The in vitro immunosuppressive effect of deoxymethylspergualin in man as compared with FK506 and cyclosporine.

The effect of deoxymethylspergualin (MeDSG) on in vitro human lymphocyte response was assessed in comparison with FK506 and cyclosporine. Peripheral blood mononuclear cells from normal human volunteers were used for assay of mixed lymphocyte reaction, cell mediated lympholysis, and blastogenesis by PHA, IL-2, and OKT3. MeDSG suppressed only allogeneic stimulation (MLR and CML) and IL-2-induced blastogenesis, not PHA- or OKT3-induced blastogenesis, although the other immunosuppressive agents showed some suppressive effect for all assays. A kinetic study of MLR showed that the suppressive activity did not decrease even when MeDSG was added at day 3 or day 4. The other agents, however, showed a weak suppressive effect when added at a later phase of MLR.     

Mechanism of suppression of cloned human suppressor T cells

Abstract We report the mechanism of suppression of suppressor T cell clone III-1-C5 using helper T cell clone III-1-B6, mitogen responses and rIL-2. Clone III-1-C5 suppressed the mixed lymphocyte reaction (MLR) by secreting alloantigen non-pecific, MHC non-restricted suppressor factor(s). Clone III-1-C5 did not suppress mitogen (PHA, Con A, PWM) response nor proliferation by exogeneous rIL-2. Clone III-1-C5 suppressed proliferation by clone III-1-B6, which augments proliferation by direct cell to cell contact with responder cells and not by soluble factors. These results indicated that suppressor T cells exhibit suppressive effects not only by inhibiting IL-2 synthesis but by inhibiting the direct effects of helper T-cells.     

Induction of suppressor T cells by donor-specific blood transfusion (DST): establishment of a human T cell hybridoma producing an MLR suppressing factor]

We established a human T cell hybridoma producing a mixed lymphocyte reaction (MLR) suppressing factor. Three weeks after DST, peripheral blood lymphocytes (PBL) were obtained from a recipient and were cultured for 3 days with mitomycin C (MMC)-treated donor PBL. These lymphocytes were fused with an azaguanine-resistant mutant of a human T cell leukemic cell line (CCRF-CEMAG). Four weeks after fusion, approximately 30% of the wells showed hybridoma cell growth. To select hybridoma clones with suppressive activity, irradiated hybridoma clones were added as regulator cells to the mixed lymphocyte culture. After the cloning, one clone causing suppression of the donor-specific MLR was established (termed HK40: %MLR suppression = 38.9%). Unstimulated supernatant of HK40 showed no suppressive effect on the specific MLR. In contrast, supernatant of HK40 cultured with donor PBL for 24 hrs, suppressed the donor-specific MLR dose-dependently. This primed supernatant of HK40 markedly suppressed the specific MLR when added added at the culture initiation. These findings indicate that functional clones causing suppression of the alloantigen-specific MLR can be generated in patients receiving DST, and suggest that these clones may be essential to the prolongation of kidney allograft survival.     

Donor bone marrow-derived chimeric cells present in renal transplant recipients infused with donor marrow. I. Potent regulators of recipient antidonor immune responses

BACKGROUND: Even though a number of transplant centers have adopted donor-specific bone marrow cell (DBMC) infusions to enhance donor cell chimerism, to date there has been no direct evidence linking chimerism with tolerance induction in human organ transplant recipients. METHODS: Cells of donor phenotype were isolated 1 year postoperatively from the peripheral blood lymphocytes and iliac crest bone marrow of 11 living-related-donor (LRD) renal transplant recipients, who had received perioperative donor bone marrow cell infusions. These recipient-derived donor (RdD) cells were characterized phenotypically by flow cytometric analysis and functionally as modulators in mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. RESULTS: The yield of RdD cells ranged from 0.1 to O.9% of the starting material with the majority being TcRalphabeta, CD3 positive T cells, a substantial percentage of which coexpressed CD28. At 1 year posttransplant almost 50% of the LRD-kidney/DBMC recipients tested so far exhibited donor-specific unresponsiveness in MLR (7/17) and CML (6/13) reactions and this trend was further enhanced at 23 years. In the recipients with residual positive antidonor immune responses, the RdD cells inhibited recipient antidonor MLR and CML responses significantly more strongly than freshly isolated and similarly treated iliac crest bone marrow cells from the donor. RdD cells also inhibited the MLR of the recipient to third party allogeneic stimulator cells; however, this nonspecific effect was significantly weaker than specific inhibition. We also established long-term bone marrow cultures stimulated every 2 weeks with irradiated alogeneic feeder cells, that had similar functional properties thus possibly providing us with an in vitro correlate the RdD cells. CONCLUSIONS: These results clearly support the notion that the infused donor cells play a positive role in the induction and/or maintenance of transplant tolerance.     

Transplantation tolerance mediated by suppressor T cells and suppressive antibody in a recipient of a renal transplant

This is a report of a patient who underwent cadaveric renal transplantation in spite of the presence of three HLA-A, B and two DR antigen mismatches between the recipient and donor. The recipient received more than 20 units of blood before transplantation. The crossmatch between the recipient's serum and the T and B cells of the donor was negative. The patient exhibited hepatic dysfunction from the early posttransplant period, which eventually led to discontinuation of azathioprine or Bredinin at one year posttransplantation. Thereafter, only betamethasone was administered once every 3 days. The patients has maintained good renal function for more than one year following withdrawal of the immunosuppressants. It appeared that transplantation tolerance was established in this patient. Therefore, we examined the mechanisms sustaining the tolerance. Both nylon-wool-adherent, alloantigen-specific suppressor T cells and nonadherent, nonspecific suppressor T cells were observed in the lymphocytes of the patient after transplantation. It was also shown that suppressive antibody was present in the serum directed toward the clone of autologous lymphocytes that reacted with the mixed lymphocyte reaction (MLR) antigen of the donor. In the inhibition test against various types of MLR antigens using this suppressive antibody, it was found that the reaction against the donor cells was suppressed when the responding cells shared the same class I antigen with the recipient. When the stimulating cells had the class II antigen of the donor, the reaction of the specific responding cells was also inhibited. These inhibiting effects were only seen when the responding cells were pretreated with the antibody, but not when stimulating cells were pretreated.     

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.     

Effects of interleukin 2 receptor b chain (P75)-specific monoclonal antibody on the generation of cytotoxic T lymphocytes and suppressor T cells in mixid lymphocyte culture

The interaction of interleukin 2 (IL-2) with its receptor (IL-2R) plays an essential role in the proliferation and differentiation of T cells. The IL-2R β-chain is considered to function directly in the intracellular signal transduction. In this study, we investigated using a newly established IL-2R β-chain-specific monoclonal antibody (MAb) (TU-25) and an IL-2R α-chain-specific MAb (H-31). The IL-2-induced proliferation of concanavalin blasts and the mixed lymphocyte reaction (MLR) were suppressed by TU-25 in combination with H-31. This combination had a greater suppressive effect than each of them alone. The generation of cytotoxic T lymphocytes (CTL) using a cell-mediated lympholysis (CML) assay, was not inhibited by TU-25 alone. TU-25 in combination with H-31 suppressed the generation of CTL completely in this assay even if recombinant IL-2 (rIL-2) was added. Although the CTL generation was inhibited, cells that suppressed a fresh MLR were preserved. Our study suggests that the combination of TU-25 with H-31 completely blocks the functional high-affinity binding site of IL-2 but does not inhibit the generation of suppressor cells. This may lead to immunosuppressive therapy using an IL-2R 13-chain-specific MAb in combination with an IL-2R α-chain-specific MAb in clinical organ transplantation.     

Nature of the suppressor cells mediating prolonged graft survival after administration of extracted histocompatibility antigen and cyclosporine

Antigen-specific suppressor T cells are induced by donor histocompatibility antigen extracted from spleen cells with 3M KCl combined with cyclosporine (Ag-CsA). A single i.v. injection of 5 mg 3M-KCl-extracted donor Buffalo (Buf, RT1b) antigen (Ag) combined with a three day course of CsA prolonged renal allograft survival in Wistar-Furth (WFu, RT1u) hosts to a greater extent (MST 26.5 days) than CsA alone (MST 11.8 days). Peripheral blood lymphocytes (PBL) or spleen cells harvested from Ag-CsA-treated recipients ten days after transplantation inhibited the mixed lymphocyte reaction (MLR) between normal responder WFu cells and irradiated Buf cells (55.6% and 64.4% suppression, respectively, P less than 0.025), but not third-party Brown-Norway (BN, RT1n) stimulator cells (13.6% and -18.3% suppression, respectively, NS). The suppressor effect was not mediated by cytolytic cells; there was neither primary nor secondary cytolytic activity against 51Cr-labeled Con-A blastoid Buf cells. The suppressor cells were neither adherent to plastic dishes nor to nylon-wool columns. PBL irradiated with 800 rads, but not 1500 rads, suppressed the MLR. A single injection of cyclophosphamide (CY, 25 mg/kg) seven days after transplantation abrogated the suppression induced by Ag-CsA treatment. Moreover, PBL from Ag-CsA recipients failed to suppress the MLR, if depleted either of all T cells by treatment with monoclonal antibody (Mab) W3/13 HLK (pan T cells; % suppression -15.8), or of cytotoxic/suppressor cells with Mab OX-8 (-19.3% suppression) together with rabbit antimouse immunoglobulin and complement. On the other hand, PBL treated with the Mab W3/25 (helper) showed suppressor cell activity (+56.4%, P less than 0.001) similar to untreated cells (62.4%, P less than 0.001). Moreover, adoptive transfer of suppressor T cells purified from pooled lymphocytes by rosetting using Mab significantly prolonged the survival of donor-specific, but not third-party, test grafts in naive secondary hosts. Thus, these studies demonstrated antigen-specific suppressor T cells mediate the long-term unresponsiveness induced by the Ag-CsA regimen.     

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34(+) stem cells and the CD34(-) early progeny like CD38(+), CD2(+), CD5(+) and CD1(+) lymphoid cells as well as CD33(+) (but CD15(-)) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell-cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34(+) cells were found to give rise both to CD3(-) TCRalphabeta(+), as well as CD3(+) TCRalphabeta(+) cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.     

Immunological analysis of mixed lymphocyte culture reaction inhibitory factor induced by donor specific blood transfusions in potential kidney transplant patients

There is now overwhelming evidence that preoperative donor specific blood transfusions (DST) improve kidney graft survival remarkably. However, the exact mechanism is as yet unknown. In this study, we compared a DST group with a non-DST and investigate the mechanism of the beneficial effect of DST by using MLR, CML, and the MLR inhibition test. In the DST group, the severity of acute rejection and MLR reactivity was decreased after DST in most cases, but there was no such tendency in CML reaction. In the MLR inhibition test, the MLR serum inhibitory factor was induced in 70% of the cases (7 out of 10) received 3 times of DST. All of them were directed against recipient cell (auto-lymphocyte) and not to the blood donor cell and its activity was found in IgG fraction. The specificity to the stimulator cell was also found in 43% (3 out of 7). The results of MLR inhibition tests were well correlated with clinical findings and changes of MLR and CML. In positive cases of MLR inhibition tests, acute rejection occurred in 14% (1 out of 7), CML decreased in all (3 out of 3) and MLR decreased in 83% (5 out of 6) after DST. On the contrary, in negative cases, acute rejection occurred in 67% (2 out of 3), CML increased in all (3 out of 3) and MLR increased in 67% (2 out of 3). These results suggested that antibodies directed against recognition sites on auto-lymphocytes, so called anti-idiotypic antibodies, will be induced by 3 DST and play an important role in the prolongation of graft survival.     

Kidney transplantation after liver transplantation from the same donor: four cases of successful steroid withdrawal

BACKGROUND: Administration of corticosteroids to kidney recipients has hampered the complete clinical success of kidney transplantation. Because most organ transplantation in Japan is living-related, we had the experience of performing kidney transplantation (KT) after liver transplantation (LT) from the same donor in four patients and successfully withdrew corticosteroid administration. METHODS: Three pediatric and one adult patient received kidney allografts from 3 to 10 months after LT from the same donor. The immunosuppressive regimen consisted of a corticosteroid and tacrolimus. The steroid was withdrawn after KT in all four patients. After complete withdrawal of the steroid, DNA was extracted from two recipients and examined by polymerase chain reaction to detect microchimerism. A mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity assay (CML) were performed to test for donor-specific hyporesponsiveness. RESULTS: Steroid withdrawal was successfully accomplished after KT in every patient. No steroid-withdrawal-associated complications were observed. In the three pediatric patients, remarkable catch-up growth was observed after steroid withdrawal. In the two patients tested, donor DNA was not detected by polymerase chain reaction, suggesting the absence of microchimerism. MLR and CML showed that recipient lymphocytes reacted against donor lymphocytes at the same level as against the third-party lymphocytes. CONCLUSION: Steroid withdrawal was successfully achieved in four kidney recipients who had received a liver allograft from the same donor. The MLR and CML findings indicated the absence of donor-specific hyporesponsiveness in vitro. Although the precise mechanism is not yet clear, KT after LT from the same donor should be considered as a method that allows steroids to be withdrawn from the immunosuppressive regimen of KT.     

The Role of Regulatory Cells in Miniature Swine Rendered Tolerant to Cardiac Allografts by Donor Kidney Cotransplantation

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25⁺ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25⁺ cells or PBL depleted of CD25⁺ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25⁺ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25⁺ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.     

The effects of perioperative administration of donor lymphocytes via portal vein in rat skin transplant model]

The effects of perioperative portal venous (P.V.) administration of donor lymphocytes on skin allograft survival were investigated in rat skin transplant model. Heterotopic skin transplantations were performed form Brown-Norway (BN, RT-1n) to Lewis (LEW, RT-1(1] male rats. P.V. administration of donor BN lymphocytes (1 x 10(8] resulted in significant prolongation of BN skin graft survival (MST = 13.4 +/- 3.9 days, p less than 0.05) compared with I.V. administration of same number of donor lymphocytes (8.6 +/- 1.2 days) or with PV administration of third party DA (RT-1a) rat's lymphocytes (7.4 +/- 0.8 days) or with untreated controls (9.0 +/- 1.4 days). These results suggested that this effect was antigen specific. P.V. administration of donor lymphocytes prevented recipient which received BN skin graft form developing delayed-type hypersensitivity responses to donor antigen. Serum from LEW recipients which induced unresponsiveness by PV administration with donor BN lymphocytes had significant antigen specific suppressor effect (77.0 +/- 5%) on the MLR proliferative reaction of LEW responder cells toward donor BN cells, but not third party DA stimulation. Moreover, this immunological unresponsiveness was transferable by the serum in kidney transplant model. These results indicate that PV administration of donor lymphocytes induces recipient's unresponsiveness to donor alloantigen in rat skin transplant model, and this effect is transferable by the suppressor factor in the serum.     

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. II. Activity and specificity of a soluble suppressor factor

Human suppressor T cells induced by autologous mixed lymphocyte reaction (AMLR) using fresh responder PBL from a renal transplant recipient and an autologous irradiated antidonor CTL line (EE-1) established from a biopsy of the patient's own allograft were studied for the production of suppressor factors. The suppressor cell lines propagated (designated TsEE) were capable of inhibiting the in vitro generation of proliferative and cytotoxic responses of responder cells from the recipient or other individuals who shared HLA-B7 with TsEE cells, regardless of the stimulatory cell phenotype. Coculture of TsEE cells with the autologous irradiated EE-1 inducer cell line in vitro yielded a soluble factor (designated TsEEF) capable of inhibiting the generation of MLR and CTL responses, as well as mitogen-induced proliferative responses to PHA and PWM in an HLA-unrestricted manner. TsEEF also inhibited the replication of lymphoblastoid T cell lines (Molt-4 and HSB) but not B cell lines (SB and JC-EBV) or PBL stimulated with the B cell mitogen LPS. Control supernatants obtained from each of the cells used to generate TsEE in AMLR (i.e., EE-PBL and the EE-1 line) cultured alone or together for 48 hr demonstrated no suppressive activity in any of these test systems. TsEEF was nontoxic for lymphoid cells, was nondialyzable (greater than 12kDa), did not act by interfering with IL-1 or IL-2 utilization, and was negative for TNF and IFN-gamma activity. Functionally, the suppressive activity of TsEEF was dose-dependent, did not shift MLR kinetics, and could be absorbed by T cells. T cells incubated with TsEEF for 4 hr were unresponsive to subsequent mitogen or MLR stimulation. These findings indicate that, whereas T suppressor cell lines propagated from the circulation of a stable renal transplant recipient demonstrate class I HLA restriction, the activity of their soluble products is not HLA-restricted, and functionally inhibits T cell proliferation.     

Expression of OKT 8 antigen and Fc gamma receptors by suppressor cells mediating specific unresponsiveness between recipient and donor in renal-allograft-tolerant patients

Renal allograft tolerant patients show specific unresponsiveness in mixed-lymphocyte culture assays when confronted with donor stimulating cells. Separation of posttransplant peripheral blood lymphocytes into Fc gamma + and Fc gamma - by rosetting and into OKT8+, OKT8- by cytofluorometry enabled the demonstration of normal responses by Fc gamma - and OKT8- cells, whereas the proliferation of OKT8+ and Fc gamma + cells was depressed specifically in the presence of donor cells. Using mixing experiments, we showed that OKT8+ and Fc gamma + posttransplant lymphocytes exert a suppressive effect specific to the donor-recipient pair on the proliferative response of the pretransplant lymphocytes.     

Macrophage-related suppressor cells in human renal transplant recipients.

In this study 66 renal transplant recipients were studied by in vitro immunologic monitoring assays in an attempt to delineate some immune reactivity parameters which migh explain the successful long-term survival of HLA incompatible allografts. Ninety percent of the recipients were found to be responsive to their specific donors in one-way mixed lymphocyte culture assays, indicating the presence of antigen reactive proliferating T cells. In contrast, studies of the in vitro generation of cytotoxic T cells showed that 73% of the long-term recipients demonstrated a marked hyporesponsiveness to the donor in cell-mediated lympholysis (CML) assays. Longitudinal studies indicated that specific CML hyporesponsiveness to the donor developed after transplantion. In the majority of recipients, CML hyporesponsiveness could be related to a suppressor cell phenomenon, since recipient mononuculear cells specifically suppressed the CML reactivity of third-party cells to the donor. Recipient CML hyporesponsiveness to the donor could be abrogated by removal of recipient adherent cells, most of which were monocytes or macrophages. The suppressor cell effect on CML is postulated to be related to macrophages which are either acting independently or under the regulation of suppressor T cells. These studies are among the first to suggest that suppressor cell regulation of relevant effector T cell activity may be important in the facilitation of successful human renal transplantation.     

Long-term survival of heart grafts in the presence of donor-pecific cytotoxic T-cell precursors (CTLp) in the peripheral blood

Abstract To monitor their immunological status we determined donor and third-party-pecific cytotoxic T-cell precursor frequencies (CTLpf) in the peripheral blood of 15 heart transplant recipients. PBL samples were obtained at different time points before and after transplantation. Donor-pecific CTLpf and third-party-pecific CTLpf were within the same range for all samples (1–1489/106 cells). The donor-pecific CTLpf were not different between patients who had never had an acute rejection (AR) and patients who had an acute rejection as diagnosed by endomyocardial biopsy (EMB). No difference was' observed between donor-pecific CTLpf of samples taken on the day of transplantation and those obtained between 3 months and 3 years after transplantation. There was also no relationship between the donor-pecific CTLpf in the PBL and the culturing success of lymphocytes from EMB taken at the same time. CTLpf were in the same range both when cultures could be propagated from the graft and when no cells grew out. We conclude that long-term graft survival is possible in the presence of CTLpf in peripheral blood.     

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.     

Thymectomy impairs but does not uniformly abrogate long-term acceptance of semi-identical liver allograft in inbred miniature Swine temporarily treated with FK506

BACKGROUND: Long-term acceptance of semi-identical orthotopic liver transplants (OLTs) in inbred swine is induced by a 12-day course of FK506. To study whether acceptance is attributable to central or peripheral immune mechanisms, the effect of complete thymectomy was determined. METHODS: Total thymectomy was performed in 15 swine 3 to 4 weeks before OLT. Twelve of these animals received a 12-day course of FK506 after OLT, and three animals did not receive immunosuppression. Five additional nonthymectomized pigs received OLT and a FK506 regimen. Graft survival, liver function, histology, and cellular and humoral responses were assessed. RESULTS: Nonthymectomized, FK506-treated animals uniformly showed long-term acceptance of OLT and developed stable donor unresponsiveness. Of the 12 thymectomized, FK506-treated pigs, seven died of non-immunologic causes within 3 postoperative months, and five maintained their OLT for more than 6 months (range 180-450 days). Among these survivors, two developed a complete anti-donor response (mixed lymphocyte reaction [MLR], cell-mediated lymphocytotoxicity [CML], and immunoglobulin [IgG] antibodies) and eventually rejected their OLT at postoperative day 180. The three remaining pigs kept their liver allografts up to 450 days and developed a donor-specific unresponsiveness (a transient anti-donor MLR was observed during the follow-up but never an anti-donor CML or IgG antibodies). All three thymectomized, untreated animals rejected their allografts acutely and displayed a complete anti-donor response (MLR, CML, and IgG antibodies). CONCLUSIONS: Complete thymectomy before OLT impaired but did not uniformly abrogate long-term acceptance of semi-identical OLT, suggesting that peripheral immune mechanisms may be sufficient to induce long-term acceptance of liver allografts in some recipients.     

Effect of blood transfusion on IL-2 production

Spleen cells from B10.A mice transfused with B10.D2 blood suppress the immune responses of normal B10.A to B10.D2 in coculture as early as 2 days posttransfusion. In addition, the ability of B10.A mice to respond in cell-mediated lymphocytotoxicity (CML) is significantly impaired as early as 2 days after B10.D2 transfusion. Experiments were performed to characterize the cells mediating the suppressive effect and to determine whether the inability of transfused mice to generate a cytotoxic response is due to an inhibition of IL-2 production. To characterize the suppressor cells, spleen cells from B10.A mice were assayed 2 or 16 days after B10.D2 transfusion for the ability to suppress mixed lymphocyte culture (MLC) and CML responses of normal B10.A mice in coculture. The putative suppressor cells were either passed over a Sephadex G-10 or nylon wool column, treated with anti-Thy antibody or left untreated before addition to the coculture. Untreated cells from transfused mice suppressed the CML response of normal B10.A both 2 and 16 days posttransfusion, while the effect on the MLC response was inconsistent. Passage of the cells over Sephadex G-10 or nylon wool before assaying abrogated the suppressive effect, while treatment with anti-Thy antibody had no effect. These results suggest that the suppressor cells appearing shortly after blood transfusion have the characteristics of macrophages and not T lymphocytes. To determine the effect of transfusion on IL-2 production, cells from transfused mice were assayed for their ability to produce IL-1 and IL-2 and for the formation of IL-2 receptors. In addition, the effect of exogenous IL-1 and IL-2 on restoring the CML response of transfused mice to normal was assayed. The production of IL-1 by transfused mice was normal, while the production of IL-2 was significantly suppressed both 2 and 16 days posttransfusion. Activated cells from normal and transfused mice showed equal ability to absorb IL-2, indicating that IL-2 receptor formation is normal after transfusion. The addition of exogenous IL-2, but not IL-1, to CML cultures containing cells from transfused mice as responders restored the response to normal. These results indicate that the inability of transfused mice to respond in CML is due, at least in part, to an inability to produce IL-2. This could be mediated by prostaglandins released by activated macrophages.