Donor Conditioning with Recipient-Derived PD-L1/PD-L2-Expressing B Cells Prevents Lethal Acute GVHD in a Fully Allogeneic Mouse Model

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Abstract

Aim

We recently found that the inoculation with Balb/c (H-2d) PD-L1/PD-L2-expressing B cells lead to persistent suppression of anti-Balb/c T cell-responses, and significantly prolonged the survival of subsequently grafted Balb/c hearts in C57BL/6 (B6) (H-2b) mouse recipients. Given the effectiveness of those B cells as regulatory antigen-presenting cells targeting alloreactive T cells, we investigated if donor pre-conditioning with recipient-derived PD-L1/PD-L2 B cells could prevent acute GVHD after fully allogeneic bone marrow transplantation (BMT)/donor lymphocyte infusion (DLI) in mice.

Methods

To induce acute GVHD, lethally irradiated BALB/c mice were transplanted with 5 × 106 bone marrow cells and 1 × 106 splenic T cells from B6 mice. Twelve days prior to the BMT/DLI, B6 donors were intravenously injected with 10 × 106 BALB/c peritoneal cavity (PerC) B cells.

Results

Mixed-lymphocyte reaction (MLR) assays using splenocytes from the B6 donor mice at 12 days after the B cell injection revealed the significantly reduced anti-Balb/c T cell-responses in the recipients of PerC B cells, as compared to those in untreated control mice. Since PerC B cells contained MHC class II+ CD80+ CD86+ PD-L1+ PD-L2+ cells among the CD5+ B-1a cell subset, PerC B cells from Balb/c mice were pre-incubated with anti-PD-L1/PD-L2 mAbs prior to injection. This treatment abrogated their immune-regulatory effects on anti-Balb/c T cells in the MLR assays, indicating that the expression of both PD-L1 and PD-L2 on allogeneic PerC B cells is required to inhibit T cell-responses to the cognate allostimulation through engaging PD-1. The difference of T cell repertoire between the conditioned and untreated B6 was investigated using mAbs directed against T cell receptor Vβ regions. The proportion of Vβ5.1/5.2+ and Vβ11+ CD8+ T cells in the spleen of the conditioned B6 mice was significantly diminished, while that of Vβ8.1/8.2+ CD8+ T cells was not, indicating the specificity of the deletion for super-antigens presented by the BALB/c B cells. The donor pre-conditioning with recipient-derived PerC B cells markedly improved clinicopathological manifestations and survival in the lethal acute GVHD model, i.e. the mean survival of recipients of BMT/DLI from the conditioned donor was 27.9 ± 16.0 days (n=8), while that of recipients of BMT/DLI from the untreated donor was 6.6 ± 0.9 days (n=5).

Conclusions

These findings demonstrate that the PerC B cells, including PD-L1/PD-L2 B-1a cells, suppress T cells responding to allostimulation by clonal deletion. Thus, our study presents what we believe to be the reversed paradigm for the prevention of lethal acute GVHD by donor pre-conditioning with those immune-regulatory B cells derived from recipients.

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