CAR Treg synergy with anti-CD154 promotes infectious tolerance and dictates allogeneic heart transplant acceptance

Abstract

Successful allograft-specific tolerance induction would eliminate the need for daily immunosuppression and improve posttransplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific chimeric antigen receptors (CAR Tregs) is a promising strategy but, as monotherapy, cannot prolong survival with allografts with multiple MHC mismatches. Using an HLA-A2-transgenic haplo-mismatched heart transplantation model in immunocompetent C57BL/6 recipients, we showed that HLA-A2-specific CAR (A2.CAR) Tregs were able to synergize with a low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited donor-specific T cell, B cell, and antibody responses and promoted a substantial increase in endogenous FOXP3+ Tregs with indirect donor specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FOXP3- A2.CAR T cells preferentially accumulated in rejecting allografts, and endogenous donor-specific responses were not controlled. This study therefore provides evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity toward allograft tolerance.

Keywords: Immunology; Therapeutics; Tolerance; Transplantation.

Figure 1. A2.CAR Tregs synergize with low-dose anti-CD154 to prolong the survival of haplo-mismatched heart grafts.

(A) Schematic of the experimental model. (B) Graft survival curve in recipients of A2.2W-OVA.F1 heart transplantation (HTx) with 3 different doses of anti-CD154. (C) Graft survival of B6 recipients of A2.2W-OVA.F1 HTx treated with low anti-CD154 (250 μg) without or with CAR Tregs or high anti-CD154 (600 μg). Survival curve for recipients receiving only 250 μg anti-CD154 is the same in B and C. Graft survival (D) and palpation scores (E) in recipients of 1.4 × 10⁶ A2.CAR Tregs + low anti-CD154 with rejected (Rej) or accepted (Acpt) grafts. Symbols are the same for D and E. Log-rank (Mantel-Cox) test for statistical significance. Each symbol represents 1 mouse. Data are presented as mean ± SEM, and statistical significance was assessed by Mann-Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Recipients of A2.CAR Tregs and low-dose anti-CD154 with Acpt allografts have low accumulation of CD4⁺Thy1.1⁺ cells and high ratios of FOXP3^pos/FOXP3^neg A2.CAR T cells.

(A–D) The absolute numbers and proportions of circulating Thy1.1⁺ A2.CAR Tregs in A2.CAR Treg recipients with Rej (filled triangles) or Acpt (unfilled triangles) grafts was quantified at the indicated weeks (A–D) or at the experimental endpoint of postoperative day (POD) 45–63 (E–L). (A) Number of CD4⁺Thy1.1⁺ cells per 100 μL blood. (B) Percentage FOXP3^pos of Thy1.1⁺ cells. (C and D) Number of FOXP3^pos or FOXP3^neg A2.CAR T cells per 100 μL blood. (E–L) At the experimental endpoint (POD 45–63), SLOs and allografts were harvested, and Thy1.1⁺ A2.CAR Tregs were quantified. (E and I) Total number of CD4⁺Thy1.1⁺ T cells from SLOs and allografts from Rej (n = 6) or Acpt (n = 7) recipients. (F and J) Percentage FOXP3^pos of Thy1.1⁺ cells. Number of FOXP3^pos (G and K) or FOXP3^neg (H and L) A2.CAR T cells recovered from SLOs and allografts. Each symbol represents 1 mouse. Data are presented as mean ± SEM, and statistical significance was assessed by Mann-Whitney test. *P < 0.05; **P < 0.01. Total number of CD4⁺Thy1.1⁺ cells from SLOs normalized to 3 × 10⁶ events. Total number of CD4⁺Thy1.1⁺ cells from allografts normalized to 1 × 10⁶ events.

Figure 3. FOXP3^pos and FOXP3^neg A2.CAR T cells in the SLOs are phenotypically distinct in recipients with Rej or Acpt allografts.

(A) Gating strategy to define FOXP3^pos and FOXP3^neg cells within CD4⁺Thy1.1⁺ A2.CAR T cells from SLOs (POD 45–63). (B) UMAP markers and experimental groups. (C and D) UMAP plots demonstrating phenotypic differences in (E) FOXP3^pos and (F) FOXP3^neg A2.CAR T cells from Rej and Acpt recipients, based on expression of FR4, CD73, PD1, SLAMF6, LAG3, and TIGIT. UMAP with heatmap and bar plots showing relative expression of indicated markers based on normalized median fluorescence intensity (MFI). Each symbol in the bar plots represents 1 mouse. Data are presented as mean ± SEM, and statistical significance was determined by Mann-Whitney test. *P < 0.05; **P < 0.01.

Figure 4. Graft acceptance in A2.CAR Tregs recipients is associated with expanded endogenous donor-specific Tregs and reduced T cell responses.

(A) Experimental groups for B and D–G. (B) Number of CD4⁺2W:I-A^bCD44^hi T cells recovered from SLOs. (C) Representative flow plots of FOXP3^pos of CD4⁺2W:I-A^bCD44^hi T cells from SLOs of the indicated experimental groups and harvested on day 45–63 after HTx. (D and E) Frequency and total number of 2W:I-A^bCD44^hi Tregs and of (F) 2W:I-A^bCD44^hi T Tconvs and (G) OVA:K^bCD8⁺ T cells. Each symbol represents 1 mouse. (A total of 7 Acpt recipients received 1.4 × 10⁶, and 1 Acpt at day 100 after HTx received 1 × 10⁶ A2.CAR Tregs.) Total 2W:I-A^bCD4⁺ T cells and OVA:K^bCD8⁺ T cells from SLOs normalized to 3 × 10⁶ events. Data are presented as mean ± SEM, and statistical significance was determined by 1-way ANOVA (#) and Mann-Whitney test (*). *P or ^#P < 0.05; ^##P < 0.01; ^####P < 0.0001.

Figure 5. Graft acceptance in A2.CAR Treg recipients is associated with reduced accumulation of endogenous T and B cells in heart allografts.

(A–E) Representative H&E-stained histological sections (scale bar: 20 μm) of naive or transplanted heart allografts from indicated groups sacrificed on days 45–63 after HTx. (F–I) Cells infiltrating the allograft were analyzed by flow cytometry. Total graft-infiltrating (F) CD8⁺ T cells, (G) CD4⁺ T cells, (H) percentage Tregs of CD4⁺ T cells, and (I) CD19⁺ B cells were normalized to 1 × 10⁶ events. Each symbol represents 1 mouse. Data are presented as mean ± SEM, and statistical significance was assessed by 1-way ANOVA. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001.

Figure 6. A2.CAR Tregs synergize with low anti-CD154 to suppress non-A2 donor-specific B cell responses.

(A) Symbols for B–D and H. (B) Quantification of IgG specific for A2, (C) donor MHC-I IgG, and (D) donor MHC-II at day 45 after HTx. (E–G) Gating strategy to track donor MHC-II I-E^d–specific GC B cells (Fas⁺GL7⁺) from (F) +A2.CAR Treg Rej or (G) +A2.CAR Treg Acpt. (H) Total GC B cells (Fas⁺GL7⁺ of I-E^d) recovered from draining lymph nodes (normalized to 2 × 10⁶ events). Each symbol represents 1 mouse. Data are presented as mean ± SEM, and statistical significance was determined by 1-way ANOVA (#) and Mann-Whitney test (*). *P or ^#P < 0.05; ^##P < 0.01; ^####P < 0.0001. DSA, donor-specific antibody.