anti-CD11b. The ability of sCD154 to bind to both CD11b⁺ cells (middle row) and CD11b⁻ cells (bottom row) in the absence of CD40 was assessed. B, CD40^−/− MACS-enriched CD11b⁺ cells were incubated with 20 μg /ml sCD154 in the presence of 50 μg of either the CD154/CD11b antagonist peptide cM7 (C-EQLKKSKTL-C), or scrambled control peptide scM7 (C-KLSLEKQTK-C) for 1h as described in Material and Methods. Surface CD154 was detected by flow cytometry. C-D, WT B6 recipients received BALB/c skin grafts plus 10⁶ DST, and either no further treatment, anti-CD154, or anti-CD40 plus either the CD154/CD11b antagonist peptide cM7 (C-EQLKKSKTL-C), or scrambled control peptide scM7 (C-KLSLEKQTK-C) as described in Material and Methods. D. Graft survival was assessed and compared by log-rank test. n=10–15/group.

Figure 3.. cM7 synergizes with anti-CD40 to inhibit recipient CD8⁺ T cell infiltration into allografts.

WT B6 recipients received BALB/c skin grafts plus 10⁶ DST, and either no further treatment or anti-CD40 plus either the CD154/CD11b antagonist peptide cM7, or scM7 control peptide as described in Methods. Animals were sacrificed on day 10 and draining lymph nodes and skin grafts were analyzed. A, Representative flow cytometry plots summary data of absolute numbers of IFN-γ-producing CD8⁺ cells following ex vivo re-stimulation. B-D. Representative flow cytometry plots and summary data of absolute numbers of skin graft-infiltrating H-2K^b+ CD3⁺ CD8⁺ T cells (B), H-2K^b+ CD11b⁺cells (C) and H-2K^b+ F4/80⁺ cells (D). Data are representative of 3 independent experiments with a total 10–15 mice/group. *p<0.05 by one-way ANOVA with Kruskal-Wallis post-test. ns, not significant.

Figure 4.. Anti-CD11b prolongs allograft survival in the context of CD40 antagonism.

WT B6 recipients received BALB/c skin grafts and 10⁶ DST, and either no further treatment, anti-CD40 alone, or anti-CD40 plus anti-CD11b (clone M1/70) on days 0, 2, 4, 6 as described in Materials and Methods. Grafts were followed for survival. Data indicate that the addition of anti-CD11b significantly improved allograft survival relative to blockade of CD40 alone (B) (p<0.05). n=10–15/group.

Figure 5.. Anti-CD154 inhibits CD11b⁺ innate immune cell infiltration into allografts in RAG^−/− mice.

B6 RAG^−/− mice received BALB/c RAG^−/− mice skin grafts and were treated with either PBS control or anti-CD154 as described in Methods. Skin grafts were harvested on day 5 post-transplant. A. Representative flow cytometry plots and summary data of frequencies of H-2K^b+ CD154⁺ cells among total live cells in naïve (ungrafted) vs. day 5 transplanted RAG^−/− BALB/c skin. Data are representative of 3 independent experiments with a total 9–12 mice/group. C. Representative flow cytometry plots and summary data of absolute numbers of infiltrating H-2K^b+ cells in allograft in untreated vs. anti-CD154 treated mice. Summary graph depicts the fold increase in number of H-2K^b+ cells in both the “No Rx” and “Anti-CD154” groups over naïve skin (set at 1). Data are cumulative of n=11 mice from 3 independent experiments. *p<0.05, ***p<0.001 by Mann-Whitney U-test.