Attenuation of donor-reactive T cells allows effective control of allograft rejection using regulatory T cell therapy

Abstract

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor-specific transfusion followed by cyclophosphamide treatment deleted 70-80% donor-reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 10(6) Tregs with direct donor reactivity or 25 × 10(6) polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8(+) T cells and concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor-reactive T cells will be an important component of Treg-based therapies in transplantation.

Keywords: CD8+ T cell; T cell deletion; Treg therapy; diabetes.

Figure 1. Tregs alone were unable to prolong islet allograft survival

B6 mice were rendered diabetic with streptozotocin before receiving 450–500 BALB/c islets under their left renal capsules. One day before transplantation, a group of mice received an intravenous infusion of 5 ×10⁶ or 25–30 ×10⁶ Tregs isolated and expanded from either 4C TCR-transgenic mice or B6 mice. Graft survival was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method. Tregs, regulatory T cells.

Figure 2. DST +CY treatment significantly reduced donor-reactive T cells

Total numbers of donor-reactive (A) and third party–reactive (B) CD4⁺ Tconv and CD8⁺ T cells in B6 mice treated with BALB/c DST and CY (100 and 200 mg/kg) were calculated from the total cellularity of spleens and lymph nodes and the frequencies of donor-reactive or third party–reactive cells (shown in Table 1). Untreated naïve B6 mice were used as controls and all numbers were normalized to the mean of the naïve B6 mice for the ease of comparison. To assess the efficacy in reducing T cells of direct versus indirect alloreactivity, B6 mice were intravenously injected with 1 ×10⁶ of each tracer CD4⁺ T cells from 4C (C, direct), TEa (D, indirect) and OT-II (E, irrelevant) TCR-transgenic mice. All the mice were subjected to BALB/c DST and CY treatment. Seven days after DST treatment, total numbers of the three tracer cell populations in spleens and lymph nodes were calculated using flow cytometry and cell counting. Data are shown as mean ±SEM. Data are representative of at least four independent experiments using one mouse per group. ^*p <0.05, ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; Tconv, T conventional.

Figure 3. Donor-antigen-reactive Tregs induced long-term islet allograft survival in DST +CY conditioned recipients

B6 mice were treated with BALB/c DST followed by CY (100 or 200 mg/kg) 2 days later. On day 4 after DST treatment, the mice were rendered diabetic with streptozotocin. On day 7 after DST, 450–500 BALB/c islets were transplanted under the left renal capsule (A). A group of the mice received an i.v. infusion of 5 ×10⁶ Tregs isolated and expanded from 4C TCR-transgenic mice 1 day before islet transplantation (B). Islets from third-party (C3H/HeJ) donors were transplanted into B6 mice treated with BALB/c DST and CY (200 mg/kg) in the presence or absence of 4C Tregs (C). CY (200 mg/kg)-treated diabetic B6 mice without DST received an i.v. infusion of 5 ×10⁶ 4C Tregs 1 day before transplantation with BALB/c islets (D). Untreated diabetic B6 mice were similarly transplanted and used as controls. Graft survival was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method, with comparisons among groups using the log-rank test. ^*p <0.05, ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; i.v., intravenous; Tregs, regulatory T cells.

Figure 4. Higher number of polyclonal Tregs was required to confer long-term protection of islet allografts

(A) B6 mice were treated with BALB/c DST followed by CY (100 or 200 mg/kg) 2 days later, and rendered diabetic with streptozotocin (5–6 mg) 4 days later. On day 6 after DST, the conditioned mice received an i.v. infusion of 5 ×10⁶ polyclonal B6 Tregs. On day 7 after DST, 450–500 BALB/c islets were transplanted under the left renal capsule. (B) B6 mice preconditioned with BALB/c DST and 200 mg/kg CY were rendered diabetic. They received an i.v. infusion of 25–30 ×10⁶ polyclonal B6 Tregs on day 6 after DST and were transplanted with 450–500 BALB/c islets 1 day later. Untreated diabetic B6 mice were similarly transplanted and used as controls. Graft survival was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method, with comparisons among groups using the log-rank test. ^*p <0.05, ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; i.v., intravenous; Tregs, regulatory T cells.

Figure 5. Prolongation of islet allograft survival in spontaneously diabetic DST +CY-treated NOD mice required combination therapy using islet autoantigen-specific and polyclonal Tregs

Total numbers of donor-reactive (A) and third party–reactive (B) CD4⁺ Tconv and CD8⁺T cells of NOD mice treated with C3H/HeJ DST and CY (200 mg/kg) were calculated from the total cellularity of spleens and lymph nodes and the frequencies of donor-reactive or third party–reactive cells (shown in Table 1). Untreated naïve NOD mice were used as controls and all numbers were normalized to the mean of the naïve NOD mice for ease of comparison. (C) Spontaneously diabetic female NOD mice received C3H/HeJ DST followed by 200 mg/kg CY 2 days later. On day 6 after DST, the mice were divided into four groups that received an i.v. infusion of 5 ×10⁶ BDC2.5 Tregs, 5 ×10⁶ polyclonal NOD Tregs, both, or no Tregs. On day 7 after DST, 450–500 C3H/HeJ islets were transplanted under the left renal capsule. Graft survival was assessed by monitoring blood glucose and calculated using the Kaplan–Meier method, with comparisons among groups using the log-rank test. ^*p <0.05, ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; i.v., intravenous; NOD, nonobese diabetic; Tconv, T conventional; Tregs, regulatory T cells.

Figure 6. Treg therapy did not induce systemic tolerance

(A) Overlaid histograms showing donor reactivity of CD8⁺T cells, CD4⁺Tconv cells and Tregs from naïve B6 mice (filled gray) and mice with long-term protected BALB/c islet grafts for more than 100 days after DST +200 mg/kg CY and Treg therapy (LTP, black line). Data are representative of four naïve B6 mice and four LTP mice. (B) Survival of BALB/c skin grafts in B6 mice with stably protected BALB/c islet grafts for more than 100 days or control age-matched naïve B6 mice. (C) Blood glucose measurement indicating the survival of the original BALB/c islet grafts in mice challenged with BALB/c skin grafts as shown in (B). The horizontal gray line indicates the threshold for rejection of islet grafts. CY, cyclophosphamide; DST, donor-specific transfusion; LTP, long-term protected; Tconv, T conventional; Tregs, regulatory T cells.

Figure 7. Treg therapy increased frequency of Tregs in the grafts early after transplantation

B6 mice preconditioned with BALB/c DST +200 mg/kg CY were transplanted with BALB/c islets either with or without receiving 5 ×10⁶ 4C Tregs as described in the legend of Figure 3. Islet grafts were collected on days 4, 6 and 14 after transplantation and analyzed using confocal immunofluorescence microscopy. (A) Representative images of grafts from Ly5.1⁺ 4C Treg-treated (right) and untreated (left) mice showing CD4 (blue), Foxp3 (green) and Ly5.1 (red) staining on day 6 after transplantation. Original magnification was 640×. (B and C) Entire graft areas were captured by 640× magnification and all the fields were stitched together after image acquisition with the aid of ImageJ software to reconstruct the entire graft section. Numbers of CD4⁺, CD4⁺Foxp3⁺ and CD4⁺Foxp3⁺Ly5.1⁺ cells in each graft section were then determined by manual counting of the reconstructed micrographs. At least three sections more than 60 μm from each other were counted for each graft and more than three mice per experimental condition were analyzed. The average percentages of total Tregs (CD4⁺Foxp3⁺) among CD4⁺ cells (B) and percentages of CD4⁺Foxp3⁺Ly5.1⁺ transferred Tregs among total Tregs (C) were calculated. The data are presented as mean ±SEM of individual graft. ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; iTx, islet transplantation; Tregs, regulatory T cells.

Figure 8. Treg therapy reduced graft-infiltrating CD8⁺T cells

Graft sections prepared as described in figure legend 7 were also analyzed for graft-infiltrating CD8⁺ T cells, CD4⁺ Tconv cells and Tregs using confocal immunofluorescence microscopy. (A) Representative images of 4C Treg-treated (left) and untreated (right) grafts showing CD4 (blue), CD8 (green) and insulin (red) staining on day 14 after transplantation. Original magnification was 640×. (B–E) Total numbers for CD8⁺ T cells (B), CD4⁺ Tconv cells (C), Tregs (D) and the ratio of CD8⁺ T cells to Tregs (E) per graft section were determined as described in figure legend 7 B and C. The data are presented as mean ±SEM of individual graft. ^*p <0.05, ^**p <0.01. A p-value <0.05 was considered statistically significant. Tconv, T conventional; Tregs, regulatory T cells.

Figure 9. Graft-infiltrating CD8⁺ T cells exhibit exhausted phenotype in Treg-treated mice

B6 mice preconditioned with BALB/c DST +200 mg/kg CY were transplanted with BALB/c islets either with or without receiving 5 ×10⁶ 4C Tregs as described in the legend of Figure 3. Graft-infiltrating CD8⁺ T cells were FACS sorted from dissociated islet grafts collected on day 6 after transplantation. Total RNA from CD8⁺ T cells was analyzed using quantitative real-time polymerase chain reaction for the mRNA levels for beta actin and genes indicated on the graph. Data presented is a summary of two independent experiments and each experiment includes 2–3 mice per group. ^**p <0.01. A p-value <0.05 was considered statistically significant. CY, cyclophosphamide; DST, donor-specific transfusion; Tregs, regulatory T cells.