Recipient Lymphocytes Research Articles

Extracorporeal Photochemotherapy after Cardiac Transplantation: A New Therapeutic Approach to Allograft Rejection

Photopheresis (ECP) is a new immunomodulatory therapy in which recipient lymphocytes are treated extracorporeally with 8-methoxypsoralen and ultraviolet light. The treatment seems to induce an inhibition of both humoral and cellular rejection after transplantation. Since recurrent rejection (RR) continues to be a severe complication after heart transplantation (HTx) and the immunosuppressive regimes used for the treatment are often associated with increased morbidity and mortality, we investigated whether ECP could have a beneficial effect on the number and severity of rejection episodes. Eleven HTX recipients (5 M and 6 F, mean age 48.5 yrs) with RR were enrolled in the study. ECP was performed at weekly intervals during the 1st month, at 2 week intervals during the 2nd and 3rd month, and then monthly for another 3 months. The fraction of biopsies (EMB) with a grade 0/1A rejection increased during ECP from 46% to 72% while the EMB showing a 3A/3B rejection decreased from 42% to 18%. It is also noteworthy that out of the 78 EMB performed during ECP only one showed a 3B rejection in comparison with 13 out of 110 EMB in the pre-ECP period. Six rejection relapses were observed in a total follow-up of 60 months, two of them occurring during the tapering of oral steroid. Four relapses were reversed by ECP, one by i.v. steroids and the last by methotrexate after the failure of both i.v. steroids and ECP. The mean doses of immunosuppressive drugs resulted lower after 6 months of ECP: steroids were reduced from 13 to 8.25 mg/day, cyclosporine from 375 to 285 mg/day, azathioprine from 55 to 35 mg/day. ECP is a well tolerated treatment. Its administration allows better RR control and significant reduction in immunosuppressive therapy.

Analysis of post-transplant immune status in recipients of liver/bone marrow allografts

The aims of this study were to assess the effect of donor bone marrow infusion on the reactivity of recipient peripheral blood lymphocytes (PBL) to mitogen and to donor and third-party cells after primary liver allotransplantation and to identify any correlation between altered immunoreactivity and HLA mismatches, occurrence of rejection, and immunosuppression. The immunoreactivity of recipient PBL toward frozen donor splenocytes was evaluated in mixed lymphocyte culture (MLC) ( n = 29) and cell-mediated lympholysis (CML) ( n = 27) assays in time intervals ranging from 0.7 to 27 months after transplant. Overall, the mean anti-donor MLC stimulation index (SI) fell from 25.6 ± 5.2 preoperatively to 4.8 ± 1.7 after transplantation ( p < 0.002), with 14 out of 29 (48.3%) patients developing donor-specific MLC hyporeactivity. HLA class II mismatches were significantly associated with recipient post-transplant immune profile ( p < 0.05): MLC donor specific hyporesponsiveness was observed in 70%, versus 37% of patients who shared a class II antigen, versus those that did not. Of the control group, 61.1% developed donor-specific nonreactivity versus 27.2% in the donor bone marrow cells (DBMC) group ( p = 0.02). Donor-specific CML hyporeactivity was observed after transplantation, independent of DBMC infusion, with mean percentage values of pre- and post-transplant donor-specific lysis of 22.4% ± 4.1% versus 3.1% ± 1.6%, p = 0.0004, respectively. Our results suggest that DBMC infusion favors development of nonspecific MLC hyporesponsiveness to donor and third-party alloantigen, with maintenance of reactivity to mitogen and no additional effect on T-cell cytotoxicity.

EARLY ACTIVE IMMUNE INTERACTION BETWEEN DONOR AND RECIPIENT LYMPHOCYTES IS ESSENTIAL FOR INDUCTION OF LONG-TERM ALLOGRAFT SURVIVAL

260 Previous studies demonstrated that posttransplant infusion of viable donor splenocytes (SC) alone could exert an active immunological effect to downregulate alloreactivity of recipient. This effect was donor-specific and might depend upon potential GvHR of infused SC. To induce long-term allograft survival, we investigated the effects of accompany use of Cyclosporine (CsA) (dose, timing) with posttranslant infusion of donor SC in a fully MHC-mismatched rat transplantation model. Methods: Cardiac allografts of LEW (RT1Al B/Dl Cl) were transplanted heterotopically into abdomens of LEW.1W (RT1Au B/Du Cu) recipients and divided into following groups. Group 1: no treatment; group 2: CsA 0.5mg/kg/day i.m., day0-5; group 3: CsA 0.5mg/kg/day i.m., day 6-10; group 4: donor SC 2×108 cells in 1 ml Ringer's solution, i.v. within 6 hours after transplantation (DSC i.v.); group 5: DSC i.v. plus CsA 0.5mg/kg/day i.m., day0-5; group 6: DSC i.v. plus CsA 0.5 mg/kg/day i.m., day 6-10; group 7: DSC i.v. plus CsA 0.5 mg/kg/day i.m., day 0-10. Graft function was detected by daily heart beat palpation. Complete cessation of cardiac pulsation was defined as rejection and was confirmed by histology. Results: See following table:TableConclusions: The findings suggest long-term allograft survival can be induced by posttransplant infusion of donor SC followed temporary use of CsA. The active suppressive effects of infused cells could be influenced by simultaneously use of CsA even in a low-dosage fashion. These support the hypothesis that early active immune interaction between donor and recipient lymphocytes is required for induction of long-term allograft unresponsiveness.

MHC ALLOPEPTIDE REACTIVE T CELL CLONES GENERATED FROM HUMAN RENAL ALLOGRAFT RECIPIENTS: INDIRECT ALLORECOGNITION IN LONG TERM ENGRAFTMENT

9 T cells obtained from patients with chronic allograft dysfunction are primed to recognize and respond to donor-specific MHC allopeptides in vitro. Class II MHC allopeptide-specific Th1 clones generated from rejecting animals exhibit a restricted TCR Vβ repertoire and transfer delayed type hypersensitivity responses in vivo indicating, the important role they play in the genesis of rejection. In order to evaluate the role of T cells primed by donor specific MHC peptides in chronic allograft dysfunction, T cell lines and clones were generated from peripheral blood lymphocytes (PBLs) of human renal allograft recipients, all of whom had been transplanted for more than one year. All patients were maintained on the same immunosuppressive protocol. Nine patients mismatched for MHC class II specificities for which peptides were available were studied. The patients were divided into 2 groups; 1. chronic allograft dysfunction (n=4) and 2. stable graft function (n=5). T cell lines were generated from PBLs by repeated stimulation with the relevant mismatched class II MHC allopeptide presented by self antigen-presenting cells. The mean proliferative response of T cells lines from "rejecting" patients was significantly greater than that of the lines from "stable" patients; (10743.0±1834.2 versus 1175.4±127.3 cpm, p<0.0001). The production of cytokines generated in response to the allopeptides was evaluated by ELISA and RNAse protection assays. Cell lines from the "rejecting" group produced the Th1 cytokine IFN-γ but not the Th2 cytokine IL-4 in culture supernatants. One of the cell lines produced TGF-β. In contrast, the "stable" group produced IL-4 but not IFN-γ in response to re-challenge with the allopeptides. These data were confirmed by RNAse protection assay. T cell clones were then generated by limiting dilution and were phenotyped using flow cytometric analysis. T cell clones from both groups were all found to be CD4+. RT-PCR transcript analysis with specific human TCR Vβ primers showed that the clones expressed a restricted TCR Vβ repertoire with some overlaps. The mean proliferative response of the "rejecting" T cell clones was also significantly greater than that of the "stable" clones; (20352.0±2678.7 versus 2873.2±348.1 cpm, p<0.0001). Interestingly, without exception, analysis of 49 T cell clones showed that all the clones from rejecting patients produced IFN-γ but not IL-4, while clones from stable patients produced IL-4 but not IFN-γ. This is the first demonstration, at the clonal level, that chronic allograft rejection in humans is associated with the presence of CD4+ MHC allopeptide-specific T cell clones that express a Th1 phenotype. In contrast, MHC allopeptide-specific T cell clones generated in the absence of rejection are associated with a Th2 phenotype that may be protective. These data provide an interesting insight into the potential mechanisms underlying chronic allograft dysfunction and more importantly suggest an innovative approach to immune surveillance.

LONG TERM IMPACT OF POSITIVE CROSSMATCH DUE TO IgG VERSUS IgM ANTIBODIES IN PRIMARY LIVER TRANSPLANTATION UNDER TACROLIMUS

766 Positive lymphocytotoxic crossmatch between recipient serum and donor lymphocytes has been demonstrated to be the cause of poor outcome in renal transplantation. Incubation of recipient serum with DTT (dithiothreitol) causes reduction of disulfide bonds of pentameric IgM complex and hence its inactivation, and has been used to distinguish between IgG positive cross match and IgM positive crossmatch. Successful renal transplant can be performed when crossmatch is positive due to non HLA IgM anti bodies. Aim of the present study is to examine the long-term impact of IgG and IgM positive crossmatch in liver transplantation under Tacrolimus. Material and Method: Between Feb. 90 and Dec. 92, 93 patients had positive crossmatch using Amos modified NIH technique. With DTT treatment 73 patients remained positive (IgG antibody positive) while 20 were rendered negative (IgM antibody positive). Characteristics of the population is shown in the (table).TableAll patients were followed until April 98, mean follow up6.8±0.9 (median 6.9; range 8.2 to 5.4) years. Episodes of rejections were recorded in first 3 months. Results: Patient and graft survivals with rejection episodes are shown below. (Table)TableConclusion: Above data suggests that the rate of rejection in IgG positive cross match is higher than IgM positive cross mach and our previously published reports. The long term 7 year patient survival is 70% and graft survival 64% under tacrolimus, which is comparable to observed in other studies.

LYMPHOCYTE-MEDIATED DENDRITIC CELL TYROSINE PHOSPHORYLATION AND MAP KINASE ACTIVATION CONTRIBUTE TO ALLOGENEIC RESPONSES IN VITRO

819 The cytokine release which follows the interaction of recipient dendritic cells (DC) and donor lymphocytes (TL) initiates the immune processes which culminate in graft rejection. Cell-cell contact between DC and TL results from a number of surface proteins, including CD86 and CD80 on DC and the receptor CD28 on TL. While CD28 co-stimulatory signals can be mediated via tyrosine phosphorylation signaling, the nature of the DC response following TL contact remains to be defined. We postulated that such contact leads to DC tyrosine phosphorylation and therefore to activation of key signaling cascades, such as the Mitogen Activated Protein (MAP) kinases. To test this hypothesis splenic TL harvested from C3H mice were layered over confluent DC isolated from C57BL/6 mice and co-cultured. TL and DC were separated at various times, and the two cell populations studied individually for signaling events. As determined by Western blot analysis, the TL stimulated a marked increase in tyrosine phosphorylation within DC that peaked by 90 minutes and then faded. By contrast, tyrosine phosphorylation in the TL population over the same time span was weak. Phosphorylated, active ERK MAP kinase isoforms were consistently upregulated in DC, with less phosphorylation of the related p38 MAP kinase. The physiologic importance of DC tyrosine phosphorylation was suggested by a 67±11% (mean±SEM, n=4) inhibition of 24-hour TNFα and 18±9% inhibition of IL-2 production when the TL/DC co-culture was exposed to the broad-spectrum tyrosine kinase inhibitor Tyrphostin AG126 (50 μM). Intriguingly, selective ERK MAP kinase inhibition with PD98059 (50 μM) inhibited production of both TNFα (45±10%) and IL-2 (39±6%), while selective p38 inhibition with SB203580 (30 μM) inhibited only TNFα (52±8%), with no effect on IL-2 release (<1%). We have previously noted that blockade of the CD86/CD28 and CD80/CD28 interaction with specific monoclonal antibodies against CD86 and CD80 abrogates cytokine generation in this model. Pretreatment of DC with the same antibodies markedly attenuated TL-induced DC tyrosine phosphorylation. These data argue that contact with allogeneic TL stimulates a time-dependent increase in DC tyrosine phosphorylation that is mediated at least in part via CD86 and CD80 and is crucial for the subsequent induction of immune responses. The ERK MAP Kinase in particular plays a key role in the allogeneic immune response, suggesting that future immunomodulatory therapies could be targeted to this pathway.

Allogeneic bronchoalveolar lavage cells induce the histology and immunology of lung allograft rejection in recipient murine lungs: role of intercellular adhesion molecule-1 on donor cells.

Intercellular adhesion molecule (ICAM)-1 expressed on accessory cells has a key role in antigen presentation. The histology and immunology of lung allograft rejection is postulated to result from donor lung accessory cells presenting alloantigens to recipient lymphocytes, and, therefore, ICAM-1 may have a crucial role in the rejection process. We have previously reported that the instillation of allogeneic (C57BL/6, I-a(b)) bronchoalveolar lavage (BAL) cells (96% macrophages, 2% dendritic cells) into the lungs of recipient BALB/c mice (I-a(d)) induced the histology and immunology of acute lung allograft rejection. Using this model, the purpose of the current study was to determine the role of ICAM-1 on donor lung cells in lung allograft rejection. BALB/c mice received allogeneic BAL cells from wild-type or ICAM-1 mutant (lacking ICAM-1 expression) C57BL/6 mice by nasal insufflation weekly for 4 weeks. Recipient mice underwent BAL and serum collection for the determination of T helper 1/T helper 2 cytokines and IgG subtypes. Lung histology was graded using standard criteria for allograft rejection. Although wild-type cells induced a lymphocytic vasculitis and bronchitis, ICAM-1 mutant allogeneic BAL cells only induced a lymphocytic vasculitis in recipient lungs. Both wild-type and ICAM-1 mutant cells induced up-regulated local interferon-gamma and IgG2a production, and deposition of IgG2a in recipient lungs. These data show that ICAM-1 on donor lung accessory cells mediates differential effects on the histology and immunology of acute lung allograft rejection.

A study of endothelial cell-lymphocyte responses in human renal transplantation.

Histological studies have demonstrated vascular damage in all types of allograft rejection. It is likely that donor endothelium suffers the major and the first insult by the recipient's immune system since, in vivo, capillary endothelium expresses human lymphocyte antigen (HLA) class I and class II antigens. The present study was designed to examine whether injury to donor endothelial cells (ECs) by recipient peripheral blood mononuclear cells (PBMCs) can be demonstrated in vitro, and whether there is a relationship between the in vitro findings and the clinical outcome of renal allografts. Twenty renal transplant recipients were included in this study, and all patients were followed up for 6 months. PBMCs were isolated from the renal transplant recipients on three occasions; in the first 24-h post-transplantation, at the beginning of the second week, and in the third week post-transplantation. Additional samples were taken at the time of any acute rejection episode. These patients received renal allografts from 15 local cadaveric donors whose ECs were isolated. Donor-specific ECs and the corresponding renal transplant recipients' PBMCs and sera were employed in proliferation and cytotoxicity assays. Our results show that donor-specific ECs consistently induced a highly significant degree of recipient lymphocyte proliferative response (p < 0.05). However, no significant correlation between acute graft rejection and the degree of donor-specific EC-induced recipient lymphocyte proliferation was found. In contrast, there was a significant correlation between lymphocyte-induced EC cytolytic effects and acute renal graft rejection (p < 0.05). When conducted in larger studies, such information can have important implications in clinical transplantation.

Decreased expression of the interleukin 2 receptor on CD8 recipient lymphocytes in intestinal grafts rendered tolerant by liver transplantation in rats

Background—In a previous study, it was shown that a spontaneously tolerated DA (RT1a) liver allograft in a PVG (RT1c) recipient was able to induce tolerance of a DA small bowel...

Risk factors for long-term renal survival after renal transplantation

Chronic progressive renal function loss is a main cause of long-term graft loss after initially successful renal transplantation. Transplanted kidneys share some risk factors for renal function loss, such as hypertension or proteinuria, with diseased native kidneys. Recently, it has been shown that renal function loss is influenced by the angiotensin-converting enzyme (ACE) (insertion/deletion [I/D]) genotype in renal disease in diseased native kidneys. This study examines whether donor or recipient ACE (I/D) genotype is a risk factor for graft loss after renal transplantation. To avoid bias by acute events, graft survival was studied, with patients dying with a functioning graft censored, starting at 12 mo after transplantation in a cohort of 367 patients transplanted between 1987 and 1994 with at least 2 yr of follow-up. Mean follow-up was 58 mo. ACE (I/D) genotype was determined by PCR on stored donor and recipient lymphocytes. Neither donor nor recipient ACE (I/D) genotype was associated with graft survival. However, Cox proportional hazards analysis identified recipient, but not donor, ACE (I/D) genotype D-allele to be independently associated with a shorter time to graft loss in subgroups of patients at high risk for graft loss defined by a creatinine clearance <50 ml/min (n = 108, P = 0.017) or proteinuria > or =0.5 g/24 h at 12 mo (n = 97, P = 0.0051) after transplantation. In conclusion, recipient ACE (I/D) genotype was associated with time to graft loss in a specific high-risk subgroup of the study population. This suggests that the effect of ACE (I/D) genotype on graft survival only becomes apparent when other risk factors are simultaneously present.

Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs.

Lung allograft rejection is believed to be initiated by donor lung accessory cells, namely macrophages and dendritic cells, interacting with recipient lymphocytes leading to up-regulated Th1 type (IFN-gamma) cellular immunity culminating in graft destruction. The purpose of this study was to determine the individual role of donor lung macrophages and dendritic cells in the rejection response. Utilizing a murine model that reproduces the immunology and histology of acute rejection, C57BL/6 mouse (I-a(b), H-2(b)) lung dendritic cells (DC-enriched lung cells), purified alveolar macrophages (I-a-negative macrophages), or various ratios of I-a-negative macrophages/DC were instilled into BALB/c mouse (I-a(d), H-2(d)) lungs followed by an assessment of local IFN-gamma production and grading of rejection pathology. The data show that DC, and not I-a-negative macrophages, induced IFN-gamma production in recipient lungs. However, the local production of IFN-gamma was not always associated with histological changes characteristic of rejection pathology. In contrast to either cell type alone, instillation of C57BL/6 I-a-negative macrophages and DC, together, were required to induce rejection pathology in BALB/c lungs. In addition, the rejection response was dependent on interactions between donor I-a-negative macrophages and DC.

Fluorescent in situ hybridization on flow-sorted cells as a tool for evaluating minimal residual disease or chimerism after allogeneic bone marrow transplantation

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

Simplified method of identifying severe combined immunodeficient (SCID) mice versus non-SCID mice by flow cytometric analysis of peripheral blood.

Several studies have utilized simple breeding strategies to create new immunodeficient mouse strains from severe combined immunodeficient (SCID) mice and non-SCID mice with secondary traits in order to evaluate the involvement of lymphocytes and immune responses in a variety of processes. We utilized a breeding strategy with C.B-17scid/scid (SCID) (H-2d) mice and SJL (H-2s) mice to generate immunodeficient mice that were histocompatible with the inbred SJL strain (H-2s) in order to evaluate the role of histocompatible recipient lymphocytes in adoptively transferred autoimmune disease mediated by SJL T lymphocytes. [SCID x SJL]F1 mice (heterozygous for H-2 loci and heterozygous for the SCID mutation) were backcrossed with SCID mice and the resulting offspring expressed a variety of phenotypes, including SCID or non-SCID and H-2s/H-2d or H-2d/H-2d. In order to screen offspring for the desired phenotype (SCID, H-2s), a flow cytometric method utilizing forward- and side-scatter parameters of peripheral blood cells was used to distinguish SCID from non-SCID animals. This method simplified the screening process and was as reliable as anti-CD3 fluorescent monoclonal antibody staining for detecting the presence (non-SCID) or absence (SCID) of T lymphocytes in peripheral blood.

PASSENGER LEUKOCYTES FROM AN ALLOGENEIC HEART GRAFT CAN INDUCE LETHAL LYMPHOID APLASIA

114 Purpose. The functional relevance of passenger leukocytes in allogeneic organ transplantation is still unclear. To study it more detail we have applied a rat transplant model where we can selectively and transiently eliminate the recipient's lymphocytes without affecting donor lymphocytes. For this purpose rat strains differing in an allomorphic form of the leukocyte common antigen(RT7a/RT7b) and an antibody against one of these antigens (RT7a) have been used. Methods. LEW·1W(RT1u, RT7a) rats were injected with anti-RT7a mAb on day -1. On day 0, these rats received either no or syngeneic (LEW·1W) or allogeneic (LEW:RT1l, RT7a or LEW·7B: RT1l,RT7b) heart grafts. In this setting both recipient's leukocytes and leukocytes of RT7a donors are eliminated by anti-RT7a mAb, while those of RT7b donors are not affected. Some of the recipients with LEW·7B grafts also received anti-CD3 mAb.Results. TableAfter anti-RT7a mAb treatment all rats showed leukopenia most markedly in T cells. Leukopenia recovered over 4-6 weeks in rats that received RT7a allografts (group 3), and these rats survived indeifnitely as rats that received no (group 1) or syngeneic grafts (group 2). The rats that receivedRT7b allografts showed further deteriorated leukopenia after 2 weeks and died of severe anemia and/or bleeding (group 4). This lethal effect was abrogated by anti-CD3 treatment on day 2 (group 5). Autopsy in group 4 showed severe atrophy of the lymphoid tissues. No evidence of graft-versus-host disease was shown. Immunohistology on day 2, but not later, revealed considerable numbers of RT1l donor cells in the lymphoid organs in group 4, while no donor cells were detected in group 3. Conclusions. The rats withRT7b allografts showed lethal lymphoid aplasia, while the rats with RT7a allografts survived indefinitely. This suggests an essential role of passenger leukocytes from the heart graft for induction of lethal lymphoid aplasia. Abrogation of this lethal effect by anti-CD3 mAb treatment indicates that donor-derived T cells play the most important role. This study shows that under certain conditions even small numbers of passenger leukocytes can have the profound functional relevance.

Characterization of an MDR1 retroviral bicistronic vector for correction of X-linked severe combined immunodeficiency

X-linked severe combined immunodeficiency (XSCID) is a hereditary disorder characterized by severe T cell lymphopenia and abnormal B cell function. The disease is caused by mutations in IL2RG, the gene encoding the interleukin-2 receptor common gamma chain (gamma c) shared by several interleukin receptors. A Harvey retroviral bicistronic vector containing an IL2RG cDNA and cDNA encoding the multidrug transporter (MDR1) was constructed to investigate the correction of XSCID. Translation of the MDR1 cDNA is achieved from an internal ribosome entry site (IRES). Mouse fibroblasts transfected or transduced with the vector expressed both membrane proteins as detected with specific monoclonal antibodies by fluorescence activated cell sorting. Two human XSCID B cell lines were transduced using a filter concentration method in combination with phosphate depletion. Significant expression of both proteins was detected by Western blot analysis. This construct might be particularly useful if high expression of gamma c is required, as might be achievable through in vivo selection for drug resistance of recipient lymphocytes.

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Role of indirect allorecognition in experimental late acute rejection.

Late acute rejection affects up to 28% of renal allograft recipients and remains a major risk factor for late graft loss. As donor-origin antigen-presenting cells are depleted with time, T-cell recognition of donor-derived alloantigenic peptides presented by self antigen-presenting cells (the "indirect pathway" of allorecognition) may play a key role in the initiation of late acute rejection episodes. To test this hypothesis, we developed a clinically relevant experimental model in the rat (Wistar-Furth/Lewis) in which allograft recipients received cyclosporine for 1 month after transplantation and were then allowed to reject the graft upon discontinuation of immunosuppression. Lymphocyte proliferation assays to synthetic class II MHC allopeptides of donor origin and also to intact donor (Wistar-Furth) cells were performed at this time. The effector mechanisms studied included delayed-type hypersensitivity (DTH) responses, lymphocyte-mediated cytotoxicity, and alloantibody production. Lymphocytes from recipients undergoing late acute rejection had marked suppression of mixed lymphocyte reaction proliferation to intact donor cells. Significant proliferation to donor-derived 25-mer polymorphic class II MHC allopeptides was elicited, however. In vivo, significant DTH responses were observed to both MHC allopeptides and intact Wistar-Furth cells. Recipient lymphocytes also exhibited significant killing of donor cells, although not third-party cells, and anti-donor alloantibodies were detected by flow cytometry. Our results indicate that T cells primed via the indirect pathway are present during acute rejection that occurs after discontinuation of cyclosporine. Mixed lymphocyte reactivity is markedly reduced at this time. Furthermore, there is an association between such allopeptide-primed T cells and the elicitation of specific DTH responses and provision of help to B cells to produce alloantibodies and activation of CD8+ T cells to become effector cytotoxic cells.

Increased inducible interleukin-2 receptor expression on CD8 lymphocytes in black recipients of renal allografts with deteriorating function

Increased inducible interleukin-2 receptor expression on CD8 lymphocytes in black recipients of renal allografts with deteriorating function

Bronchus-associated lymphoid tissue is targeted and damaged by recipient lymphocytes in long-term-surviving rat lung allograft

T HE BRONCHUS-associated lymphoid tissue (BALT) is a conspicuous part of the local immune system of the lung. In acute rejection, the BALT is the first target due to its high immunogenicity.’ Previously, we reported reduction of BALT in long-term-surviving rat lung allografts2,3 which are indefinitely accepted with a short course of treatment with cyclosporine A (CyA). These lung allografts are not maintained by classic tolerance because recipient lymphocytes could respond to donor antigens4 We hypothesized that the BALT might be damaged by immunologic events because of its high immunogenicity. Therefore, we investigated the serial histologic changes of the BALT of these lung allografts at different timepoints after transplantation.

Use of the methylxanthine derivative A802715 in transplantation immunology: II. In vivo experiments.

We have previously demonstrated in vitro that the methylxanthine derivative A802715 suppresses the cyclosporine (CsA)-resistant "signal two"-dependent pathway of T cell activation and hence acts synergistically with CsA. Here, this synergism was further investigated in vivo in rats. Primary cardiac allografts were placed in the neck, and secondary grafts were transplanted intra-abdominally. A802715 was given orally for 30 days or by continuous intravenous infusion via a mini-osmotic pump for 2 weeks. CsA was given orally for up to 30 days. T cell responses were examined in vitro using mixed lymphocyte reaction, concanavalin A whole blood, and cell-mediated lympholysis assays. In a major histocompatibility complex incompatible WKAH-->PVG combination, neither oral CsA (7.5 mg/kg/day) nor oral A802715 (100 mg/kg/day) was able to prolong graft survival. However, a combination of both drugs, given at the same dose, sustained graft survival during treatment. A similar synergism was not obtained with pentoxifylline, another methylxanthine derivative. The synergism between A802715 and CsA could be further increased by using a continuous intravenous infusion of A802715, since (1) lower doses of A802715 (20 mg/kg/day) and CsA (5 mg/kg/day) could be used, and (2) six of seven grafts survived permanently. In a major histocompatibility complex compatible Wag/Rij-->R/A combination, similar synergistic effects and permanent graft survival could also be obtained by oral A802715 (100 mg/kg/day) in combination with a low dose of CsA (2.5 mg/kg/day). In both strain combinations, long-term survivors accepted donor-type but rejected third-party second grafts in the absence of immunosuppression. This specific tolerance was not related to clonal deletion nor anergy, as recipient lymphocytes proliferated normally in the anti-donor mixed lymphocyte reaction. Instead, a defect in generating specific cytotoxic T lymphocytes was involved. A802715 synergizes with CsA in vivo to induce specific transplantation tolerance and hence should be considered as a promising new immunosuppressant.