T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.

Diversos dados na literatura têm demonstrado a participação de linfócitos B-1 em diferentes fenômenos imunológicos, tanto na resposta imune inata quanto na resposta imune adaptativa. Para melhor entendermos a ativação da resposta imune eficaz contra fungos patogênicos, pesquisamos a interação entre os linfócitos B-1 e o Paracoccidioides brasiliensis (P. brasiliensis), uma vez que este expressa moléculas antigênicas que podem ser reconhecidas pelo sistema imune. Utilizamos preparação antigênica do P. brasiliensis obtida de sua superfície leveduriforme denominada de CFA (antígeno livre da parede do fungo) e células leveduriforme do fungo. Observou-se que a maioria das células do sobrenadante da cultura celular de 4 dias de células totais aderentes peritoneais eram constituídos por linfócitos B-1; estas células expressam altos níveis de MHC-II (100%) e CD80 (90%). Contudo, não houve expressão significativa da molécula co-estimuladora CD86. Pela análise fenotípica, os linfócitos B-1 podem atuar como células apresentadoras de antígeno pois expressam CD80, CD86 e MHC-II; então realizamos o ensaio de proliferação celular utilizando linfócitos B-1 como células apresentadoras de antígenos e observamos proliferação celular de linfócitos TCD4+ significativa. Em relação às citocinas, analisamos a secreção de IL-10 e TNF-α do sobrenadante da cultura de linfócitos B-1 sem nenhum estímulo e observamos que estas células secretam tanto IL-10 quanto TNF-α; após estímulo de CFA, os valores aumentam significantemente. Analisamos a expressão de TLR-2, TLR-4, MyD88 e IL-10, por RT-PCR, dos linfócitos B-1 na presença de CFA. Encontramos expressão de TLR-2, MyD88 e IL-10 nas células com e sem estímulo. Analisamos a migração dos linfócitos B-1 da cavidade peritoneal de camundongos BALB/c após estímulo intraperitoneal (ip) com leveduras de PB. Decorridas 5 horas, foi observada grande diminuição dos linfócitos B-1 na cavidade peritoneal, que permanecia por 24 horas e 7 dias pós-infecção. Para melhor compreendermos a migração dos linfócitos B-1, foram utilizados camundongos CBA/N Xid (desprovidos de linfócitos B-1), cujo o peritônio foi reconstituído com linfócitos B-1, sendo infectados ip com leveduras de P brasiliensis. Os resultados demonstram que, após a infecção, os linfócitos B-1 migram da cavidade peritoneal para o baço. Também, observou-se aumento no número de células T com fenótipo de célula regulatória (CD4+CD25+Foxp3+). Nossos resultados sugerem que a elevada produção de IL-10 por células B-1, mediada provavelmente pela ligação de TLR-2, juntamente com a capacidade dos linfócitos B-1 em ativar linfócitos T, com a capacidade de migrar do peritônio para o baço e ativar células T regulatórias, poderia favorecer a sobrevivência do fungo no hospedeiro. / Innumerous data published in the literature have shown the involvement of B-1 cells in different immunological phenomena, both in the innate immnune response and the adaptive immune response. To better understand the activation of effective immune response against pathogenic fungi, we researched the interaction between B-1 cells and Paracoccidioides brasiliensis (P. brasiliensis), since it expresses that antigenic molecules can be recognized by the immune system. We used antigenic preparation of P. brasiliensis obtained from the surface of yeast called CFA (cell free antigen) and yeast cells of the fungus. It was observed that most cells in the cell culture supernatant 4 days of total adherent peritoneal cells consisted of B-1 cells, these cells express high levels of MHC-II (100%) and CD80 (90%). However, no significant expression of co-stimulatory molecule CD86 was observed. After phenotypic analysis, the B-1 cells can act as anyigen-presenting cells because they express C80, CD86 and MHC-II, then realized proliferation assay using B-1 cells as antigen presenting cells inducing was performed, and our results showed the significant proliferation of CD4 T cells. Regarding cytokines, we analyzed the IL-10 secretion and TNF-α in culture supernatant of B-1 cells without stimulation and found that these cells secrete both IL-10 and TNF-α, after stimulation of CFA. We analyzed the expression of TLR-2, TLR-4, MyD88 and IL-10 by RT-PCR, of the B-1 cells in the presence of CFA. We found expression of TLR-2, MyD88 and IL-10 cells with and without stimulus. We analyzed the migration of peritoneal B-1 cells after intraperitoneal (ip) infection with yeast from P. brasiliensis. After 5 hours, high decrease of B-1 cells in the peritoneal cavity was observed, which remained for 24 hours and 7 days post-infection. To better understand the migration of B-1 cells, we used mice CBA/N Xid (destitute of B-1 cells), reconstituted with B-1 cells, and infected with yeast P. brasiliensis. The results show that after the infection, the B-1 cells migrate from the peritoneal cavity to the spleen. Also, there was an increase in the number of T cells with regulatory cell phenotype (CD4+CD25+Foxp3+). Our results suggest that high production of IL-10 by B-1 cells, probably mediated by the binding of TLR-2, along with the ability of B-1 cells in activating T lymphocytes, with the ability to migrate from the peritoneum to the spleen and activate T regulatory cells, could favor the survival of the fungus in the host.

Diversos dados na literatura têm demonstrado a participação de linfócitos B-1 em diferentes fenômenos imunológicos, tanto na resposta imune inata quanto na resposta imune adaptativa. Para melhor entendermos a ativação da resposta imune eficaz contra fungos patogênicos, pesquisamos a interação entre os linfócitos B-1 e o Paracoccidioides brasiliensis (P. brasiliensis), uma vez que este expressa moléculas antigênicas que podem ser reconhecidas pelo sistema imune. Utilizamos preparação antigênica do P. brasiliensis obtida de sua superfície leveduriforme denominada de CFA (antígeno livre da parede do fungo) e células leveduriforme do fungo. Observou-se que a maioria das células do sobrenadante da cultura celular de 4 dias de células totais aderentes peritoneais eram constituídos por linfócitos B-1; estas células expressam altos níveis de MHC-II (100%) e CD80 (90%). Contudo, não houve expressão significativa da molécula co-estimuladora CD86. Pela análise fenotípica, os linfócitos B-1 podem atuar como células apresentadoras de antígeno pois expressam CD80, CD86 e MHC-II; então realizamos o ensaio de proliferação celular utilizando linfócitos B-1 como células apresentadoras de antígenos e observamos proliferação celular de linfócitos TCD4+ significativa. Em relação às citocinas, analisamos a secreção de IL-10 e TNF-α do sobrenadante da cultura de linfócitos B-1 sem nenhum estímulo e observamos que estas células secretam tanto IL-10 quanto TNF-α; após estímulo de CFA, os valores aumentam significantemente. Analisamos a expressão de TLR-2, TLR-4, MyD88 e IL-10, por RT-PCR, dos linfócitos B-1 na presença de CFA. Encontramos expressão de TLR-2, MyD88 e IL-10 nas células com e sem estímulo. Analisamos a migração dos linfócitos B-1 da cavidade peritoneal de camundongos BALB/c após estímulo intraperitoneal (ip) com leveduras de PB. Decorridas 5 horas, foi observada grande diminuição dos linfócitos B-1 na cavidade peritoneal, que permanecia por 24 horas e 7 dias pós-infecção. Para melhor compreendermos a migração dos linfócitos B-1, foram utilizados camundongos CBA/N Xid (desprovidos de linfócitos B-1), cujo o peritônio foi reconstituído com linfócitos B-1, sendo infectados ip com leveduras de P brasiliensis. Os resultados demonstram que, após a infecção, os linfócitos B-1 migram da cavidade peritoneal para o baço. Também, observou-se aumento no número de células T com fenótipo de célula regulatória (CD4+CD25+Foxp3+). Nossos resultados sugerem que a elevada produção de IL-10 por células B-1, mediada provavelmente pela ligação de TLR-2, juntamente com a capacidade dos linfócitos B-1 em ativar linfócitos T, com a capacidade de migrar do peritônio para o baço e ativar células T regulatórias, poderia favorecer a sobrevivência do fungo no hospedeiro. / Innumerous data published in the literature have shown the involvement of B-1 cells in different immunological phenomena, both in the innate immnune response and the adaptive immune response. To better understand the activation of effective immune response against pathogenic fungi, we researched the interaction between B-1 cells and Paracoccidioides brasiliensis (P. brasiliensis), since it expresses that antigenic molecules can be recognized by the immune system. We used antigenic preparation of P. brasiliensis obtained from the surface of yeast called CFA (cell free antigen) and yeast cells of the fungus. It was observed that most cells in the cell culture supernatant 4 days of total adherent peritoneal cells consisted of B-1 cells, these cells express high levels of MHC-II (100%) and CD80 (90%). However, no significant expression of co-stimulatory molecule CD86 was observed. After phenotypic analysis, the B-1 cells can act as anyigen-presenting cells because they express C80, CD86 and MHC-II, then realized proliferation assay using B-1 cells as antigen presenting cells inducing was performed, and our results showed the significant proliferation of CD4 T cells. Regarding cytokines, we analyzed the IL-10 secretion and TNF-α in culture supernatant of B-1 cells without stimulation and found that these cells secrete both IL-10 and TNF-α, after stimulation of CFA. We analyzed the expression of TLR-2, TLR-4, MyD88 and IL-10 by RT-PCR, of the B-1 cells in the presence of CFA. We found expression of TLR-2, MyD88 and IL-10 cells with and without stimulus. We analyzed the migration of peritoneal B-1 cells after intraperitoneal (ip) infection with yeast from P. brasiliensis. After 5 hours, high decrease of B-1 cells in the peritoneal cavity was observed, which remained for 24 hours and 7 days post-infection. To better understand the migration of B-1 cells, we used mice CBA/N Xid (destitute of B-1 cells), reconstituted with B-1 cells, and infected with yeast P. brasiliensis. The results show that after the infection, the B-1 cells migrate from the peritoneal cavity to the spleen. Also, there was an increase in the number of T cells with regulatory cell phenotype (CD4+CD25+Foxp3+). Our results suggest that high production of IL-10 by B-1 cells, probably mediated by the binding of TLR-2, along with the ability of B-1 cells in activating T lymphocytes, with the ability to migrate from the peritoneum to the spleen and activate T regulatory cells, could favor the survival of the fungus in the host.

Introduction Tumour-infiltrating lymphocytes (TIL) are an important prognostic indicator in high-grade serous ovarian carcinoma (HGSC). Certain types of TIL (in particular CD8+ effector T cells) predict better outcomes, whereas others (most notably CD4+CD25+FoxP3+ regulatory T cells; Tregs) predict worse outcomes. An unconventional subset of CD8+FoxP3+ T cells has been reported to be involved in autoimmunity and in immune response to several cancers. While the functional significance of CD8+FoxP3+ TIL remains poorly understood, they were associated with effective anti-tumour responses in a murine tumour model. Hypothesis CD8+FoxP3+ TIL are present in a subset of cases of HGSC and correlate with a strong immune response and increased patient survival. Experimental Design Multi-colour immunohistochemistry (IHC) was performed on a cohort of 44 primary HGSC specimens to enumerate and locate CD8+FoxP3+ TIL in comparison to CD8+FoxP3- and CD8-FoxP3+ TIL. Triple-colour IHC methodology was developed to further assess the phenotype of CD8+FoxP3+ TIL, including the measurement of additional markers CD4 and CD25 (classical markers of Tregs), Ki-67 (a marker of proliferation), and TIA-1 (a marker of cytotoxic potential). Intraepithelial versus stromal location was determined by staining adjacent sections for the epithelial marker pan-cytokeratin. Survival analysis was performed using a cohort of 188 cases of HGSC. Multi-colour staining was resolved using the Nuance™ multispectral imaging system in conjunction with Metamorph™ software. Survival analysis was performed using Kaplan-Meier and log rank tests. Results CD8+FoxP3+ cells were found in 60% of 44 cases of HGSC, in variable proportions ranging from 0.2 - 7.9% of CD8+ TIL and 0.5 – 12.7% of FoxP3+ TIL. CD8+FoxP3+ TIL were found to be either CD4+ (38.8%) or CD4- (61.2%). The majority of CD8+FoxP3+ TIL were also found to be CD25-TIA-1+Ki-67-, more closely resembling their CD8+FoxP3- counterparts. CD8+FoxP3+ TIL were found mainly in intraepithelial regions and were positively associated with patient survival (progression free survival; P = 0.0396). Conclusions CD8+FoxP3+ TIL are a component of the host immune response to HGSC. They appear to have a non-proliferative effector phenotype, consistent with an active role in the anti-tumour response. CD8+FoxP3+ TIL are associated with increased patient survival. An improved understanding of this new TIL subset may inform future immunotherapeutic strategies for this challenging malignancy. / Graduate / 0982 / sarakost@hotmail.com

Influenza virus infects the human population worldwide and causes acute respiratory disease. Currently, the primary strategy for preventing influenza is seasonal vaccination which is capable of providing protection in most populations. However, seasonal vaccines are less efficacious to immunize the elderly and poorly induce cross-protective immunity against the reassorted pandemic virus in the recipients. Neuraminidase (NA) inhibitors have also been widely utilized to limit disease outcome. The currently used NA inhibitors, nonetheless, generate the drug-resistant progeny viruses; moreover, they are unable to directly target the host immune responses which cause immunopathology in severe cases. Therefore, new strategies that provide more effective immunogenicity, cross-protection and therapies against influenza infection must be developed. In this thesis, the adjuvanticity of CpG oligodeoxynucleotide (ODN), type I interferon (IFN) and Complete Freund’s adjuvant (CFA) when coadministered with seasonal influenza vaccines in ferrets is presented. It has been found that the adjuvanted vaccines are efficacious to induce neutralizing antibody responses. Several common and distinguished signaling pathways leading to dendritic cell (DC) maturation and B cell activation have been discovered from their adjuvanticity. Furthermore, it was determined that seasonal H1N1 prior infection more effectively induces cross-protection against the newly emerged 2009 pandemic H1N1 (H1N1pdm) virus in ferrets and mice than the seasonal vaccines. The prior infection-induced cross-reactive but non-neutralizing antibodies are capable of providing substantial protection in the H1N1pdm infected mice when CD8 T cells are absent. Lastly, function of different vaccine adjuvants for controlling H1N1pdm infection in mice has been investigated. Unlike other adjuvants, CFA is capable of protecting the mice from infection through enhancement of Treg cell suppressive molecules galectin-1 and CTLA-4 which downregulated DC costimulation and effector T cell responses. Overall, this thesis has provided novel mechanistic insights for developing protective strategies against influenza infection.

Influenza virus infects the human population worldwide and causes acute respiratory disease. Currently, the primary strategy for preventing influenza is seasonal vaccination which is capable of providing protection in most populations. However, seasonal vaccines are less efficacious to immunize the elderly and poorly induce cross-protective immunity against the reassorted pandemic virus in the recipients. Neuraminidase (NA) inhibitors have also been widely utilized to limit disease outcome. The currently used NA inhibitors, nonetheless, generate the drug-resistant progeny viruses; moreover, they are unable to directly target the host immune responses which cause immunopathology in severe cases. Therefore, new strategies that provide more effective immunogenicity, cross-protection and therapies against influenza infection must be developed. In this thesis, the adjuvanticity of CpG oligodeoxynucleotide (ODN), type I interferon (IFN) and Complete Freund’s adjuvant (CFA) when coadministered with seasonal influenza vaccines in ferrets is presented. It has been found that the adjuvanted vaccines are efficacious to induce neutralizing antibody responses. Several common and distinguished signaling pathways leading to dendritic cell (DC) maturation and B cell activation have been discovered from their adjuvanticity. Furthermore, it was determined that seasonal H1N1 prior infection more effectively induces cross-protection against the newly emerged 2009 pandemic H1N1 (H1N1pdm) virus in ferrets and mice than the seasonal vaccines. The prior infection-induced cross-reactive but non-neutralizing antibodies are capable of providing substantial protection in the H1N1pdm infected mice when CD8 T cells are absent. Lastly, function of different vaccine adjuvants for controlling H1N1pdm infection in mice has been investigated. Unlike other adjuvants, CFA is capable of protecting the mice from infection through enhancement of Treg cell suppressive molecules galectin-1 and CTLA-4 which downregulated DC costimulation and effector T cell responses. Overall, this thesis has provided novel mechanistic insights for developing protective strategies against influenza infection.

T cells use their highly variable T cell receptor (TCR) to engage major histocompatibility molecules (MHC) presenting peptides on the surface of antigen presenting cells during an immune response. The TCR repertoire of developing T cells is shaped by thymic selection, resulting in a self-tolerant and foreign peptide specific naïve T cell population. However, naive T cells are alloreactive and generate immune responses towards foreign MHC alleles in clinical settings involving transplantation. While T cell immune responses towards foreign pathogens are peptide specific, the overall specificity of allo-responses is still debated. Under normal circumstances, immune system homeostasis and self-tolerance is maintained by specialized natural T regulatory cells (nTregs) that develop in the thymus. nTregs respond to self-peptide MHC they encountered in peripheral tissues with immune-suppressive activities. However, the identify of self-peptides that stimulate nTregs, specificity towards these self-peptides, and the method nTreg TCRs engage self-peptide MHC molecules is not clear. Here, we built a library of defined MHC-linked self-peptides eluted from the I-Ab MHC molecule to screen alloreactive T cells and self-reactive nTregs for activating self-peptides. We used this library to show that negative selection shapes the TCR repertoire’s specificity to self-peptides. We also provide evidence that alloreactive T cells have degenerate self and foreign peptide recognition if the foreign MHC allele is largely different from the host’s MHC allele. Finally, we identified a self-peptide that activates an nTreg, and present protein crystal structures that reveal its TCR engages self and foreign peptide MHC complexes via fairly conventional mechanisms.

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