There is documented evidence of high genetic diversity amongst African populations, but there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA genes are highly polymorphic, and encode for proteins that are part of the host defence mechanism mediated through antigen presentation to immune system effector cells. The highly polymorphic nature of HLA genes facilitates the presentation of a wide range of antigenic peptides to the immune system leading to an immune response. With the high disease burden in Africa, it is important to fully understand HLA diversity in these populations, to establish HLA-disease associations, and potentially use this data for the informed design of population-specific vaccines against the many diseases, and to improve on donor-recipient matching. The aim of this thesis is to understand HLA diversity in South African populations to support transplantation programs, add knowledge on human diversity and build a potential future resource for disease association and population studies. There is generally limited HLA data from southern African populations (Chapter 2) to support disease association studies, provide guidance in vaccine design and donor recruitment for transplantation programs. Despite being the only active bone marrow donor registry in Africa supporting transplantation programs, HLA diversity in volunteer bone marrow donors registered at the South African Bone Marrow Registry (SABMR) is largely undocumented. This study documents HLA -A, -B, -C, -DRB1 and -DQB1 allele and haplotype frequencies from a subset of 237 SABMR registered donors with the objective of highlighting HLA diversity in South Africans (Chapter 3). Additionally, mixed resolution HLA data from the National Health Laboratory Services (NHLS) and the South African National Blood Transfusion Service (SANBS) are reported (Chapter 4). A comparison of South African HLA data (NHLS and SANBS) with other global populations including sub Saharan Africans confirm the genetic diversity of South Africans. To counter the paucity of HLA data, in silico HLA imputation tools may be used to determine HLA alleles from existing whole genome sequencing (WGS) data. HLA imputation is an economically feasible typing option for resource limited settings. To support the feasibility of HLA imputation, this study describes high resolution (up to 8 digit typing) HLA alleles determined by in silico HLA imputation tools from 24 WGS of South African individuals (chapter 5). Generally, HLA diversity of South African populations is described in detail through literature meta-analysis, documentation of previously typed individuals (SANBS, NHLS and SABMR) and HLA imputation from existing next generation sequencing (NGS) data. Although results reported here are from a small subset of 237 SABMR registered donors (chapter 3), 24 WGS (chapter 5) and mixed resolution typing NHLS and SANBS data (chapter 4), allele and haplotype frequencies generated could be a useful resource for future anthropological and population genetics studies. Furthermore, these findings may better inform donor recruitment strategies for the SABMR, and disease association studies. Future study recommendations include development of an HLA diversity resource for African populations, a comparison of large SABMR dataset with other global registries, and using more robust assembly based computational tools to fully understand the HLA diversity in South Africans. / Thesis (PhD)--University of Pretoria, 2018. / South African Medical Research Council (SAMRC) in terms of the MRC’s Flagships Awards Project (SAMRC-RFA-UFSP-01-2013/STEM CELLS), the SAMRC Extramural Unit for stem cell Research and Therapy, the Institute for Cellular and Molecular Medicine of the University of Pretoria, and the National Research Foundation of South Africa. / Immunology / PhD Medical Immunology / Unrestricted

Orientador: Erick da Cruz Castelli / Resumo: O Complexo HLA (Human Leukocyte Antigen Complex) é a região mais variável do genoma humano. O gene HLA-A é o segundo mais polimórfico do grupo e responsável por codificar moléculas envolvidas no processo de apresentação antigênica e modulação das células NK (Natural Killer). Diversos estudos avaliaram a variabilidade do gene HLA-A em populações mundiais. No entanto, estes estudos focaram-se principalmente na variabilidade dos éxons, ignorando a variabilidade de íntrons e regiões regulatórias. A variabilidade do gene HLA-A é particularmente elevada no segmento que codifica a fenda de ligação a peptídeos (éxons 2 e 3), relacionado com a função de apresentação antigênica e alvo de seleção balanceadora. No presente estudo avaliamos a diversidade genética do gene HLA-A considerando um segmento contínuo que compreende o promotor estendido (1.5 Kb upstream do primeiro ATG traduzido), todos os éxons (incluindo a região 3’ não-traduzida) e íntrons por meio de sequenciamento paralelo massivo. As estratégias computacionais empregadas no estudo utilizam-se de programas de livre acesso ou desenvolvidos localmente, viabilizando um mapeamento adequado das sequências produzidas e a genotipagem/haplotipagem para genes HLA. A variabilidade detectada para o gene HLA-A em uma amostra brasileira demonstrou que os segmentos regulatórios apresentam poucas sequências diferentes, porém as sequências existentes são bastante divergentes entre si. As variantes regulatórias apresentam alto desequilíbrio ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: HLA-A is the second most polymorphic loci at the Human Leucocyte Antigen (HLA) complex and encodes a key molecule for antigen presentation and NK cell modulation. Many studies have evaluated HLA-A variability in worldwide populations focusing mainly on exons, and the regulatory segments are poorly characterized. HLA-A variability is particularly high at the segment encoding the peptide-binding groove (exons 2 and 3), which is probably related to their antigen presentation function and balancing selection acting at these segments. Here we evaluate the variability and haplotypes of the HLA-A gene considering a continuous segment encompassing the extended promoter (1.5 Kb upstream the first translated ATG), all exons and introns, and the entire 3’ untranslated region, by using massively parallel sequencing. To achieve this goal, we used a freely available bioinformatics workflow that optimizes read mapping for HLA genes and defined haplotypes using either the phase among variable sites observed directly in sequencing data or probabilistic models. The HLA-A variability detected in a highly admixed population sample from Brazil demonstrates that the HLA-A regulatory segments present few, but divergent haplotypes. The regulatory segments are in close association with the coding alleles. Introns segments are also quite variable. In addition, patterns of molecular diversity suggest that the promoter, in addition to the coding region, might be under the same selective regime, but a di... (Complete abstract click electronic access below) / Doutor

Orientador: Erick da Cruz Castelli / Resumo: O gene HLA-C está localizado dentro do Complexo Principal de Histocompatibilidade (MHC), a região mais variável do genoma humano. HLA-C codifica moléculas que participam principalmente do processo de apresentação de antígenos intracelulares aos linfócitos T citotóxicos e interage com receptores presentes nas células NK, modulando sua atividade. A variabilidade das moléculas HLA permite a apresentação de diferentes peptídeos por um mesmo indivíduo e aumenta o repertório de peptídeos apresentados por uma população. Além disso, HLA-C é expresso na interface materno-fetal pelo trofoblasto, onde possui uma importante função imunomodulatória por meio da interação com receptores KIR das células NK maternas. Devido a sua dupla função, alguns alelos de HLA-C ou combinações de alelos HLA-C/KIR têm sido associados a diversos contextos fisiológicos e patológicos. Contudo, a variabilidade desse gene tem sido explorada principalmente para os éxons, em especial os éxons 2 e 3, que codificam os domínios de ligação ao peptídeo, enquanto a variabilidade de outros segmentos gênicos importantes (como outros éxons, íntrons e regiões regulatórias) foram pouco estudadas. Este estudo avaliou a variabilidade genética e assinaturas de seleção natural ao longo do gene HLA-C em uma amostra de 418 indivíduos do Brasil e 108 indivíduos do Benin. Considerando as duas populações, detectamos 359 sítios de variação ao longo da região analisada. Os haplótipos promotores, codificadores e de 3’NT apresentaram um... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Human Leucocyte antigen-C (HLA-C) is a classical HLA class I molecule that binds and presents peptides to cytotoxic T lymphocytes in the cell surface. HLA-C has a dual function since it also interacts with KIR receptors expressed in NK and T cells, modulating their activity. The structure and diversity of the HLA-C regulatory regions, as well as the relationship among variants along the HLA-C locus, is poorly addressed, and no population-based study explored the complete HLA-C variability in different population samples. Here we first present a new molecular and bioinformatics method to evaluate the full HLA-C segment, including regulatory sequences. Then, we applied this method to survey the HLA-C diversity in two geographically distinct population samples, one admixed from Brazil (São Paulo State) and one less admixed from Benin. Our results indicate that the HLA-C promoter and 3’UTR are very polymorphic, with the presence of few but highly divergent haplotypes. However, both these regulatory regions present conserved segments that are shared among different primates. Nucleotide diversity was higher in other exonic segments rather than exons 2 and 3, and also higher in the second half of the 3’UTR region. We detected evidence of balancing selection on the entire HLA-C locus and positive selection in exon 1, for both populations. HLA-C motifs previously associated with KIR interaction and expression regulation are similar between both populations, but the frequency of amino ... (Complete abstract click electronic access below) / Doutor

Pitch Technologies specializes their work on the HLA standard, a standard that specifies data exchange between simulators. The company provides a solution for recording HLA data into a database as raw byte data entries. In this thesis, different design solutions to store and organize recorded HLA data in a manner that reflects the content of the data are proposed and implemented, with the aim of making the data possible to query and analyze after recording. The design solutions impact on storage, read- and write performance as well as usability are evaluated through a suite of tests run on a PostgreSQL database and a TimescaleDB database. It is concluded that none of the design alternatives is the best solution for all aspects, but the most promising combination is proposed.

Hintergrund: Die HLA-Komplexe sind wichtige Mittler bei Entzündungsgeschehen. Durch die Präsentation von zelleigenen oder zellfremden Peptiden werden T-Zellen aktiviert, welche über Zyto- und Chemokine weitere Entzündungszellen aktivieren, anlocken oder aber zum programmierten Zelltod der präsentierenden Zelle führen. Dabei ist die Peptidbindungsstelle der HLA-Komplexe zwar genetisch hochkonserviert, doch auch hochvariabel und so hat jeder HLA-Komplex sein eigenes Bindungsrepertoire. Beim Diabetes mellitus (DM) gibt es drei etablierte Entitäten, die mit einer Autoinflammation einhergehen: Typ 1 Diabetes mellitus (T1DM), Typ 2 Diabetes mellitus (T2DM) sowie der Schwangerschaftsdiabetes (GD). Bei T1DM kommt es zu einer Insulitis mit Aktivierung von CD8+ T-Zellen und darüber zum Untergang der β-Zellen und konsekutiv zu absolutem Insulin-Mangel. Dabei sind schon seit langem die HLA-Serotypen HLA-DR3/DR4 und HLA-DQ2/DQ8 als assoziiert bekannt. Im Falle des T2DM kommt es zur Inflammation des Fettgewebes und darüber zu einer Verminderung der Insulinsensitivität. Hierbei scheinen MHC II Moleküle eine wichtige Rolle zu spielen, da ein Fettgewebsspezifisches Knock-out der MHC II Expression zu einer Verminderung der Insulinresistenz führt. Deng et al. zeigten, dass die MHCII Expression als Reaktion auf Leptinausschüttung erhöht wird, noch bevor es zur Einwanderung der Entzündungszellen kommt. Außerdem scheint es zu einer CD4+ T-Zell getriebenen Insulitis, sowie zu autoimmun aktivierten T-Zellen zu kommen. Schwangerschaftsdiabetes geht ebenfalls mit einem Entzündungsgeschehen einher. 5 Hier zeigte eine großangelegte Studie eine Assoziation zu HLA-DQB1*02, DQB1*06:02 und DRB1*13:02. Ziel: Ziel dieser Studie war der Nachweis einer Assoziation von HLA II Allelen mit der Ausprägung und dem Bestehen eines nicht-insulin-geführten Typ 2 Diabetes mellitus. Methoden & Material: LIFE – Adult (N=4649) und Life-Heart (N=4815) sind Leipziger Bevölkerungsstudien, während die Sorbenkohorte (N=949) ursprünglich Sorben aus der Lausitz umfasst. Die Kohorten wurden umfangreich charakterisiert und die vorhandenen Single Nucleotide Polymorphism Daten aus der Sequenzierung und 1000 Genome Imputation wurden in den weiteren Analysen untersucht. Außerdem fielen alle Teilnehmer heraus, die mit Insulin behandelt wurden. Auf Basis der SNP Genotypisierung wurden schließlich HLA -Genotypen auf 4-Digit Level imputiert, deren Allelfrequenz für ein vergleichendes Phylogramm verwendet wurden. Assoziationen der einzelnen Allele mit Diabetesstatus, HbA1c, Nüchternplasmaglucose, HOMA-IR, HOMA-B und dem Stumvoll-Index in allen Kohorten und wurden mittels linearer bzw. logistischer Regression untersucht, für alle Allele, die mit einer Frequenz von mindestens 5% in den Kohorten vertreten waren. Anschließend wurde eine Meta-Analyse der Assoziationsstudien mit allen drei Kohorten durchgeführt. Dem schloss sich eine Haplotypanalyse und Epitopanalyse an. Alle Ergebnisse wurden nach Benjamini-Hochberg (FDR) gegen multiples Testen korrigiert und auf Alter, Geschlecht und BMI adjustiert. Ergebnisse: Die Allelfrequenzen der LIFE Kohorten unterschieden sich nach Kalkulation der Nei’s distance nicht signifikant von der Deutschen Knochenmarks Spender-Kohorte, während die sorbische Kohorte sich zwischen der deutschen und der tschechischen Vergleichskohorte aus der öffentlich zugänglichen Datenbank einordnen ließ. In der Allelanalyse zeigte sich, dass HLA-DQB*05:01 mit Plasmanüchternglucose und HLA-DRB1*01:01 mit Plasmanüchternglucose und HOMA-IR in der sorbischen Kohorte asoziiert war. In der LIFE-Adult Kohorte assoziierte HLA-DQB*03:02 und HLA-DRB4*01:03 signifikant mit HbA1c. In der anschließenden Meta-Analyse zeigte HLA-DRB5*01:01 den größten protektiven Effekt. Ebenfalls signifikant protektiv wirksam waren die HLA Komplexe HLA-DQA*01:02, HLA-DQB*06:01 und HLA-DRB1*15:01. Der von diesen Allelen gebildete Haplotyp ist bekannt für seine protektive Wirkung in T1DM und zeigte diesen Effekt auch in dieser Testung auf nicht-Insulin-geführten T2DM. Einen Risiko-vermittelnden Haplotyp gab es in Form des DRB1*07:01-DQA1*02:01-DQB1*03:03 Haplotypes. In der Epitopanalyse fanden sich mit A71 und R13 zwei schützende Epitope für T1DM, die in meiner Studie auch als schützende Epitope mit T2DM assoziiert waren. Diskussion: Im Endeffekt zeigte sich in dieser Studie, dass wenn es um den HLA II Locus geht, T1DM und T2DM scheinbar gemeinsame genetische Grundlagen haben. Da nicht einmal für T1DM die Wirkungsweise der HLA-Allele vollkommen verstanden ist, lässt sich nur mutmaßen, wie der kausale Zusammenhang der Ergebnisse dieser Studie aussieht. Zum Beispiel könnte es sein, dass sich aufgrund unterschiedlicher Affinität protektive und permissive Allele in ihrem Bindungsverhalten krankheitsrelevanter Epitope unterscheiden. Da sich HLA-DRB1*15:01 und HLA-DRB1*07:01 in ihrem Bindungsverhalten stark ähneln, aber konträre Effekte auf die Entwicklung eines T2DM haben, muss der Unterschied in den dem Haplotyp zugehörigen DQA1 und DQB1 Allelen liegen. Eine weitere Theorie ist das Epitope stealing, das für HLA-DQ6 im Raume steht. Dabei führte die Bindung der entsprechenden Epitope nicht zu einer CD4-Antwort und somit war das Pankreas vor autoinflammatorischen Geschehen geschützt. Zusammenfassend lässt sich schlussfolgern, dass der HLA Locus sowohl permissive als auch protektive Wirkung sowohl in der Entstehung von T1DM als auch T2DM hat.:Inhaltsverzeichnis 1 Einleitung ......................................................................................................................................... 3 1.1 Aus der Historie des Diabetes mellitus.................................................................................... 3 1.2 Diabetes Mellitus Typ 1 ........................................................................................................... 4 1.3 Diabetes Mellitus Typ 2 ........................................................................................................... 6 1.4 Major Histocompatibility Complex & Human Leucocyte Antigen ........................................... 7 1.4.1 HLA Allele ....................................................................................................................... 10 1.4.2 MHC / HLA und T1DM ................................................................................................... 14 1.4.3 MHC / HLA und T2DM ................................................................................................... 15 1.5 Gemeinsame Determinanten für T1DM und T2DM .............................................................. 16 1.6 Rationale ................................................................................................................................ 17 2 Publikation ..................................................................................................................................... 20 3 Zusammenfassung der Arbeit ....................................................................................................... 30 4 Literaturverzeichnis ....................................................................................................................... 34 References ............................................................................................................................................. 34 5 Supplements .................................................................................................................................. 39 5.1 R-Scripts ................................................................................................................................. 39 5.1.1 Errechnung der Allelfrequenzen.................................................................................... 39 5.1.2 Vorbereitung für Arlequin ............................................................................................. 42 5.1.3 PCA und Dendrogramme ............................................................................................... 48 5.1.4 Signifikanz-Plot .............................................................................................................. 53 5.1.5 Assoziationen LIFE Adult................................................................................................ 61 5.1.6 Assoziationen LIFE Heart ............................................................................................. 115 5.1.7 Assoziationen Sorben .................................................................................................. 169 5.1.8 Meta-Analyse............................................................................................................... 223 6 Abkürzungsverzeichnis ................................................................................................................ 223 7 Abbildungsverzeichnis ................................................................................................................. 226 8 Tabellenverzeichnis ..................................................................................................................... 226 9 Nachweis über die Anteile des Coautors .................................................................................... 227 10 Anlagen .................................................................................................................................... 228 10.1 Erklärung über die eigenständige Abfassung der Arbeit ..................................................... 228

HLA-DRA gene polymorphisms might play an important role in alcohol dependence (AD). We examined genetic associations of 29 single-nucleotide polymorphisms (SNPs) within the HLA-DRA gene with AD using two Caucasian samples - the Collaborative Study on the Genetics of Alcoholism (COGA) sample (660 AD cases and 400 controls) and the Study of Addiction: Genetics and Environment (SAGE) sample (623 cases and 1,016 controls). Logistic regression analysis using PLINK showed that 16 SNPs were associated with AD in the COGA sample and 13 SNPs were associated with AD in the SAGE sample (p < 0.05). The best novel signal was SNP rs2239803 associated with AD in both samples (p = 0.000817 for the COGA sample and p = 0.0026 for the SAGE sample, respectively) while one flanking SNP rs4935356 also showed strong association in both samples (p = 0.00219 and 0.0026 for the COGA and SAGE samples, respectively). Furthermore, these two SNPs revealed stronger associations in meta-analysis of these two samples (p = 8.97 × 10-6 and 2.02 × 10-5 for rs2239803 and rs4935356, respectively). In addition, the G-A haplotype from these two SNPs revealed a significant association with AD in both the COGA and SAGE samples (p = 0.0007 and 0.0019, respectively). These findings highlight the novel associations with HLA-DRA that may play an important role in the etiology of AD.

HLA-DRA gene polymorphisms might play an important role in alcohol dependence (AD). We examined genetic associations of 29 single-nucleotide polymorphisms (SNPs) within the HLA-DRA gene with AD using two Caucasian samples - the Collaborative Study on the Genetics of Alcoholism (COGA) sample (660 AD cases and 400 controls) and the Study of Addiction: Genetics and Environment (SAGE) sample (623 cases and 1,016 controls). Logistic regression analysis using PLINK showed that 16 SNPs were associated with AD in the COGA sample and 13 SNPs were associated with AD in the SAGE sample (p < 0.05). The best novel signal was SNP rs2239803 associated with AD in both samples (p = 0.000817 for the COGA sample and p = 0.0026 for the SAGE sample, respectively) while one flanking SNP rs4935356 also showed strong association in both samples (p = 0.00219 and 0.0026 for the COGA and SAGE samples, respectively). Furthermore, these two SNPs revealed stronger associations in meta-analysis of these two samples (p = 8.97 × 10-6 and 2.02 × 10-5 for rs2239803 and rs4935356, respectively). In addition, the G-A haplotype from these two SNPs revealed a significant association with AD in both the COGA and SAGE samples (p = 0.0007 and 0.0019, respectively). These findings highlight the novel associations with HLA-DRA that may play an important role in the etiology of AD.

OBJECTIVE. To determine if the levels of circulating myeloid-derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy. MATERIALS AND METHODS. We analyzed peripheral blood mononuclear cells isolated from untreated PCa patients (uPCa; N=18; mean age±SD: 72.1± 6.9 years), tPCa (N = 22; 72.8 ± 9.8 years) and age matched controls (AMC; N = 12; 68.8 ± 7.5 years). We quantified surface marker phenotype, differentiation potential, effects on T cell proliferation and intracellular cytokines. RESULTS. We observed an unexpectedly high percentage of a type of myeloid-derived suppressor cells, CD14+HLA-DR low/- monocytes, in tPCa (30.7±15.0% of CD14+ cells) relative to AMC (4.1+6.5%, P<0.0001) and uPCa (10.6 ± 14.3%, P = 0.0001). The levels of CD14+ HLA-DR low/- cells were significantly correlated with circulating PSA levels and treatment with LHRH-agonist leuprolide in combination with either an antiandrogen or dexamethasone. Monocytes from tPCa inhibited autologous T cell proliferation statistically significantly more effectively than AMC monocytes and were defective in their ability to differentiate into phenotypically mature dendritic cells. Isolated CD14+HLA-DRlow/- cells expressed higher levels of intracellular interleukin-10 and suppressed T cell proliferation more effectively than isolated CD14+HLA-DR+ cells. CONCLUSIONS. This is the first report of CD14+ cells exhibiting reduced expression of HLADRmolecules in PCa patients. These cells suppress immune cell function in vitro and, plausibly, in vivo, a finding that must be factored into the design of immunotherapy protocols for PCa patients.

Homeostasis of human T cells is regulated by many factors that control proliferation, differentiation of effector cells and generation of memory. Our current knowledge of the mechanisms controlling human T cell homeostasis in vivo is based on experiments in small animal models. However many differences exist between immune systems of mice and humans, including cell composition, function, and gene expression. Humanized mouse models have shown great value in the study of human immunobiology. I have used novel humanized mouse models to examine the role of human MHC (HLA) and human IL2 in CD8 T cell and CD4 regulatory T cell (Treg) homeostasis. To study human CD8 T cells I engrafted CD8 T cells from healthy donor PBMC into NOD-scid IL2rgnull (NSG) mice that lacked expression of murine MHC and that expressed HLA-A2. My data demonstrate that CD8 T cell survival and effector function required the presence of HLA-A2, helper function from human CD4 T cells and exogenous human IL2. To study human Treg homeostasis I used NSG mice engrafted with human fetal thymus and hematopoietic stem cells (BLT model). NSG-BLT mice support the growth of human thymic tissue and enable the efficient development of HLA-restricted Treg and conventional T cells. Using an AAV vector to express human IL2, I demonstrated that functional human Treg but not conventional T cells increased in number in NSG-BLT mice and that this coincided with increases in activated human NK cells. Overall my research has revealed that HLA and human IL2 have an essential role in human T cell survival and function in vivo.

Presentation of antigenic peptides on MHC-II molecules is essential for induction of tolerance to self and for effective immunity against foreign pathogens. The non- classical MHC-II molecule DO (HLA-DO in humans, H2-O in mice) functions in selection of MHC-II epitopes by competitively inhibiting the peptide exchange factor DM. Previous studies have suggested a role for DO in development of autoimmunity and in the immune response to retroviral infection, presumably via modulation of the MHC-II peptidome, but the precise effect of DO has been difficult to discern. Through characterization of the full spectrum of peptides from DO-sufficient and DO-deficient cells, we demonstrate that DO functions to broaden the diversity of peptide species presented on MHC-II. DO is regulated differently from other components of the MHC-II processing machinery, with expression limited to B cell and dendritic cell subsets, as well as thymic epithelial cells, suggesting a role for DO in mediating central tolerance. In a mouse model lacking DO, we show that selection of T regulatory cells (Tregs) is increased and that DO- deficient Tregs are more activated and exert greater suppressive capacity. Despite augmented Treg function, mice lacking DO display enhanced susceptibility to autoimmunity, with altered germinal center (GC) Tregs and B cells indicative of an aberrant GC reaction. These data suggest that DO expression serves to fine-tune the immunopeptidome in order to promote self-tolerance to a wide spectrum of epitopes and to select a Treg population with appropriate specificity for self- antigens.