Jean-Denis Bignon

Frequency and Clinical Implications of Development of Donor-Specific and Non–Donor-Specific HLA Antibodies after Kidney Transplantation

The involvement of immunologic and nonimmunologic events in long-term kidney allograft failure is difficult to assess. The development of HLA antibodies after transplantation is the witness of ongoing reactivity against the transplant, and several studies have suggested that the presence of HLA antibodies correlates with poor graft survival. However, they have not discriminated between donor-specific (DS) and non-specific (NDS) antibodies. A total of 1229 recipients of a kidney graft, transplanted between 1972 and 2002, who had over a 5-yr period a prospective annual screening for HLA antibodies with a combination of ELISA, complement-dependent cytotoxicity, and flow cytometry tests were investigated; in 543 of them, the screening was complete from transplantation to the fifth year postgrafting. Correlations were established between the presence and the specificity of the antibodies and clinical parameters. A total of 5.5% of the patients had DS, 11.3% had NDS, and 83% had no HLA antibodies after transplantation. NDS antibodies appeared earlier (1 to 5 yr posttransplantation) than DS antibodies (5 to 10 yr). In multivariate analysis, HLA-DR matching, pretransplantation immunization, and acute rejection were significantly associated with the development of both DS and NDS antibodies and also of DS versus NDS antibodies. The presence of either DS or NDS antibodies significantly correlated with lower graft survival, poor transplant function, and proteinuria. Screening of HLA antibodies posttransplantation could be a good tool for the follow-up of patients who receive a kidney transplant and allow immunosuppression to be tailored.

Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis

Hereditary pancreatitis (HP) is a rare inherited disorder, characterised by recurrent episodes of pancreatitis often beginning in early childhood. The mode of inheritance suggests an autosomal dominant trait with incomplete penetrance. The gene, or at least one of the genes, responsible for hereditary pancreatitis has been mapped to the long arm of chromosome 7 and a missense mutation, an arginine to histidine substitution at residue 117 in the trypsinogen cationic gene (try4) has been shown to segregate with the HP phenotype. The aim of this work was to investigate the molecular basis of hereditary pancreatitis. This study was performed on 14 HP families. The five exons of the trypsinogen cationic gene were studied using a specific gene amplification assay combined with denaturing gradient gel electrophoresis (DGGE). The present paper describes three novel mutations, namely K23R and N29I and a deletion –28delTCC in the promoter region. We also found a polymorphism in exon 4, D162D. In eight of these families we found a mutation which segregates with the disease. A segregation analysis using microsatellite markers carried out on the other families suggests genetic heterogeneity in at least one of them. Our findings confirm the implication of the cationic trypsinogen gene in HP and highlight allelic diversity associated with this phenotype. We also show that the pattern of inheritance of HP is probably complex and that other genes may be involved in this genetic disease.

HLA-DRw52a is involved in alloimmunization against PL-A1 antigen

The alloimmunization against the platelet PL-A1 antigen is strongly associated with a HLA class II structure in mothers of thrombocytopenic neonates. Most of the immunized women have first been shown to possess the DR3 specificity and subsequently the DRw52 allele. The 18 immunized mothers studied here by restriction fragment length polymorphism analysis had the DRw52a specificity at the DRB3 locus whatever their HLA-DRB1 gene product. This finding strongly suggests that the DRB3 chain is directly involved in the presentation of the PL-A1 antigen to the specific T cell. In addition, the similarities between DR3 and DRw52 structures due to a hypothetical gene conversion event should be considered in order to understand the high frequency of DR3 among the DRw52a-responding women. Alternatively, the high frequency of DR3 among the DRw52-responding mothers might be due to the high responder status associated with the former specificity.

Identification of the antibodies involved in B-cell crossmatch positivity in renal transplantation

Background. The significance of a positive B-cell crossmatch (BCM) in kidney transplantation has always been controversial in the evaluation of its implications on graft survival and specificity of the antibodies involved. Methods. We have investigated the sera of 62 recipients of a kidney allograft transplanted across a positive BCM (T negative) for the presence of autoantibodies and anti-human leukocyte antigen (HLA) class I and II antibodies, using a combination of lymphocytotoxicity, enzyme-linked immunosorbent assay (ELISA), and flow cytometry tests. The controls were the 930 patients transplanted over the same period of time with a negative T and BCM. Results. Autoantibodies were detected in 16%, and donor specific anti-HLA class II antibodies, mainly DQ, in 23% of the patients. None had antibodies against donor HLA class I. The target of the antibodies was not identified in 61%. Graft survival was comparable in the controls and in the +BCM patients, with nondonor-specific HLA reactivity. Patients with donor-specific anti-HLA class II antibodies had lower early graft survival and a higher incidence of vascular rejection. However, long-term allograft survival was similar to that of the other groups. Conclusion. These data suggest that in 77% of the patients, BCM positivity was not related with anti-HLA antibodies, and, in this case, graft survival was similar to that of the −BCM controls. In a minority of patients, anti-HLA class II antibodies were responsible for the +BCM, and their presence was associated with lower early, but not long-term, graft survival. Consequently, a +BCM should not systematically contraindicate kidney transplantation.

Non-HLA-Type Endothelial Cell Reactive Alloantibodies in Pre-Transplant Sera of Kidney Recipients Trigger Apoptosis

The vascular endothelium of transplanted organs represents an important target for allograft‐directed immune responses. Although HLA antigens expressed on graft endothelial cells (EC) can become targets of the host immune response, the role of other, non‐HLA‐encoded EC antigens has been proposed but is still unclear. The aim of this study was to investigate the presence of and to characterize anti‐EC antibodies (AECA) in 57 kidney transplant recipients according to their HLA‐immunization status. Flow cytometry in pretransplant sera was used to detect AECA reactive with surface antigens on ABO and HLA‐typed primary cultures of arterial ECs, stimulated or not with tumor necrosis factor‐α (TNFα) or interferon‐γ (IFNγ). FACS analysis revealed the presence of AECA in 47% of HLA‐sensitized (PRA = 10%: mostly IgG) vs. 16.0% in nonsensitized patients (PRA < 10%) (p < 0.02). No significant correlation was found between the presence of AECA and acute rejection occurrence and graft outcome. Non‐HLA reactive AECA are directed against TNFα‐ and IFNγ‐inducible membrane molecule(s), and react with two predominant antigens of ∼35 kDa and ∼50 kDa expressed on ECs but not on B cells. Binding of AECA decreases in vitro EC viability by 50–60% by promoting EC apoptosis, as demonstrated by DNA fragmentation assays.

Highly Altered Vβ Repertoire of T Cells Infiltrating Long-Term Rejected Kidney Allografts

Chronic rejection represents a major cause of long-term kidney graft loss. T cells that are predominant in long-term rejected kidney allografts (35 ± 10% of area infiltrate) may thus be instrumental in this phenomenom, which is likely to be dependant on the indirect pathway of allorecognition only. We have analyzed the variations in T cell repertoire usage of the Vβ chain at the complementary determining region 3 (CDR3) level in 18 human kidney grafts lost due to chronic rejection. We observed a strongly biased intragraft TCR Vβ usage for the majority of Vβ families and also a very high percentage (55%) of Vβ families exhibiting common and oligoclonal Vβ-Cβ rearrangements in the grafts of patients with chronic rejection associated with superimposed histologically acute lesions. Furthermore, Vβ8 and Vβ23 families exhibited common and oligoclonal Vβ-Jβ rearrangements in 4 of 18 patients (22%). Several CDR3 amino acid sequences were found for the common and oligoclonal Vβ8-Jβ1.4 rearrangement. Quantitative PCR showed that biased Vβ transcripts were also overexpressed in chronically rejected kidneys with superimposed acute lesions. In contrast, T lymphocytes infiltrating rejected allografts with chronic rejection only showed an unaltered Gaussian-type CDR3 length distribution. This pattern suggests that late graft failure associated with histological lesions restricted to Banff-defined chronic rejection does not involve T cell-mediated injury. Thus, our observation suggests that a limited number of determinants stimulates the recipient immune system in long-term allograft failure. The possibility of a local response against viral or parenchymatous cell-derived determinants is discussed.

An exceptional genealogy for hereditary chronic pancreatitis

Nearly one hundred families affected with hereditary chronic pancreatitis (HCP) have been reported in the literature. However, the fact that the disease involved only a few members of each family limits the informativeness of these reports and accounts for the infrequency and disappointing results of pathogenetic and genetic research. Our study concerned an exceptional HCP genealogy which would seem to provide an ideal model for the detection of a genetic anomaly linked to the expression of the disease. We studied 249 members of a family (214 still alive), covering eight generations born between 1800 and 1993. According to the customary criteria, 63 had definite and 17 probable HCP. Fifty-eight members under 18 years of age were still susceptible to developing the disease. This series confirms the mode of autosomal dominant heredity with variable penetrance. The clinical features and disease course were typical, except that symptoms tended to appear earlier. The series represents the most extensive HCP genealogy compiled and is one of the largest families studied in the field of genetic disease, regardless of etiology. Blood samples were taken from 146 subjects to facilitate pathogenetic and genetic research.

Crucial role of the third and fourth hypervariable regions of HLA-DPB1 allelic sequences in the mixed lymphocyte reaction

Since HLA-DP mismatches are known to induce proliferative response in MLR I, we investigated the real impact of the different DP alleles and the possible role of one or several hypervariable regions of the DPB allelic sequences. Accordingly, we performed MLR I between HLA-A, B, DR, DQ, and Dw identical individuals DP oligotyped after DNA amplification. A total of 23 one-DP-mismatched healthy stimulator and responder cells displaying nine different DP specificities were thus evaluated in 52 MLRs I. This allowed us to analyze the impact of amino acid composition of each of the six hypervariable regions independently of the amino acid matching or mismatching in the five others. We show here that DP combinations sharing the same amino acid sequence in the third (C) and fourth (D) hypervariable regions are associated with a low proliferative response in vitro (p less than 0.01). These data imply that a perfect HLA-DP matching may not be requisite in selecting bone marrow donors. Indeed, the choice of donors may rely on determination of these particular mismatched HVRs between the DP alleles involved especially in GvHD direction. This policy including prospective DP oligotyping should be of great interest, especially when MLRs I are false negative or nonevaluable. It will enable a better definition of which DP mismatches are acceptable in BMT.

Granulocyte antibody screening: evaluation of a bead-based assay in comparison with classical methods

BACKGROUND: Granulocyte antibodies have been implicated in allo‐ and autoimmune neutropenia and in transfusion reactions.

Interaction of anti-HLA antibodies with pig xenoantigens.

BACKGROUND Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting. METHODS Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab. RESULTS Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient. CONCLUSION Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.