Non-dividing allostimulated CD4 T cells induce specific tolerance to allogeneic skin transplants in the mouse

D Watson¹, GY Zhang¹, M Sartor¹, JF Knight¹, SI Alexander¹

1. Centre for Kidney Resarch, Children's Hospital, Westmead NSW Australia
2. Westmead Millenium Research Institute, Westmead NSW Australia

Bone marrow transplantation, co-stimulatory blockade, T cell ablation and cytokine deviation have all been used to develop specific tolerance to allogeneic grafts. We propose a novel way of creating specific tolerance to allogeneic skin grafts using allostimulated non-dividing CD4 T cells. CD4 T cells were isolated from a six day mixed lymphocyte culture after pre-staining with CFSE as a marker of cell division. Responder mononuclear cells were derived from BALB-C (H-2d) mice and stimulator mononuclear cells were derived from C57B (H-2b) mice. Dividing (D) and non-dividing (ND) CD4 T cells were isolated by flow cytometry and adoptively transferred into SCID mice on a BALB-C background. After one week skin grafts from the stimulator, responder and a third party B10.BR (H-2k) were grafted onto the reconstituted mice and control SCID mice. Graft survival was assessed on appearance. Mice reconstituted with ND CD4 T cells rejected third party grafts at the same time as mice reconstituted with D CD4 Tcells (day 16) but were tolerant to skin grafts from the stimulator strain (log rank p=0.0007). Mice reconstintuted with D CD4 T cells rejected skin grafts from both stimulator and third party strains at day 16 (log rank p=NS). Histology confirmed the absence of lymphocyte infiltration in stimulator skin grafts on mice reconstituted with ND D4 T cells, with lymphocyte infiltration in third party grafts. Lymphocyte infiltration was seen in bothth estimulator and third party skin grafts in mice reconstituted with D CD4 T cells. Analysis of the T cell repertoire in spleen samples by V beta analysis using RT-PCR and GeneScan demonstrated polyclonality in the ND CD4 T cells but restriction in the D CD4 T cells. These results suggest that specific tolerance can be achieved by selective removal of the alloreactive T cell population while retaining immune competence.

Presented at the ASN/ISN World Congress of Nephrology, San Francisco, October 2001

Correspondence
Dr Stephen Alexander
StephenA@chw.edu.au