Host-graft relationship: the systemic nature of allograft rejection

The interdependence between immunologic events occurring within acutely rejecting rat cardiac allografts and those in host lymphoid tissues were studied. To evaluate cellular dynamics of allograft infiltration, 111In-labeled thoracic duct lymphocytes from Lewis rats were administered intravenously daily (0 to 7 days after transplantation) to (Lewis X BN)F1 heart-grafted unmodified Lewis rats, sacrificed 24 hours later. Accumulation of thoracic duct lymphocytes in the allografts peaked 4 to 5 days after transplantation. To evaluate whether these changes at the graft site were sufficient to carry on the rejection response in the absence of a sustained host immunologic drive, acutely rejecting (Lewis X BN)F1 cardiac allografts were retransplanted serially at days 1 through 5 into normal syngeneic animals. All these regrafts survived greater than 100 days. Neither infusion of interleukin-2-conditioned medium (100 IU for 7 days intravenously) into regrafted hosts nor preoperative perfusion of the retransplanted hearts with interleukin-2-conditioned medium (300 IU) could complete the rejection process. Using flow cytometry analysis, we then assessed the phenotypic alterations of the mononuclear cells infiltrating the graft. The ratio of T helper: T cytotoxic/suppressor cells, which at day 3 was 1.57, inverted abruptly to 0.67 by days 5 to 6. After retransplantation a dramatic depression in T-lymphocyte subsets occurred, particularly affecting the T cytotoxic/suppressor phenotype. Trafficking studies revealed that the T cells that left the regrafts migrated mainly to spleen and mesenteric lymph nodes and away from bone marrow and peripheral blood of the syngeneic secondary recipients. Finally, histologic manifestations of acute rejection at days 4 to 5 virtually reversed themselves after regrafting. These studies emphasize the systemic nature of the rejection cascade, which depends fully on the host lymphoid system; even late changes in the graft microenvironment are not sufficient to produce final immunologic destruction.     

T suppressor cells induced by transfusions and cyclosporine. Studies in the murine cardiac allograft model

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.     

The immunosuppressive action of suppressor cells from antigen-cyclosporine-treated hosts on renal allograft survival

Systemic adoptive transfer was employed to assess the immunosuppressive efficacy of antigen-specific suppressor T (Ts) cells purified from recipients treated with 3M KCl-extracted donor histocompatibility antigen (Ag) and cyclosporine (CsA). Suppressor cells were obtained from Wistar-Furth (WFu, RT-1u) hosts treated with a single i.v. injection of 5 mg 3M KCl-extracted donor Buffalo (Buf, RT-1b) antigen combined with a three-day course of CsA, a group that displays prolonged renal allograft survival (MST 23.2 +/- 10.2 days) compared with animals treated with CsA alone (MST 12.2 +/- 2.4 days). These noncytolytic, OX-8 phenotype, 800-rad-resistant/1500-rad-sensitive, nylon-wool-nonadherent and cyclophosphamide-sensitive suppressor T cells (1 X 10(6)) were adoptively transferred ten days after transplantation into virgin, secondary syngeneic hosts-thereby prolonging Buf graft survival from 7.2 to 17.5 days. The suppressor effect was immunologically specific; adoptive transfer did not prolong the survival of third-party Brown-Norway (BN) grafts (MST 10.4 +/- 3.1 days) compared with the nontreated control group (MST 11.0 +/- 2.9 days). The potency of Ts cells purified from Ag-CsA-treated hosts to transfer unresponsiveness into normal secondary WFu hosts (MST 17.5 +/- 8.0 days) was stronger than that of Ts cells from hosts treated with CsA only (MST 10.6 +/- 2.6 days). Moreover, in vitro stimulation of monoclonal-antibody-purified Ts cells by irradiated donor Buf spleen cells potentiated the in vivo induced suppressor activity, leading to an MST of 38.1 +/- 32.6 days; indeed 3 of 12 animals (25%) displayed permanent unresponsiveness. Furthermore, Ts cells from Ag-CsA-treated hosts displayed a synergistic effect with a three-day course of CsA administration into the secondary hosts (MST 24.2 +/- 8.0 days) compared with animals only treated with CsA (MST 12.2 +/- 2.4 days, P less than 0.001). Moreover, the combination of the Ag-CsA regimen with Ts cells administered one day after transplantation caused even greater prolongation of graft survival (MST 34.2 +/- 14.2 days) compared with Ag-CsA-treated hosts (MST 23.2 +/- 10.2 days, P less than 0.025). Thus adoptively transferred antigen-specific suppressor T cells may be explored to intensify the specific immunosuppressive effect of the Ag-CsA regimen to achieve long-term unresponsiveness.     

Aorta transplantation as a model to study hyperacute, acute, and chronic rejection of xenografts

Abstract: To explore the mechanism of rejection of vascularized xenografts, we performed discordant and concordant transplantation of aortic grafts. Forty-one guinea pig-to-rat and 50 hamster-to-rat aortic transplantations were performed successfully. Recipient rats were either untreated or treated three times per week with 10 mg/kg of cyclosporine (CsA) starting the day before transplantation. Recipients were sacrificed at various timepoints after transplantation and the grafts were processed for histology and immunohistochemistry. Guinea pig aortic grafts were rejected hyperacutely in both the control and CsA treated group. Four hours after transplantation all endothelial cells and some of the medial smooth muscle cells had disappeared. Seven days after transplantation the media were totally acellular, whereas the adventitia was enlarged due to infiltrating cells, consisting mainly of macrophages. At day 56, only traces of the original graft could be identified.
Hamster aortic grafts developed signs of chronic rejection. In control rats, the intimal lesions could already be demonstrated in 17% of the rats at day 14, and in all rats from day 28. CsA treatment inhibited the development of the intimal lesions. At days 56 and 84 the difference in intimal thickness between the control group and the CsA treated group was statistically significant. The cellularity of the media of the hamster grafts remained constant during the first 21 days in both groups but declined sharply thereafter as a consequence of migration or necrosis of myocytes. The thickness of the adventitia in both groups increased after transplantation, due to infiltrating macrophages and T cells, reaching a peak at day 14. In conclusion, the aorta transplantation model provides useful information with regard to the development of chronic rejection in concordant grafts. Discordant aortic grafts are rejected hyperacutely just like discordant heart grafts.     

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

The role of TDTH and Tc populations in organ graft rejection. I. Functional analysis of graft-infiltrating T cells

To analyze the role of T cell subpopulations in the rejection of organ allografts, we developed a new model for obtaining large numbers of graft infiltrating cells (GICs). We isolated W3/25+ Th/DTH and OX8+ Ts/c from vascularized, irradiated rat spleen allografts. W3/25+ GICs obtained from spleen allografts transplanted to normal recipients were highly effective in eliciting cardiac allograft rejection when transferred to sublethally irradiated recipients, however, the OX8+ subset was incapable of eliciting rejection. On the other hand, when OX8+ GICs were obtained from spleen allografts transplanted to previously immunized recipients, they were as efficient as the W3/25+ Th/DTH subset in eliciting cardiac allograft destruction. These results indicate that the W3/25+, OX8- T cell is required for the rejection of primary organ allografts, but that the rejection of a secondary allograft by an immune recipient may be mediated, independently, by both W3/25+ and OX8+ cells.     

Phenotypic definition of primed T cells in human renal allografts. Use of the CD45R marker

Immunohistological studies indicate that T cells and macrophages are the major components of human kidney allograft infiltrates. Recent work has demonstrated a division of T lymphocytes into 2 subpopulations with distinct functions on the basis of their expression of the CD45R antigen (CD45R+ "naive" and CD45R- "memory" T cells). This study analyzes CD45R expression on circulating T cells and T cells infiltrating renal allografts in patients undergoing rejection and/or cyclosporine nephrotoxicity. The percentage of circulating T cells that expressed CD45R in patients with rejecting (63 +/- 4) or stable grafts (66 +/- 3) was not different from values obtained for normal donors (62 +/- 3). In contrast, the percentage of T cells expressing CD45R infiltrating rejecting grafts was 21 +/- 2 and was not affected by the stage of rejection; in patients with CsA toxicity the value was 22 +/- 6. The reduced proportion of T cells that expressed CD45R in the allograft may reflect a change in status from the naive state due to alloantigenic stimulation (which can be demonstrated in vitro) and/or a propensity of memory T cells to enter or be retained in an allograft.     

CD4+ T cell recognition of a single discordant HLA-A2-transgenic molecule through the indirect antigen presentation pathway induces acute rejection of murine cardiac allografts.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.     

Synergy between subtherapeutic doses of cyclosporine and immunologic enhancement in rat recipients of cardiac allografts

We have analyzed the adjunctive effect of subtherapeutic doses of cyclosporine (CsA, 1.5 mg/kg/day x 7 or 14 days) on cardiac allograft survival in actively and passively enhanced rats. This CsA dose, one tenth of the effective dose, when administered after, but not before, transplantation into enhanced hosts produced permanent graft acceptance; cardiac allografts survive c. 25 days in recipients enhanced only and 1 week in untreated animals. Adoptive transfer of spleen T cells of OX8+ or W3/25+ phenotype from long-term (greater than 200 days) graft recipients prolonged donor-specific test graft survival in naive rats (c. 16 days and c. 14 days, respectively, P less than 0.001) and delayed rejection in reconstituted B rats from 7 days to 21-23 days (P less than 0.001). Indeed, both T subsets were separately equally potent and with no overlap responsible for the suppressor activity. The phenotypic profile of the immune cells in the maintenance phase of enhanced or enhanced + CsA-treated recipients was comparable to naive or isografted controls as demonstrated by flow cytometry and immunohistologic studies. Furthermore, the activation status of the graft infiltrate in long-term survivors was similar regardless of the initial immunosuppressive protocol. CsA contributed selectively to the enhancing regimen in the induction phase of unresponsiveness, diminishing the cellularity of graft infiltrate and preventing intragraft T cell activation. These studies stress synergy between subtherapeutic doses of CsA and immunologic active/passive enhancement, 2 immunosuppressive modalities that spare T cells with suppressor capabilities but differ in the inhibition of T helper cell activation.     

Prolongation of heterotopic heart allograft survival by local delivery of continuous low-dose cyclosporine therapy

The effectiveness of local versus systemic low-dose CsA (2 mg/kg/day) therapy delivered by osmotic pump for a 14-day continuous infusion was examined in the rat model. Systemic subtherapeutic CsA treatment of WFu recipients either by oral gavage or intravenously using an osmotic pump resulted in quick rejection of BUF heart allografts within a median survival time (MST) of 8 days in comparison with untreated controls (MST = 7 days). In contrast, direct local subtherapeutic CsA delivery to BUF heart allografts produced significantly (P less than 0.01) prolonged heart allograft survivals up to MST of 40 days. Splenic T cells, isolated on days 10 to 12 from locally immunosuppressed WFu recipients, revealed a nonspecifically reduced proliferative response toward alloantigens. Coculture experiments demonstrate that these T cells have the capacity to inhibit normal T cell proliferative responses in a nonspecific fashion either by their suppressor function or more likely by carrying CsA to the culture plate. In contrast, T cells isolated from WFu recipients three weeks after transplantation and tested in vitro demonstrated the presence in alloantigen specific T suppressor cells that coincided with a decreased frequency of alloantigen-specific T cytotoxic cells and may explain the extended heart allograft survival beyond the time of CsA delivery. CsA therapy delivered directly to the graft resulted in high CsA levels within the heart graft (1108 ng/0.1 g) but subtherapeutic levels in other tissues. These results demonstrate that local drug delivery is effective in inhibiting the rejection process within the graft itself, as manifested by prolonged heart allograft survival. Further, subtherapeutic CsA therapy facilitates development of Ts cells that may be responsible for the survival of heart allografts beyond the CsA delivery time.     

Cyclosporine and experimental skin allografts. II. Indefinite survival and development of specific immunologic unresponsiveness

Immunological unresponsiveness toward skin allografts was studied in cyclosporine (CsA)-treated rats. BN skin grafts survive about 22 days and about 34 days in LEW hosts following 7 or 14 days of daily CsA treatment (15 mg/kg/day), respectively; in unmodified hosts grafts are rejected by 9 days. Indefinite (greater than 100 days) survival can, however, be produced by administering maintenance 15 mg/kg CsA every fourth day, following an initial course of the agent for 14 days. Early signs of graft rejection (hair loss, localized epidermal breakdown, and ulcerations) occurring in some animals were reversed by a CsA "pulse" (15 mg/kg/day) for 7 days, reduced gradually to the maintenance dose. CsA was equally effective when started as late as 4 days after grafting, but ineffectual when started after day 4. Once BN grafts were rejected, the agent could not prevent second-set rejection of donor-specific grafts, but significantly prolonged the survival of third-party (WF) skins. Survival of original BN grafts was unchanged by the placement of second BN grafts during both the inductive and maintenance phases; these second grafts survived as long as the original grafts. In contrast, secondary third-party (WF) grafts were promptly rejected; their destruction did not influence survival of the original grafts. Thus, indefinite survival of rat skin allografts is feasible with low maintenance doses of CsA. Graft rejection at later stages can be reversed by resuming daily therapy. Host unresponsiveness is stable and specific both during the early inductive and later maintenance phases.     

Restoration of T cell responsiveness to interleukin-2 in recipients of pancreatic islet xenografts treated with cyclosporine

The immunosuppressive mechanism of cyclosporine (CsA) was studied using pancreatic islet xenotransplantation. A dose of 40 mg/kg/day CsA significantly prolonged survival of hamster islet grafts in BDE rats to 14.0 +/- 7.1 days compared with 2.0 +/- 0.9 days in controls (P less than 0.01), and antihamster lymphocytotoxic antibody was not detected in recipient sera even after rejection. Responses of spleen cells to pokeweed mitogen (PWM) and to phytohemagglutinin (PHA) were inhibited at least 21 days after transplantation in recipients treated with CsA. The addition of exogenous interleukin-2 (IL-2) to spleen cell cultures on day 7 had no effect on the impaired response to PHA. At the time of graft rejection (day 14), the T cell response to PHA recovered with the addition of IL-2. However, significant changes were not observed in the ratio of spleen cell subpopulations, which were studied with monoclonal antibodies of W3/ 13, OX-12, W3/25, and OX-8. From these results we conclude that CsA can significantly prolong islet xenograft survival in closely related species, and inhibit the production of humoral antibodies and T cell responses. At the time of graft rejection, recipient T cell responsiveness to IL-2 was restored. This suggest that islet xenograft rejection is caused by the recovery of cellular immunity in recipients treated with CsA.     

Increased T Cell Glucose Uptake Reflects Acute Rejection in Lung Grafts

Although T cells are required for acute lung rejection, other graft–infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [¹⁸F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [¹⁸F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2‐NBDG revealed that T cells, and in particular CD8⁺ T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen‐presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.     

Acute cell-mediated rejection in orthotopic pig-to-mouse corneal xenotransplantation

Abstract: Background:  To investigate the role of T cells and natural killer (NK) cells in mediating corneal xenograft rejection in a pig-to-mouse model.
Methods:  Pig corneas were orthotopically transplanted into BALB/c, C57BL/6, nude, severe combined immunodeficiency (SCID), and NOD/SCID/γc^null (NOG) mice. Graft survival was clinically assessed by slit-lamp biomicroscopy and median survival times (MST) were calculated. The rejected grafts were histologically evaluated using antibodies against CD4, CD8, NK1.1, and F4/80.
Results:  The pig corneal xenografts were acutely rejected by BALB/c and C57BL/6 mice (MST 9.0 days), while nude, SCID and NOG mice rejected pig corneas in a more delayed fashion (MST 16.0, 16.4, and 16.9 days, respectively). The majority of infiltrating cells found in rejected grafts in C57BL/6 mice were macrophages and CD4⁺ T cells, while CD8⁺ T cells and NK cells were rarely found. The grafts in nude mice had markedly decreased inflammatory infiltration with small numbers of macrophages and CD4⁺ T cells. Infiltration was even more modest in grafts in SCID and NOG mice.
Conclusions:  T cells play an important role in acute rejection of pig corneal xenografts in mice, although acute rejection is not solely the result of T-cell-mediated immunity. NK cells are less likely to be involved in the rejection process.     

Evidence that rat renal allografts are rejected by cytotoxic T cells and not by nonspecific effectors

Infiltrating cells were harvested on day 5 from rat renal allografts (LEW X BN into LEW) that were either rejecting (untreated) or healthy (cyclosporine [CsA]-treated or passively enhanced with LEW anti-BN serum). Similar numbers of cells were harvested from rejecting (mean 11 X 10(7), range 8-15 X 10(7], CsA-treated (6 X 10(7), 5-7 X 10(7], and enhanced (9 X 10(7), 7-10 X 10(7] grafts. These cells were a heterogeneous population of mononuclear cells. Fluorescence-activated cells sorter analysis after labeling with a range of monoclonal antibodies showed that they all labeled with MRC OX-1 (against the leukocyte-common antigen), and that about one-third of infiltrating cells bound MRC OX-19 (pan-T cells) and one-third bound MRC OX-12 (B cells), with little difference between the rejecting, CsA-treated or enhanced grafts. However, the ratio of the numbers of W3/25-positive cells (helper T cells and macrophages) to MRC OX8-positive cells (cytotoxic T cells and natural killer cells) was less in untreated (mean 0.6, range 0.5-0.6) than in CsA-treated (1.3, 0.8-1.6) or enhanced (2.1, 1.8-2.4) grafts. Using a 6-hr chromium-release assay, cells from untreated, CsA-treated, and enhanced grafts showed similar levels of nonspecific cytotoxicity (against Y3 myeloma cells), but only untreated grafts showed alloantigen-specific target cell lysis (against BN concanavalin A blasts). These results suggest that specific cytotoxic T cells rather than nonspecific responses play an essential role in allograft rejection in the rat.     

The in vivo immunosuppressive action of suppressor cells from alloantigen-cyclosporine-treated mice and the capacity of spleen cells to release interleukins and gamma-interferon

Antigen-nonspecific suppressor T cells were identified in spleens of mice rendered unresponsive by sensitization of allogeneic antigen in combination with cyclosporine (CsA) treatment. Suppressor cells were obtained from C57BL/6 (B6, H-2b) mice treated with a single i.p. injection of 1 x 10(7) allogeneic P815 (H-2d) cells combined with a five-day course of CsA, a group that did not show any cytotoxic activity of spleen cells against P815 targets. These noncytolytic spleen cells displayed suppressor activity on the induction of cytotoxic T (Tc) cells of normal lymphocytes against not only P815 stimulator (80.9% suppression, P less than 0.01, responder:additional cell ratio = 2.5:1) but also third-party BW5147 (H-2k) stimulator (68.2% suppression, P less than 0.01). The unresponsive state appears to be due to suppressor T (Ts) cells that are nonadherent to plastic or nylon-wool, 1500 rads-sensitive, and Thy-1-positive. Capacities of spleen cells from CsA-P815-treated mice to release cytokines (interleukin 1 [IL-1]), interleukin 2 [IL-2], interleukin 3 [IL-3], and gamma-interferon [gamma-IFN]) were examined. Spleen cells from CsA-P815-treated B6 mice displayed 84.1%, 91.7% and 90.8% inhibition (0.35 +/- 0.07 U/ml, 1.4 +/- 0.29 U/ml, and 7.0 +/- 0.9 U/ml) of IL-1, IL-2, and gamma-IFN production compared with normal mice (2.2 +/- 0.54 U/ml, 16.9 +/- 2.1 U/ml, and 76.0 +/- 3.1 U/ml, P less than 0.01), respectively. However, IL-3 production was significantly less inhibition (46.1%, 2.35 +/- 1.0 U/ml in CsA-P815-treated mice and 4.36 +/- 1.7 U/ml in normal mice) compared with other cytokines (IL-1, IL-2, gamma-IFN). Two systems were employed to assess the immunosuppressive efficacy of antigen-nonspecific Ts cells in vivo. First, adoptive transfer (i.p.) of spleen cells harvested from CsA-P815-treated mice ten days after treatment on 3 consecutive days (days 0, 1, 2) at a 3 x 10(7) cell dose into virgin B6 mice that were immunized with P815 cells (1 x 10(7), day 0) completely inhibited the development of Tc cells against P815 targets (5% specific cytolysis, effector:target ratio [E:T] = 200). The suppressor effect was immunologically nonspecific; adoptive transfer of Ts cells from CsA-P815-treated mice also abrogated the development of Tc cells against third=party BW5147 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunologic diagnosis of kidney rejection using FACS analysis of graft-infiltrating functional and activated T and NK cell subsets

In human organ transplantation, the correct diagnosis of rejection may be difficult using clinical and/or histopathologic criteria, and immunologic assays should therefore be considered. We have applied monoclonal antibodies in 2-color flow cytometric (FACS) analysis to study phenotypic patterns of kidney-infiltrating activated and functional T and NK cell subsets in 132 posttransplantation biopsies. Viable intragraft lymphocytes and kidney tubular cells were obtained by the use of medium-sized ultra-sound guided needle biopsy. In univariate analyses, highly significant differences were observed between rejecting and stable grafts. During rejection, the proportions of HLA-DR+ kidney tubular cells, total lymphocytes, T suppressor/cytotoxic cells, and natural killer (NK)-like cells increased; the fractions of phenotypically activated, HLA-DR+ cells within the latter 2 cell populations were elevated. Further, during rejection phenotypic T suppressor inducer and T suppressor effector cells were virtually absent in kidney tissue but reappeared rapidly upon successful antirejection therapy. Multivariate analyses revealed that combinations of antibodies in FACS analysis defining functional and activated subsubsets of T and NK cells allow a precise and rapid immunologic diagnosis of kidney rejection.