Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

A evolução do conhecimento sobre o HIV e seus efeitos sobre as diferentes células do sistema imune possibilitaram a criação e o aperfeiçoamento de um grande arsenal terapêutico. Atualmente, a sobrevida de casos recém-­ diagnosticados é medida em décadas; entretanto, alguns pacientes não apresentam recuperação do sistema imune após a agressão inicial sofrida pelo vírus, a despeito de tratamento adequado. As células NK são identificadas como componentes da imunidade inata, responsáveis pelo combate a infecções virais e tumores. Elas são divididas em CD56dim e CD56hi, com diferentes capacidades citotóxicas e de produção de citocinas; uma terceira subpopulação composta por células CD56neg está presente em proporções mínimas em adultos saudáveis, porém tem maior importância em neonatos e está expandida em indivíduos cronicamente infectados pelo HIV, podendo ser identificada pelos marcadores CD7 e CD16. Dentre diversos outros, as células NK expressam receptores ativadores e inibitórios chamados KIR, que interagem com moléculas HLA, identificando células próprias e aquelas que reduzem sua expressão como mecanismo de escape imunológico; a interação entre KIR e HLA tem papel na evolução clínica da infecção por HIV/AIDS, particularmente envolvendo o receptor KIR3DL1. PD-­ 1 é um checkpoint do sistema imunológico que pode ter sua expressão aumentada em tumores e infecções virais crônicas. A expressão de PD-­1 em células T correlaciona-­se a marcadores prognósticos na infecção por HIV/AIDS; sua expressão em células NK já foi documentada, porém temos poucas informações a respeito. Este trabalho buscou detalhar a expressão de PD-­1 e seu ligante PD-­L1 em células NK e monócitos em participantes infectados pelo HIV e controles. Foram recrutados participantes diagnosticados e acompanhados desde a infecção aguda, participantes diagnosticados após um intervalo de tempo desconhecido desde a soroconversão e controles não infectados sob alto risco por exposição sexual. As amostras foram processadas a fresco no LIM-­60; PD-­1 e outros marcadores foram analisados por citometria de fluxo multicor. A expressão de PD-­1 em células NK correlacionou-­se a contagens de células T CD4+ e expressão de PD-­1 em células T nos participantes infectados; dentre estes, os participantes seguidos desde a infecção aguda tiveram menor expressão de PD-­1. Os participantes seguidos desde a infecção aguda tiveram ainda menor expressão de PD-­L1 em monócitos quando comparados aos participantes diagnosticados em fase desconhecida da doença, e também quando comparados aos controles não infectados. Houve aumento expressivo da proporção de células KIR3DL1+ entre as células CD56neg nos participantes infectados em comparação ao grupo não infectado. Concluímos que a expressão de PD-­1 em células NK está aumentada em pessoas infectadas pelo HIV e correlaciona-­se a outros parâmetros imunológicos, como contagem de células T CD4+ e expressão de PD-­1 em células T. A exaustão das células NK pode, portanto, contribuir para o dano imunológico causado pelo HIV e pode ser explorada como um alvo para novas modalidades terapêuticas / The expansion of our knowledge about the HIV and its effects on the entire immune system has led the development of a vast therapeutic arsenal. Survival for newly diagnosed cases is now measured in decades;? some patients, however, never recover full immune function following the initial aggression inflicted by HIV, despite adequate treatment. NK cells are identified as innate immunity components, responsible for fighting viral infections and tumors. They are separated in CD56dim and CD56hi cells, which present different cytotoxicity and cytokine production capacity. A third distinct subpopulation constituted by CD56neg cells can be found in minimal counts in healthy adults, but is present in newborns and is expanded in chronically HIV-­ infected subjects;? these cells can be identified as CD7+CD16+. Among others, NK cells express activating and inhibitory receptors called KIR, which interact with HLA molecules and identify \"self\" cells and cells that have downregulated its expression as an immunologic evasion strategy. Studies have documented the importance of KIR and HLA interaction in HIV/AIDS infection clinical course, particularly involving the receptor KIR3DL1. PD-­1 is an immune checkpoint that can be upregulated by tumors and chronic viral infections. PD-­ 1 expression on T cells is correlated to prognostic factors in HIV/AIDS infection; NK cells have been shown to express it, but further information is necessary. This study aimed at investigating PD-­1 and its ligand PD-­L1 expression on NK and monocytes in HIV-­infected participants and controls. We recruited a group of participants who were diagnosed during acute phase of HIV infection and have been followed ever since, a group of participants who were diagnosed after unknown interval since seroconversion, and a group of uninfected controls who have a high risk due to sexual exposure. Samples were freshly processed at LIM-­60; PD-­1 and other markers were analyzed by multicolor flow cytometry. We found PD-­1 expression on NK cells was correlated to T CD4+ cell counts and PD-­1 expression on T cells, in infected participants; among them, participants followed since acute infection expressed less PD-­1. They also expressed less PD-­L1 in monocytes, as compared to participants diagnosed after unknown interval since seroconversion, as well as compared to the uninfected group. We found significant increase in proportion of KIR3DL1-­expressing cells among CD56neg cells in infected participants compared to the uninfected group. We concluded that PD-­1 expression on NK cells is increased in people infected by HIV and correlated to other immunologic parameters such as T CD4+ counts and PD-­1 expression on T cells. NK cell exhaustion may, therefore, contribute to the immune damage induced by HIV-­1 infection and can be also explored as a target to find new ways to restore antiviral immunity

Orientadores: Cármino Antonio de Souza, Jeane Eliete Laguila Visentainer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T17:20:39Z (GMT). No. of bitstreams: 1 Cardozo_DanielaMaira_D.pdf: 3033896 bytes, checksum: b4696b2dc5fc0ec422091c74289aed9f (MD5) Previous issue date: 2013 / Resumo: No organismo humano, as moléculas HLA (Human Leukocyte Antigens) são proteínas expressas na superfície da maioria das células nucleadas e são codificadas por genes localizados no braço curto do cromossomo 6 na região do Complexo Principal de Histocompatibilidade (CPH). Essas proteínas são caracterizadas pelo alto grau de polimorfismo, e também faz a ligação com receptores KIR (Immunoglobulin-like Receptors), expressos nas células Natural Killer. Os receptores KIR, que reconhecem moléculas do complexo HLA de classe I, estão entre os principais receptores inibidores dos linfócitos NK. Células infectadas por vírus e células tumorais perdem ou têm diminuída a expressão de moléculas HLA de classe I e, por isso, são eliminadas pela ausência de ligação entre moléculas HLA e receptores KIR inibitórios. Atualmente, muitos estudos têm destacado a importância dos genes KIR e HLA no Transplante de Células Progenitoras Hematopoiéticas (TCPH). O TCPH é o tratamento de escolha para muitas doenças hematológicas e dependem de vários fatores incluindo o estágio da doença, o regime de condicionamento, a fonte de células, o grau de identidade HLA entre doador e receptor e o desenvolvimento da doença do enxerto contra o hospedeiro (DECH). Estudos recentes indicam que a presença de células NK alorreativas no enxerto representa um fator favorável à recuperação de pacientes, uma vez que essas células têm a capacidade de eliminar células tumorais residuais pela ausência ou diminuição da expressão de moléculas HLA e sem a indução da DECH. Também outros fatores podem estar envolvidos na resposta pós-transplante, como a presença e ausência de determinados alelos HLA e genes KIR, os quais podem estar ligados à melhor ou pior resposta pós-transplante. O primeiro ensaio investigou a associação entre HLA e a ocorrência da DECH aguda e crônica em pacientes que receberam transplante de células progenitoras hematopoiéticas HLA-idêntico, aparentados. No total, foram 176 pacientes que receberam o primeiro transplante entre 1997 e 2009. DECH aguda foi positivamente associada ao HLA-A10 (P = 0.0007), HLA-A26 (P = 0.002), B55 (P = 0.001), DRB1*15 (P = 0.0211) e DQB1*05 (P = 0.038), enquanto que HLA-B16 (P = 0.0333) foi mais frequente em pacientes sem DECH aguda. DECH crônica foi positivamente associada com HLA-A9 (P = 0.01) e A23 (P = 0.0292) e negativamente associada com HLA-A2 (P = 0.0031) e B53 (P = 0.0116). HLA-B35 (P = 0.0373), B49 (P = 0.0155) e B55 (P = 0.0024) foi alta em pacientes com DECH aguda grau 3 ou mais, do que os outros pacientes. Nos pacientes com DECH crônica extensa, HLA-A9 (P = 0.0004), A24 (P = 0.0059) e A26 (P = 0.0411) foi maior do que nos outros pacientes, enquanto HLA-A2 foi baixo (P = 0.0097). O objetivo do segundo ensaio foi avaliar as possíveis interações dos genes KIR e HLA com o curso clínico do transplante HLA compatível, aparentado e não depletado de linfócitos T, particularmente na doença do enxerto contra o hospedeiro (DECH) aguda e crônica, recaída, sobrevida global e sobrevida livre de evento. A maioria dos doadores (78%) apresentaram o haplótipo B do KIR enquanto que 22% apresentaram o haplótipo A. Dos pacientes que receberam o haplótipo A do doador, 90% tiveram DECH, aguda ou crônica, comparados com os que receberam o haplótipo B (58%) (dados não estatisticamente significantes). Não houve diferença significativa para recaída entre pacientes que receberam os haplótipo A ou B (27% vs 23%). Não houve diferença no desenvolvimento da DECH e recaída para os pacientes homozigotos (C1C1 ou C2C2) e heterozigotos (C1C2) e nem para aqueles com HLA-Bw4 presente e ausente. Também, a sobrevida global não foi diferente para os grupos de pacientes analisados. No entanto, houve forte correlação entre o grupo de pacientes heterozigotos para HLA-C (C1C2) e a incidência de DECH aguda e recaída. A SLE foi maior nos pacientes heterozigotos que não desenvolveram DECHa (p<0,0001). Resultados mostraram que as variantes de HLA podem influenciar na ocorrência de DECH em transplante alogênico, com doadores relacionados, HLA-idênticos, tanto como fatores de proteção, quanto como fatores de susceptibilidade. Ainda, a interação KIR/HLA tem impacto significante no resultado dos transplantes relacionados, HLA compatível, sem depleção de linfócitos T, influenciando na incidência de recaída e na ocorrência da DECH. Resultados mostraram que para o grupo heterozigoto (C1C2) a maioria dos pacientes não desenvolveu DECH aguda e apresentou maior SLE, sugerindo um possível efeito protetor para esse grupo / Abstract: In the human organism, the HLA (human leukocyte antigens) are proteins expressed on the surface of most nucleated cells and are encoded by genes located on the short arm of chromosome 6 in the region of the Major Histocompatibility Complex (MHC). These proteins are characterized by a high degree of polymorphism, and also make the connection with KIR (Immunoglobulin-like Receptors), expressed in Natural Killer cells. KIR receptors that recognize HLA molecules of class I are among the major inhibitory receptors of NK-cells. Virus infected cells and tumor cells have lost or diminished expression of HLA class I molecules and therefore are eliminated by the absence of binding between HLA molecules and inhibitory KIR receptors. Currently, many studies have highlighted the importance of KIR and HLA genes in Hematopoietic Stem Cell Transplantation (HSCT). HPCT is the treatment of choice for many hematological malignancies and depends on various factors including stage of disease, the conditioning regimen, the source of cells, the degree of identity between donor and recipient HLA and development of chronic graft-versus-host (GVHD). Recent studies indicate that the presence of alloreactive NK cells in the graft is a factor aiding the recovery of patients, since these cells have the ability to eliminate residual tumor cells by the absence or diminution of expression of HLA molecules and without inducing GVHD. Also other factors may be involved in response post-transplant, as the presence or absence of certain HLA genes and KIR, which can be connected to a better or worse response after transplantation. The first trial investigated the association between HLA and the occurrence of acute and chronic GVHD in patients receiving hematopoietic stem cell transplant HLA-identical related. In total, 176 patients who received a first transplant between 1997 and 2009. GVHD was positively associated with HLA-A10 (P = 0.0007), HLA-A26 (P = 0.002), B55 (P = 0.001), DRB1 * 15 (P = 0.0211) and DQB1 * 05 (P = 0.038), while that HLA-B16 (P = 0.0333) was more frequent in patients without acute GVHD. Chronic GVHD was positively associated with HLA-A9 (P = 0.01) and A23 (P = 0.0292) and negatively associated with HLA-A2 (P = 0.0031) and B53 (P = 0.0116). HLA-B35 (P = 0.0373), B49 (P = 0.0155) and B55 (P = 0.0024) was high in patients with acute GVHD grade 3 or more, than the other patients. In patients with extensive chronic GvHD, HLA-A9 (P = 0.0004), A24 (P = 0.0059) and A26 (P = 0.0411) was greater than in the other patients, whereas HLA-A2 was low (P = 0.0097). The objective of the second test was to evaluate the possible interactions of KIR and HLA genes with the clinical course of the transplant HLA compatible related and not depleted of T lymphocytes, particularly in chronic graft versus host disease (GVHD) acute and chronic relapse, survival overall and event-free survival. Most donors (78%) presented the KIR B haplotype while 22% were haplotype A. Of the patients who received the donor haplotype A, 90% had GvHD, acute or chronic, compared with those who received the haplotype B (58%) (data not statistically significant). There was no significant difference in relapse between patients who received the haplotype A or B (27% vs 23%). There was no difference in the development of GVHD and relapse for patients homozygous (C1C1 or C2C2) and heterozygous (C1C2) and not for those with HLA-Bw4 present and absent. Also, the overall survival was not different for the groups of patients studied. However, there was strong correlation between the group of patients heterozygous for HLA-C (C1C2) and the incidence of acute GVHD and relapse. The SLE was higher in patients who did not develop GVHD heterozygotes (p <0.0001). Results showed that the HLA variants may influence the occurrence of GVHD in allogeneic transplantation with related donors, HLA-identical, both as protective factors, such as susceptibility factors. Furthermore, the interaction KIR / HLA has a significant impact on the outcome of transplantation related HLA-compatible, without depletion of T cells, influencing the incidence of relapse and the occurrence of GVHD. Results showed that for the heterozygous group (C1C2) most patients did not develop acute GVHD and showed higher SLE, suggesting a possible protective effect for this group / Doutorado / Clinica Medica / Doutora em Clínica Médica

Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS126-34. We predicted higher frequencies of NS126-34-specific CD8+ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8+T cells. We generated a tetramer against this epitope (B57-NS126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34-specific, other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8+ T cells. In vitro stimulation of CD8+ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation. During the course of our analysis we found substantial binding of B57-NS126-34 TET to CD8- cells. We demonstrated that the B57-NS126-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS126-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1+ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness. Our data identified CD71 as biologically useful marker to study DENV-specific CD8+ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).