Jean-Marie Tiercy

Nomenclature for factors of the HLA system, 2010

The WHO Nomenclature Committee for Factors of the HLA System met following the 14th International HLA and Immunogenetics Workshop in Melbourne, Australia in December 2005 and Buzios, Brazil during the 15th International HLA and Immunogenetics Workshop in September 2008. This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports (1–18).

A Totally Synthetic Polyoxime Malaria Vaccine Containing Plasmodium falciparum B Cell and Universal T Cell Epitopes Elicits Immune Responses in Volunteers of Diverse HLA Types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT*)4-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T*, which was originally defined using CD4+ T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T*-specific cellular responses with high anti-repeat Ab titers suggests that the T* epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.

A synthetic malaria vaccine elicits a potent CD8+ and CD4+ T lymphocyte immune response in humans. Implications for vaccination strategies

We report the first synthetic peptide vaccine eliciting strong CD8+ and CD4+ T lymphocyte responses in humans. The vaccine, representing the C‐terminal region of the circumsporozoite protein of Plasmodium falciparum (amino acids 282–383) was well tolerated and strong sporozoite‐specific antibodies were elicited. In addition, robust lymphocyte proliferation responses were equally elicited with concomitant in vitro production of IFN‐γ, crucial in the elimination of the parasite. Most importantly, we also observed the development of CD8+ T lymphocyte responses decisive in the immunity to malaria. The latter finding opens new, possibly safer, avenues for vaccination strategies when a CD8+ T cell response is needed.

Oligonucleotide geriotyping of HLA polymorphism on microtitre plates

Molecular analysis of mutations and polymorphisms that are of medical importance requires both accuracy and simplicity. In organ transplantation there is a need for an HLA typing procedure that combines the remarkable accuracy of oligonucleotide genotyping with the simplicity of conventional serological typing. We describe a simple semiautomated method of HLA class II typing consisting of an oligonucleotide hybridisation assay done on microtitre plates followed by automatic colorimetric reading. Individual HLA-DR generic typing for 30 DR specificities, including subtypes of DR1, DR2, DR13, DR14, and DR52, is done on a single plate. The entire typing assay can be completed in less than 4 hours. The procedure has been validated on more than a thousand haplotypes in prospective DR typing of kidney transplant patients, leukaemic patients, and their potential donors. The simplicity of this assay makes it suitable for routine laboratory use. It can be applied to genetic testing in general, including the testing of patients with multiple mutations.

Natural killer cell receptor repertoire and their ligands, and the risk of CMV infection after kidney transplantation.

Cytomegalovirus (CMV) infection is the most common viral complication after solid organ transplantation (SOT). Whilst current immunosuppression is known to impair antiviral‐specific T‐cell immunity in SOT, a potential role for natural killer (NK) cells not affected by immunosuppressive therapy remains to be determined. To address this, we compared the genotype of the NK immunoglobulin‐like receptor (KIR) genes and their HLA cognate ligands to the rate of CMV infection in 196 kidney transplant recipients. We have shown that the absence of the HLA‐C ligand for inhibitory KIR and the presence of activating KIR genes in the recipients were both associated with a lower rate of CMV infection after transplantation. In a cohort of 17 recipients with acute CMV infection, NK cells were phenotyped over a period of time after diagnosis by their expression profile of C‐type lectin receptors and capacity to secrete IFN‐γ. The increased expression of the activating C‐type lectin receptors NKG2C and NKG2D was paralleled by the decreased IFN‐γ secretion during the early phase of CMV infection. In conclusion, our findings suggest that KIR/HLA genotype and expression of NKG2C and NKG2D might play a significant role in regulating NK cell function and anti‐CMV immunity after kidney transplantation.

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.

An extended HLA-DQ-DR haplotype rather than DRB1 alone contributes to RA predisposition

Abstract In the present study, we tested our hypothesis on the role of a DQ-DR haplotype in rheumatoid arthritis (RA) predisposition. Using two groups of patients and controls, one from The Netherlands and one from Switzerland, we found that DQA1*0301-homozygous and DQA1*0301//DQA1*0101/04-heterozygous individuals are highly predisposed to RA in both populations, while DQA1*0101/04-homozygous are not. The DQA1*0301-DRB1*0403/06/07 and DQA1*0301-DRB1*0901 haplotypes are not associated with RA by themselves but strongly increase the risk of developing disease in DQA1*0301- and DQA1*0101/04-heterozygous. DRB1 alleles carrying the motif DERAA in their third hypervariable region, i.e., *0103, *0402, *1102, *1103, *1301, and *1302, provide a long-lasting protection against RA in DQA1*0101/04- but not in DQA1*0301-positive individuals. These data show that considering both DQ and DR gives a better distinction between patients and controls than the shared epitope hypothesis.

Oligotyping of HLA-A2, -A3, and -B44 subtypes. Detection of subtype incompatibilities between patients and their serologically matched unrelated bone marrow donors.

We have set up a simple PCR-SSO oligotyping procedure that is able to discriminate ten HLA-A2 (2 PCR/11 probes), two HLA-A3 (1 PCR/1 probe), and two HLA-B44 subtypes (1 PCR/2 probes). The frequency of these subtypes has been determined in a large panel of local blood donors and leukemic patients in combination with their unrelated potential donors. A*0201 and A*0301 were the predominant subtypes (> 95%) for A2 and A3, respectively. B*4402 occurred twice as frequently as B*4403. A2 and B44 subtype mismatches were analyzed in a group of 30 patients and their 116 unrelated potential donors who were matched serologically (low-stringency matching: AB without splits, DR1-10). For seven patients (23%) at least one A2- or B44-subtype-mismatched donor was found. For two of these patients (7%), the subtype-mismatched donor would have been considered as compatible on the basis of high stringency matching (AB splits, DRB1 subtypes, DRB3/B5). For one patient of Mediterranean origin, all five donors recruited from a north European registry (matched with high stringency) appeared to be subtype incompatible (A*0201/A*0205). The rather low percentage of A2- and B4-subtype mismatches in DRB1/B3/B5 matched combinations confirms the significance of linkage disequilibria of HLA antigens. Because unrelated donor selection is done through international registries, however, class I subtyping might be necessary when individuals originate from different geographic areas.

The frequency of pretransplant donor cytotoxic T cell precursors with anti-host specificity predicts survival of patients transplanted with bone marrow from donors other than HLA-identical siblings

Transplantation with bone marrow from other than genotypically HLA-identical donors is associated with an increased incidence and severity of graft-versus-host disease (GvHD). The precise influence of HLA incompatibilities is not easy to analyze as even perfectly matched, HLA-identical unrelated donors might still express HLA differences that remain undetected by conventional typing. To measure T cell activity against serologically detectable and nondetectable HLA antigens, we analyzed the frequencies of CTL precursors (CTLp) between 11 unrelated HLA-matched and five related haploidentical donor/recipient pairs in graft-versus-host direction. Our results show that whenever HLA class I disparities could be identified by serology, high precursor frequencies (1/28,000–1/94,000) were measured. In contrast, in donor/ recipient pairs that differed for class II only, no precursors were detected. CTLp were elevated in two out of eight fully matched donor/recipient combinations. These combinations displayed activities as high (1/21,000; 1/ 52,000) as the combinations that were serologically HLA class I disparate. The incompatibilities detected by the cellular assay were highly significant for the clinical results after transplantation. High CTLp frequencies before transplantation correlated with unfavorable clinical results independent of the incidence of detected HLA differences. Five out of the six patients with high (>1/ 100,000) CTLp frequencies died within 120 days after transplantation. GvHD IV was the cause of death for all (3/5) patients who had received an unmanipulated bone marrow. In the group with intermediate or undetectable CTLp frequencies, eight out of 10 patients are alive, seven (CTLp frequency undetectable) without GvHD more severe than grade II, while one patient (CTLp frequency = 1/180,000) suffered from GvHD grade III. One patient rejected the graft and was rescued by an autologous BMT.

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.