L’activité anti-tumorale des cellules NK est régulée par des récepteurs de surface activateurs et inhibiteurs qui reconnaissent des ligands exprimés par les cellules cibles. Afin d’augmenter le potentiel cytotoxique des cellules NK face à des cellules cancéreuses autologues, Innate Pharma a développé des Ac bloquant les interactions entre certains récepteurs inhibiteurs de la cellule NK, les KIRs, et leurs ligands, des molécules du CMH de classe I.Dans un premier temps, nous avons mis en place un modèle tumoral d’efficacité des Ac anti-KIR dans des souris transgéniques pour l’expression d’un KIR. Ce modèle nous a permis de démontrer l’efficacité thérapeutique anti-tumorale du blocage des interactions entre les KIRs exprimés par les cellules NK et les molécules HLA exprimées par la tumeur. Ce modèle nous a également permis de tester la combinaison de l’Ac anti-KIR avec un Ac thérapeutique utilisé dans le traitement des lymphomes B non-hodgkiniens, rituximab. Utilisé en combinaison, ces deux Ac augmentent significativement la survie de souris greffées avec une lignée tumorale d’origine B humaine par rapport à chacun des deux traitements seuls.Ces données précliniques nous amènent à proposer une stratégie thérapeutique pour le traitement de tumeurs hématologiques, basée sur la combinaison d’un Ac anti-KIR avec un Ac induisant de l’ADCC afin de potentialiser la réponse anti-tumorale des cellules NK. / NK cell anti-tumoral activity is regulated by inhibitory and activating receptors interacting with target cell ligands. Innate Pharma has developed anti-KIR Abs (IPH2101 and lirilumab), directed against inhibitory NK cell receptors interacting with HLA molecules, in order to increase NK cell activity against autologous tumor cells.We have set up a preclinical model to assess anti-KIR anti-tumoral efficacy in transgenic mice expressing a KIR receptor. With this model, we have also shown the therapeutic benefit of combining lirilumab with rituximab, a therapeutic Ab mediating ADCC.These results support the rationale of combining anti-KIR Ab with Ab mediating ADCC as a therapeutic strategy for hematological malignancies.

Orientadores: Carmino Antonio de Souza, Jeane Eliete Laguila Visentainer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T20:55:46Z (GMT). No. of bitstreams: 1 Marangon_AmandaVansan_D.pdf: 2274304 bytes, checksum: 433df5157fab3588b51689f24c4b86cf (MD5) Previous issue date: 2014 / Resumo: O Linfoma difuso de grandes células B (LDGCB) representa o subtipo mais prevalente de linfoma maligno não-Hodgkin, sendo responsável por 30-40% de todos os casos de LNH. O LDGCB não tem etiologia e patogênese bem definidas, porém o seu desenvolvimento parece estar relacionado a respostas imunes ineficazes, devido a frequente associação desse linfoma com estados de imunossupressão. Os fatores genéticos envolvidos no desenvolvimento e evolução da doença não são bem entendidos. Nesse contexto, o objetivo desse trabalho foi avaliar a influência dos genes KIR, dos ligantes HLA e do polimorfismo em genes de citocinas na susceptibilidade ou resistência ao desenvolvimento de LDGCB, bem como na evolução clínica e resposta ao tratamento. Para tanto, foram selecionados 112 pacientes com diagnóstico de LDGCB e 292 doadores de sangue e medula óssea como grupo controle. As tipificações dos genes KIR e dos ligantes HLA foram realizadas com a técnica de PCR-SSOP e a tipificação de citocinas foi realizada com a técnica PCR-SSP. As análises estatísticas foram realizadas pelo pacote estatístico "R" versão 3.0.2 para o programa Windows e os valores de P<0,05 foram considerados significativos. A distribuição dos genes KIR nos grupos estudados mostrou uma menor frequência do gene KIR2DL2 nos pacientes quando comparados aos controles (45,5% vs 58,1%; P=0,036), essa associação mostrou-se significativa também na combinação de KIR2DL2 com C1 (33,0%vs 45,9%; P=0,026) sugerindo um papel de proteção desse gene ao desenvolvimento de LDGCB. Em relação à evolução clínica da doença, os ligantes HLA-Bw4 e HLA-Bw4 80I foram mais frequentes nos pacientes com estádios mais avançados da doença (64,7% vs 40,9%; P=0,020 e 44,1% vs 25,0%; P=0,046, respectivamente) sugerindo que a presença desses ligantes pode ser fator de prognóstico ruim ao LDGCB. Em relação à resposta terapêutica, o gene KIR2DL3 foi associado positivamente ao tratamento do LDGCB, pois esse gene foi mais frequente nos indivíduos com resposta completa que nos indivíduos não respondedores (88,3% vs 71,0%; P=0,044). A respeito dos genes reguladores de citocinas, o genótipo IFN-gama-874/A:A foi associado positivamente ao LDGCB, sendo encontrado mais frequente nos pacientes que nos controles (50,9% vs 27,9%; P=0,001). Contrariamente os genótipos: IFN-gama-874/T:A, IL10-819/C:C e IL10-592/C:C foram menos frequentes nos pacientes que nos controles (P=0,001; P=0,025; P=0,025). Ademais, o genótipo IL10-1082/G:G foi relacionado a maior sobrevida livre de progressão. Os resultados encontrados sugerem que os genes KIR, os ligantes HLA e os genes de citocinas parecem ter envolvimento na proteção, susceptibilidade, evolução clínica e resposta ao tratamento do LDGCB / Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of malignant non-Hodgkin lymphoma and affects approximately 30-40 % of all cases. The DLBCL has no clearly defined etiology and pathogenesis, but its development seems to be related to ineffective immune responses due to frequent association of lymphoma with immunosuppression. Genetic factors involved in the development and progression of the disease are not well understood. The aim of this study was to evaluate the influence of KIR genes, HLA ligands and cytokine polymorphisms in the susceptibility or resistance to the development of DLBCL, as well as influence in the clinical course and response to treatment. To this end, we selected 112 patients with DLBCL and 292 bone marrow donors as control group. The typing of KIR genes and HLA ligands were performed by PCR-SSOP and typing of cytokine genes was performed by PCR-SSP technique. Statistical analyzes were performed by the statistical package " R " version 3.0.2 for Windows program. P values < 0.05 were considered significant. The distribution of KIR genes in both groups showed a lower frequency of the KIR2DL2 gene in patients compared to controls (45.5% vs 58.1% P=0.036), this association was significant also in combination KIR2DL2 with C1 (33.0% vs 45.9%, P=0.026) suggesting a protective role of this gene to the development of DLBCL. Regarding the clinical course of the disease, HLA-Bw4 and HLA-Bw4 80I ligands were more frequent in patients with more advanced stages of the disease (64.7% vs 40.9%, P=0.020 and 44.1% vs 25 0%, P=0.046, respectively) suggesting that the presence of these ligands may be poor prognostic factor to DLBCL. In regard to treatment response, the KIR2DL3 gene was positively associated with the treatment of DLBCL, because this gene was more frequent in individuals with complete response than in nonresponders individuals (88.3% vs 71.0%, P=0.044 ). Regarding the cytokine genes , IFN -gamma-874/A genotype:A/A was positively associated with DLBCL, it was more frequently in patients than in controls (50.9% vs 27.9%, P=0.001). On other hand genotypes: IFNG -874 /T:A, IL10-819/C:C and IL10 -592 /C:C were less frequent in patients than in controls (P=0.001, P=0.025, P=0.025 respectively). Moreover, the genotype IL-1082/G:G was related to increased progression-free survival. The results suggest that the KIR genes, HLA ligands and cytokine genes seem to be involved in the protection, susceptibility, clinical course and response to treatment of DLBCL / Doutorado / Clinica Medica / Doutora em Clínica Médica

KirBac channels are prokaryotic homologs of eukaryotic inwardly-rectifying potassium channels, which have served as models for gaining insight into the structure of eukaryotic channels. This thesis focuses on the structure-function relationship in these channels. The first part of this study concerns a novel KirBac channel, KirBac9.2, which contains a unique amino acid sequence in the place of the canonical GYG selectivity filter. Although expressed and purified in a stable and functional form, the protein did not form well-diffracting crystals. Functional studies suggest that KirBac9.2 is non-selective for monovalent cations and a random mutagenesis screen identified a number of activatory mutants in the cytoplasmic domains of the channel. A full electrophysiological investigation of KirBac9.2 channel function is beyond the scope of this study. However, initial studies suggest that it is possible to record currents from KirBac9.2 channels reconstituted into lipid bilayers. The second part of this thesis investigates KirBac3.1, which is a classical KirBac channel containing the consensus GYG sequence for potassium selectivity. Five high resolution structures of a mutant channel are reported, which suggest a new feature in the gating mechanism of KirBac3.1 where a rotation of the cytoplasmic domains is linked to a change in the electrostatic environment of the cytoplasmic cavity. In addition, a functional study of the KirBac3.1 showed that the channel is highly pH sensitive.

Since its beginning, graft rejection remains the key problem of solid organ transplantation. This reaction of the recipient's immune system against mismatched antigens of the transplanted organ causes graft damage and consequently loss of its function. Rejection involves cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Among the genetic factors which may have a prognostic value in rejection risk evaluation are the Human Leukocyte Antigens (HLA) genotype, the Killer Immunoglobuline-like Receptor (KIR) gene repertoir, cytokine and other gene polymorphisms. These factors could be screened for before transplantation to find the best possible combination of genetic characteristics of the donor and recipient and to reveal patients with "risky" genotypes, who may need more intensive immunosuppression and more careful post-transplant follow-up. Molecular factors, such as HLA and non-HLA antibodies, soluble CD30 molecule (sCD30), Hepatocyte Growth Factor (HGF) and other cytokines, measured before and/or after transplantation in the recipient's blood may be helpful for rejection risk estimation and may also be used as post-transplant rejection onset markers. In our study, we focused on some of the above mentioned factors. We found that ethnicity plays a significant role in the...

[Truncated abstract] Natural killer (NK) cell alloreactivity can be exploited in haploidentical haematopoietic stem cell transplantation (HSCT) to improve graft survival, reduce graft versus host disease and decrease leukaemic relapse. NK cells lyse cells that have reduced expression of class I HLA molecules. In an allogeneic setting, donor NK cells may be activated by the absence of donor (self) class I HLA molecules on recipient cells; the absence of self-epitopes being detected by inhibitory KIR receptors on donor NK cells. The way in which genetic polymorphism of the receptors and ligands affects NK allorecognition of missing self, has not been fully elucidated. HLA-C molecules are divided into two groups, C1 and C2, with KIR2DL1 recognising cells expressing C2 and KIR2DL2 and KIR2DL3 recognising cells expressing C1. Donor NK cells expressing KIR2DL2 or KIR2DL3 can be alloreactive towards a recipient if they lack the C1 epitope and donor NK cells expressing KIR2DL1 can be alloreactive towards a recipient if they lack the C2 epitope. KIR3DL1 recognises the Bw4 epitope present on one-third of HLA-B alleles and certain HLA-A alleles. NK cells from donors expressing KIR3DL1 can be alloreactive towards recipients whose cells lack Bw4. Mismatches of KIR related HLA epitopes does not always results in NK alloreactivity. Therefore it is not possible to reliably predict NK alloreactivity based solely on the donor's HLA type and KIR repertoire and the recipient's HLA type. ... All Bw4-positive HLA-B alleles, with the exception of HLA-B*1301 and B*1302, protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors whose only Bw4 positive allele was HLA-A*2402 but not in donors whose only Bw4 positive HLA allele was HLA-B*1301 or B*1302. Finally this thesis demonstrated that an activating KIR can control NK cell alloreactivity. Donors who are C2 negative and KIR2DS1 positive had NK cells that expressed the activating receptor KIR2DS1 and were capable of lysing cells expressing the C2 epitope. More so, KIR2DS1 dependent NK clones were shown to override inhibitory signals generated by NKG2A interacting with its ligand, HLA-E. The identification of these NK clones has important implications for haploidentical HSCT in that recipient expressing all three NK epitopes, C1, C2 and Bw4 were previously thought to be resistant to alloreactive NK cells controlled by inhibitory receptors. Such patients may be amenable to haploidentical HSCT from C2 negative, KIR2DS1 positive donors. These results will improve the ability to predict NK cell alloreactivity based on a donor's HLA type and KIR repertoire and the recipient?s HLA type.

Los objetivos de este trabajo han sido estudiar la expresión de receptores de células NK (NKR), en particular CD94/NKG2C, en relación con la infección por citomegalovirus humano (HCMV). Los resultados descritos constituyen la primera evidencia de que la infección por HCMV modifica el repertorio de NKR. El incremento en la proporción de células CD94/NKG2C+ en donantes seropositivos para HCMV sugiere que participan en la respuesta al patógeno. El receptor CD94/NKG2C no sólo estimula las funciones efectoras y la proliferación de las células NK, sino que también activa a una subpoblación minoritaria de células T CD8+. El estudio de la expansión in vitro de la subpoblación NK CD94/NKG2C+ tras la interacción con fibroblastos infectados por el HCMV, sugiere que el propio receptor está implicado en la proliferación. / The main goals of this work have been to study the influence of human cytomegalovirus (HCMV) infection on the NK cell receptor (NKR) repertoire, mainly on the CD94/NKG2C receptor. Our observations provide a first evidence indicating that human cytomegalovirus (HCMV) may selectively shape the NKR repertoire. The increased proportions of NKG2C+ cells in HCMV-seropositive donors suggest a role in the response against the virus. The CD94/NKG2C receptor triggers the effector functions and proliferation not only in NK cells but also in a subset of CD8+ T lymphocytes. The stimulation of PBL from HCMV+ donors with virus-infected fibroblasts elicited a preferential expansion of CD94/NKG2C+ NK cells; studies carried out in this experimental system suggest that the receptor is involved in driving the proliferation.

Natural killer (NK) cells can kill virus-infected cells without prior antigenic exposure, and are therefore important for controlling viral replication prior to the onset of adaptive immune responses. Primate NK cells express activating and inhibitory killer-cell immunoglobulin-like receptors (KIRs) that bind to specific major histocompatibility complex (MHC) class I molecules. The importance of KIR interactions with MHC class I in human immunodeficiency virus (HIV) pathogenesis is demonstrated by the association of select KIR and MHC class I genotypes with delayed progression to acquired immunodeficiency syndrome (AIDS).

PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1-2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1-2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1-2 crystallographic structures were solved at 2.02A and 2.19A in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1-2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1-2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40A resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes.

Nosso estudo teve como um dos objetivos analisarmos a frequência do polimorfismo dos genes KIR na população caucasóide do Rio Grande do Sul e comparamos com resultados de outros Estados do Brasil e também de outros países. Em um segundo momento, avaliar a associação entre os genes KIR em pacientes com diabete Mellitus tipo 1 (DM1) e controles saudáveis. As células natural killer (NK) fazem parte do sistema imune inato e reconhecem as moléculas HLA (antígeno leucocitário humano) de classe I em células-alvo através de seus receptores de membrana. Os receptores principais das células NK são conhecidos como receptores killer do tipo imunoglobulina (KIR) e estão localizados no cromossomo 19q13.4. Estão divididos em grupos funcionais inibidores e ativadores. No primeiro artigo, o objetivo era de analisar os genes KIR e HLA -A, -B e -Cw em 200 indivíduos saudáveis do Rio Grande do Sul pela técnica PCR-SSP. A população do Sul do Brasil demonstrou similaridades com Estados mais próximos geograficamente, e diferenças principalmente com o norte e nordeste. O gene KIR2DS5 foi o menos frequente na população estudada, e a interação KIR ⁄HLA foi mais comum na associação 2DS1-⁄2DL1+⁄C2+. No segundo artigo, analisamos 15 genes KIR e os alelos do sistema HLA classe I em 248 pacientes caucasóides brasileiros com DM1 e em 250 controles saudáveis, usando a técnica de PCR com primers específicos (PCR-SSP). O genótipo 2DL1/C2+ foi mais comum em controles (p=0.001), assim como o haplótipo KIR2DL2/DR3/DR4+ e KIR2DL2/DR3+ (p<0.001; p<0.001).

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2011 / Made available in DSpace on 2012-10-26T05:49:47Z (GMT). No. of bitstreams: 1 294074.pdf: 1739239 bytes, checksum: f9dbbd3514607cc628d58c10c60f0629 (MD5)