The ability of members of the hsp70 family to bind to peptides in vivo and in vitro suggests that they may be involved in the processing of antigens for binding to Major Histocompatibility (MHC) class I and/or class n molecules. The aims of this thesis have been to provide evidence for the involvement of hsp70s in antigen processing and to characterise the binding of peptides by hspTOs by structural and functional studies. Firstly, the peptide-binding domains of two hsp70s, hsp70hom and PBP74, were expressed in isolation from the rest of the molecule for structure determination. Both of these hsp70s were implicated in antigen processing: hsp70hom in the class I pathway, due to its cytoplasmic localisation and constitutive expression, and the presence of its gene in the MHC; and PBP74 in the class n pathway because published work indicated that it was localised to endosomes and that antibodies against it inhibited antigen processing. The expression and purification of both peptide-binding domains was very successful, and one dimensional NMR experiments indicated that they were folded. However, it was not possible to determine their structures by NMR spectroscopy or X-ray crystallography because they aggregated in solution at high concentrations. Instead, the structure of the C-terminal region of hsp70hom, which includes its peptidebinding domain, was modelled based on the known structure of the equivalent portion of dnaK, the hsp70 of E.coli. The structure of hsp70hom is predicted to be very similar to that of dnaK, and modelling studies suggest that it is likely to bind peptides in a closely related fashion. The modelling of complexes between hsp70hom and two peptides suggest that the peptide-binding groove is very versatile, accounting for the broad peptide-binding specificity of hsp70s. The interactions of hsp70hom and PB74 with peptides were investigated using plate binding assays and isothermattitration calorimetry. A biotinylated peptide bound to the peptide-binding domain of hsp70hom, immobilised in plastic wells, with a Kd of <25 μM, which is within the range of Kds reported for other hsp70-peptide complexes (0.1-100 μM). In solution, isothermal titration calorimetry showed that the binding of peptides to the peptide-binding domains of hsp70hom and PBP74 was likely to be entropically rather than enthalpically driven, and, therefore, the interactions involved are likely to be predominantly hydrophobic. Secondly, PBP74, an hsp70 thought to be involved in the class II antigen processing pathway in endosomes, was localised by immunofluorescence microscopy. It was shown to be a mitochondrial protein, and is, therefore, unlikely to be involved in antigen processing. The presence of other members of the hsp70 family in lysosomes purified from a B cell line by Percoll density gradient centrifugation was investigated using antibodies that reacted with many Afferent members of the hsp70 family. No hsp70s were detected in these late endocytic compartments, even after heat shock or serum starvation. However, the presence of an hsp70 in endosomes, or of a member of this family not detected by the antibodies used, in lysosomes, cannot be ruled out. A third approach investigated the induction of the three hsp70 genes found in the MHC by four cytokines. The hsp70-l and hsp70-2 genes are induced at the mRNA level by IFN-γ and IL- 1, while TNF induces hsp70-2 alone. This data supports a role for the heat-inducible hsp70 in MHC class I antigen processing, as it appears to be coregulated with known members of this antigen processing pathway. The expression of hsp70hom was unaffected by any of the four cytokines examined. In addition, the mitochondrial hsp70 (which is not encoded in the MHC) appears to be induced by IFN-γ at the protein level. The research presented in this thesis provides a greater understanding of the peptide-binding properties of two hsp70s. Further work is necessary to show conclusively whether any of the hsp70s is involved in antigen processing.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Adequate cell production for adoptive cell transfer therapies such as Chimeric Antigen Receptor (CAR)-T cell therapy remains a critical barrier to treatment for indications that fail to achieve clinical success. One such disease is Chronic Lymphocytic Leukemia (CLL), a B-cell lymphoma with their characteristically exhausted T cells, marked by a progressive loss of the ability to secrete cytokines and proliferate, as well as an increase in the expression of checkpoint inhibitor molecules such as PD-1. The goal of this thesis is to characterize the functional differences or specific biomarkers within the CLL patient population that is indicative of the proliferation outcomes. Conventional clinical markers such as Rai stage or PD-1 expression alone were inadequate to describe the complex variability among patients. In order to better characterize exhaustion using microscopy-based cell function assays, we developed a sample sparing microscopy chamber that requires as little as 1000 cells per sample. The microscopy chambers were mass produced via injection molding, and made compatible with the antibody microcontact printing technique developed in the Kam lab. The chambers typically reduced cell usage per experiment by 20-fold. This reduction allowed us to measure IL-2 secretion, T cell arrest response to activating antibody patterns (pattern alignment), and motility of scarce human samples simultaneously from a single experiment. Results from these functional readouts along with other clinical markers were used as inputs for a multifactor exploratory analysis to cluster patients according to their functional similarities from the combination of responses in an unbiased manner. The resulting clusters based on the combination of the top 3 parameters IL-2, pattern alignment, and PD-1 resulted in better separation of patient groups and provided a basis for predicting max doubling outcomes from these inputs. We further used motility measurements as a way to understand initial T cell response to activation before the stop response, which was measured as pattern alignment previously. The time it takes for cells to come to a stop at the signal was most informative for translating T cell activation response to a stop response, and eventually to downstream effector functions of cytokine secretion and proliferation. The results of this work provide a powerful framework to describe different donors, and can be applied to cells from additional donors to guide future cell expansion studies.

No description available.

No description available.

A toxoplasmose, causadas por protozoário Apicomplexa, Toxoplasma gondii, é amplamente disseminada e pouco sintomática. A infecção crônica mantém cistos residuais por toda a vida, mas protege da reinfecção. Neste cenário complexo, vacinas com cistos residuais não são factíveis e vacinas de subcomponentes resultam em baixa proteção. A radiação ionizante foi usada para aprimoramento de imunógenos tanto vivos ou de subcomponentes. O uso da radiação gama em extratos solúveis de taquizoítos de T. gondii foi eficiente na proteção de camundongos contra a infecção utilizando diferentes cepas, sem adjuvantes e de fácil conservação. O antígeno irradiado passa por alterações físicas sem adição de novas moléculas, com agregação de proteínas, quebras de cadeias e reações oxidativas, que podem melhorar seu direcionamento a receptores celulares em células apresentadoras de antígeno (APC). Os extratos irradiados apresentaram alterações estruturais mínimas afetando 60% das proteínas, mas com manutenção das características antigênicas e imunogênicas. O extrato irradiado a 1500Gy (STag 1500Gy) induziu maior proteção e maior resposta humoral que o extrato nativo (p<0.05), mais evidente na dose de 10?g/animal, com altos índices de anticorpos IgG específicos e maior maturação da afinidade de IgG específica, com eficiência similar ou inferior em doses maiores. Animais imunizados com STag 1500Gy apresentaram maiores proporções de linfócitos B e T CD4+ de memória, enquanto que a imunização com taquizoítos íntegros irradiados mostrou aumento de linfócitos T CD8+, ambas muito maiores que a induzida por STag nativo. Construímos STag marcados por via biossintética ou por acoplamento a diferentes marcadores não-oxidativos. STag3H 1500Gy apresentou captação maior e mais duradoura por macrófagos, sem degradação como STag3H nativo. O uso de STags fluorescentes, mostrou que a maior ligação do STag 1500Gy não é relacionada a susceptibilidade a proteases, dada a mesma sensibilidade dos extratos para as peptidases testadas, além de permanecer na célula por muito mais tempo. Na presença de bloqueadores de receptores Scavengers Dextran sulfato (SRA) e Probucol (CD36), a ligação e captação por macrófagos do STag 1500Gy foi mais afetada por inibidores de radicais oxidados (CD36) do que por inibidores de radicais negativos (SRA). Em macrófagos peritoneais de camundongos deficientes do receptor Scavenger CD36 (KOCD36-/-), observamos uma cinética inversa a que foi obtida em macrófagos normais, com menor incorporação do STag 1500Gy, fato comprovado tanto por ensaios quantitativos como por ensaios em células individuais por citometria de fluxo. Os animais KOCD36-/- não mostram produção significativa de IgG específica em todos os imunógenos usados, e foram altamente suscetíveis ao desafio com cepas viáveis de T. gondii agressivas ou cistogênicas. O transplante de macrófagos peritoneais normais \"primados\" com STag 1500Gy em animais KOCD36-/- mostrou aumento de IgG específica em soro de animais recipientes. A melhor imunogenicidade dos antígenos irradiados deve ser relacionada a captação de proteínas oxidadas via CD36, que dirige estes antígenos para via intracelular favorável à sua apresentação para resposta imune adaptativa. Nossos resultados mostram que a radiação ionizante foi capaz de modificar proteínas dos STag tornando seu processamento por células imunes mais eficiente, sem a adição de adjuvantes ao processo. / Toxoplasmosis, caused by the protozoan Apicomplexa, Toxoplasma gondii, is widely disseminated and little symptomatic. Chronic infection maintains residual cysts throughout life, but protects from reinfection. In this complex scenario, vaccines with residual cysts are not feasible and vaccines of subcomponents result in low protection. Ionizing radiation was used for enhancement of either live or subcomponent immunogens. The use of gamma radiation in soluble extracts of T. gondii tachyzoites was efficient in protecting mice against infection using different strains, without adjuvants and easy management. The irradiated antigen undergoes physical changes without addition of new molecules, with protein aggregation, chain breaks and oxidative reactions, which can improve its targeting to cellular receptors in antigen-presenting cells (APCs). The irradiated extracts showed minimal structural alterations affecting 60% of the proteins, but with maintenance of the antigenic and immunogenic characteristics. The extracts irradiated at 1500Gy (STag 1500Gy) induced greater protection and higher humoral response than the native extract (p <0.05), more evident at a dose of 10?g/animal, with high specific IgG antibody levels and increased maturation of specific IgG affinity, with similar or lower efficiency at higher doses. Animals immunized with STag 1500Gy presented higher proportions of memory lymphocytes B and CD4+ while immunization with irradiated intact tachyzoites showed an increase in CD8+ lymphocytes, both much larger than that induced by native STag. We construct STag labeled by biosynthetic pathway or by coupling to different non-oxidative markers. STag3H 1500Gy showed greater and longer uptake by macrophages, with no degradation as STag3H native. The use of fluorescent STags showed that the greater binding of the STag 1500Gy is not related to the loss of protease susceptibility, given the same sensitivity of the extracts for peptidases, besides remaining in the cell for much longer time by fluorescence. In the presence of Scavengers receptor blockers Dextran sulfate (SRA) and Probucol (CD36), binding and uptake of STag 1500Gy in macrophages was more affected by oxidized radical (CD36) inhibitors than by negative radical inhibitors (SRA). In peritoneal macrophages of Scavenger receptor CD36 deficient mice (KOCD36-/-), we observed an inverse kinetics, with less incorporation of STag 1500Gy, as evidenced by both quantitative assays and individual cells by cytometry flow, compared to wild type macrophages. KOCD36-/- animals did not show significant production of specific IgG in all immunogens used, and were highly susceptible to challenge with viable strains of aggressive or cistogenic T. gondii strains. Transplantation of normal peritoneal macrophages \"primed\" with STag 1500Gy induced increase of specific IgG in sera from CD36-/-recipient animals. The best immunogenicity of the irradiated antigens should be related to the uptake of oxidized proteins via CD36 in APCs, which directs these antigens to the intracellular route favorable to their presentation for adaptive immune response. Our results show that ionizing radiation was able to modify STag proteins, making its processing by immune cells more efficient without the addition of adjuvants to the process.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.