MHC Class II DRB genotyping is highly predictive of in-vitro alloreactivity in the common marmoset.

The common marmoset (Callithrix jacchus) is emerging as a promising alternative pre-clinical model for transplantation and immunological research. It is therefore important to establish a rapid and reliable method of confirming alloreactivity between donor-recipient pairs. In this study of a large marmoset colony (n=49), we firstly characterised MHC Class II genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using, for the first time in this species, sequence-based allelic typing techniques. Exon 2 was amplified using M13-tailed PCR primers specific for known marmoset alleles, and sequenced using universal M13 sequencing primers and dye terminator cycle sequencing. Twenty-six genotypes involving monomorphic Caja-DRB*W1201, 8 Caja-DRB*W16 and 5 Caja-DRB1*03 alleles were observed. Two new DRB*W16 alleles were identified. Subsequently we investigated whether matching at MHC-DRB loci alone could accurately predict in-vitro alloreactivity as assessed by mixed lymphocyte reactions. Peripheral blood mononuclear cells (PBMC) isolated from fully and partially DRB-matched and fully mismatched animal pairs were mixed and co-cultured for T-cell proliferation. PBMC co-cultured from fully or partially mismatched pairs exhibited significant T cell proliferation above single cell controls (p<0.01). Mixed PBMC from fully DRB-matched pairs exhibited no proliferation over controls (p=0.3). Thus using Caja-DRB genotyping, suitably alloreactive donor-recipient pairs can be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.

Human plasmacytoid dendritic cells regulate immune responses to Epstein-Barr virus (EBV) infection and delay EBV-related mortality in humanized NOD-SCID mice.

Epstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease (PTLD), which is a leading cause of cancer death in recipients of transplants. We investigated the role of plasmacytoid dendritic cells (PDCs) in the development of EBV infection and the onset of lymphoproliferative disease (LPD) in humanized NOD-SCID mice and studied the effect of EBV on PDC function. NOD-SCID mice reconstituted with PDC-depleted peripheral blood mononuclear cells (PBMCs) from EBV IgG+ human donors had significantly enhanced mortality from disseminated EBV infection (median survival, 43 days) compared to PBMC-only mice (median survival, 72 days; log-rank P<.05). Mice reconstituted with PDC-enriched PBMCs challenged with EBV exhibited delayed mortality from EBV-LPD (median survival, 80 days) compared to PBMC-only mice challenged with EBV (median survival, 50 days; log-rank P<.05). EBV-stimulated pDCs produced interferon alpha (IFN-alpha) and promoted the activation of natural killer cells and IFN-gamma-producing CD3+T cells. PDC activation of CD3+T cells in response to EBV stimulation was dependent on cell-to-cell contact, in part mediated by toll-like receptor 9 (TLR-9) signaling that was inhibited by chloroquine and TLR-9 inhibitory CpG. Thus, PDCs play an important role in anti-EBV cellular immune responses that may be targets for manipulation in novel strategies for the treatment of PTLD.