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Long-term survival of parathyroid allografts after preoperative treatment of recipients with cyclosporine A
Abstracts
175 nificant n u m b e r of potential graft recipients may develop such unresponsiveness prior to transplantation.
DISTINCTIVE T CELLS IN BONE MARROW TRANSPLANT RECIPIENTS: EXPRESSION OF HLA-DQ MOLECULES ON A SUBPOPULATION OF T LYMPHOCYTES. Howard M. Gebel, Herbert Kaizer, and Alan L. Landay; Rush Medical Center. Chicago, IL It is well known that bone marrow transplant recipients have a severely depressed immune system up to 1 yr posttransplant. The mechanism behind this immunodeficiency is not established. Since class II molecules can play such an important role in both the inductive and effector phases of an immune response, we followed the expression of both H L A - D R and H L A - D Q expression on T lymphocytes from bone marrow transplant (BMT) recipients. Peripheral blood mononuclear cells were obtained from 18 B M T patients 1 month posttransplant. We determined that 57%-+ 8 % o f T lymphocytes from the patients expressed H L A D R molecules (normal range: 2 - 3 % ) . In contrast, only 10% _+2 % of the T cells also expressed H L A - D Q molecules (normal range: 1 - 2 % ) . Further analysis of these T cells revealed that < 1% of the cells expressed receptors for interleukin2 (IL-2). If T cells from B M T patients were stimulated with either concanavalin A (Con A) or phytohemagglutinin (PHA), < 3 % of the stimulated cells expressed either receptors for IL-2 or H L A - D Q molecules. In contrast, Con A and P H A stimulation of lymphocytes from normal subjects resulted in expression of IL-2 receptors on 7 0 % of the T cells. A m o n g T lymphocytes from normal donors, Con A did not induce expression of H L A - D Q molecules; P H A stimulation induced 3 5 % of normal T lymphocytes to express H L A - D Q . Interestingly, when a mitogenic dose (200 ng/ml) of the monoclonal antibody O K T 3 was used to stimulate T cells from either normal subjects or B M T recipients, there was a dramatic difference. In normal subjects, O K T 3 induced 7 4 % of the T cells to express IL-2 receptors; none of these cells expressed H L A - D Q . In contrast, after stimulation of T cells from B M T recipients with O K T 3 , 35% of the T cells expressed receptors for IL-2 and 3 7 % of the cells expressed H L A - D Q . It is apparent that T cells from normals and B M T patients respond differently to various mitogens. These differential responses may provide clues to the mechanisms behind the protracted immunodeficiency that occurs in B M T recipients. (Supported in part by a grant from the Leukemia Foundation.)
LONG-TERM SURVIVAL OF PARATHYROID ALLOGRAFTS AFTER PREOPERATIVE TREATMENT OF RECIPIENTS WITH CYCLOSPOR1NE A. A.D. Bloom, Steven G. Economou, and H.M. Gebel; Rush Medical Center, Chicago, IL Short-term, preoperative cyclosporine A (CyA) therapy may p r o m o t e long-term graft survival in animals. In this investigation, we transplanted allogenic rat parathyroid glands into untreated recipients or recipients that were treated prior to transplant with a 3-day course of CyA Lewis x Brown Norway (RTI~'") rats were used as donors and Wistar Furth (RT1 u) rats were used as recipients. Parathyroidectomized recipients were fed a calcium-deficient diet immediately postparathyroidectomy to expedite hypocalcemia. Hypocalcemia (Ca < 6 . 0 mg/dl) was d o c u m e n t e d in all recipients by a serum calcium determination. A successful graft was defined as a calcium value < 7 . 0 mg/dl; rejection occurred when the graft first met the criteria for function and then dropped below 7.0 mg/dl. CyA was diluted with castor oil (20 mg/ml) and administered at a dose of 30 mg/kg subcutaneously once a day for 3 days before transplantation. Both parathyroid glands from a single donor were transplanted into the hamstring muscle of one
176
Annual AACHT Meeting, t985 recipient. Recipients receiving no prior treatment (n = 7) all rejected their grafts by day 16. In CyA-treated recipients (n = 7), all grafts were functional at day 42 and one graft still functioned at day 70. Prolongation of allograft survival with high dose preoperative CyA demonstrates the efficacy of immunosuppressive drugs given only at the time o f initial antigen presentation. Both in vitro and il, vivo studies indicate that CyA must be present at the initiation o f the antigen signal to interrupt the rejection process. We propose that pregraft treatment is sufficient to promote long-term survival without continuous immunosuppressiv¢therapy. Presently, long-term survival of histoincompatible allografts in humans is dependent upon lifetime immunosuppression. The hazards of such therapy are well documented. An obvious goal of transplant surgeons is to perform allotran-splants with little, if any, immunosuppression. This study demonstrates that shortterm treatment with a potent immunosuppressive reagent can prolong graft survival. Future studies will address the dose and kinetics of the preoperative CyA therapeutic regimen.
FUNCTIONAL LYMPHOCYTE SUBSETS INFILTRATING RENAL ALLOGRAFTS. James T. Kurnick, Lenora A. Boyle, Frederic I. Preffer, Carol P. Leary, and Robert C. Colvin: Department of Pathology, Massachusetts General Hospital. Boston. MA In order to dissect the repertoire of lymphocytes that infiltrate human renal allografts, we have developed an in vitro culture system for propagation of activated lymphocytes present within needle biopsy fragments of atlografts undergoing episodes of relection. As previously described (J Immunol 134:258, 19857. the culture o f biopsy fragments in interleukin-2 (IL-2) results in the proliferation of activated lymphocytes which exude from the cut surface o f the tissue. The cells thus cultured can be shown to react to donor antigens in both mixed lymphocyte culture and in celt mediated lympholysis. We have now sorted subsets o f functional lymphocytes and cloned individual cell progeny in order to discern the diversity o f cells which infiltrate the rejecting allograft tissue. Double stain analyses demonstrate that both Leu 2 + (mostly Leu 15 ) and Leu 3 + (mostly Leu 8 - ) cells are present in the graft, and a discrete population of Leu 2 + / Leu 3 + cells can also be isolated. Sorted subsets of Leu 2 +/3 - cells both killed and proliferated in response to donor antigens, while Leu 3 + / 2 - cells prolit~ erated but did not lyse donor lymphocyte targets. The Leu 2 +/3 + subset neither killed nor responded in MLR. The Leu 2 +/3 -~ cells did not suppress the MLR reactivity o f the other subsets. All three subsets could produce IL-2 in response to PHA. The Leu 2 + and Leu 3 ~- cells produced interferon, while the Leu 2 + / 3 0- did not. O f 63 clones from one allograft, 21 were Leu 2 +/Leu 3 and 42 were Leu 3 + / L e u 2 . N o Leu 2 + / 3 - clones were isolated from limiting dilution of unsorted tymphocytes, although Leu 2 +/3 + clones were obtained from single cell dilutions o f presorted lymphocytes. Most o f the Leu 2 ~- clones were donor specific, and could both kill and respond in MLR. Some Leu 2-~ clones were cytotoxic to both donor lymphocytes and K562 targets, while others killed only the donor. Two Leu 2 + clones killed K562 but not donor targets. Only about 30% o f the Leu 3 + clones were donor specific. Most o f the specific clones proliferated in MLR, but were not cytotoxic. A few Leu 3 + clones were also cytolytic. About 3 0 % of the Leu 3 + cells that were not donor-specific lysed K562 targets. N o known function has been noted for over 30% of the Leu 3 -~ clones and all o f the Leu 2 +/3 + clones. Further phenotypic and functional analyses are proceeding to determine the fine specificity o f donor reactive and nonreactive clones. Possible regulatory interactions between specific and donornonspecific lymphocytes will be investigated.
175 nificant n u m b e r of potential graft recipients may develop such unresponsiveness prior to transplantation.
DISTINCTIVE T CELLS IN BONE MARROW TRANSPLANT RECIPIENTS: EXPRESSION OF HLA-DQ MOLECULES ON A SUBPOPULATION OF T LYMPHOCYTES. Howard M. Gebel, Herbert Kaizer, and Alan L. Landay; Rush Medical Center. Chicago, IL It is well known that bone marrow transplant recipients have a severely depressed immune system up to 1 yr posttransplant. The mechanism behind this immunodeficiency is not established. Since class II molecules can play such an important role in both the inductive and effector phases of an immune response, we followed the expression of both H L A - D R and H L A - D Q expression on T lymphocytes from bone marrow transplant (BMT) recipients. Peripheral blood mononuclear cells were obtained from 18 B M T patients 1 month posttransplant. We determined that 57%-+ 8 % o f T lymphocytes from the patients expressed H L A D R molecules (normal range: 2 - 3 % ) . In contrast, only 10% _+2 % of the T cells also expressed H L A - D Q molecules (normal range: 1 - 2 % ) . Further analysis of these T cells revealed that < 1% of the cells expressed receptors for interleukin2 (IL-2). If T cells from B M T patients were stimulated with either concanavalin A (Con A) or phytohemagglutinin (PHA), < 3 % of the stimulated cells expressed either receptors for IL-2 or H L A - D Q molecules. In contrast, Con A and P H A stimulation of lymphocytes from normal subjects resulted in expression of IL-2 receptors on 7 0 % of the T cells. A m o n g T lymphocytes from normal donors, Con A did not induce expression of H L A - D Q molecules; P H A stimulation induced 3 5 % of normal T lymphocytes to express H L A - D Q . Interestingly, when a mitogenic dose (200 ng/ml) of the monoclonal antibody O K T 3 was used to stimulate T cells from either normal subjects or B M T recipients, there was a dramatic difference. In normal subjects, O K T 3 induced 7 4 % of the T cells to express IL-2 receptors; none of these cells expressed H L A - D Q . In contrast, after stimulation of T cells from B M T recipients with O K T 3 , 35% of the T cells expressed receptors for IL-2 and 3 7 % of the cells expressed H L A - D Q . It is apparent that T cells from normals and B M T patients respond differently to various mitogens. These differential responses may provide clues to the mechanisms behind the protracted immunodeficiency that occurs in B M T recipients. (Supported in part by a grant from the Leukemia Foundation.)
LONG-TERM SURVIVAL OF PARATHYROID ALLOGRAFTS AFTER PREOPERATIVE TREATMENT OF RECIPIENTS WITH CYCLOSPOR1NE A. A.D. Bloom, Steven G. Economou, and H.M. Gebel; Rush Medical Center, Chicago, IL Short-term, preoperative cyclosporine A (CyA) therapy may p r o m o t e long-term graft survival in animals. In this investigation, we transplanted allogenic rat parathyroid glands into untreated recipients or recipients that were treated prior to transplant with a 3-day course of CyA Lewis x Brown Norway (RTI~'") rats were used as donors and Wistar Furth (RT1 u) rats were used as recipients. Parathyroidectomized recipients were fed a calcium-deficient diet immediately postparathyroidectomy to expedite hypocalcemia. Hypocalcemia (Ca < 6 . 0 mg/dl) was d o c u m e n t e d in all recipients by a serum calcium determination. A successful graft was defined as a calcium value < 7 . 0 mg/dl; rejection occurred when the graft first met the criteria for function and then dropped below 7.0 mg/dl. CyA was diluted with castor oil (20 mg/ml) and administered at a dose of 30 mg/kg subcutaneously once a day for 3 days before transplantation. Both parathyroid glands from a single donor were transplanted into the hamstring muscle of one
176
Annual AACHT Meeting, t985 recipient. Recipients receiving no prior treatment (n = 7) all rejected their grafts by day 16. In CyA-treated recipients (n = 7), all grafts were functional at day 42 and one graft still functioned at day 70. Prolongation of allograft survival with high dose preoperative CyA demonstrates the efficacy of immunosuppressive drugs given only at the time o f initial antigen presentation. Both in vitro and il, vivo studies indicate that CyA must be present at the initiation o f the antigen signal to interrupt the rejection process. We propose that pregraft treatment is sufficient to promote long-term survival without continuous immunosuppressiv¢therapy. Presently, long-term survival of histoincompatible allografts in humans is dependent upon lifetime immunosuppression. The hazards of such therapy are well documented. An obvious goal of transplant surgeons is to perform allotran-splants with little, if any, immunosuppression. This study demonstrates that shortterm treatment with a potent immunosuppressive reagent can prolong graft survival. Future studies will address the dose and kinetics of the preoperative CyA therapeutic regimen.
FUNCTIONAL LYMPHOCYTE SUBSETS INFILTRATING RENAL ALLOGRAFTS. James T. Kurnick, Lenora A. Boyle, Frederic I. Preffer, Carol P. Leary, and Robert C. Colvin: Department of Pathology, Massachusetts General Hospital. Boston. MA In order to dissect the repertoire of lymphocytes that infiltrate human renal allografts, we have developed an in vitro culture system for propagation of activated lymphocytes present within needle biopsy fragments of atlografts undergoing episodes of relection. As previously described (J Immunol 134:258, 19857. the culture o f biopsy fragments in interleukin-2 (IL-2) results in the proliferation of activated lymphocytes which exude from the cut surface o f the tissue. The cells thus cultured can be shown to react to donor antigens in both mixed lymphocyte culture and in celt mediated lympholysis. We have now sorted subsets o f functional lymphocytes and cloned individual cell progeny in order to discern the diversity o f cells which infiltrate the rejecting allograft tissue. Double stain analyses demonstrate that both Leu 2 + (mostly Leu 15 ) and Leu 3 + (mostly Leu 8 - ) cells are present in the graft, and a discrete population of Leu 2 + / Leu 3 + cells can also be isolated. Sorted subsets of Leu 2 +/3 - cells both killed and proliferated in response to donor antigens, while Leu 3 + / 2 - cells prolit~ erated but did not lyse donor lymphocyte targets. The Leu 2 +/3 + subset neither killed nor responded in MLR. The Leu 2 +/3 -~ cells did not suppress the MLR reactivity o f the other subsets. All three subsets could produce IL-2 in response to PHA. The Leu 2 + and Leu 3 ~- cells produced interferon, while the Leu 2 + / 3 0- did not. O f 63 clones from one allograft, 21 were Leu 2 +/Leu 3 and 42 were Leu 3 + / L e u 2 . N o Leu 2 + / 3 - clones were isolated from limiting dilution of unsorted tymphocytes, although Leu 2 +/3 + clones were obtained from single cell dilutions o f presorted lymphocytes. Most o f the Leu 2 ~- clones were donor specific, and could both kill and respond in MLR. Some Leu 2-~ clones were cytotoxic to both donor lymphocytes and K562 targets, while others killed only the donor. Two Leu 2 + clones killed K562 but not donor targets. Only about 30% o f the Leu 3 + clones were donor specific. Most o f the specific clones proliferated in MLR, but were not cytotoxic. A few Leu 3 + clones were also cytolytic. About 3 0 % of the Leu 3 + cells that were not donor-specific lysed K562 targets. N o known function has been noted for over 30% of the Leu 3 -~ clones and all o f the Leu 2 +/3 + clones. Further phenotypic and functional analyses are proceeding to determine the fine specificity o f donor reactive and nonreactive clones. Possible regulatory interactions between specific and donornonspecific lymphocytes will be investigated.