Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.

<p>Southern Africa is facing one of the most serious HIV epidemics. This project contributes to the HIVNET, Network for Prevention Trials cohort for vaccine development. HIV’s biology and rapid mutation rate have made vaccine design difficult. We examined HIV-1 subtype C diversity and how it relates to CTL epitope location along viral gag sequences. We found a negative correlation between codon sites under positive selection and epitope regions / suggesting epitope regions are evolutionarily conserved. It is possible that epitopes exist in non-conserved regions, yet fail to be detected due to the reference strain diverging from the circulating viral population. To test if CTL clustering is an artifact of the reference strain, we calculated differences between the gag codons and the reference strain. We found a weak negative correlation, suggesting epitopes in less conserved regions maybe evading detection. Locating conserved and optimal epitopes that can be recognized by CTLs is essential for the design of vaccine reagents.</p>

Base teórica: A sepse é uma síndrome heterogênea, definida como disfunção orgânica que ameaça à vida, causada por uma resposta desregulada do hospedeiro à infecção. É um problema de saúde mundial, graças à sua alta prevalência, morbimortalidade associada, além de custos para seu tratamento. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). A intensidade da resposta à infecção pode variar entre indivíduos, logo pode-se considerar que esta seja determinada por bases genéticas, e estas influenciem na ocorrência de sepse e variabilidade nos desfechos. Objetivos: Avaliar a associação entre os genes KIR e os ligantes HLA em pacientes críticos, comparando pacientes com sepse e controles não sépticos internados na mesma UTI. Métodos: Foi examinado o polimorfismo de 16 genes KIR e seus ligantes HLA em 271 pacientes críticos, caucasóides, sendo 211 pacientes com sepse e 60 controles, pela técnica de PCR-SSO e PCR-SSP, respectivamente. Resultados: Os genes ativadores KIR2DS1 e KIR3DS1 foram mais frequentes nos controles que nos pacientes com sepse (41,23% versus 55,00%, e 36,49% versus 51,67%; p = 0.041 e 0,025, respectivamente). Estes resultados fornecem informação inicial sobre o papel de polimorfismos de KIR na sepse, sugerindo que este possa ser um potencial marcador diagnóstico ou prognóstico da doença. / Background: Sepsis is a heterogeneous syndrome, defined a life-threatening organic dysfunction caused by a dysregulated host response to infection. Sepsis is a global health problem, due to its high prevalence, associated morbidity and mortality, and costs for its treatment. Cells Natural Killer (NK) cells are part of the innate immune system that recognize HLA class I molecules on target cells via membrane receptors called killer cell immunoglobulin-like receptors (KIR). The intensity of the response to an infection may vary among individuals and might be influenced genetic features affecting sepsis occurrence and variability in outcomes. Objectives: To evaluate the association between KIR genes and HLA ligands in critically ill patients, comparing patients with sepsis and without sepsis admitted to the same ICU. Methods: We examined the polymorphism of 16 KIR genes and their HLA ligands in 271 critically ill patients, Caucasians, and 211 patients with sepsis and 60 controls by PCR-SSO and PCR-SSP, respectively. Results: Activating KIR2DS1 and KIR3DS1 genes were more common in controls than in patients with sepsis (41.23% versus 55.00% and 36.49% versus 51.67%, p = 0.041 and 0.025, respectively). These results provide initial information on the role of polymorphism of KIR in sepsis, suggesting that this may be a potential diagnostic or prognostic marker of the disease.

Despite the success of vaccines in routine use worldwide, there are substantial challenges hampering our ability to develop vaccines against extant diseases including malaria and tuberculosis. Novel approaches are urgently required to help us understand immunological correlates of protection against disease and facilitate our understanding of the impact of human genetic variation on the success of diverse vaccines. To identify host genetic factors responsible for variation in antibody responses against vaccine antigens delivered routinely to infants worldwide I performed a genome-wide association study (GWAS) involving 2,499 infants recruited from three diverse sites across Africa. I identified strong genetic associations between variants in the class II major histocompatibility complex (MHC) locus and responses against five antigens: pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin; diphtheria toxin (DT); and hepatitis B surface antigen. To characterise these associations at the gene and allelic level I developed a large, high-resolution (6-digit 'G') population-specific human leukocyte antigen (HLA) imputation reference panel including 697 individuals from the vaccine GWAS typed at 11 genes, highlighting the diversity of HLA across the African continent. Using this panel I imputed HLA into the remaining GWAS dataset to fine-map the associations to specific HLA alleles, amino acid and single nucleotide polymorphism sites; some of which were found to be African specific. I then used these HLA association findings observed with PT response to correlate, through genetics, this trait with susceptibility to whooping cough in an independently recruited and analysed set of cohorts from the UK. I further used these genetic correlations to demonstrate the relevance of levels of PT-specific circulating follicular helper T-cells and TRBV29-1 T-cell receptor gene expression levels in the development of this protective immune response against PT. By using HLA-peptide binding studies I also demonstrate the diversity of mechanisms that are involved in HLA-disease association, showing that the breadth and affinity of DT-peptide binding are increased with HLA-DRB1 alleles associated with increased DT antibody responses. Taken together, these data represent the first comprehensive genetic association study of multiple vaccine responses undertaken in African infants. These results highlight the importance of human genetics in modulating protective responses against vaccine antigens and demonstrate how such associations can be harnessed to understand biological mechanisms of protective efficacy in greater detail that may in turn facilitate future vaccine development.

Base teórica: A sepse é uma síndrome heterogênea, definida como disfunção orgânica que ameaça à vida, causada por uma resposta desregulada do hospedeiro à infecção. É um problema de saúde mundial, graças à sua alta prevalência, morbimortalidade associada, além de custos para seu tratamento. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). A intensidade da resposta à infecção pode variar entre indivíduos, logo pode-se considerar que esta seja determinada por bases genéticas, e estas influenciem na ocorrência de sepse e variabilidade nos desfechos. Objetivos: Avaliar a associação entre os genes KIR e os ligantes HLA em pacientes críticos, comparando pacientes com sepse e controles não sépticos internados na mesma UTI. Métodos: Foi examinado o polimorfismo de 16 genes KIR e seus ligantes HLA em 271 pacientes críticos, caucasóides, sendo 211 pacientes com sepse e 60 controles, pela técnica de PCR-SSO e PCR-SSP, respectivamente. Resultados: Os genes ativadores KIR2DS1 e KIR3DS1 foram mais frequentes nos controles que nos pacientes com sepse (41,23% versus 55,00%, e 36,49% versus 51,67%; p = 0.041 e 0,025, respectivamente). Estes resultados fornecem informação inicial sobre o papel de polimorfismos de KIR na sepse, sugerindo que este possa ser um potencial marcador diagnóstico ou prognóstico da doença. / Background: Sepsis is a heterogeneous syndrome, defined a life-threatening organic dysfunction caused by a dysregulated host response to infection. Sepsis is a global health problem, due to its high prevalence, associated morbidity and mortality, and costs for its treatment. Cells Natural Killer (NK) cells are part of the innate immune system that recognize HLA class I molecules on target cells via membrane receptors called killer cell immunoglobulin-like receptors (KIR). The intensity of the response to an infection may vary among individuals and might be influenced genetic features affecting sepsis occurrence and variability in outcomes. Objectives: To evaluate the association between KIR genes and HLA ligands in critically ill patients, comparing patients with sepsis and without sepsis admitted to the same ICU. Methods: We examined the polymorphism of 16 KIR genes and their HLA ligands in 271 critically ill patients, Caucasians, and 211 patients with sepsis and 60 controls by PCR-SSO and PCR-SSP, respectively. Results: Activating KIR2DS1 and KIR3DS1 genes were more common in controls than in patients with sepsis (41.23% versus 55.00% and 36.49% versus 51.67%, p = 0.041 and 0.025, respectively). These results provide initial information on the role of polymorphism of KIR in sepsis, suggesting that this may be a potential diagnostic or prognostic marker of the disease.

Magister Scientiae - MSc / Southern Africa is facing one of the most serious HIV epidemics. This project contributes to the HIVNET, Network for Prevention Trials cohort for vaccine development. HIVÂ’s biology and rapid mutation rate have made vaccine design difficult. We examined HIV-1 subtype C diversity and how it relates to CTL epitope location along viral gag sequences. We found a negative correlation between codon sites under positive selection and epitope regions; suggesting epitope regions are evolutionarily conserved. It is possible that epitopes exist in non-conserved regions, yet fail to be detected due to the reference strain diverging from the circulating viral population. To test if CTL clustering is an artifact of the reference strain, we calculated differences between the gag codons and the reference strain. We found a weak negative correlation, suggesting epitopes in less conserved regions maybe evading detection. Locating conserved and optimal epitopes that can be recognized by CTLs is essential for the design of vaccine reagents. / South Africa

No description available.

Carcinomatose péritonéale est un terme utilisé pour désigner la dissémination métastatique généralisée du cancer dans la cavité péritonéale. Il se caractérise par l’accumulation de liquide appelé « ascite » et est considéré comme étant au stade terminal du cancer, car il est difficile à traiter. L'ascite accumulée dans la PC comprend des cellules tumorales, cytokines et cellules immunitaires. Les cellules cancéreuses expriment des protéines spécifiques qui les aident à supprimer les cellules immunitaires et à survivre, appelées points de contrôle immunitaires. Des points de contrôle immunitaires sont présents pour réguler le système immunitaire et sont cruciaux contre la tolérance de soi. PD-1 / PD-L1 et CTLA-4 sont des voies de contrôle immunitaire bien établies adaptées au cancer pour échapper à l'immunité. Récemment, HLA-G a été reconnu comme un point de contrôle et il a été constaté que la survie globale était diminuée dans plusieurs types de cancers solides.Au cours de ma thèse, nous avons évalué l'expression de HLA-G dans la carcinomatose ovarienne. Nous avons constaté que les cellules cancéreuses dans l'ascite de presque tous les patients atteints de carcinomatose ovarienne exprimaient HLA-G. De plus, des taux croissants de sHLA-G1 et de HLA-G5 ont été trouvés dans les ascites. Cette présence de sHLA-G s'est révélée être corrélée positivement avec les Tregs et en corrélation négative avec les cellules T cytotoxiques (CD8) et les cellules NK. De plus, nous avons constaté que les ascites peuvent induire l’expression de HLA-G dans des «Hospicells» via des cytokines inflammatoires. Parmi les cytokines inflammatoires, le TGF-β et IL-1β ont une importance capitale dans l’induction de HLA-G. En outre, nous avons constaté que IL-1β implique la voie NF-κB. Dans une cohorte distincte de carcinomatose péritonéale, composée de patients atteints de PC d'origine différente, nous avons constaté que le groupe de cellules cancéreuses dans l'ascite avait une expression génique hétérogène de PD-L1, CTLA-4 et HLA- G. En outre, nous avons constaté que tous les patients présentaient des taux solubles de HLA-G et PD-L1 dans leur ascite. Cependant, seulement 5 patients présentaient des taux de CTLA-4 solubles dans leur ascite. De plus, nous avons trouvé une très forte corrélation positive entre le niveau de gène de PD-L1 et de CTLA-4, alors qu'aucune corrélation n'a été trouvée pour HLA-G avec PD-11 et CTLA-4 suggérant que HLA-G agit indépendamment des deux points de contrôle immunitaires. En outre, nous avons évalué l'expression de ces points de contrôle immunitaires par des nodules de cancer présents sur la membrane péritonéale. Nous avons trouvé une faible expression de HLA-G et PD-L1, mais la moitié des échantillons étaient fortement positifs pour sHLA-G. Nous avons également constaté que le sHLA-G pouvait être absorbé par l'ascite par la couche mésothéliale. Cette sHLA-G absorbée peut fournir un environnement immunosuppresseur pour la fixation des grappes de cellules cancéreuses à la membrane péritonéale. In vitro, nous avons constaté que l'ascite peut exercer une action immunosuppressive et retarder la lyse des cellules cancéreuses par les cellules immunitaires.De plus, nous avons constaté que la différenciation des cellules cancéreuses se traduit par une augmentation des propriétés immunosuppressives par une expression accrue de HLA-G ou PD-L1. En outre, l'expression de HLA-G et PD-L1 dépend de la phase du cycle cellulaire. Les cellules cancéreuses, si elles sont bloquées dans les cellules mitotiques, expriment des niveaux élevés de HLA-G et de PD-L1, tandis qu'une expression plus faible a été observée en phase G1. Par conséquent, nous suggérons d’éviter l’utilisation d’inhibiteurs de la mitose car ils pourraient augmenter la suppression immunitaire du cancer. De plus, le Ki-67 étant directement lié à l'index mitotique, nous suggérons de développer une échelle de Ki-67 pour évaluer le profil d'immunosuppresseur des patients cancéreux. / Peritoneal carcinomatosis (PC) is a term used for widespread metastatic dissemination of cancer to the peritoneal cavity. It is characterized by the accumulation of fluid called “ascites” and is considered a terminal stage of cancer, as it is hard to treat. The overall survival rate for untreated patients is six-months. However, owing to modern techniques like HIPEC, the survival rate can be increased up to five years. The ascites accumulated in PC, consists of tumor cells, cytokines and immune cells. Cancer cells express specific proteins to suppress immune cells activity and their attack, known as immune checkpoints. PD-1/PD-L1 and CTLA-4 are well established immune checkpoint pathways adapted by cancer in evading immunity. Recently, HLA-G has been recognized as an immune checkpoint and has been found to decrease overall survival in several types of solid cancers. We evaluated the expression of HLA-G in ascites from ovarian carcinomatosis. We found that HLA-G is expressed by cancer cells in ascites from all of the patients(n=16) with ovarian carcinomatosis. Moreover, increased levels of sHLA-G1 and HLA-G5 were found in ascites. This presence of sHLA-G isoforms was found to be positively correlated with Tregs and negatively correlated with cytotoxic T-cells (CD8) and NK-cells suggesting the role of HLA-G in immune suppression. Further, we found that ascites can induce the expression of HLA-G in “Hospicells” via inflammatory cytokines. Among the inflammatory cytokines, TGF-β and IL-1β are of crucial importance in HLA-G induction with IL-1β being more potent compared to TGF-β. Further, we found that IL-1β induces HLA-G expression through NF-κB pathway.In a separate cohort of peritoneal carcinomatosis(n=27), consisting of patients with cancer from a different origin, we found that cancer cell cluster in ascites (n=23) had a heterogeneous gene expression of PD-L1, CTLA-4 and HLA-G. Further, we found that all of the patients presented soluble levels of HLA-G in their ascites. However, one patient was negative for soluble PD-L1 and only 5 patients presented soluble CTLA-4 levels in their ascites. This heterogeneity explains why some of the patients respond to immune therapy while others don’t. This also suggests the need for prescreening patients before immune therapy. Moreover, we found a very strong positive correlation (rs=0.793) between gene level of PD-L1 and CTLA-4, while no correlation was found for HLA-G with PD-L1 and CTLA-4 suggesting that HLA-G acts independently of both the immune checkpoints. Also, we evaluated the expression of these immune checkpoints by cells in peritoneal tissue (n=20). We found low expression of HLA-G and PD-L1, but the majority of the samples were found strongly positive for sHLA-G presence. This sHLA-G can provide an immune-suppressive environment for the attachment of the cancer cell clusters to the peritoneal membrane to form cancer nodule. Additionally, we developed an in-vitro cytotoxicity assay to show that the ascites can provide the immune-suppressive action by interfering with immune cell interaction and delaying the lysis of cancer cells by the immune cells.In parallel, we found that the differentiation of the cancer cells results in increased expression of immune checkpoints like HLA-G or PD-L1. This may render these cells more immune resistant and can protect against immune attack. However, in-vivo mice model is needed to study the oncogenic potential of these differentiated cells. Further, we report that the expression of HLA-G and PD-L1 is dependent on the cell cycle phase. The cancer cells, if blocked in mitotic phase express high levels of HLA-G and PD-L1, while lowest expression was observed in G1-phase. Therefore, we suggest avoiding the use of mitotic inhibitors as it may increase the immune suppression of cancer. Moreover, as Ki-67 is directly related to the mitotic index, we suggest developing a Ki-67 scale to evaluate the immune-suppressive profile of cancer patients.

O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.

O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.