J. L. Vicario

Bi-directional allelic recognition of the human minor histocompatibility antigen HB-1 by cytotoxic T lymphocytes

Human minor histocompatibility antigens (mHag) are target antigens of the graft‐versus‐leukemia response observed after allogeneic HLA‐identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage‐specific mHag HB‐1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA‐B44. The HB‐1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR‐RFLP assay to perform HB‐1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA‐B44‐matched sibling pairs for HB‐1H is 2.8%, whereas recipient disparity for HB‐1Y is expected to be 12.4%. Therefore, we addressed whether the HB‐1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA‐B44 molecules and that the H/Y substitution has noinfluence on formation of epitope precursor peptides by 20 S proteasome‐mediated degradation. More directly, CTL recognizing the naturally presented HB‐1Y peptide could be generated from a HB‐1H homozygous donor using peptide‐pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB‐1 allelic homologues. HB‐1 is the first human mHag described that induces bi‐directional allogeneic CTL responses that may contribute to a specific graft‐versus‐leukemiaresponse following allogeneic stem cell transplantation.

Allelic and haplotypic HLA frequency distribution in Spanish hematopoietic patients. Implications for unrelated donor searching

Histocompatibility criteria for unrelated donor selection are based on high-resolution definition of HLA genes. In spite of the expansion of the unrelated donor registries, HLA matching remains a problem for many patients because of the great diversity of HLA alleles and haplotypes. The availability of matched donors at an allelic level depends on the frequency of the patients alleles and haplotypes. Therefore, data regarding HLA distribution for each population are needed in order to evaluate the donor searching approach and, may be, even the therapeutic strategy. In the present report, we have analyzed 253 haematological Spanish patients awaiting unrelated haematopoietic stem cell (HSC) donors. HLA allele and haplotype frequencies have been defined at high resolution for the first time in this population. Significant differences in HLA distribution have been reported when comparing two patient groups, one that received full-match (10/10) unrelated donors and one that did not. Factors like rare alleles, presence of B*510101 (because of the association with multiple HLA-C alleles), as well as infrequent B-C and DRB1-DQB1 associations, showed a negative value for finding a suitable donor, whereas the presence of one of the six-gene haplotypes with a frequency ≥ 0.9% in our sample was a positive factor influencing donor searching. These differences will be useful in donor searching advising and in the use of different therapeutic strategies.

Genetic variation in the nuclear factor κB pathway in relation to susceptibility to rheumatoid arthritis

Objective: To examine genetic association between rheumatoid arthritis (RA) and known polymorphisms in core genes of the nuclear factor (NF)κB pathway, the major intracellular pathway in RA pathogenesis. Methods: Discovery and replication sample sets of Spanish patients with RA and controls were studied. A total of 181 single nucleotide polymorphisms (SNPs) uniformly spaced along the genomic sequences of 17 core genes of the NFκB pathway (REL, RELA, RELB, NFKB1, NFKB2, NFKBIA, NFKBIB, NFKBIE, IKBKA, IKBKB, IKBKE, IKBKAP, KBRAS1, KBRAS2, MAP3K1, MAP3K14, TAX1BP1) were studied by mass spectrometry analysis complemented with 5′-nuclease fluorescence assays in the discovery set, 458 patients with RA and 657 controls. SNPs showing nominal significant differences were further investigated in the replication set of 1189 patients with RA and 1092 controls. Results: No clear reproducible association was found, although 12 SNPs in IKBKB, IKBKE and REL genes showed significant association in the discovery set. Interestingly, two of the SNPs in the IKBKE gene, weakly associated in the discovery phase, showed a trend to significant association in the replication phase. Pooling both sample sets together, the association with these two SNPs was significant. Conclusion: We did not find any major effect among the explored members of the NFκB pathway in RA susceptibility. However, it is possible that variation in the IKBKE gene could have a small effect that requires replication in additional studies.

Influence of HLA DRB1 alleles in the susceptibility of rheumatoid arthritis and the regulation of antibodies against citrullinated proteins and rheumatoid factor

IntroductionThe purpose of this study was to investigate the association between HLA-DRB1 alleles with susceptibility to rheumatoid arthritis (RA) and production of antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF).MethodsWe studied 408 patients (235 with RA, 173 non-RA) and 269 controls. ACPA, RF and HLA-DR typing were determined.ResultsWe found an increased frequency of HLA DRB1 alleles with the shared epitope (SE) in ACPA-positive RA. Inversely, HLA DRB1 alleles encoding DERAA sequences were more frequent in controls than in ACPA-positive RA, and a similar trend was found for HLA DR3. However, these results could not be confirmed after stratification for the presence of the SE, probably due to the relatively low number of patients. These data may suggest that the presence of these alleles may confer a protective role for ACPA-positive RA. In RA patients we observed association between SE alleles and ACPA titers in a dose-dependent effect. The presence of HLA DR3 or DERAA-encoding alleles was associated with markedly reduced ACPA levels. No association between RF titers and HLA DR3 or DERAA-encoding alleles was found.ConclusionsHLA DRB1 alleles with the SE are associated with production of ACPA. DERAA-encoding HLA-DR alleles and HLA DR3 may be protective for ACPA-positive RA.

Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation.

The main objective of the present study was to determine the role of CD34+ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G‐CSF were analysed. Using dual‐colour flow cytometry, different subpopulations of CD34+ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34+CD38− cells and CD34+CD38+ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34+ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 106 CD34+CD38− cells for neutrophil engraftment, and 2.0 × 106 CD34+CD38+ cells for platelet recovery. It is suggested that the analysis of CD34+ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34+ cells are collected.

High frequency of the HLA-DRB1*0405-(Dw15)-DQw8 haplotype in Spaniards and its relationship to diabetes susceptibility.

A study of DR4 subtypes has been done in Spanish unrelated controls and insulin-dependent diabetics by using dot blot hybridization with specific DR4B1 exon-2 oligonucleotides and automated dideoxy DNA sequencing. Dw15-DQw8 is the predominant DR4 subtype present in our normal population (37%); this DR4 frequency characteristic singles out our population from all other Caucasoids tested so far and may also be a marker of the original Iberian paleo-North African population. Dw15-DQw8 is not significantly increased in our insulin-dependent diabetics sample and despite its relative high frequency in the control population it does not have a bearing in lowering insulin-dependent diabetes mellitus frequency of DR4-positive Spaniards. In addition, no particular DR4 split is by itself significantly increased in Spanish diabetics; this may indicate that selective diabetogenic environmental factors may be working upon DR4-positive individuals, but on genes (or gene products) other than DR or at least not upon the polymorphic sites of DRB1 exon-2 products.

Genetic variation in the NFκB pathway in relation to susceptibility to rheumatoid arthritis

Objective: To examine genetic association between rheumatoid arthritis (RA) and known polymorphisms in core genes of the nuclear factor (NF)κB pathway, the major intracellular pathway in RA pathogenesis. Methods: Discovery and replication sample sets of Spanish patients with RA and controls were studied. A total of 181 single nucleotide polymorphisms (SNPs) uniformly spaced along the genomic sequences of 17 core genes of the NFκB pathway (REL, RELA, RELB, NFKB1, NFKB2, NFKBIA, NFKBIB, NFKBIE, IKBKA, IKBKB, IKBKE, IKBKAP, KBRAS1, KBRAS2, MAP3K1, MAP3K14, TAX1BP1) were studied by mass spectrometry analysis complemented with 5′-nuclease fluorescence assays in the discovery set, 458 patients with RA and 657 controls. SNPs showing nominal significant differences were further investigated in the replication set of 1189 patients with RA and 1092 controls. Results: No clear reproducible association was found, although 12 SNPs in IKBKB, IKBKE and REL genes showed significant association in the discovery set. Interestingly, two of the SNPs in the IKBKE gene, weakly associated in the discovery phase, showed a trend to significant association in the replication phase. Pooling both sample sets together, the association with these two SNPs was significant. Conclusion: We did not find any major effect among the explored members of the NFκB pathway in RA susceptibility. However, it is possible that variation in the IKBKE gene could have a small effect that requires replication in additional studies.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Immunogenetics of Systemic Lupus Erythematosus in Spanish Patients: Differential HLA Markers

HLA-DR3 antigen included in the compound phenotype B18BfF1 (but not the one linked to the B8BfS compound phenotype) was found to be significantly increased in our SLE patients. It is remarkable that in our Southern-Mediterranean population, B18BfF1DR3 individuals (but not B8BfSDR3) are prone to SLE with renal disease, in contrast with other Northern European and Caucasoid populations. Also, patients with autoantibodies to Ro/La have a significant increase of the B8DR3 compound phenotype. Production of autoantibodies against Ro alone was associated to DR2 and production of anti-Sm/nRNP to DR3 (either B18BfF1 or B8BfS associated) only in the subgroup without renal disease. The distinctive HLA and autoimmune associations to SLE with and without renal disease suggests that both clinical forms may not share a common identical pathogenesis.

Long-Term Hematopoietic Engraftment after Autologous Peripheral Blood Progenitor Cell Transplantation in Pediatric Patients: Effect of the CD34+ Cell Dose

Background and Objectives: We analyzed the relationship between long-term hematopoietic recovery and the number of CD34+ cells infused in order to determine the optimal dose of CD34+ cells for rapid and stable engraftment. Patients and Methods: Between November 1993 and December 1998, 96 consecutive autologous transplantations were performed in 92 pediatric patients with different malignancies. Peripheral blood progenitor cells (PBPC) were mobilized by G-CSF alone (12 μg/kg/day s.c., Neupogen®; Amgen, Thousand Oaks, Calif., USA) and collected using a Cobe Spectra blood cell separator (Cobe, Denver, Colo., USA) through a central venous catheter with double lumen. The CD34+ cell contents of apheresis products were assessed by means of flow-cytometric analysis using an Epics Elite flow cytometer (Coulter, USA). Results: The median number of CD34+ cells infused was 3.2 × 106/kg (range 0.17–44.4). The median times for short-term engraftment (neutrophil count >0.5 × 109/l and platelet count >20 × 109/l) was 9 (range: 7–16) and 13 days (range: 7–91), respectively. The median times for long-term engraftment (platelet count >50 × 109/l and >100 × 109/l) was 21 (range: 10–249) and 45 days (range: 12–288). When the infused CD34+ cell dose was ≥5 × 106/kg (median 7.99, range 5.01–44.4), there was a statistically significant increase in the rate of short- and long-term hematopoietic recovery compared to patients transplanted with a lower number of CD34+ cells (p < 0.0001). The earlier recovery in the high CD34+ cell group resulted in less transfusional support, fewer days on intravenous antibiotics and shorter hospitalization. Conclusions: This study confirms that G-CSF-mobilized PBPC provide rapid short- and long-term hematopoietic engraftment in pediatric patients undergoing autologous transplantation if a CD34+ cell dose ≥5.0 × 106/kg is infused. As this PBPC dose seems to have clinical and potentially economic implications, it should be considered the optimal dose for apheresis.