An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(+) iTreg in the absence and presence of CTLA-4 Ig

Abstract

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc-mediated deletion of CD154-expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti-CD154-induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti-CD154 therapy, two alternative means of targeting the CD40-CD154 pathway were used: a nonagonistic anti-CD40 antibody and an Fc-silent anti-CD154 domain antibody. We compared these therapies to an Fc-intact anti-CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti-CD40 mAbs as well as Fc-silent anti-CD154 domain antibodies were equivalent to Fc-intact anti-CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen-specific T cells and promote the conversion of Foxp3(+) iTreg. Importantly, iTreg conversion observed with Fc-silent anti-CD154 domain antibodies was preserved in the presence of CTLA4-Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

Keywords: Alloreactivity; CD8+ T-lymphocytes; costimulation; regulatory T cells.

Figure 1. Anti-CD154 domain antibodies are functionally equivalent to Fc-intact anti-CD154 mAbs

(A) On day 0, B6 mice were transplanted with BALB/c skin grafts and were treated with 10⁷ BALB/c DST and/or 250 µg of CTLA4-Ig, MR-1, 7E1-G2b or CD154 dAbs where indicated. (B) Mice treated with CTLA4-Ig alone rejected skin grafts with an MST of 15.5 d. Addition of MR-1 prolonged MST to 33 days, whereas addition of 7E1-G2b or CD154 dAbs led to an MST of 37 and 31 days, respectively. Data are cumulative of three independent experiments with a total of fourteen to fifteen mice per group. *** = p < 0.001 compared to CTLA4-Ig alone. Comparisons between MR-1, 7E1-G2b and CD154 dAbs were not statistically significant.

Figure 2. Activation and cytokine production of alloreactive T cells are diminished in the presence of anti-CD154 dAbs

(A) Representative histograms of CD44 expression on splenic CD4⁺ and CD8⁺ T cells isolated from grafted mice at day 14. (B) Frequencies of CD44^hi cells among CD4⁺ and CD8⁺ T cell populations. Treatment with MR-1, 7E1-G2b or CD154 dAbs results in the reduction of CD44 expression on both CD4⁺ and CD8⁺ cells. (C) Representative flow plots of intracellular cytokine staining of recipient splenocytes, gated on CD8⁺ T cells. 10⁶ recipient splenocytes were incubated with 2×10⁶ BALB/c stimulator splenocytes for 5 hours and then fixed and permeabilized before being stained for FACS analysis. (D) Frequencies and absolute numbers of cytokine-producing CD8⁺ T cells. Treatment with MR-1, 7E1-G2b or CD154 dAbs results in the reduction of percentage of multicytokine producing CD8⁺ T cells. Data are cumulative of two separate experiments with a total of 8 to 10 mice per group. ** p < 0.01, *** p < 0.001.

Figure 3. Anti-CD154 dAbs inhibit donor-reactive T cell expansion in response to a minor antigen

(A) Mice were adoptively transferred with 1.5×10⁶ of each CD45.2⁺ CD8⁺ OT-I and Thy1.1⁺ CD4⁺ OT-II T cells 2 days before transplantation. On day 0, mice were transplanted with mOVA SG and were treated with 10⁷ mOVA DST and/or 250 µg of MR-1, 7E1-G2b or CD154 dAbs where indicated. Mice were sacrificed at day 11. (B-D) Representative flow plots of CD8⁺ T cells (B) and the frequencies and absolute numbers of antigen-specific CD8⁺ and effector CD4⁺ T cells (C) in the draining lymph nodes at day 11. DST treatment alone moderately diminished the frequency of antigen-specific CD8⁺ T cells, but the addition of MR-1, 7E1-G2b or the CD154 dAbs resulted in a significant reduction in the frequencies of these cells in both organs. Data are representative of four independent experiments with a total of fourteen to sixteen mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4. Anti-CD154 dAbs reduce numbers of functional antigen-specific cells and prevent differentiation into multiple cytokine producers

(A) Representative flow plots of intracellular cytokine staining of splenic antigen-specific CD8⁺ T cells harvested at day 11 and stimulated for 4 hours in vitro with SIINFEKL peptide. (B) Percentages and absolute numbers of IFN-γ producing antigen-specific CD8⁺ T cells. Compared to DST alone, treatment with MR-1, 7E1-G2b or CD154 dAbs significantly reduced the frequency and absolute numbers of IFN-γ-producing antigen-specific CD8⁺ T cells in the spleen. (C) Frequencies of double cytokine producing antigen-specific CD8⁺ T cells within each treatment group. Treatment with MR-1, 7E1-G2b or CD154 dAbs resulted in the reduction of percentage of multicytokine producing antigen-specific CD8⁺ T cells. Data are representative of three independent experiments with a total of eleven to twelve mice per group. *** p < 0.001.

Figure 5. Administration of Fc-silent anti-CD154 dAbs results in antigen-specific iTreg conversion

(A) Representative flow plots of CD25⁺ Foxp3⁺ iTreg among antigen-specific Thy1.1⁺ CD4⁺ T cells in draining lymph nodes at day 11. (B) Summary data of percentages of CD25⁺ Foxp3⁺ cells within the antigen-specific CD4⁺ T cell compartment. Treatment with MR-1, 7E1-G2b or CD154 dAbs resulted in a significant increase in the percentage of antigen-specific iTregs. (C) Treg:Effector cell ratio for antigen-specific CD4⁺ and CD8⁺ T cells in each treatment group. Treatment with MR-1, 7E1-G2b or CD154 dAbs results in a significant increases in the ratio of Tregs to both CD4⁺ and CD8⁺ antigen-specific effector cells. Data are cumulative of two separate experiments with a total of eight to nine mice per group. *** p < 0.001.

Figure 6. Fc-Silent Anti-CD154 dAbs results in iTreg generation even in the presence of CTLA4-Ig

(A) Representative flow plots of total CD4⁺ T cells (top panels) and antigen-specific CD4⁺ (Thy1.1⁺) T cells (bottom panels) in draining lymph nodes at day 11, with gates on CD25⁺ Foxp3⁺ cells. (B) Percentages of CD25⁺ Foxp3⁺ cells within the total CD4⁺ T cell compartment and the antigen-specific CD4⁺ T cell compartment. (C) Representative flow plots of Helios expression on total CD4⁺ T cells (left panel), as well as on total endogenous CD4⁺ CD25⁺ Foxp3⁺ Treg (upper right panels) and antigen-specific Thy1.1⁺ iTreg (lower right panels). Data are cumulative of four separate experiments with a total of ten to fourteen mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.