L’objectif général de cette thèse était d’étudier le rôle de la protéine soluble HLA-G (sHLA- G) dans la variabilité des réponses à l’infection par P. falciparum chez la femme enceinte et son nouveau-né pendant ses deux premières années de vie. Précisément, nous avons étudié, chez les mères pendant la grossesse et leurs nourrissons de la naissance à 2 ans, les relations entre les niveaux de sHLA-G et l’infection palustre au niveau périphérique et placentaire. Nous avons également étudié les polymorphismes génétiques situés dans la région 3’UTR du gène HLA- G chez les mères et leurs nourrissons par la détermination des fréquences alléliques, génotypiques et haplotypiques afin évaluer l’impact de ces polymorphismes sur la cinétique d’expression de sHLA-G dans un contexte d’infection palustre. Nos résultats ont montré une association entre le niveau élevé de sHLA-G chez les nourrissons et l’augmentation du risque d’infection palustre au cours du trimestre suivant. Ces niveaux élevés de sHLA-G dans le sang de cordon ont été également associés au faible poids de naissance du nouveau-né. De même, nous avons trouvé une forte corrélation entre les niveaux de sHLA-G maternels dans le sang périphérique à l’accouchement et ceux de l’enfant dans le sang du cordon à la naissance. Nous avons aussi montré l’existence de trois profils d’expression de sHLA-G chez les individus inclus dans l'étude. Certains individus expriment la protéine à chaque prélèvement (HLA-G ++) alors que d’autres l’expriment par intermittence (HLA-G +-) ou ne l’expriment pas (HLA-G --). Le risque de développer un accès palustre chez les mères était respectivement trois fois plus élevé (p=0,001, OR=3,47;p=0,008, OR=3,14) chez celles appartenant au groupe HLA-G (++) et HLA-G (+-) que le groupe HLA-G (--). L’analyse génétique de la région 3’UTR du gène nous a permis de mettre en évidence huit sites polymorphes dans cette région et de construire six haplotypes correspondant (UTR 1, 2, 3, 4, 5, 6). Nous avons aussi montré chez les mères une association entre l’allèle T en position +3001 (C/T) et une expression plus fréquente de sHLA-G tandis que l’allèle C en position +3003 (T/C) et l’haplotype UTR-4 ont été associés à une expression moins fréquente de la protéine. Ces associations n’ont pas été mises en évidence chez les enfants. L’ensemble de ces résultats suggère l'implication de sHLA-G dans la sensibilité à l'infection palustre. Cette sensibilité serait, en partie, corrélée à l’inhibition des réponses anticorps spécifiquement dirigées contre P. falciparum. sHLA-G pourrait donc à terme devenir un bio-marqueur de susceptibilité au paludisme chez la femme enceinte et chez le nouveau-né au cours des premières années de vie. / The general objective of this thesis was to study the role of soluble HLA-G protein (sHLA-G) in the variability of individual response to malaria during pregnancy and during the first 2 years of infant life. Actually, we assessed the relationships between sHLA-G and malaria infection in peripheral and placental blood. We also investigated the effect of polymorphisms in the 3’UTR region of HLA-G gene in 400 mothers and their infants on the kinetic of sHLA-G expression three times during pregnancy and at 6, 9, 12, 18, 24 months of life in a context of malaria infection. Our results showed that high levels of sHLA-G increased the risk of malaria at the subsequent trimester in infants and were associated with low birth weight. We also showed a strong correlation between the plasmatic sHLA-G level of the mothers at delivery and those of newborns in cord blood. We found that the risk of developing malaria in mothers was respectively three fold higher in the HLA-G (++) (OR=3.47; p=0.001) and HLA-G(+-)(OR=3.14, p=0.008) groups compared to HLA-G (--) group. Besides, we described eight polymorphic sites in the 3’UTR corresponding to six haplotypes (UTR 1, 2, 3, 4, 5, 6) and showed in mothers, an association between the allele T at position +3001 (C/T) and a higher frequency of sHLA-G expression. However, the allele C at position +3003 (T/C) and UTR-4 were associated to a lower frequency of sHLA-G expression. In infants, no association was observed between alleles or haplotypes and expression of the soluble protein. Overall, these results suggest that sHLA-G is implicated in malaria susceptibility. This could be partly, related to the inhibition of P. falciparum-specific antibody responses. Therefore, sHLA-G might be useful as a bio- marker of malaria susceptibility during pregnancy and during the first years of infancy.

O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

The human leukocyte antigen (HLA) is the most polymorphic region in the human genome and accounts for more than 10% of human diversity. This region plays an important role in matching donors and recipients for transplantation. The South African Bone Marrow Registry (SABMR) does not reflect the demographics of the South African population. The large number of polymorphisms resulting from HLA diversity in the Black South African population and their limited representation in the SABMR reduce the chances of finding adequate matches between donors and recipients in this group. Umbilical cord blood is an alternative to bone marrow for the treatment of fatal diseases. Less strict HLA matching is required due to the naive nature of the T cells in cord blood. A public umbilical cord blood bank is a necessity in trying to cater for the diverse population in South Africa. However, the ethnic diversity of the South African population poses a great challenge in constituting a public umbilical cord blood bank that is representative of the entire population. The Roche designed next generation sequencing (NGS) high resolution (HR) HLA typing kit enables sequencing of additional HLA exons and could improve the degree of matching between individuals to ultimately decrease adverse reactions. An extensive study of the literature was performed to establish the demographics, linguistics, and HLA diversity of the South African population to determine how a public cord blood bank should be constituted. In addition, HLA genotyping was performed by 454 NGS on 20 samples that had previously been HLA typed by conventional methods. The 454 NGS technique made use of a Roche designed medium and high resolution HLA typing kit to genotype the samples. It was possible to assign accurate genotypes to 95.5% of the loci of interest for the total number of 20 samples using the MR kit, compared with 98.5% using the HR kit. In conclusion, the present study indicates the extreme HLA diversity in the South African population, and therefore, recommends constituting the first public umbilical cord blood bank in Gauteng on the basis of race or major ethnic groupings. A minimum number of 10 000 cord blood units is needed to initiate the bank. Furthermore, the 454 NGS platform together with the HR HLA typing kit display potential as an alternative method to be used in a public cord blood bank, as well as routine clinical and diagnostic laboratories, to ultimately improve HLA matching between donors and recipients. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Immunology / unrestricted

O presente estudo tem como objetivo investigar a frequência dos diversos polimorfismos dos genes KIR (Killer Immunoglobulin-like Receptors) e HLA C1 e C2 em um grupo de pacientes com câncer de mama e comparar com um grupo controle de indivíduos sadios. As células natural killer (NK) são linfócitos que diferem das células T e B e que fazem parte da imunidade natural, reconhecendo as moléculas HLA (Antígenos Leucocitários Humano) de classe I em células infectadas por vírus ou em células tumorais, através de seus receptores de membrana. Os principais receptores das células NK são conhecidos como receptores KIR, sendo codificados por genes localizados no cromossomo 19q13.4 e classificados em grupos funcionais supressores e ativadores. Neste estudo, analisamos 15 genes KIR e alelos do sistema HLA de classe I em 230 pacientes caucasóides e em 278 controles, usando a técnica de PCR com primers específicos (PCR-SSO e PCR-SSP). Nossos resultados demonstraram uma frequência maior do genótipo supressor 2DL2 (P<0,001) em pacientes com câncer de mama, quando comparados ao grupo controle. Os genes HLA-C2 e HLA-BW4 não apresentaram diferenças significantes entre os grupos. Contudo, o gene HLA-C1 foi observado em maior frequência nos pacientes com câncer de mama. Considerando que estes achados sugerem uma potencial associação entre o sistema de genes KIR, HLA classe I e o câncer de mama, estudos adicionais sobre este tema são necessários. / We investigated the frequency of various KIR (Killer Immunoglobulin-like Receptors) and HLA C1 and C2 gene polymorphisms in a group of patients with breast cancer and healthy controls. Natural Killer (NK) cells are lymphocytes that differ from T and B cells and are part of the innate immune system, recognizing class I Human Leukocyte Antigens (HLA) molecules on target cells (virus-infected as well as cancer cells), through specific cell surface receptors. KIR comprises the main class of NK receptors, being encoded by genes located in chromosome 19q13.4. They possess both suppressor and activating functional groups. Fifteen KIR genes and class I HLA alleles obtained from 230 Caucasians patients, as well as 278 controls were studied, using PCR techniques with specific primers (PCR-SSO and PCR-SSP). Our results showed a higher frequency of suppressor genotype 2DL2 (P<0,001) in patients with breast cancer as compared to controls. No significant difference between HLA-C2 and HLA-BW4 alleles were observed between the study groups. Notably, a higher frequency of HLA-C1 gene was noted in patients with breast cancer. Our results suggest a potential association between KIR genes, HLA class I and breast cancer, deserving further investigation.

An individual's Human Leukocyte Antigen (HLA) type is an essential immunogenetic parameter, influencing susceptibility to a variety of autoimmune and infectious diseases, to certain types of cancer and the likelihood of adverse drug reactions. I present and evaluate two models for the accurate statistical determination of HLA types for single-population and multi-population studies, based on SNP genotypes. Importantly, SNP genotypes are already available for many studies, so that the application of the statistical methods presented here does not incur any extra cost besides computing time. HLA*IMP:01 is based on a parallelized and modified version of LDMhc (Leslie et al., 2008), enabling the processing of large reference panels and improving call rates. In a homogeneous single-population imputation scenario on a mainly British dataset, it achieves accuracies (posterior predictive values) and call rates >=88% at all classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DQA1, HLA-DQB1, HLA-DRB1) at 4-digit HLA type resolution. HLA*IMP:02 is specifically designed to deal with multi-population heterogeneous reference panels and based on a new algorithm to construct haplotype graph models that takes into account haplotype estimate uncertainty, allows for missing data and enables the inclusion of prior knowledge on linkage disequilibrium. It works as well as HLA*IMP:01 on homogeneous panels and substantially outperforms it in more heterogeneous scenarios. In a cross-European validation experiment, even without setting a call threshold, HLA*IMP:02 achieves an average accuracy of 96% at 4-digit resolution (>=91% for all loci, which is achieved at HLA-DRB1). HLA*IMP:02 can accurately predict structural variation (DRB paralogs), can (to an extent) detect errors in the reference panel and is highly tolerant of missing data. I demonstrate that a good match between imputation and reference panels in terms of principal components and reference panel size are essential determinants of high imputation accuracy under HLA*IMP:02.

In den humanen Leukozyten-Antigenen (HLA) wird die wichtigste genetische Ursache von rheumatoider Arthritis gesehen. Es wurden bisher mehrere Mechanismen beschrieben, wie diese HLA-Moleküle die Entstehung und den Verlauf der Erkrankung beeinflussen. Im Rahmen dieser Arbeit wurde die differentielle Expression von HLA-DRB-Genen in unterschiedlichen Antigen-präsentierenden Zellen als möglicher Mechanismus untersucht. Dabei wurden strukturelle Unterschiede zwischen den Promotoren des krankheitsassoziierten HLA-DR4-Haplotyps und den neutralen Haplotypen DR7 und DR9 eingehender betrachtet. Allen drei Haplotypen ist gemein, daß sie das DRB4-Gen als zweites funktionelles DRB-Gen tragen, wobei das DRB4-Gen entweder den DRB4A oder den -B-Promotor besitzt. Um den Einfluß einzelner Promotorelemente auf die mit dem Luziferase-Assay bestimmten Transkriptionsaktivitäten näher zu untersuchen, wurde mit Hilfe der surface plasmon resonance die Bindung der Transkriptionsfaktoren aus den Zellkernlysaten von der humanen Monozytenzellinie THP-1 und von der humanen B-Lymphom-Zellinie BJAB an die unterschiedlichen S-, X-, Y-, CCAAT- und TATA-Boxen analysiert. Es konnte gezeigt werden, daß die unterschiedliche Expression von DRB4A und DRB4B durch die ubiquitäre TATA-Box vermittelt wird. Dagegen wurde die INF-gamma-Stimulation der HLA-DR-Expression von THP-1- aber auch von BJAB-Zellen durch die für die HLA-DR-Promotoren spezifische X-Box vermittelt. Bei der Analyse von DR4-, DR7- und DR9-positiven Patienten einer bereits gut charakterisierten RA-Kohorte stellte sich heraus, daß der DRB4B-Promotor, welcher im Vergleich zu DRB4A eine höhere transkriptionelle Aktivität besitzt, mit einem schweren Krankheitsverlauf assoziiert ist, so daß eine erhöhte HLA-DR-Expression den Krankheitsverlauf negativ zu beeinflussen scheint. / Disease associated human leukocyte antigen (HLA) genes have been identified in humans where they are assumed to promote the susceptibility and/or progression of rheumatoid arthritis. Several mechanisms have been described how these HLA haplotypes impact on the disease. Among them the differential expression of HLA-DRB molecules in different types of antigen-presenting cells, which was investigated here in detail. The promoters of the disease associated HLA-DR4 to the neutral DR7 and DR9 haplotypes were analyzed for sequence polymorphisms resulting in functional differences. All three haplotypes carry as a second functional DRB gene the DRB4 gene, which is regulated by the DRB4A or -B promoter. To determine the impact of the promoter elements on the transcriptional activities measured by luciferase assay the surface plasmon resonance technology was employed. To this end, nuclear extracts from the monocytic cell line THP-1 and from the B lymphoma cell line BJAB were used to analyze their binding to the various S-, X-, Y-, CCAAT-, and TATA boxes. It could be demonstrated that the differential expression of DRB4A and -B was regulated via the ubiquitous TATA box. By contrast, the INF-gamma stimulation of HLA expression in THP-1 and BJAB was mediated via the unique X box. Analyzing the DR4, DR7 and DR9 positive patients of an RA cohort, the DRB4B promoter, which has a higher transcriptional activity than the DRB4A promoter, is associated with radiographic progression of RA. This data is thus indicative of an impact of elevated HLA-DR expression on the progression of the disease.

Made available in DSpace on 2014-12-17T14:16:34Z (GMT). No. of bitstreams: 1 HeglaynePVS_DISSERT.pdf: 2645894 bytes, checksum: da2c7ec39b2e87b75a199511857a4e83 (MD5) Previous issue date: 2013-03-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Of all of the genes associated with the development of Diabetes mellitus type 1 (T1D), the largest contribution comes from the genes in the Human Leukocyte Antigen (HLA) region, mostly the class II DR e DQ genes. Specific combinations of alleles DRB1, DQA1 and DQB1 constituting haplotypes, and further, a combination of more than one haplotype, providing multilocus genotypes are associated with susceptibility, protection and neutrality to DM1. Thus, the aim of present study was to verified the association of polymorphisms of HLA genes class II with susceptibility to type 1 diabetes mellitus (T1D). Ninety-two patients with T1D and 100 individuals normoglycemics (NG) aged between 6 and 20 years were studied. Genomic DNA was obtained from peripheral whole blood, collected in EDTA tube, using the extraction kit Illustra Triple Prep?, GE Healthcare. For HLA typing was used DNA LABType system by One Lambda kit applying Luminex? technology to the method of PCRSSO typing reverse. The alleles DRB1*03:01, *04:05, *04:01, *04:02, DQA1*03:01g, *05:01g, DQB1*02:01g, *03:02, the haplotypes DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g-DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g-DQB1*03:02 and DR3-DQ2/DR4-DQ8 genotype were significantly associated with the chance of developing T1D. The alleles DRB1*11:01, *15:03, *15:01, *13:01, DQA1*01:02, *04:01g, *01:03, DQB1*06:02, *03:01g, *06:03, *04:02, the haplotypes DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 and DRX-DQX/DRX-DQX genotype, formed by other than the DR3-DQ2 or DR4-DQ8 haplotypes, were significantly associated with T1D protection Despite the major racial Brazilian, even at the regional level, these results are similar to the majority of alleles, genotypes and haplotypes of HLA class II-related susceptibility or resistance to T1D, extensively described in the literature for Caucasian population. Children with age at diagnosis less than 5 years of age had significantly higher frequency of the heterozygous genotype DR3-DQ2/DR4-DQ8 compared to children with age at diagnosis than 5 years old. These results also demonstrate strong association of the genetic profile of the class II HLA for this age group, possibly associated with the severity and rapid progression to the onset of T1D. The knowledge of HLA class II genes may be useful in genetic screens that allow the prediction of T1D / De todos os genes j? relacionados com o desenvolvimento do Diabetes mellitus tipo 1 (DM1), a maior contribui??o vem da regi?o do genoma onde est?o localizados os genes do Ant?geno Leucocit?rio Humano (HLA), sobretudo os genes da classe II do HLA: DR e DQ. Espec?ficas combina??es de alelos DRB1, DQA1 e DQB1 formando hapl?tipos, e ainda, a combina??o de mais de um hapl?tipo, formando gen?tipos multilocus s?o associados com a susceptibilidade, neutralidade e prote??o ao DM1. Dessa forma, o objetivo do estudo foi verificar a associa??o dos polimorfismos dos genes do complexo HLA classe II com a susceptibilidade ao DM1, em pacientes do Rio Grande do Norte. Foram estudados 92 indiv?duos com DM1 e 100 indiv?duos normoglic?micos (NG), com idade entre 6 e 20 anos. O DNA gen?mico foi obtido a partir do sangue total perif?rico, coletado em tubo com EDTA, utilizando o kit de extra??o Illustra Triple Prep?, GE Healthcare. Para a tipagem do HLA foi utilizado o sistema DNA LABType atrav?s de kits One Lambda, que aplica a tecnologia Luminex? ao m?todo de tipagem por PCR-SSO reverso. Os alelos DRB1*03:01, *04:05, *04:01, *04:02; DQA1*03:01g, 05:01g; DQB1*02:01g, *03:02; os hapl?tipos DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g- DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g- DQB1*03:02 e o gen?tipo heterozigoto, DR3-DQ2/DR4-DQ8 foram significativamente associados com a chance de desenvolvimento do DM1. J? os alelos DRB1*11:01, *15:03, *15:01, *13:01; DQA1*01:02, *04:01g, *01:03; DQB1*06:02, *03:01g, *06:03, *04:02; os hapl?tipos DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 e o gen?tipo DRX-DQX/DRX-DQX, formado por outros hapl?tipos que n?o DR3-DQ2 ou DR4-DQ8, foram significativamente associados a prote??o ao DM1. Apesar da grande miscigena??o racial brasileira, at? em n?vel regional, estes resultados s?o semelhantes a maioria dos alelos, hapl?tipos e gen?tipos de HLA classe II relacionados ? susceptibilidade ou prote??o ao DM1, extensivamente descritos na literatura para a popula??o caucasiana. Crian?as com idade ao diagn?stico inferior a 5 anos de idade apresentaram significativamente maior frequ?ncia do gen?tipo heterozigoto DR3-DQ2/DR4-DQ8, quando comparada ?s crian?as com idade ao diagn?stico superior a 5 anos de idade. Esses resultados demonstram tamb?m forte envolvimento do perfil gen?tico da classe II do HLA para esta faixa et?ria, que estaria relacionada possivelmente com a gravidade e a r?pida progress?o para o in?cio do DM1. O conhecimento dos genes HLA de classe II pode ser ?til em triagens gen?ticas que possibilitem a predi??o do DM1

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Le système de différenciation entre le « soi » et le « non-soi » des vertébrés permet la détection et le rejet de pathogènes et de cellules allogéniques. Il requiert la surveillance de petits peptides présentés à la surface cellulaire par les molécules du complexe majeur d’histocompatibilité de classe I (CMH I). Les molécules du CMH I sont des hétérodimères composés par une chaîne lourde encodée par des gènes du CMH et une chaîne légère encodée par le gène β2-microglobuline. L’ensemble des peptides est appelé l’immunopeptidome du CMH I. Nous avons utilisé des approches en biologie de systèmes pour définir la composition et l’origine cellulaire de l’immunopeptidome du CMH I présenté par des cellules B lymphoblastoïdes dérivés de deux pairs de fratries avec un CMH I identique. Nous avons découvert que l’immunopeptidome du CMH I est spécifique à l’individu et au type cellulaire, qu’il dérive préférentiellement de transcrits abondants, est enrichi en transcrits possédant d’éléments de reconnaissance par les petits ARNs, mais qu’il ne montre aucun biais ni vers les régions génétiques invariables ni vers les régions polymorphiques. Nous avons également développé une nouvelle méthode qui combine la spectrométrie de masse, le séquençage de nouvelle génération et la bioinformatique pour l’identification à grand échelle de peptides du CMH I, dont ceux résultants de polymorphismes nucléotidiques simples non-synonymes (PNS-ns), appelés antigènes mineurs d’histocompatibilité (AMHs), qui sont les cibles de réponses allo-immunitaires. La comparaison de l’origine génomique de l’immunopeptidome de soeurs avec un CMH I identique a révélé que 0,5% des PNS-ns étaient représentés dans l’immunopeptidome et que 0,3% des peptides du CMH I seraient immunogéniques envers une des deux soeurs. En résumé, nous avons découvert des nouveaux facteurs qui modèlent l’immunopeptidome du CMH I et nous présentons une nouvelle stratégie pour l’indentification de ces peptides, laquelle pourrait accélérer énormément le développement d’immunothérapies ciblant les AMHs. / The self/nonself discrimination system of vertebrates allows detection and rejection of pathogens and allogeneic cells. It requires the surveillance of short peptides presented by major histocompatibility class I (MHC I) molecules on the cell surface. MHC I molecules are heterodimers that consist of a heavy chain produced by MHC genes and a light chain encoded by the β2-microglobulin gene. The peptides presented by MHC I molecules are collectively referred to as the MHC I immunopeptidome. We employed systems biology approaches to define the composition and cellular origin of the self MHC I immunopeptidome presented by B lymphoblastoid cells derived from two pairs of MHC-identical siblings. We found that the MHC I immunopeptidome is subject- and cell-specific, derives preferentially from abundant transcripts, is enriched in transcripts bearing microRNA response elements and shows no bias toward invariant vs. polymorphic genomic sequences. We also developed a novel personalized approach combining mass-spectrometry, next-generation sequencing and bioinformatics for high-throughput identification of MHC I peptides including those caused by nonsynonymous single nucleotide polymorphisms (ns-SNPs), termed minor histocompatibility antigens (MiHAs), which are the targets of allo-immune responses. Comparison of the genomic landscape of the immunopeptidome of MHC-identical siblings revealed that 0.5% of ns-SNPs were represented in the immunopeptidome and that 0.3% of the MHC I-peptide repertoire would be immunogenic for one of the siblings. We discovered new factors that shape the self MHC I immunopeptidome and present a novel strategy for the identification of MHC I-associated peptides that could greatly accelerate the development of MiHA-targeted immunotherapy.

La présentation d'antigène par les molécules d'histocompatibilité majeure de classe I (CMHI) permet au système immunitaire adaptatif de détecter et éliminer les agents pathogènes intracellulaires et des cellules anormales. La surveillance immunitaire est effectuée par les lymphocytes T CD8 qui interagissent avec le répertoire de peptides associés au CMHI présentés à la surface de toutes cellules nucléées. Les principaux gènes humains de CMHI, HLA-A et HLA-B, sont très polymorphes et par conséquent montrent des différences dans la présentation des antigènes. Nous avons étudié les différences qualitatives et quantitatives dans l'expression et la liaison peptidique de plusieurs allotypes HLA. Utilisant la technique de cytométrie de flux quantitative nous avons établi une hiérarchie d'expression pour les quatre HLA-A, B allotypes enquête. Nos résultats sont compatibles avec une corrélation inverse entre l'expression allotypique et la diversité des peptides bien que d'autres études soient nécessaires pour consolider cette hypothèse. Les origines mondiales du répertoire de peptides associés au CMHI restent une question centrale à la fois fondamentalement et dans la recherche de cibles immunothérapeutiques. Utilisant des techniques protéogénomiques, nous avons identifié et analysé 25,172 peptides CMHI isolées à partir des lymphocytes B de 18 personnes qui exprime collectivement 27 allotypes HLA-A,B. Alors que 58% des gènes ont été la source de 1-64 peptides CMHI par gène, 42% des gènes ne sont pas représentés dans l'immunopeptidome. Dans l'ensemble, l’immunopeptidome présenté par 27 allotypes HLA-A,B ne couvrent que 17% des séquences exomiques exprimées dans les cellules des sujets. Nous avons identifié plusieurs caractéristiques des transcrits et des protéines qui améliorent la production des peptides CMHI. Avec ces données, nous avons construit un modèle de régression logistique qui prédit avec une grande précision si un gène de notre ensemble de données ou à partir d'ensembles de données indépendants génèrerait des peptides CMHI. Nos résultats montrent la sélection préférentielle des peptides CMHI à partir d'un répertoire limité de produits de gènes avec des caractéristiques distinctes. L'idée que le système immunitaire peut surveiller des peptides CMHI couvrant seulement une fraction du génome codant des protéines a des implications profondes dans l'auto-immunité et l'immunologie du cancer. / Antigen presentation by major histocompatibility complex class I (MHCI) molecules allows the adaptive immune system to detect and eliminate intracellular pathogens or abnormal cells. Immune surveillance is executed by CD8 T cells that monitor the repertoire of MHCI-associated peptides (MAPs) presented at the surface of all nucleated cells. The primary human MHCI genes, HLA-A and HLA-B, are highly polymorphic and consequentially demonstrate differences in antigen presentation. We investigated qualitative and quantitative differences in expression and peptide binding. Using quantitative flow cytometry we establish clear hierarchy of expression for the four HLA-A,B allotypes investigated. Our results are consistent with an inverse correlation between expression and peptide diversity although further work is necessary to solidify this hypothesis. The global origins of the MAP repertoire remains a central question both fundamentally and in the search for immunotherapeutic targets. Using proteogenomics, we identified and analyzed 25,172 MAPs isolated from B lymphocytes of 18 individuals who collectively expressed 27 HLA-A,B allotypes. While 58% of genes were the source of 1-64 MAPs per gene, 42% of genes were not represented in the immunopeptidome. Overall, we estimate the immunopeptidome presented by 27 HLA-A,B allotypes covered only 17% of exomic sequences expressed in subjects’ cells. We identified several features of transcripts and proteins that enhance MAP production. From these data we built a logistic regression model that predicts with high accuracy whether a gene from our dataset or from independent datasets would generate MAPs. Our results show preferential selection of MAPs from a limited repertoire of gene products with distinct features. The notion that the immune system can monitor MAPs covering only a fraction of the protein coding genome has profound implications in autoimmunity and cancer immunology.