R. Duquesnoy

16th IHIW: a website for antibody-defined HLA epitope Registry.

The concept that HLA antibodies are specific for epitopes rather than HLA antigens is important not only for the determination of mismatch acceptability for sensitized patients but also for a better understanding of the antibody response to an HLA mismatch. Numerous publications describe epitope‐specific antibodies, but there is no standardized information about the repertoire of clinically relevant HLA epitopes. Under auspices of the 16th IHIW, we have developed a website‐based registry of antibody‐verified HLA epitopes. Epitope notations are based on HLA molecular modelling of amino acid residues in polymorphic sequence positions. Informative epitope‐specific antibodies had been induced by a transplant, transfusion or pregnancy and were monoclonal antibodies or eluates of sera absorbed with single HLA alleles. Antibody reactivity was determined in binding assays with single‐allele panels. Antibody producer/immunizer HLA types enhanced the characterization of specific epitopes. The Registry also includes epitopes described in original research publications. Based on the extent of antibody reactivity information, we assigned epitope status as confirmed (well documented) or provisional (more data are needed). At present, the Registry has 69 HLA‐ABC, 53 DRB1/3/4/5, 17 DQ, 8 DP and 22 MICA antibody‐verified epitopes and will be updated on a quarterly basis. Laboratories worldwide continue to submit data about previously unreported antibody‐specific epitopes. For each epitope, the website shows its amino acid composition and HLA alleles that share the epitope. Links show antibody reactivity patterns, sensitization information and references. Other links show molecular modelling of corresponding structural epitopes and polymorphic residue information for epitope‐carrying alleles. The website will also have a link to epitope frequency information in different populations. Search functions will list mismatched epitopes on mismatched alleles for selected HLA types. The HLA Epitope Registry will become a valuable resource for researchers interested in HLA compatibility at the epitope level and investigating antibody responses to HLA mismatches.

ISOLATION AND PRIMARY CULTURES OF HUMAN INTRAHEPATIC BILE DUCTULAR EPITHELIUM

SummaryA technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments.

Sensitivity of activated human lymphocytes to cyclosporine and its metabolites

Alloreactive T cells generated as clones from mixed lymphocyte cultures, or propagated from heart or liver transplant biopsies, were tested for secondary proliferation measured in the primed lymphocyte test in the presence of Cyclosporine A and metabolites fractionated from human bile. Significant differences were observed in Cyclosporine A sensitivity between various cell cultures ranging as high as 100-fold. The liver is the primary site of Cyclosporine A metabolism, which yields a number of hydroxylated and N-dimethylated derivatives that are eventually secreted into the bile. Bile was collected from adult liver transplant patients on Cyclosporine A therapy and following extraction with diethyl ether, separated by high pressure liquid chromatography. Thirteen fractions were tested for their effect on lymphocyte proliferation in concanavalin A activation, mixed lymphocyte cultures and primed lymphocyte test assays. The strongest immunosuppressive effect was found with fraction 8, which contained metabolite M17, which has a single hydroxylation in position 1. Only three other fractions 9, 10, and 13, which contained metabolites M1, M18, and M21, respectively, exhibited immunosuppressive activity, albeit much lower than that of Cyclosporine A. Differences in Cyclosporine A sensitivity among alloreactive T cells followed similar patterns with Cyclosporine A metabolites. Thus, the assessment of the Cyclosporine A effect must consider differences in drug sensitivity of lymphocytes involved in transplant immunity and the generation of metabolites with immunosuppressive activity.

First report on the antibody verification of HLA-ABC epitopes recorded in the website-based HLA Epitope Registry

The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

Relationship between the diagnosis, preoperative evaluation, and prognosis after orthotopic liver transplantation

The purpose of this study was to identify which of the biochemical, immunological, or functional parameters derived before surgery as part of a systemic evaluation were helpful in predicting the frequency of rejection episodes, the chance of survival, and the cause risk of death (should death occur) of patients after orthotopic liver transplantation (OLTx). Ninety-eight adult patients who had an extensive preoperative protocol evaluation were studied before OLTx. The biochemical parameters assessed were albumin, prothrombin time, bilirubin, and ICG clearance. The immunologie parameters assessed included total lymphocytes, T3 cells, T4 cells, T8 cells, and the T4/T8 ratio. The degree of histocompatibility antigen (HLA) matching between the donor and- the recipient was also evaluated in 80 of the 98 patients studied.Most postoperative deaths occurred within 12 weeks of the procedure (24%; 24 of 98 patients); 13 patients (13%) died within the first 6 postoperative weeks, of either bacterial or fungal sepsis. An additional 14 patients (14%) died after the initial 6 postoperative weeks due, primarily of an acquired viral and/or protozoan infection (p < 0.01).During the first 6 weeks, survival was better for patients with cholestatic liver disease (ChLD, 93%, n = 45) and miscellaneous liver diseases (MISC, 100%, n = 10) than it was for those with parenchymal liver diseases (PLD, 77%, n = 43).Although albumin, prothrombin time, T4/T8 ratios, and per cent T8 cells were statistically different in patients with PLD as compared with those with ChLD, these parameters, as well as the per cent T4 cells, serum bilirubin level, per cent retention of ICG at 15 minutes, and the plasma ICG disappearance rate were not found to be of substantial help in predicting patient survival or nonsurvival.Moreover, neither the degree of HLA matching nor the number of rejection episodes differed between surviving and nonsurviving patients.The results of this study suggest that patients with PLD are at increased risk of early postoperative death after OLTx because of bacterial and/or fungal sepsis, as compared with patients operated upon for ChLD. Better pre-, intra-, and postoperative predictors of risk of death and complications are needed to reduce the early mortality observed after orthotopic liver transplantation.

Progress report on the ASHI/CAP proficiency survey program in histocompatibility testing. I: HLA-A,B,C typing, antibody screening, and lymphocytotoxicity crossmatching

The Histocompatibility Survey Program was organized in 1982 as a joint project by the ASHI and CAP to evaluate laboratory performance in HLA typing, lymphocytotoxicity crossmatching, and antibody analysis. This report summarizes the experience with the HS surveys on HLA class I serology. During a 12-year period, the number of participating laboratories increased from 150 to 285 and HLA typing was done with 90 survey specimens representing 20 HLA-A and 35 HLA-B antigens. Most unsplit antigens were correctly identified in more than 90% of the laboratories. For many antigens, a high percentage of participants reported a split and there was generally a high consensus of a correct assignment. Nevertheless, several antigens were difficult to define, as shown by low consensus rates. During recent years, the assignments of Bw4/6 and HLA-C antigens have significantly improved. Lymphocytotoxicity crossmatching was analyzed for 138 cell-serum combinations tested by an average of 143 laboratories. Comparisons between four techniques (basic NIH, Amos modified, LI, and AHG) showed consistent results (greater than 90% crossmatch compatibility or incompatibility) for 71% of the cell-serum combinations. The crossmatch results with the remaining combinations were more variable for one or more of the crossmatch techniques. Serum antibody identification showed a continued improvement during recent years, and the average consensus for assigning acceptable antibody specificity reached 88%. A performance grading system based on a 90% consensus rate among participants is used to satisfy requirements for laboratory accreditation.

Species differences in sensitivity of T lymphocytes to immunosuppressive effects of FK 506.

FK 506 is a novel immunosuppressive drug isolated from Streptomyces tsukubaensis in 1984 (1). Initial in vitro studies have indicated that FK 506 is a potent inhibitor of murine and human mixed lymphocyte reactions and can prevent the generation of cytolytic cells. FK 506 suppresses T cell–dependent responses and this effect appears to be mediated through the inhibition of interleukin-2 release and diminished expression of the IL-2 receptor on activated T cells (2, 3). The strong immunosuppressive effect of FK 506 has been observed during primary activation of lymphocytes, such as the mixed leukocyte reaction and the secondary proliferation of alloreactive T lymphocytes generated from MLR or propagated from organ transplant biopsies as measured by the primed lymphocyte test (4). n nAlthough both FK 506 and cyclosporine exhibit similar mechanisms of action, the immunosuppressive effect of FK 506 is several-hundred-fold greater than that of CsA. Synergism between the two drugs has been observed in vivo and in vitro, as demonstrated by significant immunosuppressive effects of combinations of drugs at low doses that are individually ineffective (5, 6). Synergism has also been noted between FK 506 and azathioprine (7). n nIn vivo studies have also established the effectiveness of FK 506 in several allograft models. Todo et al. showed that FK 506 greatly prolongs allograft survival of heterotopic heart transplants in rats (8–10), and renal and liver transplants in dogs (11). In these experiments, FK 506 was found to be effective at dose levels of 1 – 1.5 mg/kg/day—however, preliminary studies with kidney transplants performed in baboons indicated that this dose of FK 506 was ineffective in prolonging graft survival (12). In vitro studies were conducted to quantitate the immunosuppressive effects of FK 506 on the MLR with lymphocytes from baboons, dogs, rats, and humans. n nHeparinized blood was collected from normal dogs (n = 9), rats (n = 3), baboons (n = 10) and humans (n = 8). Mononuclear cells were isolated by Ficoll-Hypaque sedimentation and uni-directional MLR cultures were set up in triplicate with equal numbers (106/ml) of responders and irradiated stimulators in 10% autologous serum and incubated for 6 days at 37°C and 5% CO2. During the final 20 hr of incubation, each culture was labeled with 1 µCi of [3H]-thymidine (ICN Radiochemicals, 1 mCi/ml), harvested, and counted in a liquid scintillation counter. The dose effect of FK 506 and CsA on MLR activity was measured at different concentrations of the drug ranging from 0.03–10 ng/ml for FK 506 to 3.5–2500 ng/ml for CsA. For each culture the ID50 dose was calculated as the concentration of FK 506 or CsA that caused a 50% inhibition of the MLR. n nFigure 1 illustrates, that the ID50 of FK 506 was about 10-fold higher for baboon lymphocytes (3.85±2 ng/ml) than for lymphocytes from the other three species (rats 0.33±0.2 ng/ml; humans 0.29±0.2 ng/ml; dogs 0.59±0.58 ng/ml). These differences in FK 506 sensitivity were statistically significant (P<0.001). Similar differences between ID50 values were observed for CsA, although their magnitudes were somewhat lower (Fig. 2). The ID50 of CsA was Significantly higher for baboons (212±161 ng/ml) than for rats (45±4 ng/ml); humans (50.5±28.5 ng/ml) and dogs (40.8±18.5 ng/ml). These data also extend previous observations that FK 506 is 100–300 times more potent in inhibiting MLR than CsA (4). n n n nFIGURE 1 n nID50 values for FK 506 in MLR cultures from rats, human, dogs, and baboons. Each point represents one experiment done in triplicate. Statistical analysis of the ID50 values was done using the unpaired Students t test. The analysis was done for baboons ... n n n n n nFIGURE 2 n nID50 values for CsA in MLR cultures from rats, humans, dogs, and baboons. Each point represents one experiment done in triplicate. n n n nComparable differences in FK506 sensitivity were observed in vivo in different allograft models. Table 1 summarizes the survival rates of kidney transplants in baboons versus kidney transplants in dogs and cardiac transplants in rats. In the baboon and dog models FK 506 was administered orally, in the rat model the drug was administered intramuscularly. Successful prolongation of RT-I-incompatible cardiac allograft in rats was achieved at FK506 doses of 1.28 mg/kg/day with a mean survival of 99 days (10), whereas untreated allografts only survived 6 days. At daily doses of 1.5 mg/kg/day, FK 506 prolonged allogeneic renal transplants from mongrel to beagle dogs from 14 days to 61 days (11). However, a similar dose of FK 506 (2 mg/kg/day) induced a modest and insignificant prolongation of renal transplant survival in baboons (16 days versus 9 days for untreated controls). Considerably higher doses of FK 506 (12 and 18 mg/kg/day) were necessary for baboons to achieve allograft survivals comparable to those observed in rats and dogs at lower doses (Table 1). n n n nTABLE 1 n nEffect of FK 506 on allograft survival in the three animal species n n n nThe results of these in vivo and in vitro studies demonstrate species differences in sensitivity in the immunosuppressive effects of FK 506. The baboon model requires almost tenfold higher doses of FK 506 than the rat and dog models to achieve optimal immunosuppression. The in vitro assays indicate that FK 506 sensitivity of human lymphocytes is more similar to that for dogs and rats rather than baboons. These findings suggest that FK 506 at doses of 1.5 mg/kg/day might provide effective immunosuppression of human allografts. However only clinical trials can establish the optimal administration of this drug. n nIn transplant patients the optimization of immunosuppressive treatment depends on the administration of appropriate doses that have minimal side effects and the monitoring of drug levels. Pharmacokinetic and pharmacodynamic evaluations have aided in developing individualized immunosuppressive therapy (13). Furthermore drug sensitivity of in vitro lymphocyte activation seems useful, and considerable variations have been seen between different individuals (14). In this study species differences in FK 506 sensitivity of MLR reflect the efficacy of FK 506 treatment in prolonging allograft survival. Thus in vitro studies of drug sensitivity of lymphocyte activation are useful in determining optimal drug doses to ensure successful transplantation.

Propagation Of Lymphocytes Infiltrating Human Liver Allografts: Correlation With Histologic Diagnosis Of Rejection

In this study, we have investigated whether lymphocyte growth from 196 liver transplant biopsies is correlated with the histologic diagnosis of graft rejection. One fragment of each biopsy was cultured in IL-2 to expand activated T cells, and the remainder of the biopsy was analyzed histologically. A significantly higher number of biopsies with rejection grew lymphocytes when compared to those biopsies showing no evidence of rejection (P = 0.009). Lower growth rates were observed with biopsies taken from patients on OKT3 therapy (12% vs. 29% from patients not on OKT3), so when the data were reanalyzed after omitting those biopsies we observed an even stronger correlation of growth with rejection (P = 0.001). In spite of the fact that hepatitis biopsies are also infiltrated by lymphoid cells, the frequency of lymphocyte growth (16%) was similar to that observed for the biopsies with no rejection or hepatitis (19%) and much lower than for biopsies with rejection (42%). Over all, 82% of the cultures tested were positive for donor PLT reactivity, suggesting that current culture conditions favor alloreactive lymphocyte proliferation. The size of the biopsy cultured was found to be an important factor in the propagation of biopsy-infiltrating lymphocytes. Only 25% of smaller (less than 3 mm long) biopsies with rejection grew lymphocytes compared to 91% of the large-sized biopsies (greater than 5 mm long). It is likely that culture techniques will need to be modified in order to successfully propagate infiltrating lymphocytes that recognize antigens other than alloantigens.

First report on the antibody verification of MICA epitopes recorded in the HLA epitope registry

The International Registry of HLA Epitopes (http://epregistry.com.br) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three‐dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty‐one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. The Registry is still a work‐in‐progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

Update of the HLA class I eplet database in the website based registry of antibody-defined HLA epitopes

Eplets are small configurations of polymorphic amino acid residues on human leukocyte antigen (HLA) molecules and are considered as essential components of HLA epitopes recognized by antibodies. This report describes a new design of the eplet repertoire in HLA-ABC alleles used in Luminex kits for antibody testing. There were three steps (1): identify all combinations of polymorphic residues with HLA molecular modeling within a 3-Å radius, (2) determine polymorphic residue compositions of 3u2009Å patches from amino acid sequences of HLA alleles in Luminex panels and (3) annotate eplets from one or more patches present on one allele or shared by the same group of alleles. There are now 270 HLA-ABC eplets in the Registry, of which 219 are in antibody-accessible positions on the molecular surface and 51 are defined solely by residue polymorphisms located below the molecular surface. Each eplet has a list of Luminex and non-Luminex alleles for which mismatch acceptability can be determined.