Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade

Abstract

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4(+) and CD8(+) T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4(+) and CD8(+) TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8(+) T-cell expansion, delaying differentiation into IFN-γ(+) TNF(+) multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4(+) CD25(hi) Foxp3(+) T-cell population, which did not arise from endogenous natural T(reg) but rather were peripherally generated from naïve Foxp3(-) precursors.

Fig. 1.

CD154 blockade and DST prolongs SG survival. (A) Mice were adoptively transferred with 1.5 × 10⁶ of each OT-I and OT-II T cells 2 d before transplantation. On day 0, mice were transplanted with mOVA SG and were treated with 10⁷ mOVA DST and/or 500 μg of MR-1 where indicated. (B) Untreated mice rejected SGs with an MST of 13.5 d. Anti-CD154 monotherapy led to bimodal survival of SGs, with 50% of mice rejecting grafts with an MST of 15 d and 50% demonstrating indefinite survival (P = 0.0027). DST monotherapy resulted in to an MST of 18 d (P = 0.031), whereas combined anti-CD154/DST led to an indefinite survival of SGs (P < 0.0001). Data are cumulative of two independent experiments with five mice per group.

Fig. 2.

CD154 blockade delays expansion of antigen-specific CD8⁺ T cells but does not alter activation status at peak of response. Mice were treated as described in Fig. 1 and killed at the indicated time points. (A) Concatenated flow plots of CD8⁺ splenocytes. Gates shown are on donor-reactive CD8⁺ (Thy1.1⁺) T cells. (B) Frequencies of donor-reactive CD8⁺ splenocytes are shown. OT-I T-cell populations in untreated mice peaked at days 7–10 with 2.27% ± 0.27% and 2.98% ± 0.69%, respectively. Compared with untreated controls, anti-CD154 treatment delayed expansion of T cells (D10: 3.47% ± 1.37%), whereas DST accelerated expansion of OT-I T cells (D4: 0.32% ± 0.04% vs. 5.08% ± 0.98%; P = 0.006). Combined treatment minimally expanded T cells (D4: 1.39% ± 0.95%). (C) Activation markers on OT-I T cells on day 7 in the spleen. CD40/CD154 blockade reduced CD44 and CD62L up-regulation, while promoting an increase in KLRG-1 expression. Data are summarized from three experiments with three mice per group. Values are mean ± SEM. *P < 0.05; ***P < 0.001.

Fig. 3.

CD154 blockade delays donor-reactive CD8⁺ T cells differentiation into multifunctional cytokine-producing cells. Mice were treated as described in Fig. 1 and killed at the indicated time points. (A) Concatenated flow plots of intracellular cytokine staining in splenic OT-I T cells after stimulation for 4 h in vitro with SIINFEKL peptide. (B) Cytokine production by splenic OT-I T cells, summarized from three experiments with three mice per group. Untreated mice developed into dual cytokine producers at day 7 (6.22 × 10⁴ ± 0.29 × 10⁴). DST accelerated T-cell production of TNF and IFN-γ (D4: 4.90 × 10⁴ ± 2.14 × 10⁴). Anti-CD154 delayed T-cell differentiation (D10: 1.15 × 10⁵ ± 0.65 × 10⁵). Anti-CD154/DST inhibited differentiation (D4: 2.08 × 10⁴ ± 1.98 × 10⁴). (C) Day 10 in vivo cytotoxicity assay. Peptide-coated targets and unpulsed control targets were labeled with different concentrations of carboxyfluorescein succinimidyl ester and adoptively transferred into recipients. After 18 h, the ratio of unpulsed targets to peptide-pulsed targets remaining was assessed by flow cytometry. Relative to untreated controls, anti-CD154 treatment did not alter killing of target cells (91.01% ± 0.82% vs. 66.59% ± 11.03%; P = 0.379). DST treatment significantly impaired killing (55.52% ± 8.28%; P = 0.003). Anti-CD154/DST significantly impaired cytolysis (28.26% ± 12.78%; P = 0.001). Data are summarized from two experiments with five mice per group. Values are mean ± SEM.

Fig. 4.

Anti-CD154 and DST treatment reduces antigen-specific CD4⁺ T-cell accumulation and promotes Foxp3⁺ T_reg cell migration to the graft. Mice were treated as described in Fig. 1 and killed at the indicated time points. (A) Day 7 concatenated flow plots of CD4⁺ T cells in draining LNs, with gates identifying OT-II (Thy1.1⁺) T cells. (B) Donor-reactive CD4⁺ T cells in the draining LNs. Anti-CD154 monotherapy reduced OT-II T-cell levels compared with untreated controls (0.54% ± 0.17% vs. 2.10% ± 0.55%; P = 0.054). DST monotherapy reduced OT-II T-cell levels (0.57 ± 0.07%; P = 0.051). Anti-CD154/DST significantly reduced OT-II levels (0.20% ± 0.02%; P = 0.026). (C) Frequency of total T_reg (both transgenic and endogenous) cells in the draining LNs over time. (D) Day 7 Foxp3⁺ cells in SGs. Foxp3⁺ cells were counted in 10 high-power fields (HPFs; 400× magnification). Anti-CD154/DST treatment significantly increased Foxp3⁺ cell infiltration compared with untreated controls (14.2 ± 4.78/HPF vs. 4.95 ± 0.22/HPF; P < 0.0005). Data are summarized from three experiments with three mice per group. Values are mean ± SEM.

Fig. 5.

Anti-CD154 and DST treatment promote conversion of donor-reactive CD4⁺ T cells into CD25⁺ Foxp3⁺ iT_reg. (A) Day 7 concatenated flow plots of OT-II (Thy1.1⁺) T cells in LNs, with gates on CD25⁺ Foxp3⁺ OT-II cells. (B) Longitudinal analysis of Foxp3⁺ OT-II T cells. On day 7, the frequency of T_reg in untreated mice was 0.88% ± 0.44% that of OT-II T cells. Anti-CD154 (4.68% ± 1.49%; P = 0.071) and DST (2.27% ± 1.37%; P = 0.389) monotherapies slightly increased the frequency of Foxp3-expressing T cells within the OT-II T-cell compartment. Anti-CD154/DST significantly increased Foxp3 expression in OT-II T cells compared with untreated controls (10.61% ± 3.28%; P = 0.042). On day 14, both anti-CD154 and anti-CD154/DST reached similarly high levels of iT_reg conversion (16.57% ± 2.68% and 16.17% ± 10.53%, respectively; P = 0.973). Data are summarized from three experiments with three mice per group. (C and D) B6.SJL (CD45.1⁺) were adoptively transferred with OT-I and RAG^−/− OT-II T cells and treated with anti-CD154/DST. On day 7, draining LNs were analyzed for Foxp3-expressing OT-II T cells. (C) Representative flow plots of CD45.2⁺ RAG^−/− OT-II T cells; gate represents CD25⁺ Foxp3⁺ iT_reg cells. (D) Combined CD154 blockade and DST significantly increased the conversion of OT-II T cells into Foxp3⁺ iT_reg compared with untreated controls (6.452% ± 1.552% vs. 0.559% ± 0.225%; P = 0.0035). Data are summarized from two experiments with between three and five mice per group. (E) Relative frequencies of Thy1.1⁺ OT-I T cells compared with peripherally converted Thy1.1⁺ OT-II iT_reg from untreated LNs (Left) and anti-CD154/DST–treated LNs (Right). OT-I T cells are measured on the left y-axis, and the OT-II T_reg are measured on the right y-axis. Data are summarized from three experiments with three mice per group. Values are mean ± SEM.