CD40Ig treatment results in allograft acceptance mediated by CD8CD45RC T cells, IFN-gamma, and indoleamine 2,3-dioxygenase

Abstract

Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8(+)CD45RC(low) subset resulted in donor-specific long-term survival, whereas CD8(+)CD45RC(low) T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8(+)CD45RC(low) T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-gamma. Neutralization of IFN-gamma or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40-CD40 ligand (CD40-CD40L) interactions induces allospecific CD8(+) Tregs that maintain allograft survival. CD8(+)CD45RC(low) T cells act through IFN-gamma production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8(+) Tregs may promote local graft immune privilege through IDO expression.

Figure 1. CD8⁺ T cells mediate transfer of transplantation tolerance after CD40Ig treatment.

Cells from control rats that had rejected their grafts or from CD40Ig-treated recipients were injected i.v. the day of transplantation into LEW.1A recipients that received LEW.1W heart transplants (day 0) and that were sublethally irradiated (4.5 Gy, at day –1). (A) Total splenocytes (50 × 10⁶; n = 6) or splenocytes depleted of T cells (n = 2), CD4⁺ cells (n = 2), CD8⁺ cells (n = 4), or NK cells (n = 2) were injected. (B) Splenocytes (50 × 10⁶; n = 6), purified T cells (n = 3), CD8⁺ T cells (n = 4), or CD4⁺ T cells (n = 2) were injected. Graft survival was assessed by abdominal palpation of cardiac beating. (C) Grafts were either untreated (n = 9; thick black line), treated with a control mAb (3G8) (n = 6, gray line), transduced with Addl324 (5 × 10¹⁰ IP; n = 9; dotted line), transduced with AdCD40Ig (n = 27, black line with open circles), or transduced with AdCD40Ig and injected with a depleting anti-CD8 mAb (OX8) (n = 12; black line with open triangles) or an anti-MHC class I mAb (OX18) (n = 3; black line with filled diamonds). A group of control animals received the control mAb (3G8) in addition to CD40Ig (n = 6; black line with open diamonds). **P < 0.01, ***P < 0.0001 versus animals injected with AdCD40Ig or AdCD40Ig and 3G8.

Figure 2. CD8⁺CD45RC^low T cells mediate transfer of transplantation tolerance after CD40Ig treatment.

(A) CD8⁺CD45RC^low and CD8⁺CD45RC^high T cells were sorted to greater than 99% purity from spleens. (B) Cells were purified from naive, CD40Ig-treated, or adoptively transferred recipients 120 days after transplantation. A total of 2.5 × 10⁶ cells of each cell population was injected i.v. the day of transplantation of LEW.1W hearts into naive LEW.1A recipients sublethally irradiated (4.5 Gy) at day –1. Graft survival was assessed by abdominal palpation of cardiac beating. Black line, CD8⁺CD45RC^low T cells from CD40Ig-treated animals (n = 4). Dotted line, CD8⁺CD45RC^low T cells from adoptively transferred animals (n = 2). Gray line, CD8⁺CD45RC^high T cells from CD40Ig-treated animals (n = 4). Dashed gray line, CD8⁺CD45RC^low T cells from naive animals (n = 4). P < 0.01 for CD8⁺CD45RC^low from CD40Ig-treated animals versus CD8⁺CD45RC^high and CD8⁺CD45RC^low from naive animals.

Figure 3. Expression of IFN-γ and regulatory molecules by CD8⁺CD45RC^low T cells.

(A) Intracytoplasmic analysis of IFN-γ production in total splenocytes and in CD8⁺CD45RC^low and CD8⁺CD45RC^high cells from CD40Ig-treated or adoptively transferred animals. Cells were harvested, stimulated for 7 hours using phorbol myristate acetate and ionomycin, and analyzed with a FITC-labeled anti–IFN-γ mAb. Results are expressed as percent of positive cells. Each symbol indicates a value for an individual animals. Lines indicate mean ± SD. ATS, adoptively transferred. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Quantitative analysis of transcript accumulation was performed after isolation in CD8⁺CD45RC^low or CD8⁺CD45RC^high T cells isolated from CD40Ig-treated recipients or in CD8⁺CD45RC^low cells from naive animals. Results are expressed in arbitrary units of molecules normalized to HPRT ± SD. GITR, glucocorticoid-induced tumor necrosis factor receptor family–related gene. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4. Analysis of the TCR repertoire in CD8⁺CD45RC^low T cells.

CD8⁺CD45RC^low or CD8⁺CD45RC^high T cells were purified from spleen of CD40Ig-treated or naive animals. A qualitative and quantitative analysis of the TCR Vβ transcriptome was performed as described in Methods. Each histogram represents a qualitative immunoscope analysis of the Vβ11 family repertoire of 1 animal. The histograms display the intensity of fluorescence in arbitrary units as a function of runoff CDR3 Vβ11 length in nucleotides. The CDR3 length distribution was unaltered (gaussian) in CD8⁺CD45RC^low from all naive animals or CD8⁺CD45RC^high from CD40Ig-treated recipients and altered (non-gaussian) in spleen CD8⁺CD45RC^low T cells from all CD40Ig-treated recipients with a predominant CDR3 of always the same length (27 nucleotides, arrows).

Figure 5. Grafts accepted long term after CD40Ig treatment showed strong accumulation of transcripts for regulatory molecules.

Heart grafts from syngeneic donors or from allogeneic donors were harvested 120 days after transplantation. Recipients from allogeneic donors were treated with CD40Ig. (A) Heart total RNA was analyzed using real-time quantitative RT-PCR. Symbols indicate values for individual animals in arbitrary units. *P < 0.05, and **P < 0.01 versus syngeneic animals. (B) Confocal analysis of allogeneic or syngeneic (inserts) grafts using anti-CD8α (red) or anti–IFN-γ (green) antibodies. Merge analysis shows IFN-γ expression almost exclusively restricted to CD8⁺ cells (arrows), whereas some cells are IFN-γ^– (arrowheads). Identical results were obtained with 3 grafts from CD40Ig-treated animals and from adoptively transferred grafts. Original magnification, ×400.

Figure 6. IFN-γ and IDO mediate the effect of CD40Ig and of CD8⁺CD45RC^low T cells.

(A) Graft survival after 1-MT or anti–IFN-γ mAb administration beginning the day of transplantation (d0) to CD40Ig-treated animals or to animals receiving adoptive transfers at 120 days after transplantation (d120). Heartbeat of nonrejected grafts after 1-MT or anti–IFN-γ administration was + compared with +++ in CD40Ig-treated or adoptively transferred recipients. Arrow indicates adoptive transfer and beginning of treatment with 1-MT. (B) Representative Western blot analysis of IDO expression in cardiac grafts from syngeneic, CD40Ig-treated, and adoptively transferred recipients at day 120. Histograms show quantification of IDO normalized to tubulin in 3–4 samples per group. **P < 0.01, syngeneic versus adoptively transferred grafts. (C) Confocal analysis of grafts from adoptively transferred or syngeneic recipients using anti-IDO (green) and anti-CD31 (red) antibodies. Merge analysis shows IDO expression almost exclusively restricted to ECs. The inserts represent staining using an irrelevant mAb (3G8) or rabbit serum. Identical results were obtained with 2 other grafts from adoptively transferred animals and 2 grafts from CD40Ig-treated animals. Original magnification, ×400. (D) Kynurenine in cultured supernatants. CD8⁺CD45RC^low or CD8⁺CD45RC^high T cells from CD40Ig-treated recipients (n = 2 in each group) were cultured for 2 days with ECs from LEW.1W animals in the presence of anti–IFN-γ or control mAb (3G8) at 20 μg/ml. Levels are expressed as μM ± SD. *P < 0.05. (E) Quantitative RT-PCR analysis of IDO mRNA in ECs cultured for 2 days with CD8⁺CD45RC^low or CD8⁺CD45RC^high T cells from CD40Ig-treated recipients (n = 3 in each group). **P < 0.01 for CD8⁺CD45RC^low versus CD8⁺CD45RC^high.

Figure 7. IDO gene transfer into the graft but not at a distant site results in long-term allograft survival.

AdIDO or Addl324 (2 × 10¹⁰ IP) were injected into the cardiac grafts or intramuscularly into the hind limb the day of transplantation. Rejection was defined when beating of the transplanted heart could no longer be detected. P < 0.001, AdIDO graft versus Addl324 and AdIDO i.m.