CD80 and CD86 polymorphisms in populations of various ancestries: 5 new CD80 promoter alleles

CD80 and CD86 are closely linked genes on chromosome 3 that code for glycoproteins of the immunoglobulin superfamily, expressed on the surface of antigen-presenting cells. These costimulatory molecules play essential roles for stimulation and inhibition of T cells through binding to CD28 and CTLA-4 receptors. In this study, CD80 promoter and CD86 exon 8 polymorphisms were analyzed to investigate the genetic diversity and microevolution of the 2 genes. We genotyped 1,124 individuals, including Brazilians of predominantly European, mixed African and European, and Japanese ancestry, 5 Amerindian populations, and an African sample. All variants were observed in Africans, which suggests their origin in Africa before the human migrations out of that continent. Five new CD80 promoter alleles were identified and confirmed by cloning and sequencing, and promoter 2 is most likely the ancestral allele. Nucleotide -79 is monomorphic in 4 Amerindian populations, where the presence of the -79 G allele is probably the result of gene flow from non-Amerindians. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Tolerance and immunity to human tumor-associated antigens

Tumor-assoziierte Antigene (TAA) repräsentieren wichtige Zielstrukturen in zytotoxischen T-Zell (ZTL)-basierten Immuntherapien zur Behandlung maligner Erkrankungen. Die Tatsache, dass TAA nicht spezifisch nur in Tumoren sondern auch in nicht-transformierten Zellen vorhanden sind, kann infolge verschiedener Toleranz-Mechanismen zur Eliminierung von ZTL führen, deren T-Zell-Rezeptoren eine hohe Affinität für TAA besitzen. Entsprechend erfordert die Entwicklung effektiver Immuntherapeutika die genaue Analyse des verfügbaren T-Zell-Repertoires mit Spezifität für ein gegebenes TAA.Die Arbeit fokusierte das Tyrosinase (369-377) ZTL-Epitop, das im Komplex mit HLA-A*0201 (A2.1) auf der Zell-Oberfläche von malignen Melanomen und verschiedenen nicht-transformierten Zellen präsentiert wird. Es wurde gefunden, dass sowohl das humane als auch das murine Tyrosinase (369-377)-spezifische ZTL-Repertoire durch Selbst-Toleranz kompromittiert ist und dass diese Toleranz weder durch Verwendung einer bestimmten Peptid-Variante noch durch Interferenz mit CD4+CD25+ regulatorischen T-Zellen oder CTLA-4 umgangen werden kann. Diese Ergebnisse wurden anschließend auf ein anderes Krankheitsmodell, das Multiple Myelom (MM), adaptiert. Unter Umgehung von Selbst-Toleranz in A2.1-transgenen Mäusen wurde gezeigt, dass Transkriptionsfaktoren, die die terminale Differenzierung von B-Zellen in maligne und nicht-maligne Plasmazellen diktieren, als MM-assoziierte ZTL-Epitope dienen können.Diese Arbeit bietet einen bedeutenden und innovativen Beitrag zur Gestaltung von Tyrosinase-basierten Melanom- und MM-reaktiven Immuntherapien.

Molecular characterization of the immune modulatory function of matrix metalloproteinase-7, a factor in the tumour micromilieu

Matrix metalloproteinases are the components of the tumour microenvironment which play a crucial role in tumour progression. Matrix metalloproteinase-7 (MMP-7) is expressed in a variety of tumours and the expression is associated with an aggressive malignant phenotype and poor prognosis. A role for MMP-7 in the immune escape of tumours has been postulated, but the mechanisms are not clearly understood. The present study was focused on identifying physiological inactivators of MMP-7 and also to unravel the mechanisms involved in MMP-7 mediated immune escape. This study shows that human leukocyte elastase (HLE), secreted by polymorphonuclear leukocytes cleaves MMP-7 in the catalytic domain as revealed by N-terminal sequencing. Further analysis demonstrates that the activity of MMP-7 was drastically decreased after HLE treatment in a time and dose dependent manner. MMP-7 induces apoptosis resistance in tumour cells by cleaving CD95 and CD95L. The effect of HLE on MMP-7 mediated apoptosis resistance was analysed. In vitro stimulation of apoptosis by anti-Apo-1 (anti-CD95 antibody) and the chemotherapeutic drug doxorubicin is reduced by MMP-7. Also tumour specific cytotoxic T cells do not effectively kill tumour cells in the presence of MMP-7. This study revealed that HLE abrogates the negative effect of MMP-7 on apoptosis induced by CD95 stimulation, doxorubicin or cytotoxic T cells and restores apoptosis sensitivity of tumour cells. To gain insight into the possible immune modulatory functions of MMP-7, experiments were performed to identify new immune relevant substrates. The human T cell line, Jurkat, was selected for these studies. Hsc70 which is involved in uncoating of clathrin vesicles was found in the supernatants of the MMP-7 treated cells indicating a modulatory role of MMP-7 on endocytosis. Further studies demonstrated that MMP-7 leads to decreased clathrin staining in HEK293, HepG2, Jurkat, CD4+ T cells and dendritic cells. Results also show MMP-7 treatment increased surface expression of cytotoxic T lymphocyte associated protein-4 (CTLA-4) which accumulated due to inhibition of the clathrin mediated internalization in CD4+CD25+ cells.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

The additional facet of immunoscore: immunoprofiling as a possible predictive tool for cancer treatment

Recent investigations of the tumor microenvironment have shown that many tumors are infiltrated by inflammatory and lymphocytic cells. Increasing evidence suggests that the number, type and location of these tumor-infiltrating lymphocytes in primary tumors has prognostic value, and this has led to the development of an 'immunoscore. As well as providing useful prognostic information, the immunoscore concept also has the potential to help predict response to treatment, thereby improving decision- making with regard to choice of therapy. This predictive aspect of the tumor microenvironment forms the basis for the concept of immunoprofiling, which can be described as 'using an individual's immune system signature (or profile) to predict that patient's response to therapy' The immunoprofile of an individual can be genetically determined or tumor-induced (and therefore dynamic). Ipilimumab is the first in a series of immunomodulating antibodies and has been shown to be associated with improved overall survival in patients with advanced melanoma. Other immunotherapies in development include anti-programmed death 1 protein (nivolumab), anti-PD-ligand 1, anti-CD137 (urelumab), and anti-OX40. Biomarkers that can be used as predictive factors for these treatments have not yet been clinically validated. However, there is already evidence that the tumor microenvironment can have a predictive role, with clinical activity of ipilimumab related to high baseline expression of the immune-related genes FoxP3 and indoleamine 2,3-dioxygenase and an increase in tumor-infiltrating lymphocytes. These biomarkers could represent the first potential proposal for an immunoprofiling panel in patients for whom anti-CTLA-4 therapy is being considered, although prospective data are required. In conclusion, the evaluation of systemic and local immunological biomarkers could offer useful prognostic information and facilitate clinical decision making. The challenge will be to identify the individual immunoprofile of each patient and the consequent choice of optimal therapy or combination of therapies to be used.

Glucocorticoid alterations in the human type-1/type-2 cytokine balance and the role of CD28/B7 costimulation

We have recently reported that psychological stress is associated with a shift in the human type-1/type-2 cytokine balance toward a type-2 cytokine response. The mechanisms of these cytokine alterations are unknown, but likely involve glucocorticoid (GC) modulation of cytokine production. Therefore we sought to characterize the effects of GC on the in vitro human type-1/type-2 cytokine balance. We hypothesized that GC induce a type-2 cytokine shift through modulation of critical regulatory cytokines and alterations in the CD28/B7 costimulatory pathway. ^ We first sought to characterize the effect of the GC, dexamethasone (DEX), on type-1 (IFN-γ, IL-12) and type-2 (IL-4, IL-10) cytokine production by human peripheral blood mononuclear blood cells (pBMC) stimulated with a variety of T-lymphocyte and monocyte stimuli. DEX, at concentrations mimicking stress and supraphysiologic levels of cortisol, decreased IFN-γ and IL-12 production and increased IL-4 and IL-10 production, indicating a shift in the type-1/type-2 cytokine balance toward a type-2 response. Furthermore, both CD4+ and CD8+ T-lymphocytes were susceptible to the cytokine modulating effects of DEX. Furthermore, in the absence of the monocyte, the DEX-induced alterations in T-lymphocyte cytokine production were reduced, indicating that the interaction between the monocyte and T-lymphocyte plays a significant role. ^ We next determined the role of regulatory cytokines, known to modulate the type-1/type-2 cytokine balance, in the DEX-induced cytokine alterations. The addition of the recombinant IL-12p70 and IFN-γ, but not the neutralization of IL-4, IL-10 or IL-13 using monoclonal antibodies, attenuated the DEX-induced type-1/type-2 cytokine alterations. These data suggest that the DEX-induced cytokine alterations are mediated, at least in part, through the initial inhibition type-1 cytokines. Lastly, we investigated the role of the CD28/B7 costimulatory pathway in these cytokine alterations. DEX decreased the expression of CD80 and CD86 on THP-1 cells, a monocyte cell line, and the expression of CD28 and CTLA-4 on PHA-stimulated pBMC. The DEX-induced decrease in CD28 and CTLA-4 expression was attenuated by rhIL-12. Finally, CD28 activation attenuated the DEX-induced decrease in IFN-γ production, suggesting that modulation of the CD28/B7 costimulatory pathway may contribute to the DEX-induced type-1/type-2 cytokine alterations. ^

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

Quantitative Association Tests of Immune Responses to Antigens of Mycobacterium Tuberculosis: A Study of Twins in West Africa

There is now considerable evidence that host genetic factors are important in determining the outcome of infection with Mycobacterium tuberculosis (MTB). The aim of this study was to assess the role of several candidate genes in the variation observed in the immune responses to MTB antigens. In-vitro assays of T-cell proliferation, an in-vivo intradermal delayed hypersensitivity response; cytokine and antibody secretions to several mycobacterial peptide antigens were assessed in healthy, but exposed, West African twins. Candidate gene polymorphisms were typed in the NRAMP1, Vitamin D receptor, IL10, IL4, IL4 receptor and CTLA-4 genes. Variants of the loci IL10 (-1082 G/A), CTLA-4 (49 A/G) and the IL4 receptor (128 A/G) showed significant associations with immune responses to several antigens. T-cell proliferative responses and antibody responses were reduced, TNF-alpha responses were increased for subjects with the CTLA-4 G allele. The T-cell proliferative responses of subjects with IL10 GA and GG genotypes differed significantly. IL4 receptor AG and GG genotypes also showed significant differences in their T-cell proliferative responses to MTB antigens. These results yield a greater understanding of the genetic mechanisms that underlie the immune responses in tuberculosis and have implications for the design of therapeutic interventions.

Regulatory T-cells infiltrate periodontal disease tissues

CD4(+) CD25(+) regulatory T ( Tr) cells are critical in regulating the immune response and thereby play an important role in the defense against infection and control of autoimmune diseases. Our previous studies demonstrated the involvement of autoimmune responses in periodontitis. The aim of this study was to identify CD4(+) CD25(+) Tr cells in periodontitis tissues and compare them with those in gingivitis tissues. Immunohistological analysis of CD4, CD25, and CTLA- 4 and the gene expression analysis of FOXP3, TGF- beta 1, and IL-10 on gingival biopsies revealed the presence of CD4(+) CD25(+) Tr cells in all tissues. In periodontitis, the percentage of CD4(+) CD25(+) Tr cells increased with increasing proportions of B-cells relative to T- cells. FOXP3, a characteristic marker for CD4(+) CD25(+) Tr cells, TGF- beta 1 and IL-10 were expressed more highly in periodontitis compared with gingivitis. These findings suggest that CD4(+) CD25(+) Tr cells and possibly other regulatory T- cell populations do exist and may play regulatory roles in periodontal diseases.

Moléculas coestimulatórias na leishmaniose visceral

Visceral leishmaniasis (VL) is endemic in many countries, including Brazil. The protozoan Leishmania infantum, is the etiological agent of VL, and is transmitted by the bite of female sandflies during the blood meal. The majority of subjects when exposed to the parasite do not develop the disease, because of development of Th1 cellular responses. Those who have develop signs of VL such as fever, weight loss, hepatosplenomegaly, have impairment of the cellular immune response, specific to the Leishmania antigens. We evaluated whether the specififc anergy during symptomatic VL, may be associated with changes in T cells costimulatory molecules or their ligands in CD14+ monocytes. There is an increase in CTLA-4 porcentage on CD4+ T lymphocytes (p=0.001) and ICOS on CD4+ and CD8+ T cells (p=0.002 to CD4+ and p=0.003 to CD8+), after stimulation by soluble Leishmania antigen (SLA) during active visceral leishmaniasis, and that there is a higher percentage of these molecules ex vivo, when comparing symptomatic to recovered individuals (p=0.04 to CTLA-4 in CD4+, and p=0.001 to ICOS in CD4+ and p=0.026 to CD8+). Moreover, we found a high gene expression of CTLA-4, OX-40 and ICOS during active VL. CD40, CD80, CD86, HLA-DR and ICOSL molecules do not suffer changes during disease. There is IFN-γ production by the peripheral blood cells, after SLA stimulation, by peripheral blood cells in symptomatic subjects; however, there is a decrease of the ratio IFN-γ/IL-10, which is reversed after clinical recovery. The impairment of some costimulatory molecules pathways during symptomatic VL could inhibit the ability of phagocytes to kill Leishmania and could facilitate their survival and the proliferation inside macrophages.

Immunotherapy for the treatment of poor prognosis cancers

Cancer is amongst the leading causes of death worldwide and the number one cause in the developed world. Every year there are close to 10 million cancer related deaths and this corresponds to hundreds of millions of euro in health care costs and lost productivity, placing a substantial drain on the economy. The efficacy of traditional treatment modalities for cancer therapy, such as surgery, radiotherapy and chemotherapy has plateaued, and while they are undoubtedly effective at prolonging patient lifespan, there is a high rate of adverse side effects and fatal reoccurrence. Currently, there is a huge amount of interest in the areas of cancer immunosurveillance and cancer immuno-editing, which explain some of the complex interactions between the host immune system and cancer. If left unchecked, cancerous malignancies have the ability to generate an immunosuppressive microenvironment, effectively shielding themselves from elimination and promoting tumour growth and progression. To overcome this, the potential of the immune system must be harnessed and the work undertaken in this thesis sought to contribute to this goal. Focus was placed on using novel therapies, combining tumour ablation with immune-modulating antibodies to maximise tumour elimination in an immune dependent manner, to overcome immunosuppression and promote immune activation. Chapter 2 focuses on the use of ECT as a method of tumour ablation and its effects on the immune system. ECT proved to be effective at inhibiting the tumour growth both in vitro and in vivo, and conferred significant survival advantages in both small and large animal models. More importantly, ECT proved to cause tumour death in an immune dependent manner, displaying the hallmarks of Immunogenic Cell Death, increases in immune cell infiltration and generating tumour-specific immune responses. Chapter 3 focuses on combining ECT with immune checkpoint blockade inhibitors; anti- CTLA-4 and anti-PD-1. Both combinations proved to be effective at inhibiting both primary and distal tumour growth, indicating the generation of tumour specific immune responses and prolonged animal survival. In addition, the treatments caused increases in the levels of certain intra-tumoural immune cell subsets and modulated the cytokine profile of treated animals in a way that was favourable overall. Chapter 4 focuses on the combining ECT with an anti-iCOS agonist antibody, capable of causing immune co-stimulation. This novel combinational therapy proved to be the most effective by far, with a high cure rate achieved across a number of different in vivo tumour models. Total regression was seen in both primary and distal tumours, as well as spontaneous metastases, with the tumour specific immune response generated conferring total protection to animals on tumour rechallenge. Overall the data presented here adds further insight into the area of cancer immunotherapy with some of the novel combinational therapies demonstrating substantial clinic potential.

Traitement de la maladie du greffon contre l’hôte chronique par la photophérèse extra corporelle au TH9402 : m écanisme de régulation de la maladie du greffon contre l’hôte chronique par les cellules T régulatrices

La maladie du greffon contre l’hôte (GVHD) est la principale cause de mortalité et de morbidité suite aux greffes de cellules souches hématopoïétiques. Plusieurs patients demeurent réfractaires aux traitements actuels ce qui rend nécessaire le développement de nouvelles stratégies afin de combattre cette maladie. Dans l’étude qui suit, nous avons utilisé un nouvel agent thérapeutique, le TH9402, une molécule photosensible et démontré qu’elle permet, lorsqu’exposée à la lumière visible (514 nm), d’éliminer sélectivement les cellules T activées in vivo tout en préservant les cellules T au repos et les cellules T régulatrices (Tregs). Les Tregs ainsi préservés peuvent abroger la réponse alloréactive par la sécrétion d’IL-10 ou par contact cellule-cellule via un mécanisme impliquant le CTLA-4. Nous avons découvert que la signalisation du CTLA-4 était associée à une hausse de la population Treg in vitro. Cette hausse est due à la conversion de cellules T CD4+CD25- en Tregs et non à une prolifération sélective des Tregs. Dans la deuxième partie de cette étude, nous avons démontré que la signalisation de CTLA-4 était associée à une augmentation de l’expression de la protéine Indoleamine 2,3 dioxygenase (IDO). Ces effets nécessitent la déplétion du tryptophane ainsi que de la protéine de phase aigue GCN2. Finalement, nous avons observé que l’infusion de cellules traitées au TH9402 chez des patients souffrant de GVHD chronique est associée à une augmentation de la population Treg chez ces patients sans causer de lymphopénie ni de diminution de la réponse immunitaire dirigée contre les antigènes viraux. Ces résultats suggèrent que le traitement au TH9402 pourrait représenter une approche particulièrement intéressante pour le traitement de la GVHD chronique réfractaire aux traitements actuels. De plus, l’augmentation de l’expression d’IDO pourrait être utilisée comme valeur prédictive de la réponse du patient au traitement. Ceci pourrait permettre d’améliorer la qualité de soins ainsi que de la qualité de vie des patients souffrant de GVHD chronique.

The Role of HBD-2 and HBD-3 in Human T Cell Development

Human β-defensins (hBDs) are a family of cationic peptides able to directly kill a wide range of microorganisms including bacteria, fungi and viruses. In addition to their antimicrobial activities, defensins also contribute to the modulation of both the host innate and adaptive immunity. In this project, we demonstrate that the αCD3/28 co-stimulation of human CD4+ T cells in the presence of 10μg/ml hBD-2 or hBD-3 together causes an up-regulation in numbers of CD4+CD69+CD25+ and CD4+CD69-CD25+ T cell subsets, indicating that the treatment of hBD-2 and 3 enhances CD4+ T cell activation. Consistent with this finding, proliferation assay using CFSE suggests that hBD-2 and hBD-3 treatment in vitro induces the proliferation of CD4+ T cells following by 96hrs culture. Analysis of expression of the regulatory T cells (Tregs) specific marker, FoxP3, reveals a shift in the CD4+CD127-CD25+ Treg subset at 18hrs. However, at the later time point, we found that the percentage of FoxP3+cells decreased in the CD4+CD127-CD25+ Treg population, whereas the presence of the FoxP3+CTLA-4+ Treg subset increased. These data indicate that Treg suppressive function may be potentially defective following the co-incubation of purified T cells with either hBD-2 or hBD-3 for 42hrs in vitro due to the apparent loss of FoxP3 expression. We further characterise the role of hBD-2 and hBD-3 in driving human CD4+ T cells polarisation. Our in vitro data suggests that treatment with hBD-2 and hBD-3 can not only induces effector T cell (Teff) differentiation into RORγt+T-bet+ (Th17/Th1) cells, but can also trigger the differentiation of Treg expressing RORγt and T-bet rather than the master controller of Treg function, FoxP3. This apparent plasticity of T cell phenotype allows them to convert from Treg to Th1/17-like effector T cell phenotype following 18hrs in culture. By 42hrs in culture, treatment with hBD-2 and hBD-3 induced both Teff cell and Treg cell differentiation towards the Th17-like phenotype. Compared with the treatment with hBD-2, treatment with hBD-3 induced a more pronounced effect to increase levels of RORγt in CD4+ T cells. This elevated expression may, in turn, be responsible for the induction of higher IL-17A secretion. Consistent with this idea, it was found that treatment with hBD-3 but not hBD-2 was capable of inducing the higher level of secretion of IL-17A. Additionally, treatment with hBD-3 induced an increased expression of IL-6, which is capable of driving the differentiation of naïve T cells towards IL-17-producing Th17 cells. Functionally, using the Treg suppression assay, the data suggested that hBD-2 may dampen down Treg cell ability to induce suppression of Teff cell activity. Interestingly, co-culture with hBD-2 would also appear to increase Teff cell resistance to Treg immunoregulation in vitro. Further investigation using microarray gene analysis revealed chemokine C-C motif ligand 1 (CCL1) as potential genes responding to hBD-2 treatment. The blockade of CCL1 has been reported to inhibit Treg suppressive function. Thus, this study explored the function of these antimicrobial candidates in regulating CD4+ T cell plasticity which could result in hBD-2 and hBD-3 being able to regulate its own production, but also may regulate Treg and Teff cell development and function, thus strengthening the link between innate and adaptive immunity

Doença enxerto-versus hospedeiro: determinação da expressão de genes e alterações de vias associadas ao sistema imunológico usando metodos da biofísica

O transplante de medula óssea (TMO) é um procedimento terapêutico importante em casos relacionados à pacientes com leucemia ou linfoma. Em decorrência desse processo, uma reação conhecida como doença enxerto-versus-hospedeiro (GVHD) pode ocorrer em pacientes susceptíveis como conseqüência da presença de células imunocompetentes do doador. Entretanto, não existe um modelo para descrever completamente as ações relacionadas ao mecanismo imunológico da GVHD desde a fase que inicializa a doença até a fase efetora. O Objetivo geral deste estudo é a investigação da resposta imunológica considerando-se o sistema HLA (antígenos leucocitários humano) em pacientes que desenvolveram a GVHD em decorrência do TMO. O National Cancer Institute (NCI) – Pathway interaction Database e Reactome foram usados como bases de dados com o objetivo de se estudar a expressão de genes e vias relacionados às Classes I e II do sistema HLA (antígenos leucocitários humano). O estudo considerou a mudança de expressão de genes relacionados às 17 vias do sistema imunológico com potencialidade para se expressar em pacientes que desenvolveram a GVHD associada à TMO. Dados referentes aos transcriptomas foram obtidos utilizando-se a plataforma GPL570 Affymetrix Genoma Humano U133 Plus. A atividade relativa foi usada para determinar as alterações das vias em amostras de GVHD em relação ao controle. As análises foram realizadas utilizando-se o software Via Complex e Bioconductor. Observou-se aumento significativo da expressão de genes ralacionados às vias do sistema imune adaptativo, antígenos associados às Classe I e II do HLA, fosforilação de CD3 e CD247, sinalização dos receptores de células T em CD4+ nativas e ativação de NF-kapa β nas células B. Também observou-se alterações significativas na mudança de expressão dos genes associados às vias relacionadas à super família de moléculas B7:CD28\CTLA-4 quando comparadas ao controle. Isso pode indicar a necessidade de geração de um segundo sinal co-estimulador em GVHD, acionado pelas moléculas dessa super família. O aumento da expressão do gene CD69 nas amostras experimentais caracteriza a ativação celular e, portanto, a sinalização de estímulos em GVHD. Os achados obtidos neste estudo contribuem para melhor elucidar o mecanismo imunopatogênico associado à GVHD. P

Traitement de la maladie du greffon contre l’hôte chronique par la photophérèse extra corporelle au TH9402 : m écanisme de régulation de la maladie du greffon contre l’hôte chronique par les cellules T régulatrices

La maladie du greffon contre l’hôte (GVHD) est la principale cause de mortalité et de morbidité suite aux greffes de cellules souches hématopoïétiques. Plusieurs patients demeurent réfractaires aux traitements actuels ce qui rend nécessaire le développement de nouvelles stratégies afin de combattre cette maladie. Dans l’étude qui suit, nous avons utilisé un nouvel agent thérapeutique, le TH9402, une molécule photosensible et démontré qu’elle permet, lorsqu’exposée à la lumière visible (514 nm), d’éliminer sélectivement les cellules T activées in vivo tout en préservant les cellules T au repos et les cellules T régulatrices (Tregs). Les Tregs ainsi préservés peuvent abroger la réponse alloréactive par la sécrétion d’IL-10 ou par contact cellule-cellule via un mécanisme impliquant le CTLA-4. Nous avons découvert que la signalisation du CTLA-4 était associée à une hausse de la population Treg in vitro. Cette hausse est due à la conversion de cellules T CD4+CD25- en Tregs et non à une prolifération sélective des Tregs. Dans la deuxième partie de cette étude, nous avons démontré que la signalisation de CTLA-4 était associée à une augmentation de l’expression de la protéine Indoleamine 2,3 dioxygenase (IDO). Ces effets nécessitent la déplétion du tryptophane ainsi que de la protéine de phase aigue GCN2. Finalement, nous avons observé que l’infusion de cellules traitées au TH9402 chez des patients souffrant de GVHD chronique est associée à une augmentation de la population Treg chez ces patients sans causer de lymphopénie ni de diminution de la réponse immunitaire dirigée contre les antigènes viraux. Ces résultats suggèrent que le traitement au TH9402 pourrait représenter une approche particulièrement intéressante pour le traitement de la GVHD chronique réfractaire aux traitements actuels. De plus, l’augmentation de l’expression d’IDO pourrait être utilisée comme valeur prédictive de la réponse du patient au traitement. Ceci pourrait permettre d’améliorer la qualité de soins ainsi que de la qualité de vie des patients souffrant de GVHD chronique.