Presque toutes les cellules nucléées expriment des peptides associés au CMH I (HLA I chez l’humain)(MAP) qui sont échantillonnés à partir du protéome cellulaire et transportés vers la surface cellulaire pour inspection par les lymphocytes T CD8. En tant que tel, la collection de MAP à la surface des cellules, ou immunopeptidome, informe les lymphocytes T CD8 de l’état cellulaire interne. L’immunosurveillance du cancer repose sur la capacité des lymphocytes T CD8 à reconnaître les MAP anormaux sur les cellules tumorales et à les éliminer tout en épargnant les cellules saines. Par conséquent, l’existence du cancer indique que bien souvent, les lymphocytes T CD8 spécifiques à la tumeur sont impuissants, dysfonctionnels ou incapables d’exercer leur fonction. Les vaccins anticancéreux peuvent actionner la destruction des tumeurs en stimulant la reconnaissance des MAP anormaux. Toutefois, le développement de vaccins anticancéreux efficaces est entravé par le manque de MAP exploitables, ou antigènes tumoraux (TA), exprimés exclusivement sur les cellules tumorales. La recherche et l’identification de TA ont été largement limitées aux MAP dérivés de mutations non synonymes situées dans des exons canoniques codant pour des protéines. Ces régions génomiques ne représentent que 2% du génome humain. Le fait que les MAP puissent potentiellement dériver de la traduction non canonique de toutes les régions génomiques n’a été pleinement compris que récemment. Ici, nous avons utilisé la protéogénomique pour découvrir des TA exploitables dérivés de produits de traduction canoniques et non canoniques partagés au sein ou entre divers types de cancers humains. Premièrement, nous avons utilisé des cellules souches pluripotentes induites (iPSC) pour identifier les MAP associés à la pluripotence (paMAP) étant partagés par les cellules cancéreuses. Les antigènes pluripotents sont exprimés dans les tissus embryonnaires et absents des tissus adultes sains, mais anormalement réexprimés par les cellules cancéreuses. Ainsi, bien qu'ils ne soient pas mutés, les paMAP constituent des cibles idéales et spécifiques au cancer. Nous avons identifié un ensemble de 48 paMAP dérivés de transcrits codants et non codants (48 %) impliqués dans le maintien de la pluripotence et exprimés de manière aberrante dans plusieurs types de cancer. Ainsi, bien qu’elles proviennent de différents types de cellules et de tissus, des tumeurs 4 distinctes convergent vers un programme transcriptionnel associé à la pluripotence. En effet, l’expression des paMAP dans les cancers est corrélée à l’hypométhylation récurrente de leurs gènes sources, la présence d’aberrations génomiques courantes et l’adoption par les tumeurs de stratégies d’évasion immunitaire communes. Enfin, comme plusieurs paMAP sont immunogènes, leur utilisation comme cibles dans des vaccins anticancéreux pourrait entrer en synergie avec les inhibiteurs disponibles des voies d'évasion immunitaire et améliorer le traitement de plusieurs cancers agressifs. Ensuite, nous avons évalué l’ensemble des TA ayant un potentiel thérapeutique dans deux types de tumeurs présentant une charge mutationnelle particulièrement élevée, le mélanome et le cancer du poumon non à petites cellules (NSCLC). Nous avons constaté que les TA mutés (mTSAs) représentent une minorité (1 %) des TA exploitables dans ces deux types de cancer. Cela peut s'expliquer par une faible expression d'ARN de la plupart des mutations non synonymes ainsi que par leur localisation en dehors des régions génomiques les plus efficaces pour la génération de MAP. En revanche, 99 % des TA dérivent de séquences génomiques non mutées spécifiques au cancer (aeTSA), surexprimées dans le cancer (TAA) ou spécifiques à la lignée cellulaire d'origine (LSA, exprimés par les mélanocytes ou par les cellules épithéliales pulmonaires, pour le mélanome et le NSCLC, respectivement). Tout comme les paMAP, environ 50 % des aeTSA identifiés dans le mélanome et le NSCLC proviennent de séquences non canoniques et sont régulés de manière épigénétique. Alors que les mTSA sont exclusivement spécifiques à chaque patient patient, les aeTSA sont partagés entre les échantillons tumoraux. De plus, leur absence dans les tissus normaux, leur abondance et leur capacité à activer les lymphocytes T CD8 en font des cibles idéales pour traiter les mélanomes et les NSCLC. En conclusion, cette thèse fournit un aperçu de la biogenèse de différents types de TA dans diverses cohortes de patients et ouvre la voie au développement d’immunothérapies ciblées et efficaces contre une grande variété de cancers. / Nearly all nucleated cells express MHC I (HLA I in humans)-associated peptides (MAPs) which are sampled from the cellular proteome and transported to the cell surface for inspection by CD8 T cells. As such, the collection of cell-surface MAPs, or the immunopeptidome, informs CD8 T cells on the inner cell state. Cancer immunosurveillance relies on the capacity of CD8 T cells to recognize abnormal MAPs on tumor cells and eliminate them while sparing healthy cells. Hence, the existence of cancer indicates that tumor-specific CD8 T cells are underpowered, dysfunctional or inhibited from exerting their function. Anti-cancer vaccines can boost tumor killing by stimulating the recognition of abnormal MAPs. The development of effective anti-cancer vaccines is limited by the identification of actionable MAPs, or tumor antigens (TAs), expressed exclusively on tumor cells. The TA search space has been largely limited to MAPs derived from non-synonymous mutations in canonical protein-coding exons which represent a mere 2% of the human genome. That MAPs can derive from the non-canonical translation of potentially all genomic regions has only recently been fully appreciated. Herein, we used proteogenomics to discover actionable TAs derived from canonical and non-canonical translation products shared within or across different types of human cancer. First, we used induced pluripotent stem cells (iPSCs) to identify pluripotency-associated MAPs (paMAPs) shared by cancer cells. Pluripotency antigens are restricted to embryonic tissues and absent from healthy adult tissues but abnormally re-expressed by cancer cells, which makes them ideal tumor-specific targets despite being unmutated. We identified a set of 46 paMAPs derived from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance and aberrantly expressed in multiple cancer types. Thus, despite originating from different cell types and tissues, distinct tumor types converged towards a pluripotency-associated transcriptional program. Indeed, the expression of paMAPs across cancers correlated with recurrent source gene hypomethylation, genomic aberrations, and immune evasion properties. Several paMAPs were immunogenic, thus their targeting could synergize with available inhibitors of immune evasion pathways to improve the outcome of multiple aggressive cancers. 7 Next, we evaluated the actionable TA landscape of two tumor types with particularly high mutational load, melanoma and non-small cell lung cancer (NSCLC). We found that mutated TAs (mTSAs) represent a minority (1%) of actionable TAs in both cancer types, which can be explained by a low RNA expression of most non-synonymous mutations and their localization outside genomic regions proficient for MAP generation. By contrast, 99% of TAs derived from unmutated genomic sequences specific to cancer (aeTSAs), overexpressed in cancer (TAAs), or specific to the cell lineage of origin (LSAs, expressed by melanocytes or by lung epithelial cells, for melanoma and NSCLC LSAs, respectively). As for paMAPs, around 50% of aeTSAs in melanoma and NSCLC were non-canonical and were epigenetically regulated. Whereas mTSAs were exclusively patient-specific, aeTSAs were shared among tumor samples and exhibited all characteristics of targetable TAs, including tumor-specificity, high abundance, and immunogenicity. Altogether, this thesis provides insights into the biogenesis of different TA types in various patient cohorts and paves the way for the development of effective TA-based immunotherapies against a large variety of cancers.

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária / Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária / Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation