Histoincompatibilities in ABDR-matched unrelated donor recipient combinations.

To get an insight into the degree of major histocompatibility mismatches in donor/recipient (D/R) combinations who were 'ABDR-matched' by serology for class I and by oligotyping for DR1-14 (low resolution typing), we performed additional HLA testing using a combination of molecular, biochemical and cellular techniques. For class II we used extended oligotyping, discriminating all the common DRB1/B3/B5-subtypes. For class I (-subtypes) we used oligotyping (HLA-A2,-A3,-B35,-B41,-B44), sequencing (HLA-B35,-B41,-Cw16), isoelectrofocusing (IEF), primary cytotoxic T lymphocyte (CTL) assays and class I-subtype specific T cell clones. In addition, all combinations were serologically typed for HLA-C. This high resolution typing by the combination of techniques revealed numerous histoincompatibilities. Fifty-three per cent of all 'ABDR-matched' combinations tested (n = 198) appeared to be DR incompatible. Moreover, independent of the presence of a class II mismatch, 47% of the donors tested (n = 131) displayed pretransplant cytotoxic activity against the patient. This activity was found to be rigorously correlated with the presence of class I incompatibilities, predominantly HLA-A,-B subtypes and HLA-C. Thus, although the D/R pairs had been originally matched for AB including serological splits and by generic class II typing, only 28% of the pairs were in fact ABCDR identical. As many as 38% of the D/R pairs were mismatched for one, 14% for two, 13% for three and 6% for four A, B, C or DRB1 antigens. We conclude that the presence of such a high number of histoincompatibilities in a group of relatively well matched D/R pairs will severely hinder the analysis of the role of HLA in marrow transplantation and that conclusions from studies in which D/R pairs are matched by conventional typing must be interpreted with extreme caution.

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 DRB1/DRB3/DRB5-subtype-matched by oligotyping (n = 62) were considered, 71% were B35-subtype-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.

HLA-B35-subtype mismatches in ABDR serologically matched unrelated donor-recipient pairs.

We have characterized HLA incompatibilities in a group of 17 B35-positive patients who were ABDR matched (AB serology and oligotyping for DR1-14) with their 28 (unrelated) potential bone marrow donors. High-resolution oligotyping for DR subtypes disclosed that nine combinations were in fact DR mismatched. Cytotoxic T-lymphocyte (CTL) activity was detected in nine combinations (32%). In the group matched for DR subtypes, three (16%) of 19 combinations were CTL positive. Patient-specific cytotoxic activity appeared to be directed against HLA C (two cases) or against a subtype of B35. In the group of DR-subtype-mismatched combinations, CTL activity was found in six (67%) of nine pairs. In all four cases that were studied in detail, however, CTL reactivity appeared to be directed against a variant subtype of B35. We have studied the B35 incompatibilities recognized in five different combinations by specificity analysis of the B35-specific CTLs and by partially sequencing of relevant segments of B35 exon 3. Preliminary data show that, within this relatively small Caucasoid group, at least five B35-variant subtypes could be distinguished. This would make B35 an antigen that will be frequently subtype mismatched, in particular when DR matching is done with low resolution (DR1-14) only.

Cloning of genes involved in myo-inositol transport in a Pseudomonas sp.

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.