Michel Jeannet

DNA typing of DRw6 subtypes: Correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes

Among MHC class II antigens, the DRw6/Dw6 complex represents a special situation where typing on a routine basis is often troublesome, mainly because monospecific alloantisera are rare and individual subtypes numerous. We demonstrate here that the use of oligonucleotide DNA typing permits an analysis of the polymorphism within DRw6 haplotypes and provides a molecular basis for correlations with functional data. Synthetic oligonucleotide probes, most of them locus- and allele-specific, were derived from the DNA sequences of three alleles of locus DRB1 and three alleles of locus DRB3. These probes allow the positive identification of distinct DRw6 subtypes. As analyzed on a panel of 26 well-defined DRw6 cell lines, oligotyping allows a direct and absolute correlation with the DRw13 serologic specificity and with the cellularly defined Dw9,Dw16,Dw18, and Dw19 specificities. Correlations of the polymorphism at the DRB1 locus with the polymorphism at the DRB3 locus (DRw52 alleles) allow us to identify preferential allelic associations such as DRw13-Dw18-DRw52a/52b, DRw13-Dw19-DRw52c, and DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least DRw14-Dw9-DRw52b. In view of the absolute segregation of the DRw52c allele with the DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least in part, epitopes on the DRw52c allele. The identification of DRw6 subtypes, as well as of other HLA class II subspecificities, by oligotyping can now complement and possibly replace serologic and cellular typing. It represents a particularly useful contribution to the optimization of class II matching in the case of bone marrow transplantation with unrelated donors.

APPLICATION OF HLA-DR OLIGOTYPING TO 110 KIDNEY TRANSPLANT PATIENTS WITH DOUBTFUL SEROLOGICAL TYPING

In renal transplantation a good HLA-DR match is associated with a higher success rate of graft outcome. However, due to a number of technical problems, reliable serological DR typing cannot always be obtained, and the very large number of HLA-DR alleles now discovered renders such DR matching more difficult. In view of the medical importance of HLA class II polymorphism in transplantation immunology, we have developed a simple HLA-DR oligotyping procedure on PCR-amplified DNA, by hybridization with 14 synthetic oligonucleotide probes able to recognize all major HLA-DR specificities. In particular, the probes used in this study allow the unambiguous discrimination of the DRw11, w12, w13, w14-Dw9 specificities or of rare alleles such as DR-Br or DRw13-DwHAG, which are very often difficult or impossible to identify by serology. In order to explore the potential of this methodology, we have analyzed by oligotyping 110 kidney transplant patients with doubtful or unreliable serological assignment, or with DR blank alleles. Comparison between serology and oligotyping shows that in 66.3% of the patients we observed an excellent correlation. About half these patients are homozygotes, as ultimately verified by RFLP typing. In 26.4% of the patients however, at least one HLA-DR antigen was discrepant, and in 7.3% of the cases oligotyping resolved uninterpretable serology. Almost all of the discrepancies were due to errors in allele assignment within the DRw52 group, mostly in the case of DRw13 alleles. This study confirms the expected qualitative advantage of the oligotyping technique and its simplicity as compared with the RFLP DNA typing procedure. Large scale application of the oligotyping methodology will therefore be beneficial to optimize HLA-DR matching in organ transplantation, particularly in high responders with first kidney graft rejection.

HA-1 and the SMCY-derived peptide fidsyicqv (H-Y) are immunodominant minor Histocompatibility antigens after bone marrow transplantation.

BACKGROUND Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens. METHODS We determined the specificity of cytotoxic T-cell clones isolated during acute GVHD or during bone marrow graft rejection in patients (n=14) transplanted with marrow from donors who were histoincompatible for different minor and/or major histocompatibility antigens. RESULTS We found a clear hierarchy among the different types of histoincompatibilities. In three combinations mismatched for a class I allele, all 27 clones isolated during GVHD were specific for the incompatible HLA molecule. In the 11 class I-identical combinations, 14 different mHags were recognized. The mHag HA-1, known to have a significant impact on the development of GVHD, was recognized in the two HA-1-incompatible combinations. In one of these combinations, which was sex mismatched, all 56 clones analyzed were directed against HA-1, demonstrating the dominance of this mHag. In the four HA-1-compatible, sex-mismatched combinations, the anti-H-Y response was directed against one immunodominant epitope rather than against multiple Y-chromosome-encoded epitopes. All male specific cytotoxic T lymphocytes (n=15) recognized the same high-performance liquid chromatography-purified peptide fraction presented by T2 cells. Moreover, all cytotoxic T lymphocytes tested (n=6) were specific for the SMCY-derived peptide FIDSYICQV, originally described as being the H-Y epitope recognized in the context of HLA-A*0201. CONCLUSIONS Some histocompatibility antigens are recognized in an immunodominant fashion and will therefore be recognized in the majority of mismatched combinations. Only for such antigens, correlations between mismatches and the occurrence of GVHD or graft rejections will be found.

Donor Lymphocyte Infusion for the Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

The results of donor lymphocyte infusion (DLI) for treatment of relapse after bone marrow transplantation (BMT) are reviewed. Durable complete remission can be achieved at the molecular level for a majority (more than 70%) of patients with CML, when treated at early relapse. Results are less favourable for acute leukemias, although useful responses have been reported. Data are scarce though promising for myelodysplastic syndromes and multiple myeloma. Major treatment-associated toxicities are GVHD and bone marrow aplasia. The latter complication can be predicted by evaluating the level of residual donor-derived hematopoiesis. Modification of infused cells (CD8 negative selection or transduction with a suicide gene), addition of peripheral blood stem cells, and early implementation of escalating doses may counteract the complications and increase the response rate. Response rate is variably influenced by the presence of chronic GVHD after initial BMT, T-cell depleted BMT, underlying disease and stage at relapse, and the level of mixed chimerism. DLI is a direct demonstration of the graft-versus-leukemia effect (GVL). Because GVL after BMT is sometimes the predominant cause of cure, it may be advisable in such situations to redirect the conditioning regimens for BMT towards engraftment and less immediate cytotoxicity.

T-cell repertoire complexity after allogeneic bone marrow transplantation

We analyzed the T-cell repertoire in patients transplanted with bone marrow from an HLA identical sibling by determining the TCR diversity through Vbeta-CDR3-size spectratyping with Vbeta/Cbeta- and Vbeta/Jbeta-specific primers. Using the Vbeta/Cbeta primers, we observed limited TCR diversity only in recipients of a T-cell-depleted graft, whereas the TCR diversity of patients transplanted with an unmanipulated graft seemed to be indistinguishable from the one of a normal individual. However, with Vbeta/Jbeta-specific primers, increase of the resolution by approximately 10-fold also allowed the detection of imbalances in the TCR repertoire of recipients of an unmanipulated graft. This demonstrates that when high numbers of T cells are cotransfused with marrow, the TCR repertoire is more complete but still not as complete as in normal individuals, thereby emphasizing the important role of coinfused mature T cells in the restoration of the T-cell compartment after bone marrow transplantation.

Complete analysis of HLA-DQB1 polymorphism and DR-DQ linkage disequilibrium by oligonucleotide typing

HLA class II polymorphism is functionally important in the control of immune responses, in transplantation immunology, and in the suceptibility to autoimmune diseases. HLA-DQA1 and -DQB1 genes exhibit a larger degree of allelic polymorphism than usually recognized by routine serology. We have therefore performed an extensive analysis of DQB1 polymorphism by oligotyping. A set of 12 oligo probes was hybridized on polymerase chain reaction-amplified DNA, thus allowing the detection of 12 DQB1 alleles, as demonstrated in homozygous as well as in heterozygous individuals. This highly sensitive detection system is particularly relevant within the DQw1 specificity where the 7 allelic sequences can easily be identified. The DQ-DR linkage disequilibrium was analyzed by oligotyping of 80 Caucasoid heterozygous individuals (160 haplotypes), and very tight associations were observed between DRB1 and DQB1 alleles. Five DRB1 alleles, DR-BON, DR4/Dw4 or Dw14, DR7, DRw8.3, and DRw11, however, can be associated with different DQB1 alleles. Moreover the DRB1 and DQB1 oligotyping analysis performed on 20 randomly chosen DRw8 Caucasoid individuals showed a high prevalence of the DRB1*0801-DQB1*0402 haplotype. By combining the analysis of allelic variations at DRB1, DRB3, and DQB1 loci, we can detect 33 different DR-DQ combinations in our panel of Caucasoid individuals. We now apply DQB1 oligotyping on a routine basis for optimal matching of unrelated donors for bone marrow transplantation.

Pretransplant cytotoxic donor T‐cell activity specific to patient HLA class I antigens correlating with mortality after unrelated BMT

Unrelated bone marrow transplantation (BMT) is associated with increased post‐transplant complication rates, partly because more transplantation antigens are mismatched than in HLA‐identical related BMT. We have shown previously that the cytotoxic T‐lymphocyte precursor (CTLp) test performed before transplantation specifically detects HLA class I mismatches demonstrating its usefulness for the identification of new HLA class I alleles. In this study we analysed the clinical relevance of the CTLp test in 41 patients who underwent unrelated BMT between 1990 and 1994. All patient–donor pairs were HLA‐A, ‐B, ‐DR compatible as defined by AB‐serology and oligotyping for DR1‐14. The host‐reactive CTLp test was performed using previously frozen peripheral blood mononuclear cells (PBMC) as stimulators and PHA blasts as target cells. We found 10 CTLp‐positive and 31 CTLp‐negative patient–donor pairs. Between the two groups there were no significant differences for age, diagnosis, sex, preconditioning and GvHD prophylaxis. The clinical results for the CTLp positive and the CTLp negative transplants were: severe acute GvHD III–IV 67% and 26% (P = 0.0315), transplant‐related mortality 60% and 26% (P = 0.0085), and patient survival at 3.5 years 10% and 54% (P = 0.0006).

Heterogeneity of HLA-B35 : oligotyping and direct sequencing for B35 subtypes reveals a high mismatching rate in B35 serologically compatible kidney and bone marrow donor/recipient pairs

We used a simple HLA-B35 PCR/SSO-oligotyping procedure, combined with exon 3 direct sequencing for the analysis of B35 subtype frequencies in our population, and for the evaluation of the degree of B35-subtype compatibility in serologically matched unrelated bone marrow and kidney transplant pairs. B*3501 was the most frequent allele (0.6), followed by B*3503 (0.19), B*3502 (0.13), B*3508 (0.07), and B*3505 (<0.01). HLA-B35-subtype matching of donors and recipients was strongly dependent on the stringency of ABDRB1 matching. Among 10 kidney donor/recipient pairs, only 30% were B35-subtype-matched. Due to the lack of ABDRB1 haplotype matching, this low degree of matching was not better than what would be expected on the basis of the subtype frequency distribution in the population. In contrast, HLA-B35 subtype compatibility was higher in unrelated bone marrow donor/recipient pairs that were serologically ABDR-matched: 30 of the 62 (48.4%) B35-positive combinations tested were B35-subtype-compatible. When all patient/donor plus donor/donor combinations (n=160) were taken into account, 46% of the ABDR-matched pairs were B35-subtype-compatible. When only pairs that were DRBl/DRB3/DRB5-subtype-matched by oligotyping (n=62) were considered, 71% were B35-sub-type-compatible. The fact that a significant percent of patient/donor pairs matched by conventional HLA-typing are found incompatible, as shown here for B35, explains the difficulties in assessing the beneficial effect of HLA matching in transplantation.

High-resolution histocompatibility testing of a group of sixteen B44-positive, ABDR serologically matched unrelated donor-recipient pairs. Analysis of serologically undisclosed incompatibilities by cellular techniques, isoelectrofocusing, and HLA oligotyping.

We have characterized HLA incompatibilities in a group of 16 B44-positive patients who were serologically ABDR matched with their 23 (unrelated) potential bone marrow donors. After analysis with a combination of cellular techniques, IEF for HLA-A/B and oligotyping for class II and HLA-B44, 44% of the patients revealed one or more HLA incompatibility with at least one of their potential donors. CTL activity was detected in 12 of the 22 combinations tested. CTL incompatibility occurred more frequently in DR subtype-mismatched combinations, but CTL reactivity was always directed against class I. To characterize these incompatibilities between matched unrelated individuals, we analyzed the specificity of T-cell clones from seven primary CTL cultures. In three combinations, CTL reactivity was directed against a subtype of B44. In two combinations, the CTL reactivity was directed against a non-B44 class I subtype. In two of seven combinations, the CTLs recognized an antigen that, though unconditionally associated with B4403, was expressed by 60% of the B4403+ cells only. Because all 12 of these B4403+ targets recognized could be typed for one HLA-C allele only (Cwl-Cw8), we believe that this alloreactivity might be directed against a serologically undefined Cw antigen.

Transfusion-associated graft-versus-host disease in a patient treated with Cladribine (2-chlorodeoxyadenosine): demonstration of exogenous DNA in various tissue extracts by PCR analysis.

Transfusion-associated graft-versus-host disease can occur in both immunocompetent and immunocompromised hosts. Cladribine is a synthetic analogue of adenine used in the treatment of lymphoid malignancies, commonly associated with a decrease in T lymphocytes. Cladribine was given for a low-grade non-Hodgkins lymphoma with thrombocytopenia as the main side-effect. Six units of pooled non-irradiated platelets were transfused from six unrelated donors; 10 d later a clinical picture typical of graft-versus-host disease resulted. Polymerase chain reaction of the highly polymorphic DNA minisatellites and HLA-DR oligotyping were used to demonstrate the exogenous DNA. In the patients blood and tissues, only the pattern of donor 5 was found. The patient (DRB1*0301/1101; DRB3*0101/02) and this donor (DRB1*0301/1104; DRB3*02) by chance shared a partial common haplotype. This complication highlights the sensitivity of DNA minisatellite analysis. It further raises the question of transfusion and of prophylactic irradiation of all blood products in immunosuppressed patients and those treated with cladribine. This case represents a previously unreported situation where an immunosuppressed patient was able to eliminate cells from five totally HLA-DR dissimilar donors but not from one heterozygous donor with strong HLA-DR similarity.