Mixed Lymphocyte Culture Tests Research Articles

In vitro testing of immunosupressive effects of mesenchymal stromal cells on lymphocytes stimulated with alloantigens

Mesenchymal stromal cells (MSC) derived from adult bone marrow or adipose tissue offer the potential to open a new frontier in medicine. MSC are involved in modulating immune response and tissue repair in vitro and in vivo. Experimental evidence and preliminary clinical studies have demonstrated that MSC exhibit an important immunomodulatory function in patients with graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. The immunosuppressive properties of MSC have already been exploited in the clinical setting. However the precise mechanisms are being still investigated. We examined the immunosuppressive function of MSC by coculturing them with stimulated HLA incompatible allogeneic lymphocytes in a mixed lymphocyte culture test. The metabolic and proliferative activity of lymphocytes was determined by MTT test. After stimulation with alloantigens the presence of MSC caused significant decrease of absorbance levels by 62% (P<0.01), 26% (P<0.01) and 6% (P=0.0437) in comparison to positive control depending on the MSC/lymphocyte ratio (1:5, 1:50, 1:500). The mitogenic stimulation of lymphocytes with fMLP or PHA was also significantly reduced during MSC cocultivation. The absorbance was reduced by 42% (P<0.001) and 67% (P<0.001). Allogeneic bone marrow is an ideal source of MSC for clinical application. The experiments confirmed the dose-dependent inhibitory effect of MSC on lymphocyte proliferation triggered by cellular or mitogenic stimulation. The mixed lymphocyte culture test offers a simple method for characterization and verification of the immunosuppressive potential of MSC, being prepared for clinical use.

The predictive value of stimulation index calculated by modified mixed lymphocyte culture in the detection of GVHD following hematopoietic stem cell transplantation.

Mixed lymphocyte culture (MLC) is one of the routine tests performed prior to hematopoietic stem cell transplantation (HSCT) as a predictive assay for assessing the quality of donor matching and graft-versus-host disease (GVHD). The stimulation index is one of the formulas of the MLC test, and it is used for evaluation of matching between donor and recipient. Modified MLC (mMLC) test is produced by adding various cytokines to the MLC test, and increased sensitivity has been reported with this modification. The importance of the stimulation index values in MLC and mMLC tests was evaluated in 59 patients who received HSCs from human leukocyte antigen-identical sibling donors. In the mMLC test, cytokines were added as interleukin (IL)-2, IL-2 + IL-4 and IL-2 + interferon (IFN)-gamma + tumor necrosis factor (TNF)-alpha. Stimulation index values in mMLC test were compared with stimulation index values in MLC test. Twenty-three (39%) patients developed GVHD. When evaluated in terms of stimulation index >1 patients, in MLC, 55% of the patients developed GVHD (p=0.229), whereas these values were 75% in the IL-2 added mMLC test (p=0.035), 100% in the IL-2 + IL-4 added mMLC test (p=0.076) and 85.7% in the IL-2 + IFN-gamma + TNF-alpha added mMLC test (p=0.015). mMLC increased the sensitivity of the test. The relation between the positive results and evidence of GVHD after transplantation was found significant.

Delayed Recovery of Accelerated Acute Rejection

AbstractAccelerated acute rejection (AAR) after kidney transplantation may occur because of the pre‐sensitization of the host cell against the graft antigens. We report the case of a 49‐year‐old female, under chronic hemodialysis therapy who received a living transplant from her husband. Both of them had the same blood type and the result of the mixed lymphocyte culture test was acceptable before transplantation. The operation was smooth and a combination therapy with cyclosporine, prednisolone, and mycophenolate mofetil (MMF) was given. However, oliguria developed on the second day after transplantation. After excluding the possibility of occlusive lesion, the first course of pulse steroid therapy was given under the impression of AAR, supported by renal biopsy. After the second course of pulse steroid and OKT3 therapy, her renal function did not recover. Thereafter, she received regular hemodialysis therapy and cyclosporine was replaced by tacrolimus. Two months later, her urine amount increased dramatically. The renal biopsy revealed changes of interstitial cells infiltration without acute rejection. The patient's serum creatinine level declined to 1.76 mg/dl 6 months later, and was maintained at around 1.1 mg/dl after 4.5 years. This is a demonstration of a delayed recovery of AAR combined with the suspicion of cyclosporine‐related nephrotoxicity. Protocol repeated biopsies may be required in those patients who do not recover after an initial rejection to detect early second pathologies.

Dose-dependent induction of IL-6 by plant-derived proteasesin vitro

Oral administration of proteases such as bromelain and papain is commonly used in patients with a wide range of inflammatory conditions, but their molecular and cellular mechanisms of action are still poorly understood. The aim of our study was to investigate the impact of these proteases on the release of interleukin-6 (IL-6) and other cytokines in the recently described modified mixed lymphocyte culture (MMLC) test system which is based on the mutual interaction of cells of the innate and adaptive immunity. Bromelain and papain enhanced IL-6 production dose-dependently up to 400-fold in MMLC before and up to 30-fold after neutralization of LPS content of proteases using polymyxin B, indicating that IL-6 induction by protease treatment was attributable to both protease action and LPS content of enzyme preparations. The production of IFNgamma and IL-10 was not altered by bromelain or papain, indicating a selective and differential immune activation. Both proteases impaired cytokine stability, cell proliferation and expression of cell surface molecules like CD14 only marginally, suggesting no impact of these mechanisms on protease-mediated cytokine release. These findings might provide the mechanistic rationale for the current use of proteases in wound healing and tissue regeneration since these processes depend on IL-6 induction.

RhG-CSF effect on mixed lymphocyte cultures and circulating soluble HLA antigen levels in volunteer stem cell donors

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a cytokine widely used in the procurement of peripheral blood stem cells (PBSC) from donors for allogeneic hematopoietic cell transplantation. Therefore, we were interested in its immediate and long-term effects on cellular and soluble factors known to be involved in the immune response. We studied 35 PBSC donors by mixed lymphocyte culture (MLC) and lymphocyte transformation test (LTT), and 41 for soluble plasma factors (soluble human leukocyte antigen [sHLA]-G, -class I, -DR, and interleukin [IL]-10) pre and 5 days post initial rhG-CSF administration, respectively. In addition, 10 donors were reexamined at an average of 2 months (3-16 weeks) post-rhG-CSF. At 5 days post-rhG-CSF the donors presented a significant (p < 0.05) decrease of MLC, LTT mitogen, and recall antigen reactions. Plasma levels of sHLA-G, -class I, -DR, and IL-10 (p < 0.005 each) were significantly increased. The changes in IL-10 but not in sHLA were significantly (p < 0.05) correlated with LTT responses. In the 2-month follow-up there was no significant difference in alloreactivity and LTT reactions as compared to the pre-rhG-CSF results. The results generated after 3 to 16 weeks did not depend on the time point of investigation. Consistently, soluble factors decreased to pre-rhG-CSF levels. rhG-CSF administration suppresses cellular immune functions within 5 days and increases sHLA and IL-10 plasma levels. These immunomodulatory effects appear to be short-term only and vanished at an average of 2 months after rhG-CSF application.

Human liver allograft acceptance and the "tolerance assay": in vitro anti-donor T cell assays show hyporeactivity to donor cells, but unlike DTH, fail to detect linked suppression.

Human allograft acceptance is associated with immune regulation, characterized by donor-antigen-linked suppression of delayed-type hypersensitivity (DTH). We wished to determine if "classical" in vitro assays of alloreactivity could also detect linked suppression and thus be useful in the clinical diagnosis of active immune regulation. We analyzed peripheral blood mononuclear cells from a group of eight liver transplant recipients, one of whom had stopped all immunosuppression 4.5 years ago yet continues to have good graft function (graft acceptor). The regulator phenotype was defined as the ability to suppress a DTH response to a recall antigen in the presence of donor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro mixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specifically hyporesponsive to donor compared with third-party B-LCL stimulators. However, in contrast to the linked suppression of DTH seen when a given B-LCL expressed donor-type HLA-B antigens, there was no evidence of linked suppression in vitro, either in CTL, proliferative, or interferon-gamma cytokine release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor beta or interleukin-10, which could restore a strong DTH response to donor B-LCL. We conclude that DTH analysis can readily detect donor antigen-linked suppression in liver transplant recipients. CTL and MLC tests failed to do so. These findings may be relevant to the development of a tolerance assay suitable for use in clinical trials.

Vascular structure and microcirculation of experimental pancreatic carcinoma in rats.

To study angiogenesis and microcirculation in experimental pancreatic carcinoma. Open experimental study. 2 University hospitals, Germany and USA. 16 male Lewis rats. Induction of a duct-like pancreatic cancer in the pancreas and peritoneum by interposition of fragments of tumour between 2 inert transparent polymethylmethacrylate plates. Microcirculation in the tumour and interaction between leucocytes, tumour, and endothelium investigated by intravital microscopy. The density of vessels in the carcinoma was significantly less than in normal pancreatic tissue (p = 0.0004). The vasculature of the tumour was characterised by a lack of differentiation in architecture of vessels, formation of sinusoidal and lacunar vessels and sudden changes in diameter of vessels. Red blood cell velocity differed among tumour vessels, but mean values were similar to those of normal exocrine pancreas. The mixed lymphocyte culture test indicated that the cell line DSL6A was immunogenic. However, high-affinity leucocyte-endothelium-interaction was significantly reduced in the tumour's microcirculation after both orthotopic and heterotopic implantation (p = 0.002). Rates of apoptosis were suppressed in heterotopic tumours compared with orthotopic ones. Tumour growth was faster in heterotopic tumours. Experimental duct-like pancreatic carcinoma can be differentiated from normal pancreas by: chaotic arrangement of vessels with loss of differentiation of architecture and heterogeneous distribution; the formation of sinusoidal or lacunar vessels; lower vascular density with similar erythrocyte velocity; increase in mean diameter of vessels; reduced leucocyte-endothelium interaction despite confirmed immunogeneity independent of wall shear rates. The site of implantation influences apoptosis and growth rates.

Induction of specific inhibition of alloreactivity in beagle dogs by intrathymic injection of donor splenocytes

Abstract The aim of this study was to investigate whether intrathymic injection (ITI) of donor splenocytes in dogs might lead to specific immu-nomodulation as assessed by mixed lymphocyte culture (MLC) tests. Two groups of five beagles each were used. Group 1 contained animals that were 2 years old, group 2 consisted of animals that were 6 months old. One animal was splenectomized per experimental group and 1 × l09 sple nocytes were injected into the thymic lobes or thymic remnant of the four remaining dogs. On the day of ITI the dogs were treated subcutaneously with a single dose of 2 ml/kg anti-lymphocyte serum (ALS). In group 1 the thymus of all dogs was found to be atrophic. ITI in this group did not result in a decreased immunoreactivity but rather in an enhanced immune response. In group 2 the thymus was still clearly present and ITI was easy to perform. ITI induced a significant reduction of specific MLC reactivity at 1 week after treatment. The effect was transient and not significantly further diminished at week 2. These results indicate that ITI is a techni cally feasible procedure in a preclini-cal animal model. It may induce temporary sensitization as well as immu-nosuppression, possibly depending on the age of the recipient.

Lack of correlation of MLC reactivity with acute graft-versus-host disease and mortality in unrelated donor bone marrow transplantation

Acute graft-versus-host disease (AGvHD) is a significant cause of morbidity and mortality in patients receiving a bone marrow transplant from an unrelated donor, and in an effort to reduce this problem, donors are selected for the least possible HLA incompatibility with the recipient. Selection criteria have included minimal incompatibility for the HLA-A, -B, and -DR loci and low reactivity in mixed lymphocyte culture (MLC); however, the value of MLC reactivity for prediction of development of AGvHD has been questioned. We therefore examined the correlation of MLC reactivity with AGvHD in recipients of unrelated bone marrow transplants. Reactivity in the GvH direction was assessed as relative response (RR) of donor lymphocytes to recipient stimulator lymphocytes. In 126 transplanted pairs with technically satisfactory MLC tests, the RR was divided into quartiles (0–1, 2–5, 6–16, and 17–117% RR). HLA-DRB1 incompatibilities were more frequent in the highest quartile ( p < 0.001); there were no significant differences among quartiles in donor or recipient age, diagnosis, or frequency of HLA-A or -B incompatibility. Incidence of AGvHD during the first 100 days post-transplant was assessed by Kaplan-Meier analysis. There was no significant difference in incidence of AGvHD among quartiles for the entire group of 126 pairs, for a subset with hematologie. malignancy, for a subset selected by a more stringent standard for “technically satisfactory” MLC, or for a subset matched for A, B, and DRB1. The MLC response of donor lymphocytes to recipient stimulator lymphocytes is thus not predictive of development of AGvHD in our patient population receiving unrelated donor bone marrow. Since there was no difference in mortality related to high and low MLC responses, our data also suggest that MLC results are not predictive of survival in this population. Human Immunology 49, 49–55 (1996)

Direct activation of human responder T cells by porcine stimulator cells leads to T cell proliferation and cytotoxic T cell development

Abstract: Functional activities of highly purified T human responder lymphocytes reactive against porcine stimulator cells were studied to evaluate whether porcine stimulator cells can directly activate a xenospecific cellular immune response. Mixed lymphocyte culture (MLC) tests revealed that CD4+ human responder cells proliferate when stimulated in vitro with porcine cells, a reaction similar to that of alloresponses regarding magnitude and tempo. A direct pathway of activation was verified by the requirement for adherent porcine stimulator cells and the partial blocking by monoclonal antibodies against porcine major histocompatibility complex (MHC) class II antigens. An analysis of proliferative CD4+, CD3+, CD16‐, and 56‐ human T cell clones revealed that some clones were seemingly recognizing allele‐specific determinants, whereas others could be restimulated by a wide range of porcine stimulator cells. Cytotoxic T cells (CTLs) were generated following direct recognition of pig cell ligands by human T cells in the absence of autologous antigen‐presenting cells (APCs). Although the identification of target antigen(s) on the pig cell recognized by the CTL warrants some discussion, the pattern of killing exhibited by the CTLs indicates the recognition of porcine polymorphic determinant(s). The implications of these findings for cellular reactivity against porcine transplants are discussed.

Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: matching for transplants from related haploidentical donors.

The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-DR region by HTC typing. Donor antirecipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II-IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.

The changing activities of a regional marrow donor program.

A regional marrow donor program was established in 1982. Following the establishment of the National Marrow Donor Program (NMDP) in 1987, the activities of this regional program changed. To better understand the changes that occurred in the regional marrow donor program, its donor recruitment and marrow collection activities through 1991 were studied retrospectively. Data analyzed included the total number of potential donors, the number and types of potential donors recruited each year, the number of searches performed, the number of samples collected for HLA-DR typing and mixed lymphocyte culture testing, and the number of transplants in both programs from 1987 through 1991. Statistical analysis was performed by using chi-square. Initially, only persons who donated platelets by apheresis were enrolled into the program. In 1986, the regional program's first drive to recruit people who were not apheresis donors occurred. The number of such drives increased each year, and in 1991, 12 drives occurred, which resulted in the recruitment of 1313 potential marrow donors. From 1987 to 1991, the number of potential donors in the regional program grew from 3252 to 9146, but the proportion of apheresis donors in the program decreased. In 1987, 91.9 percent of marrow donors at the regional center had been apheresis donors, but in 1991, 41.7 percent had been apheresis donors. The number of marrows donated at the regional center increased from 11 in 1987 to 29 in 1989, but then fell to 24 per year in 1990 and 1991. The decrease in the number of donations at the regional program was due to the rapid growth in the NMDP file of potential marrow donors and the selection of donors whose HLA antigens were more compatible with those of the transplant recipients. In 1989, the regional program contained 4.6 percent of all HLA-A,B-typed and 11.2 percent of all HLA-A,B,DR-typed potential donors in the NMDP and collected 15.3 percent of all marrows. However, in 1991, the regional program contained 2.0 percent of HLA-A,B-typed donors and 4.1 percent of HLA-A,B,DR-typed donors and collected 5.3 percent of marrows. In 1987, 18 percent of the people who donated marrow at the regional center were phenotypically HLA-A,B,DR identical with the recipient, but in 1991, 92 percent of donor-recipient pairs were phenotypically HLA-A,B,DR identical. Recruitment activities became an increasingly larger part of the Regional Marrow Donor Program's activities. Increasing the size of the file of potential donors was necessary to maintain a constant number of donations. Persons who were not regular blood donors were an important part of the marrow donor program.

The immunogenicity of glycerol-preserved donor skin

Previous clinical observations have suggested that the application of glycerol-preserved donor skin as a temporary wound dressing provokes a weaker rejection reaction than fresh, vital donor skin. Like others, we frequently observed that considerable parts of the allodermis not only remained on the wound for an extended period of time, but even became re-epithelialized in some cases. In order to quantify this effect, we applied the mixed lymphocyte culture (MLC) test in a rat model, using the two highly inbred, histoincompatible rat strains DA and Lewis as donor and recipient respectively. Using the methodology of the Euro Skin Bank, Beverwijk, The Netherlands, split thickness skin, excised from the back of the rats, was equilibrated in 98 per cent glycerol. The immunological reaction after grafting vital DA-skin, glycerolized DA-skin onto Lewis rats, and vital as well as glycerolized Lewis-skin onto Lewis rats was compared. The results of these experiments do not support the clinical observations that the glycerolization procedure results in decreased immunogenicity of donor skin.

Analysis of mixed lymphocyte culture testing to determine predictability of uninterpretable results: [formula omitted], S Shepherd, J Wisecarver, R Rubocki, Transplant Immunology, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE

Analysis of mixed lymphocyte culture testing to determine predictability of uninterpretable results: [formula omitted], S Shepherd, J Wisecarver, R Rubocki, Transplant Immunology, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE

Immunological Consequences of Exposure to Pentachlorophenol

Evaluation of lymphocyte phenotype frequencies, functional responses, serum immunoglobulin levels, and autoantibodies was completed for 38 individuals (i.e., 10 families) who were exposed to pentachlorophenol (PCP) in manufacturer-treated log houses. Comparison of subjects with controls revealed that the exposed individuals had activated T-cells, autoimmunity, functional immunosuppression, and B-cell dysregulation. Autoimmunity was evidenced by elevation of TA1 phenotype frequencies and a 21% incidence of anti-smooth muscle antibody. Functional immunosuppression was evidenced by the significantly reduced responses to all mitogens tested and to allogeneic lymphocytes in the mixed lymphocyte culture test. There was a significant elevation of CD10, and an 18% increase or decrease in serum immunoglobulins was noted. A striking anomaly was the enhanced natural killer activity found in exposed females but not in males.

Comparison of the effectiveness of potential marrow donors recruited from apheresis donors or whole blood donors and through appeals to the general public

Marrow transplants with phenotypically HLA-matched, unrelated donors have been used effectively to treat a number of diseases. Many blood centers have recruited HLA-typed apheresis blood donors into marrow donor registries. However, to build larger registries so that more patients may be treated with unrelated donor marrow transplants, whole blood donors and people who do not donate blood have been added to the registries. The marrow donor program at our blood center had 2844 potential donors, of whom 1725 were also apheresis donors, 608 were whole blood donors, and 511 were recruited from the general public as a result of community appeals for marrow donors for a specific patient. Over a 9-month period, 297 potential donors were asked to donate blood samples for HLA-DR typing or mixed lymphocyte culture (MLC) testing or to participate in an informational session, undergo a medical evaluation, and sign a statement indicating an intention to donate marrow for a specific patient. Overall, these requests were successfully completed by 75.5 percent of apheresis donors, 87.2 percent of whole blood donors, and 78.1 percent of the potential donors recruited through community appeals. Furthermore, there was no difference among the three groups in the portion of people who donated blood samples for HLA-DR typing or MLC testing. Fifteen of 18 apheresis donors who were found to match a specific donor signed a statement of intent to donate marrow, 2 apheresis donors were deferred for medical reasons, and 1 decided not to donate.(ABSTRACT TRUNCATED AT 250 WORDS)

PCR‐SSO TYPING FOR DR4‐Dw SUBTYPES: APPLICATION TO UNRELATED BONE MARROW TRANSPLANT DONOR SELECTION

Thirty-seven DR4-positive patient-unrelated bone marrow donor pairs previously DR/DQ restriction fragment length polymorphism (RFLP) typed and tested in mixed lymphocyte culture (MLC), have been DR4-Dw subtyped retrospectively using sequence specific oligonucleotide probes. We found that DR4-Dw subtyping substantially increased the accuracy of pre-MLC matching and could potentially accelerate donor searches by avoiding unnecessary MLC tests on Dw-mismatched donors.

FK506 suppression of heart and liver allograft rejection. II: The induction of graft acceptance in rats.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.

Serial Mixed Lymphocyte Culture Responses in Pigs after Liver or Kidney Auto- or Allotransplantation

In order to investigate further the peculiarly limited response of the transplanted pig liver to rejection, this study investigated serial mixed lymphocyte culture (MLC) responses and suppression of phytohaemagglutinin (PHA)-stimulated lymphocytes by serum from transplanted animals. The responses of lymphocytes from liver allografted pigs were compared with those from sham-operated, liver-autografted or kidney auto- or allografted animals for up to 22 weeks postoperatively. Lymphocytes from operated animals were added to cultures of 2 'universal' donors which were not anaesthetised at any time and which remained lymphocyte donors for the period of each operated pig's survival. After liver allograft, a sustained stimulation of MLC responses was noted which contrasted with the minimal random stimulatory activity seen in the other groups. The response was not seen in lymphocytes from animals subjected to liver autograft. There was a surprising variability in the week-to-week MLC tests of individual animals for which no cause could be found. The serum from liver auto- and allografted pigs had suppressive action on the PHA stimulation of lymphocytes. There was no correlation between the length of survival and the intensity of the pre-operative MLC response. Whilst the biochemical data of liver allografted pigs showed evidence of ongoing hepatocyte damage, the histological changes were less obvious especially in the long-surviving group. This study highlights again the ability of unimmunosuppressed pigs to accept liver allograft and seems to associate this with sustained hepatocyte damage and stimulation of the MLC.

Matching for Supertypic DR Epitopes Does Not Reduce Mixed Lymphocyte Culture Reactivity

It was recently demonstrated that matching for the supertypic DR specificities DRw52 and DRw53 significantly improved graft survival in a group of retrospectively typed kidney transplant patients. We investigated whether this phenomenon was also reflected in vitro in the mixed lymphocyte culture (MLC) test system. MLC matrices were set up, but no effect of the matching between responder and stimulator for DRw52 and DRw53 was observed, although matching for the DR 'private' specificities (DR1-DRw10) was significant. In responder-stimulator combinations, too, which were mismatched for 1 DR antigen only, it did not matter whether or not the mismatched antigen on the stimulator was DRw52 or DRw53 compatible to the responder. These findings imply that matching for supertypic DR epitopes does not necessarily lead to reduced MLC reactivity.