Morphologic studies of acute rat cardiac allograft rejection across an isolated major histocompatibility complex class I (RT1A) disparity.

To define the morphologic correlates of acute rat cardiac allograft rejection across an isolated major histocompatibility complex (MHC) class I disparity, rejecting PVG.R1 cardiac allografts transplanted to (PVG x WF)F1 recipients were studied from days 4-8 posttransplantation. Documented ultrastructural tracer techniques as well as immunohistologic and immunoelectron microscopic methods were employed for morphologic analysis. Using intravenously administered horseradish peroxidase as a tracer probe for cell membrane permeability dysfunction, it was shown that severe diffuse loss of integrity of the microvascular endothelium preceded functional rejection, providing strong evidence that the allograft microcirculation is a central target of graft destruction. Also, rejection was associated with localized cardiac myofiber alterations prior to development of significant endothelial changes, indicating that cardiac muscle cells are additional cellular targets of immunologic injury. The ultrastructural features of progressive endothelial and myofiber injury, the predominance of MRC OX8+ lymphocytes and MRC OX6+ macrophages sequestered within the grafts, and the pattern of donor class I expression by allograft endothelium and cardiac myofibers were similar to those observed in rejecting allografts in full MHC-disparate combinations. Since it has been previously shown that MRC OX8+ class I-reactive T cells are absolutely required for rejection in this isolated class I-disparate model, the morphologic data raise the possibility that the OX8+ T lymphocyte subpopulation may also play a highly significant role in rat cardiac allograft rejection across a full MHC disparity.

Immune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic-suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat.

The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells (7-9 days, n = 6) or inocula (5 X 10(6) cells) depleted of SIg+ cells (10-13 days, n = 5), but additional depletion of cytotoxic T cells and their precursors (OX8+) resulted in marked prolongation of graft survival (n = 4, 4 X 10(6) cells, 45-67 days). Reducing the reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells (OX8-, SIg-) prolonged graft survival to that observed in unreconstituted recipients (generally greater than 100 days). Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate (stimulation index [SI] 1.01, P = NS) on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated (SI 4.25, P less than .001) and generated greater cytotoxic T lymphocyte (CTL) activity (P less than .001) under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency. Further, though PVG-RT1r1 heart grafts were rejected in 10-12 days (n = 3) by (PVG X WF)F1 recipients, (PVG-RT1r1 X WF)F1----(PVG X WF)F1 heart grafts (RT1Aa disparity) survived greater than 100 days despite an equivalent alloimmune response, and this was shown to correlate with a reduced sensitivity of (PVG-RT1r1 X WF)F1 target cells to lysis by CTL. These data, therefore, strongly suggest that the pivotal role of CTL in the rejection of class-I-disparate heart grafts is, in fact, related to their function in direct cell-mediated cytolysis.

Immune mechanisms in organ allograft rejection. VI. Delayed-type hypersensitivity and lymphotoxin in experimental renal allograft rejection.

The cellular requirements for renal allograft rejection have been reassessed in a rat adoptive transfer model, preceding studies having shown that transplanted kidneys may be rejected in the absence of cytotoxic T cells or specific antibody. Unilaterally nephrectomized, sublethally irradiated (780 rads) LEW recipients of renal allografts from irradiated WF donors, were selectively reconstituted with spleen cells from sensitized syngeneic donors and subjected to delayed nephrectomy of the residual native kidney 3 days posttransplantation. In some experiments the reconstituting inocula were depleted of SIg+ cells (anti-Ig column) or additionally depleted of cytotoxic T cells and their precursors reactive with monoclonal OX8 (rosette depletion). Depleting the reconstituting inocula of SIg+ cells as well as cells reactive with monoclonal OX8 failed (n = 4) to alter the tempo of rejection, as demonstrated by a mean serum creatinine +/- SD on day 8 of 5.4 +/- 3.8 vs. 6.4 +/- 4.2 in recipients (n = 8) reconstituted with unfractionated inocula. These data support a link between DTH and graft rejection, so additional studies were performed to characterize rat lymphotoxin (LT), one of the potential mediators of DTH-induced tissue injury, and to demonstrate the presence of LT in rejecting rat renal allografts. Rat LT, generated in vitro by stimulating spleen cells from specifically sensitized rats with keyhole limpet hemocyanin (100 micrograms m/ml), was shown on gel filtration to have an MW of approximately 50,000. In-vitro-generated rat LT was shown to be heat stable (70 degrees C for 15 min) and soluble in 40% (NH4)2SO4. Rat LT eluted as a single peak on DEAE anion exchange chromatography (0-0.15 M, NaCl osmotic gradient), supporting the existence of but a single molecular form. LT was isolated from rejecting renal allografts on day 6 after renal transplant but undetected (less than 1 unit) in residual native kidneys. This study, therefore, provides substantial support for a link between DTH and renal allograft rejection. Lymphotoxin, one of the potential mediators of tissue injury in this model system, has been partially characterized and demonstrated to be present in rejecting rat renal allografts.