Intracellular MHC class II controls regulatory tolerance to allogeneic transplants

Abstract

MHC class II (MHCII) genes have been implicated in the regulation of T lymphocyte responses. However, the mechanism of MHCII-driven regulation remains unknown. Matching for MHCII between donors and recipients of allografts favors regulatory T cell tolerance to transplants and provides a unique opportunity to study this regulation. In this study, we investigated MHCII regulation using transfer of donor MHCII genes in recipients of cardiac allografts. Transfer of MHCII IA(b) genes in the bone marrow of CBA mice (H-2(k)) prior to the grafting of IA(b+) fully allogeneic C57BL/6 (B6, H-2(b)) heart transplants resulted in donor-specific tolerance associated with long-term survival of B6, but not third-party, allografts without sustained immunosuppression. Strikingly, the majority of accepted heart transplants (>170 d) were devoid of allograft vasculopathy. Further studies indicated that intracellular IA(b) initiated the tolerogenic process, which was mediated by regulatory T cells (Tregs) that polarized antigraft responses to Th2 cytokine producers. This mechanism seems to be unique to MHCII genes, because previous MHC class I gene-based therapies failed to produce Tregs. These results demonstrate the key role of MHCII in the induction of Tregs. They also underscore a potential mechanism of specific inactivation of T cells in this model; when activated by IA(b+) grafts, IA(b)-specific Tregs repress the entire alloresponse to C57BL/6 transplants (including MHC I and minor Ags), thus mediating T cell tolerance.

FIGURE 1

Conditioning protocol for IA^b gene therapy. CBA (IA^k, IE^k) recipients were conditioned with anti-CD4, anti-CD8 mAbs (days −3 and −1) and Busulfex (days −4, −3, −2, and −1) prior to transplantation of syngeneic BMCs that were transduced with the IAb.RV or GFP.RV vector (Fig. 2A). Eight weeks after BM transplantation, animals received B6 hearts (IA^b, IE⁰) with anti-CD8. For adoptive transfers, tolerant (POD 170) and naive CBA mice received DST at days −21 and −14 relative to the adoptive transfer. Purified CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells from tolerant or naive CBA mice were infused at day 0 into naive CBA recipients, together with 1 mg of anti-CD8. Animals were transplanted with B6 cardiac allografts the following day.

FIGURE 2

Survival of IA^b+ cardiac transplants in IAb-CBA and GFP-CBA recipient mice. A, The IAb-RV vector contained cDNA for the IAα^b- and IAβ^b-chains, spaced by an IRES. Transcription was under long terminal repeat promoter control (arrow). Intact vector transcripts were detected by RT-PCR (double arrowheads). The IAb-GFP.RV vector was constructed on the same backbone with the addition of the GFP sequence that was fused, in frame, to that of I-Aα^b. The GFP.RV vector included the GFP sequence. B, IA^b-transduced CBA mice (n = 15) received B6 (n = 11) or third-party BALB/c hearts (n = 4) that were monitored for survival. Controls were CBA mice (n = 7) engineered with the GFP.RV vector and transplanted with B6 hearts. Representative histology of two long-term accepted B6 hearts from IAb-CBA mice (C, D) and a B6 transplant rejected by a control GFP-CBA mouse (E) (Verhoeff elastic stain, original magnifications ×200 (C), ×320 (D), and ×640 (E) at POD 177, 175, and 23, respectively). CAV lesions were quantified as detailed in Materials and Methods.

FIGURE 3

T cell alloresponses in IAb-CBA mice. IL-2 (A, D), IFN-γ (B, E), and IL-4 (C, F) ELISPOT assays were performed on splenocytes from naive CBA (white bars) and IAb-CBA (black bars) mice. Assays were done 8 wk after BM transplantation, prior to heart transplantation (n = 3) and 10 d after transplantation of B6 heart grafts (n = 3). Stimulator cells were irradiated splenocytes from BALB/c or B6 mice. MEM: control medium. Results are mean ± SD.

FIGURE 4

Time course analysis of IA^b transcription in vivo. A, RT-PCR analysis of IA^b and control CD2 gene transcription in tissues from long-term tolerant CBA #13 (POD 176). Proviral IA^b sequences were detected by annealing to a [³²P]-IRES–specific oligonucleotide. B, IA^b transgene transcription in PBMCs, lymph nodes (LN), spleen, and thymus. Two rejector IAb-CBA (Rej; tested 11 wk after BMC infusion) and six long-term tolerant IAb-CBA (Tol; 30–32 wk) mice were monitored by RT-PCR for proviral IA^b transcription. *Number of animals positive for IA^b/number tested. Rej, rejector IAb-CBA mouse; Tol, long-term tolerant IAb-CBA mouse.

FIGURE 5

Patterns of IA^b transgene expression in APCs. A, BM Sca1⁺ progenitors from CBA mice transduced with the empty (mock) or IAb.RV (IAb) vectors were analyzed 48 h after transduction for IA^b expression. B, Subsets of DCs, derived from BMCs from CBA mice, were sorted as iDCs (CD11c⁺IAk^loCD86^lo) or mDCs (CD11c⁺IAk^hiCD86^hi). Each subset was transduced with the GFP.RV vector and analyzed 5 d later for GFP expression. Data presented are from one of two experiments. C, 1: Unsorted DC cultures from CBA mice were transduced with the IAb-GFP.RV vector and tested for surface expression of recipient (IE^k, dotted line) and vector-derived MHCII (IA^b, black line) 7 d posttransduction. Shaded curve represents Ig isotype control. 2, 3: GFP expression in the iDC and mDC subsets that were derived from the same bulk DC culture transduced with the IAb-GFP.RV vector. Representative results from one of three experiments. 4, 5: DCs and L cells transduced with IAb-GFP.RV were analyzed for expression of the IA^b-GFP fusion protein 1 wk after transduction. Similar data were obtained in four additional experiments using DC or other APC lines. 6: Real-time RT-PCR analysis of GFP transcripts in transduced DC and L cells from panels 4 and 5. 7: The IA^d+ IE^d+ lymphoblastic cell line A20 was transduced with the IAb-GFP.RV and tested 4 d later for endogenous (IA^d, fine dotted line) and ectopic (IA^b, bold dotted line) expression. Shaded curve represents staining with isotype control (Ctr). 8: A20 cells from panel C7 were also tested for GFP expression in the absence (−) or presence (+) of monensin.

FIGURE 6

Tregs from tolerant recipients transfer IA^b-induced tolerance to B6 heart grafts. CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were purified from naive CBA mice, CBA mice infused with a donor-specific transfusion (CBA+DST), or long-term tolerant IAb-CBA mice treated with DST (IAb-CBA+DST). Each T cell subset was injected separately into naive CBA mice that also received a dose of anti-CD8 mAb and B6 heart grafts on the following day. The effects of adoptive transfer of CD4⁺CD25⁻ (white columns) and CD4⁺CD25⁺ (gray columns) T cells on graft survival were monitored. Each point represents survival data from a single mouse. *Survival time (days; mean ± SD).

FIGURE 7

MHCII gene therapy to induce Treg-mediated tolerance to vascularized transplants. Transduction of BM-derived APCs from recipient mice (H-2^k) with the IAb.RV (retro IA^b) vector leads to IA^b peptides (pep) presented on MHCII^k heterodimers. These MHCII/peptide complexes participate in the thymic differentiation of IA^b-specific Tregs (GT Tregs). Self-Tregs would differentiate on self-MHCII peptides/MHCII complexes. In these animals, transplanted hearts from B6, but not from third-party BALB/c donors, provide IA^b signals for the in situ activation of GT Tregs via the direct (D) or indirect (I) presentation pathways. In turn, locally activated GT Tregs repress the entire Th1 antigraft alloresponse (spreading tolerance) and prevent rejection.