Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts

Abstract

The almost uniform failure in transplant patients of tolerance-inducing regimens that have been found to be effective in rodents, has made it necessary to examine large animal models before testing of new approaches clinically. Miniature swine have been shown to share many relevant immunologic parameters with humans, and because of their reproducible genetics, have proved extremely useful in providing such a large animal model. We have previously shown that indefinite systemic tolerance to renal allografts in miniature swine is induced in 100% of cases across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-d course of Cyclosporine A (CyA), in contrast to irreversible rejection observed uniformly without CyA treatment. In the present study, we have examined the role of the thymus during the induction of tolerance by performing a complete thymectomy 21 d before renal transplantation. This analysis demonstrated a striking difference between thymectomized and nonthymectomized animals. Thymectomized swine developed acute cellular rejection characterized by a T cell (CD25(+)) infiltrate, tubulitis, endothelialitis and glomerulitis, and anti-donor CTL reactivity in vitro. Nonthymectomized and sham thymectomized animals had a mild T cell infiltrate with few CD25(+) cells and no anti-donor CTL response in vitro. These results indicate that the thymus is required for rapid and stable induction of tolerance.

Figure 1

Schematic diagram of the origin of available homozygous porcine MHC haplotypes. Partially inbred SLA^aa, SLA^cc, and SLA^dd haplotypes were derived from the original founder miniature swine. Recombination events between the MHC class I and class II haplotypes have been identified and maintained as homozygous, recombinant haplotypes SLA^ff, SLA^gg, SLA^hh, SLA^jj, and SLA^kk.

Figure 2

Macroscopic findings of a completely resected thymus from a thymectomized animal.

Figure 3

Clinical course of thymectomized animals (a) and nonthymectomized animals (b). D, died; S, killed due to progressive uremia; *, No. 11561, 11574. These pigs were excluded from this study on POD 42 (see text).

Figure 4

Representative histological findings of thymectomized animals and nonthymectomized animals. (a) A thymectomized animal on POD 8; diffuse and moderate mononuclear cell infiltration is seen with diffuse tubulitis. Glomeruli show typical acute allograft glomerulopathy (PAS ×200). (b) A nonthymectomized animal on POD 8; mild and focal mononuclear cell infiltration is seen with mild focal tubulitis (PAS ×200). (c) Immunohistochemistry for CD25 on POD 8 in a thymectomized animal, and (d) a nonthymectomized animal. Many infiltrating mononuclear cells are seen expressing CD25 in the thymectomized animal (c), whereas only a few of these cells are seen in nonthymectomized animal (d) (×600). (e) Thymectomized animal on POD 60 shows chronic transplant glomerulopathy with diffuse interstitial fibrosis (PAS ×200), and (f) nonthymectomized animal show a normal glomerular structure on POD 60 (PAS ×200).

Figure 5

Phenotype of GIC from typical thymectomized and nonthymectomized animals. Flow cytometric analysis of the CD25 expression (closed histogram) on GIC on POD 8 prepared from renal biopsies taken from thymectomized (a) and nonthymectomized (b) animals is represented. The negative control antibody staining is also shown (open histogram). The CD8 versus CD4 dot-plot analysis of the GIC on POD 8 is shown for a thymectomized (c) and nonthymectomized animal (d). The analysis of the phenotype shows the large number of CD8 single positive and CD4/8 double positive GIC in both animals.

Figure 6

CTL antidonor reactivity on POD 30 in thymectomized animals and nonthymectomized animals. PSL at an E/T ratio of 100:1 was significantly higher in thymectomized animals when compared to nonthymectomized animals.