Equal Expansion of Endogenous Transplant-Specific Regulatory T Cell and Recruitment Into the Allograft During Rejection and Tolerance

Abstract

Despite numerous advances in the definition of a role for regulatory T cells (Tregs) in facilitating experimental transplantation tolerance, and ongoing clinical trials for Treg-based therapies, critical issues related to the optimum dosage, antigen-specificity, and Treg-friendly adjunct immunosuppressants remain incompletely resolved. In this study, we used a tractable approach of MHC tetramers and flow cytometry to define the fate of conventional (Tconvs) and Tregs CD4⁺ T cells that recognize donor 2W antigens presented by I-A^b on donor and recipient antigen-presenting cells (APCs) in a mouse cardiac allograft transplant model. Our study shows that these endogenous, donor-reactive Tregs comparably accumulate in the spleens of recipients undergoing acute rejection or exhibiting costimulation blockade-induced tolerance. Importantly, this expansion was not detected when analyzing bulk splenic Tregs. Systemically, the distinguishing feature between tolerance and rejection was the inhibition of donor-reactive conventional T cell (Tconv) expansion in tolerance, translating into increased percentages of splenic FoxP3⁺ Tregs within the 2W:I-A^b CD4⁺ T cell subset compared to rejection (~35 vs. <5% in tolerance vs. rejection). We further observed that continuous administration of rapamycin, cyclosporine A, or CTLA4-Ig did not facilitate donor-specific Treg expansion, while all three drugs inhibited Tconv expansion. Finally, donor-specific Tregs accumulated comparably in rejecting tolerant allografts, whereas tolerant grafts harbored <10% of the donor-specific Tconv numbers observed in rejecting allografts. Thus, ~80% of 2W:I-A^b CD4⁺ T cells in tolerant allografts expressed FoxP3⁺ compared to ≤10% in rejecting allografts. A similar, albeit lesser, enrichment was observed with bulk graft-infiltrating CD4⁺ cells, where ~30% were FoxP3⁺ in tolerant allografts, compared to ≤10% in rejecting allografts. Finally, we assessed that the phenotype of 2W:I-A^b Tregs and observed that the percentages of cells expressing neuropilin-1 and CD73 were significantly higher in tolerance compared to rejection, suggesting that these Tregs may be functionally distinct. Collectively, the analysis of donor-reactive, but not of bulk, Tconvs and Tregs reveal a systemic signature of tolerance that is stable and congruent with the signature within tolerant allografts. Our data also underscore the importance of limiting Tconv expansion for high donor-specific Tregs:Tconv ratios to be successfully attained in transplantation tolerance.

Keywords: allospecific T cells; conventional T cells; costimulatory blockade; immunosuppression; murine heart transplant; regulatory T cells; transplant tolerance; transplantation immunology.

Figure 1

Increased donor-specific regulatory T cell (Treg) percentages in the spleens of costimulatory blockade-induced tolerant recipients is due to inhibition of conventional T cell (Tconv) expansion and modest accumulation of Tregs. C57BL/6 recipients were transplanted with heterotopic heart allografts from Act.2W-OVA⁺ BALB/c × C57BL/6 F1 donors. Recipients were either given anti-CD154 on days 0, 7, and 14 post-transplantation plus donor splenocyte infusion on day 0 (αCD154/DST), or were untreated (No Rx). On day 7 or day 30 post-transplantation, mice were sacrificed and their splenocytes were analyzed. (A) Cartoon depicting the experimental protocol. (B) Sample gating strategy of CD4⁺ splenocytes for 2W:I-A^b (2W) Tregs. (C) Percentage 2W-specific cells among CD4⁺ T cells. (D) Total number of 2W-specific CD4⁺ T cells in the spleen. (E) Total number of 2W-specific FoxP3^–CD4⁺ Tconv in the spleen. (F) Total number of 2W-specific FoxP3⁺CD4⁺ Tregs in the spleen. (G) Percentage of FoxP3⁺ Tregs among 2W-specific CD4⁺ T cells. (H) Percentage of FoxP3⁺ Tregs among all splenic CD4⁺ T cells. **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA when comparing between naïve and individual time points and by two-tailed t-test when comparing between treatment groups. Mean ± SEM is shown, and each point represents one animal from 4–5 replicate experiments per time point (n = 8–13).

Figure 2

Rapamycin and cyclosporine A (CsA) inhibit expansion of allospecific conventional T cell (Tconv) but do not promote regulatory T cell (Treg) accumulation in costimulatory blockade-induced tolerant recipients. C57BL/6 recipients were transplanted with heterotopic heart allografts from Act.2W-OVA⁺ BALB/c × C57BL/6 F1 donors as described in Figure 1. Some recipients were untreated (No Rx), or treated with αCD154/DST also received daily injections of 2.5 mg/kg rapamycin or 50 mg/kg CsA, and all recipients were sacrificed on 30 days post-transplantation. Data for the naïve and anti-CD154/DST groups are from Figure 1. (A) Cartoon depicting experimental design. (B) Total splenic CD4⁺FoxP3^– Tconv and (C) CD4⁺FoxP3⁺ Tregs. (D) Percentage of FoxP3⁺ cells among CD4⁺ T splenocytes. (E) Splenic 2W:I-A^b(2W)-specific Tconvs and (F) Tregs were enumerated. (G) Percentage of Tregs among 2W-specific CD4⁺ T splenocytes. *p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA. Mean ± SEM is shown, and each point represents one mouse from 3–4 replicate experiments (n = 7–19). The Naïve, No Rx, and αCD154/DST groups are from Figure 1.

Figure 3

Divergent effect of transient vs. continuous CTLA4-Ig on allospecific conventional T cell (Tconv) and regulatory T cells (Tregs). C57BL/6 recipients were transplanted with heterotopic heart allografts from Act.2W-OVA⁺ BALB/c × C57BL/6 F1 donors. Recipients were untreated (No Rx), treated with anti-CD154/DST, CTLA4-Ig on days 0 and 2 post-transplantation, or twice a week from day 0 to day 30 post-transplantation (CTLA4-Ig D0⁺ and D0-30) and sacrificed at either D30 or D60 post-Tx (date of sacrifice in parenthesis). Data for naïve and 30 days post-transplantation anti-CD154/DST groups are from Figures 1 and 2. (A) Splenic CD4⁺FoxP3^– Tconv and (B) CD4⁺FoxP3⁺ Tregs were enumerated. (C) Percentage of FoxP3⁺ cells among CD4⁺ T splenocytes. (D) Splenic 2W:I-A^b(2W)-specific Tconv and (E) 2W-specific Tregs were enumerated. (F) Percentage of FoxP3⁺ Tregs among 2W-specific CD4⁺ T splenocytes. *p < 0.01, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA, ^##p < 0.01 by two-tailed ranked t-test. Mean ± SEM is shown, and each point represents one mouse from 2–4 replicate experiments (n = 4–19). The Naïve, No Rx, and αCD154/DST groups are from Figure 1.

Figure 4

Regulatory T cells (Tregs) infiltrate comparably into allografts in rejection and tolerance while conventional T cell (Tconv) infiltration is reduced in tolerance. C57BL/6 recipients were as described in Figure 1. On day 7 or day 30 post-transplantation, mice were sacrificed and their grafts and graft-infiltrating cells were analyzed by flow cytometry (A–D) or immunohistochemistry (E–F). (A) Gating strategy for graft-infiltrating CD4⁺ Tregs. (B) Total number of graft-infiltrating FoxP3^–CD4⁺ Tconv and (C) FoxP3⁺CD4⁺ Tregs. (D) Percentage of FoxP3⁺ cells among CD4⁺ graft-infiltrating T cells. (E) Total number of FoxP3⁺ cells per cm² from entire heart histology sections and (F) Ratio of FoxP3⁺:CD4⁺ cells from matched subsections of sequentially cut histology sections (four subsections at 10× magnification per mouse). *p < 0.01, **p < 0.01, ***p < 0.001 by one-way ANOVA. Mean ± SEM is shown, and each point represents one mouse from 7–8 replicate experiments (n = 13).

Figure 5

Donor-specific 2W:I-A^b regulatory T cells (Tregs) infiltrate comparably into allografts in rejection and tolerance while 2W:I-A^b conventional T cell (Tconv) infiltration is reduced in tolerance. Graft-infiltrating cells were analyzed from recipients as described in Figure 4. (A) Gating strategy for 2W:I-A^b(2W)-specific FoxP3⁺CD4⁺ graft-infiltrating Tregs. (B) Graft-infiltrating 2W-specific CD4⁺FoxP3^– Tconv and (C) 2W-specific FoxP3⁺ Tregs were enumerated. (D) Percentage of FoxP3⁺ cells among graft-infiltrating 2W-specific CD4⁺ T cells. **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA. Mean ± SEM is shown, and each point represents one animal from 7–8 replicate experiments (n = 12–13).

Figure 6

Phenotypic analysis of donor-specific 2W:I-A^b regulatory T cells (Tregs) isolated from recipients with rejecting and tolerant allografts at day 7 post-transplantation. C57BL/6 recipients transplanted with heterotopic heart allografts from Act.2W-OVA⁺ BALB/c × C57BL/6 F1 donors, were untreated (No Rx) or treated with αCD154/DST, and sacrificed on day 7 post-transplantation. Sample gating strategies (A,D,G,J), percentage of cells positive (B,E,H,K), and mean fluorescent intensity (C,F,I,L) relative to naïve 2W:I-A^b-specific FoxP3⁺ or FoxP3⁻ cells of (A–C), Helios; (D–F), Neuropilin-1; (G–I), CD25; and (H–L), CD73; in naïve, acutely rejecting (No Rx), and αCD154/DST-treated (+Rx) animals. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA. Mean ± SEM is shown, and each point represents one animal from three replicate experiments (n = 4–6/group).