Esteban Arrieta-Bolaños

HLA-A, -B, -C, -DQB1, and -DRB1,3,4,5 allele and haplotype frequencies in the Costa Rica Central Valley Population and its relationship to worldwide populations.

The human leukocyte antigen (HLA) system is the most polymorphic in humans. Its allele, genotype, and haplotype frequencies vary significantly among different populations. Molecular typing data on HLA are necessary for the development of stem cell donor registries, cord blood banks, HLA-disease association studies, and anthropology studies. The Costa Rica Central Valley Population (CCVP) is the major population in this country. No previous study has characterized HLA frequencies in this population. Allele group and haplotype frequencies of HLA genes in the CCVP were determined by means of molecular typing in a sample of 130 unrelated blood donors from one of the countrys major hospitals. A comparison between these frequencies and those of 126 populations worldwide was also carried out. A minimum variance dendrogram based on squared Euclidean distances was constructed to assess the relationship between the CCVP sample and populations from all over the world. Allele group and haplotype frequencies observed in this study are consistent with a profile of a dynamic and diverse population, with a hybrid ethnic origin, predominantly Caucasian-Amerindian. Results showed that populations genetically closest to the CCVP are a Mestizo urban population from Venezuela, and another one from Guadalajara, Mexico.

RAET1/ULBP alleles and haplotypes among Kolla South American Indians

NK cell cytolysis of infected or transformed cells can be mediated by engagement of the activating immunoreceptor NKG2D with one of eight known ligands (MICA, MICB and RAET1E-N) and is essential for innate immunity. As well as diversity of NKG2D ligands having the same function, allelic polymorphism and ethnic diversity has been reported. We previously determined HLA class I allele and haplotype frequencies in Kolla South American Indians who inhabit the northwest provinces of Argentina, and were found to have a similar restricted allelic profile to other South American Indians and novel alleles not seen in other tribes. In our current study, we characterized retinoic acid early transcription-1 (RAET1) alleles by sequencing 58 unrelated Kolla people. Only three of six RAET1 ligands were polymorphic. RAET1E was most polymorphic with five alleles in the Kolla including an allele we previously described, RAET1E*009 (allele frequency (AF) 5.2%). Four alleles of RAET1L were also found and RAET1E*002 was most frequent (AF=78%). Potential functional diversity only affected RAET1E and RAET1L, which were in linkage disequilibrium indicating a selective advantage. The results suggest that limited RAET1 polymorphism in the Kolla was not detrimental to human survival but still necessary and may affect disease susceptibility or severity.

Human Leukocyte Antigen Profiles of Latin American Populations: Differential Admixture and Its Potential Impact on Hematopoietic Stem Cell Transplantation

The outcome of hematopoietic stem cell transplantation (HSCT) is shaped by both clinical and genetic factors that determine its success. Genetic factors including human leukocyte antigen (HLA) and non-HLA genetic variants are believed to influence the risk of potentially fatal complications after the transplant. Moreover, ethnicity has been proposed as a factor modifying the risk of graft-versus-host disease. The populations of Latin America are a complex array of different admixture processes with varying degrees of ancestral population proportions that came in different migration waves. This complexity makes the study of genetic risks in this region complicated unless the extent of this variation is thoroughly characterized. In this study we compared the HLA-A and HLA-B allele group profiles for 31 Latin American populations and 61 ancestral populations from Iberia, Italy, Sub-Saharan Africa, and America. Results from population genetics comparisons show a wide variation in the HLA profiles from the Latin American populations that correlate with different admixture proportions. Populations in Latin America seem to be organized in at least three groups with (1) strong Amerindian admixture, (2) strong Caucasian component, and (3) a Caucasian-African gradient. These results imply that genetic risk assessment for HSCT in Latin America has to be adapted for different population subgroups rather than as a pan-Hispanic/Latino analysis.

High-resolution HLA allele and haplotype frequencies in majority and minority populations of Costa Rica and Nicaragua: Differential admixture proportions in neighboring countries

The HLA system shows the most extensive polymorphism in the human genome. Allelic and haplotypic frequencies of HLA genes vary dramatically across human populations. Due to a complex history of migration, populations in Latin America show a broad variety of admixture proportions, usually varying not only between countries, but also within countries. Knowledge of HLA allele and haplotype frequencies is essential for medical fields such as transplantation, but also serves as a means to assess genetic diversity and ancestry in human populations. Here, we have determined high‐resolution HLA‐A, ‐B, ‐C, and ‐DRB1 allele and haplotype frequencies in a sample of 713 healthy subjects from three Mestizo populations, one population of African descent, and Amerindians of five different groups from Costa Rica and Nicaragua and compared their profiles to a large set of indigenous populations from Iberia, Sub‐Saharan Africa, and the Americas. Our results show a great degree of allelic and haplotypic diversity within and across these populations, with most extended haplotypes being private. Mestizo populations show alleles and haplotypes of putative European, Amerindian, and Sub‐Saharan African origin, albeit with differential proportions. Despite some degree of gene flow, Amerindians and Afro‐descendants show great similarity to other Amerindian and West African populations, respectively. This is the first comprehensive study reporting high‐resolution HLA diversity in Central America, and its results will shed light into the genetic history of this region while also supporting the development of medical programs for organ and stem cell transplantation.

Alloreactive T Cell Receptor Diversity against Structurally Similar or Dissimilar HLA-DP Antigens Assessed by Deep Sequencing

T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vβ immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2u2009±u20099.9 vs. 61.5u2009±u20097.7%, respectively, pu2009<u20090.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vβ level, with both alloantigens eliciting specific responses from all TCR-Vβ specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vβ specificities against DPB1*09:01. In line with these observations, TCRB-CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 [0.39 (0.36–0.45) and 0.39 (0.30–0.46), respectively], or in the cumulative frequencies of the 10 most frequent responding clones (55–67 and 58–62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of TCRB immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.

Dissecting Genetic Control of HLA-DPB1 Expression and Its Relation to Structural Mismatch Models in Hematopoietic Stem Cell Transplantation

HLA expression levels have been suggested to be genetically controlled by single nucleotide polymorphisms (SNP) in the untranslated regions (UTR), and expression variants have been associated with the outcome of chronic viral infection and hematopoietic stem cell transplantation (HSCT). In particular, the 3′UTR rs9277534-G/A SNP in HLA-DPB1 has been associated with graft-versus-host-disease after HSCT (Expression model); however its relevance in different immune cells and its mode of action have not been systematically addressed. In addition, there is a strong though not complete overlap between the rs9277534-G/A SNP and structural HLA-DPB1 T cell epitope (TCE) groups which have also been associated with HSCT outcome (TCE Structural model). Here we confirm and extend previous findings of significantly higher HLA-DPB1 expression in B cell lines, unstimulated primary B cells, and monocytes homozygous for rs9277534-G compared to those homozygous for rs9277534-A. However, these differences were abrogated by interferon-γ stimulation or differentiation into dendritic cells. We identify at least seven 3′UTR rs9277534-G/A haplotypes differing by a total of 37 SNP, also characterized by linkage to length variants of a short tandem repeat (STR) in intron 2 and TCE group assignment. 3′UTR mapping did not show any significant differences in post-transcriptional regulation assessed by luciferase assays between two representative rs9277534-G/A haplotypes for any of eight overlapping fragments. Moreover, no evidence for alternative splicing associated with the intron 2 STR was obtained by RT-PCR. In an exemplary cohort of 379 HLA-DPB1 mismatched donor-recipient pairs, risk prediction by the Expression model and the Structural TCE model was 36.7% concordant, with the majority of discordances due to non-applicability of the Expression model. HLA-DPB1 from different TCE groups expressed in the absence of the 3′UTR at similar levels by transfected HeLa cells elicited significantly different mean alloreactive CD4+ T-cell responses, as assessed by CD137 upregulation assays in 178 independent cultures. Taken together, our data provide new insights into the cell type-specific and mechanistic basis of the association between the rs9277534-G/A SNP and HLA-DPB1 expression, and show that, despite partial overlap between both models in HSCT risk-prediction, differential alloreactivity determined by the TCE structural model occurs independently from HLA-DPB1 differential expression.

In silico prediction of nonpermissive HLA-DPB1 mismatches in unrelated HCT by functional distance

In silico prediction of high-risk donor-recipient HLA mismatches after unrelated donor (UD) hematopoietic cell transplantation (HCT) is an attractive, yet elusive, objective. Nonpermissive T-cell epitope (TCE) group mismatches were defined by alloreactive T-cell cross-reactivity for 52/80 HLA-DPB1 alleles (TCE-X). More recently, a numerical functional distance (FD) scoring system for in silico prediction of TCE groups based on the median impact of exon 2-encoded amino acid polymorphism on T-cell alloreactivity was developed for all DPB1 alleles (TCE-FD), including the 28/80 common alleles not assigned by TCE-X. We compared clinical outcome associations of nonpermissive DPB1 mismatches defined by TCE-X or TCE-FD in 8/8 HLA-matched UD-HCT for acute leukemia, myelodysplastic syndrome, and chronic myelogenous leukemia between 1999 and 2011 (N = 2730). Concordance between the 2 models was 92.3%, with most differences arising from DPB1*06:01 and DPB1*19:01 being differently assigned by TCE-X and TCE-FD. In both models, nonpermissive mismatches were associated with reduced overall survival (hazard ratio [HR], 1.15, P < .006 and HR, 1.12, P < .03), increased transplant-related mortality (HR, 1.31, P < .001 and HR, 1.26, P < .001) as well as acute (HR, 1.16, P < .02 and HR, 1.22, P < .001) and chronic (HR, 1.20, P < .003 and HR, 1.22, P < .001) graft-versus-host disease (GVHD). We show that in silico prediction of nonpermissive DPB1 mismatches significantly associated with major transplant outcomes is feasible for any DPB1 allele with known exon 2 sequence based on experimentally elaborated FD scores. This proof-of-principle observation opens new avenues for developing HLA risk-prediction models in HCT and has practical implications for UD searches.