936
T CELL RECONSTITUTION AND THE MECHANISM OF
TOLERANCE INDUCTION BY ALLOGENEIC THYMOKIDNEY TRANSPLANTS IN MINIATURE
SWINE
We have previously demonstrated that thymectomy prior to
kidney transplant interfered with the induction of tolerance, while
thymectomized (Thyx) recipients receiving composite thymokidney allografts
accepted their grafts indefinitely. The purpose of the present study was to
examine the immunological function of the vascularized thymic graft in an
allogeneic thymokidney. Materials and Methods. In order to create
vascularized thymic graft donors, juvenile swine received autologous thymic
grafts under the renal capsule (thymokidney, ThyK). Vascularized donor ThyK in
residence for 3 months in donors were transplanted into class I mismatched
recipients (n=3) with a 12 day course of CyA. Two recipients were Thyx 3 weeks
prior to ThyKTx. One recipient was immunoincompetent with low CD4 single
positive (SP) cells following a Thyx 4 years before ThyKTx. Control Thyx
animals (n=5) received a kidney graft alone across the same MHC barrier with a
12-day course of CyA. ThyK biopsy was performed on POD14,30,60 and >100 to
assess sequential changes in the thymic grafts and to examine thymopoiesis.
Anti-allo responses were examined by cell-mediated lympholysis (CML) and mixed
lymphocyte reaction (MLR). MHC restriction was tested using trinitrophenyl
(TNP) CML assay. Flow cytometric analysis (FACS) was performed for phenotypic
analysis of T cells in peripheral blood and lymph node. Renal graft function
was assessed by creatinine and histology. Results: 1) All
juvenile thymic tissue engrafted under the renal capsule with normal thymic
structure; 2) All recipients of kidney grafts alone showed
unstable renal function with the persistence of anti-donor CTL. In contrast,
all recipients of ThyK showed stable renal function, donor specific
unresponsiveness and no evidence of rejection by histology; 3) Recipient type
dendritic cells migrated in the thymic graft by POD14, and recipient type class
I positive thymocytes appeared in the thymic medulla by POD60, indicating
thymopoiesis in the thymic grafts; 4) One animal was tested for MHC restriction
of T cells 3 months after ThyKTx. T cells were restricted by both donor and
recipient MHC; 5) The animal thymectomized 4 years prior to ThyKTx developed
low numbers of CD4 SP cells 3 years after Thyx and lost anti-allo MLR
responses. However, from POD30 after the ThyKTx, CD45RA+/CD4 SP cells (thymic
derived naive T cells) increased and correlated with total CD4 SP cells, that
reached levels similar to those of the naïve animal by POD80; 6) In
addition, immune response assessed by MLR was fully restored 3 months after the
ThyK graft [anti-third party stimulation index=3 (before ThyKTx) vs. 115
(3 months after ThyKTx)], indicating that the thymic graft had resulted in
return of immunocompetence. Conclusion. To our knowledge these studies
provide the first evidence of a functional vascularized thymic graft (composite
ThyK allografts) reconstituting T cells and leading to a return of an
immunocompetent state as well as inducing transplant tolerance in a large
animal model.