A indução de tolerância imunológica ao aloenxerto, no contexto clínico, permanece um grande desafio para pesquisa científica de tradução. A retirada da imunossupressão em indivíduos transplantados leva à rejeição do enxerto, na grande maioria dos casos. Entretanto, um grupo muito raro de indivíduos transplantados, chamados de tolerantes operacionais (TO), consegue manter a função estável do enxerto após a retirada dos imunossupressores. O estudo desses indivíduos pode contribuir para melhor compreensão dos mecanismos envolvidos na tolerância ao enxerto em humanos, assim como, para a determinação de biomarcadores desse estado de homeostase. Nosso objetivo foi determinar se o estado de tolerância operacional no transplante renal induz um perfil diferencial do componente humoral da resposta imune. Para tal, analisamos o perfil de reatividade de autoanticorpos dirigidos a peptídeos da proteína de choque térmico 60 (HSP60), de alo e autoanticorpos dirigidos às moléculas HLA, o repertório do receptor de células B (BCR) e o perfil funcional de células B supressoras CD19+CD24hiCD38hi (Bregs), comparativamente, nos indivíduos com: TO (n=5), Rejeição Crônica (RC, n=13), função estável do enxerto usando doses habituais de imunossupressores (Est, n=19) e nos indivíduos saudáveis (Sau, n=11). Não observamos um perfil diferencial claro de alo/autorreatividade de anticorpos dirigidos aos peptídeos da HSP60, nem às moléculas HLA, que diferenciasse os grupos do estudo. O estado de tolerância operacional apresentou uma diversidade do repertório do receptor de células B similar à observada em Sau e Est, enquanto o grupo RC teve uma menor diversidade desse repertório. Além disso, o grupo TO apresentou uma expansão de clones linfócitos B com expressão de 2 tamanhos distintos de CDR3 (de 16aa, família VH3 isotipo IgM, e de 5aa, família VH1 isotipo IgG), diferenciando-os dos grupos Sau, RC e Est (p<0,01 e p<0,05; e p<0,01, respectivamente para VH3M e VH1G). Os números de células B com fenótipo imunorregulador CD19+CD24hiCD38hi (Bregs) circulantes, no grupo TO e Sau, foram similares, enquanto o grupo RC apresentou menores números (p<0,05). Funcionalmente, após estímulo via CD40, o grupo TO teve capacidade de gerar células Breg ativadas para STAT3 semelhante ao grupo Sau, enquanto na rejeição crônica esta capacidade foi menor (p<0,05). Concluímos que o estado de tolerância operacional envolve, principalmente, a manutenção do perfil do componente imune humoral, similar ao apresentado por indivíduos saudáveis, em contraste com o estado de rejeição crônica. Além disso, o estado de tolerância foi o único que apresentou expansões expressivas de determinados tamanhos de CDR3, se destacando de todos os grupos. A expansão diferencial desses clones de células B pode ter uma relevância funcional no estado de tolerância operacional, além de potencial valor para o diagnóstico desse estado. Esses dados, em conjunto, nos indicam que a preservação do componente humoral da resposta imune desempenha um papel importante neste estado de homeostase no transplante humano / Operational tolerance in human kidney transplantation: repertoire of B lymphocytes and alo and autoantibodies Sta individuals (p<0.01 and p<0.05; and p<0.01, respectively for VH3M and VH1G). The circulating B cell numbers with the suppressive phenotype CD19+CD24hiCD38hi (Bregs) were similar between the OT and HI groups, while CR presented lower numbers (p<0.05). In addition, the OT group exhibited a similar capacity of generate activated cells for STAT3 to HI, whereas the CR group exhibited an impaired capacity (p<0.05). We conclude that the operational tolerance state involves the maintenance of the B cell compartment profile similar to the one observed in healthy individuals, in contrast with chronic rejection. In addition, the state of operational tolerance was the only one exhibiting expressive expansions of specific CDR3 lengths, which differentiated OT from all other groups. This indicates that the expansion of B cell populations expressing specific CDR3 lengths could play a relevant role in operational tolerance and may be potential biomarkers for OT. Taken together, we suggest that the preservation of the B cell component of the immune response can play an important role in this homeostatic state in human transplantation

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Made available in DSpace on 2016-12-06T17:07:09Z (GMT). No. of bitstreams: 1 CesarMartins.pdf: 970860 bytes, checksum: 6ad6b24dacadddaad307316c6a4edb43 (MD5) Previous issue date: 2001-12-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The purpose of this study was to analyze the ultimate strength and displacement behavior of healed 3.5-mm segmental radius defects in seventeen adult male rabbits in a fourty-two days follow-up. The defect was filled out with biomaterials (ten radius) such as bone morphogenetic protein, inorganic bovine bone matrix, organic bovine bone matrix and bovine collagen in one group (group I). Group II (seven radius) received fresh-frozen allograft bone obtained from a rabbit bone-banking created for this study. The opposite limbs were the control group (group III) with seventeen radius. The three point bending and the displacement were recorded in all specimens studied. The one-way analysis of variance (p£ 0.05) and the variation coefficient in the whole three groups. Significant difference in the ultimate strengh were found between group I and III (p= 0.0009). The variable ultimate strength of group I has presented a coefficient of variation of 87%, group II has presented a oefficient of variation of 66% and the control group a coefficient of variation of 31%. Group I in the variable displacement has presented a coefficient do variation of 70%, group II has presented a coefficient of variation of 60% and group III 33%. Beetwen groups I and II the least variated group was group II in both variables. However, four in ten non-unions were recorded in the biomaterial group. All allograft group specimens have shown bone healing in the follow-up period. This study do not demonstrate the effectiveness of bovine origin biomaterials as suitable on bone formation. Clinical Relevance: Segmental Bone loss due to trauma, tumor resection or other causes are one of the most challenging problems in orthopaedic surgery. Several materials have been used as bone regenerator, even thou bone tissue has a very known potential of healing. Allograft bone tissue is very attractive possibility but presents a high disease transmission risk. Although autograft tissue has a short stock tissue for some situations in the daily orthopaedic activity, it remains the gold standard for most surgeons. In some cases, bovine origin biomaterials are the only choice available to solve the bone loss situations. / O presente estudo tem como objetivo identificar a resistência mecânica óssea e o deslocamento do osso neoformado utilizando-se biomateriais de origem bovina e enxerto homólogo no preenchimento de uma falha de 3,5 mm no rádio em 17 dos 19 coelhos adultos machos da raça nova zelândia, com massa de 3,5 a 5,0 Kg. Os biomateriais são substâncias biologicamente ativas, que apresentam efeito osteogênico, isto é, ativam a formação de osso novo nos organismos vivos. Neste estudo foram utilizados três grupos: grupo I com enxerto ósseo orgânico e inorgânico bovino, pool de proteína morfogenética bovina e colágeno bovino, como biomateriais em dez animais. Dois animais foram submetidos à retirada das asas ilíacas para posterior congelamento e implantação em sete animais formando o grupo II, ou enxerto homólogo. O grupo III ou controle consistiu nos rádios não operados dos 17 animais. Após 42 dias, os animais foram sacrificados e os rádios foram submetidos ao teste de flexão em três pontos, com a aplicação da carga no sítio da regeneração, sendo avaliados a carga máxima e o deslocamento neste ensaio mecânico. O teste estatístico realizado foi ANOVA - um caminho, com p£0,05 para três amostras, post-hoc de Scheffé e o coeficiente de variação. Quanto à carga máxima para a ruptura, o grupo I (biomateriais) apresentou uma média de 77,1 N ± 67,2N e um CV (coeficiente de variação) de 87%, o grupo II (enxerto homólogo) apresentou uma média de 154,8 N ±103,2 N, com um CV de 66%, já o grupo controle obteve uma média de 201,8 N ± 63,0N e um CV de 31%, que caracterizou uma diferença estatisticamente significante entre o grupo I com o grupo controle (p=0,0009). Quanto a variável deslocamento, o grupo I apresentou uma média de 1,0 mm ± 0,7mm com um CV de 70%, o grupo do II apresentou uma média de 1,0 mm ± 0,6 mm com um CV de 60% e o grupo III uma média de 0,9 mm ± 0,3 mm e CV de 33%, não demonstrando diferença estatisticamente significante (p=0,49). Quatro espécimes do grupo dos biomateriais que fizeram parte do estudo estatístico apresentaram não consolidação na falha criada, por outro lado os espécimes preenchidos com enxerto homólogo consolidaram em todos os casos. Em conclusão, os enxertos homólogos apresentaram consolidação satisfatória demonstrando um pico de carga máxima com valores próximos aos do grupo controle. Porém, os biomateriais demonstraram uma variação intragrupo muito alta devido às não consolidações, com média de carga máxima muito baixa.

O osso alógeno fresco congelado e o Bio-Oss® (OBM) são materiais conhecidos como substitutos ao osso autógeno em cirurgias de levantamento de seio maxilar. O objetivo deste estudo foi avaliar clínica, histológica e histomorfometricamente o processo de reparo de enxertos alógenos com e sem a utilização de OBM, utilizados em técnicas de levantamento de seio maxilar em humanos. Neste estudo do tipo prospectivo, randomizado, tipo série de casos, comparativo, foram operados 34 seios maxilares de pacientes de ambos os sexos, que apresentavam um rebordo ósseo residual com altura máxima de 5 mm na região de seios maxilares para reconstrução em altura para cirurgias de levantamento de seio maxilar prévia à reabilitação por implantes. Os pacientes foram divididos em a) Grupo Controle: Dezessete (17) seios maxilares enxertados somente com a utilização de osso alógeno e b) Grupo Teste: 17 seios maxilares reabilitados com osso alógeno e OBM, na proporção de 2:1. Seis meses após a enxertia, no momento da instalação dos implantes, amostras ósseas foram coletadas por meio de trefinas para análise histológica e histomorfométrica. Os dados da investigação foram submetidos ao teste t de Student para amostras independentes empregado para comparações entre os dois grupos, Mann-Whitney e testes de correlação foram aplicados. 29 pacientes com uma média de idade de 51,32 anos (± 6,44), foram divididos em grupos controle (17) e teste (12), sendo 34 seios avaliados no total. Não houve diferença estatística entre os grupos com relação à idade (p = 0,23) e ao gênero (p = 0,56). Mediana do torque de inserção dos implantes foi de 32N para o grupo controle, e 45N para o grupo teste (p < 0,0001). Taxa de sucesso no grupo controle foi de 93,02% e 100% no grupo teste. Análise histológica apresentou no grupo controle osso alógeno residual com lacunas osteocíticas vazias e padrão lamelar; osso neoformado com lacunas osteocíticas com osteócitos viáveis e padrão imaturo; osteoblastos em íntimo contato matriz osteóide, formando pontes entre os blocos de osso alógeno e osso neoformado; osteoclastos em proximidade às áreas remodelação óssea; ausência de sinais de infiltrado inflamatório; tecido conjuntivo; e no grupo teste, todos os ítens acima e osso bovino mineral. Histomorfometria: material enxertado remanescente (p = 0,74); osso alógeno remanescente (35.78% ± 6.21% grupo controle, 19.72% ± 10.42% grupo teste; p < 0,0001); OBM remanescente no grupo teste (14,78% ± 8,67); osso neoformado (11.94% ± 1.71% grupo controle, 25.79% ± 8.76% grupo teste; p < 0,001); osso total (47.72% ± 5.6% grupo controle, 58.96% ± 8.1% grupo teste; p < 0,001); tecido conjuntivo (52.27% ± 5.6% grupo controle, 41.45% ± 8.4% grupo teste; p < 0,01). Adicionar OBM ao osso alógeno em cirurgias de levantamento de seio maxilar mostrou-se uma técnica de enxertia eficaz para a instalação de implantes. Resultou em maior torque de inserção, porcentagens maiores de osso neoformado e osso total, permitindo a instalação de implantes e reabilitação protética funcional. / Allograft fresh frozen bone and Bio-Oss are knows as autogenous bone materials substitutes in maxillary sinus lifting. The aim of this study was to evaluate clinical, histological and histomorphometrically the process of repair with and without the association of BBM in maxillary sinus augmentation. In this prospective, randomized, comparative case series study, 34 maxillary sinuses were augmented, which had a residual bone ridge with a maximum height of 5 mm in maxillary sinus reconstruction region at the time for surgery of maxillary sinus prior to rehabilitation by implants. Patients were divided into a) control group: seventeen (17) grafted maxillary sinuses with only allograft bone and b) test group: 17 maxillary sinuses rehabilitated with allograft bone and BBM in a 2:1 ratio. Six months after grafting, at time of implant placement, bone samples were collected using a trephine burr for histological and histomorphometrical analysis. The research data were subjected to Student\'s t test for independent samples used for comparisons between two groups, Mann - Whitney and correlation tests were applied. 29 patients with a mean age of 51.32 years (± 6.44), were divided into control group (17) and test (12), with 34 maxillary sinuses evaluated in total. There was no statistical difference between the groups regarding to age (p = 0.23) and gender (p = 0.56). Median insertion torque of the implants was 32N for the control group and 45N for the test group (p < 0.0001). Survival rate in the control group was 93.02% and 100% in the test group. Histological analysis showed, at the control group, residual allograft bone with empty osteocytic lacunae and lamellar pattern; newly formed bone with osteocytic lacunae with viable osteocytes and immature pattern; osteoblasts in close contact with osteoid matrix, forming bridges between the blocks of allograft bone and new bone formation; osteoclasts in bone remodeling surrounding areas; no evidence of inflammatory infiltrate; connective tissue; and in the test group, all the items above and bovine bone mineral. Histomorphometry: graft remaining material (p = 0.74); remaining allogenous bone (35.78% ± 06.21% control group, 19.72% ± 10:42% test group, p < 0.0001); BBM remaining in the test group (14.78% ± 8.67); newly formed bone (11.94% ± 1.71% control group, 25.79% ± 8.76% test group, p < 0.001); total bone (47.72% ± 5.6% control group, 58.96% ± 8.1% test group, p < 0.001); connective tissue (52.27% ± 5.6% control group, 41.45% ± 8.4% test group, p < 0.01). Adding bovine bone mineral to allogenous bone in maxillary sinus surgery proved to be an effective technique of grafting for implant placement. It resulted in higher insertion torque, higher percentages of new bone formation and total bone, allowing installation of implants and functional loading.

Ainda que resulte na sobrevida do paciente, o tratamento atual de queimaduras profundas e extensas é imperfeito. A contratura cicatricial e o aspecto rendilhado persistentes na área enxertada produzem resultados estéticos e funcionais pobres. A principal dificuldade no adequado restabelecimento do tegumento destes pacientes é o déficit de derme, decorrente da reposição das perdas com enxertos relativamente finos. Neste trabalho são relatados quatro casos, decorrentes da familiaridade obtida em nossa prática clínica com o emprego de pele preservada em glicerol, nos quais foi utilizada a enxertia combinada, isto é, derme alógena residual sobreposta por enxertos de espessura parcial autógenos, como forma de reposição de matriz dérmica. Os tegumentos resultantes da enxertia combinada mostraram-se estáveis e duráveis, dentro do período de seguimento dos casos apresentados. / Although it results in the patient\'s survival, the current treatment of deep and extensive burns is imperfect. Scar contracture and the persistent lacy aspect in the grafted area produce aesthetic and functional poor results. The main difficulty in the appropriate re-establishment of the tegument of these patients is the deficit of dermis, due to the replacement of the skin losses with relatively thin grafts. In this work, decurrent of the familiarity obtained in our clinical practice with glycerol preserved cadaver skin, four cases are reported, which the combined grafting was used, that is, residual allogeneous dermis put upon by split thickness autogenous grafts, as form of replacement of dermal matrix. The resulting teguments of the combined grafting were shown stable and durable, inside the follow-up period of the presented cases.

La greffe cardiaque est le traitement ultime de l’insuffisance cardiaque. Le rejet aigu pose plusieurs problématiques, en particulier sa survenue imprévisible même sous traitement immunosuppresseur, et un diagnostic histologique qui nécessite des biopsies endomyocardiques (BEM) invasives répétées, et qui souffre de nombreuses limites. Le besoin de critères diagnostiques et prédictifs, idéalement non invasifs, nous a conduits à étudier le rejet aigu de greffe cardiaque sur le plan moléculaire. Nous avons caractérisé les profils d’expression génique (PEG) myocardiques et sanguins lors de différentes phases du rejet cellulaire (RC) et du rejet médié par les anticorps (RMA), par analyse sans a priori des transcriptomes sur puce à ADN. Par une première étude des PEG myocardiques menée sur une collection historique de BEM, nous avons montré la modification des PEG tissulaires lors du RC. Pour le même grade histologique, deux profils de RC aux degrés d’activation immunitaire différents ont été identifiés. De plus, les PEG myocardiques étaient modifiés dès un mois avant la survenue d’un RC, quand l’analyse histologique ne montrait encore aucune anomalie. Par une seconde étude conduite sur une collection prospective de BEM et échantillons sanguins, nous avons confirmé les résultats de la première étude, et de plus montré l’existence de modulations des PEG également dans le sang périphérique, aussi bien pendant un épisode de RC qu’un mois avant. Enfin pour la première fois la modulation tissulaire et périphérique des PEG a été montrée dans le RMA en transplantation cardiaque. L’existence de voies modulées dans les deux types de rejet devrait conduire à la recherche de biomarqueurs. / Heart transplantation is the last treatment in case of terminal heart failure. Acute rejection after heart transplantation raises several issues due to its occurrence despite immunosuppressive therapies and the requirement of invasive and repeated endomyocardial biopsies (EMB) that have several histological grading limitations. The need of non-invasive diagnostic and predictive criteria led us to study the acute rejection of cardiac allograft using a molecular approach. We characterized myocardial and peripheral blood gene expression profiles (GEP) during acute cellular rejection (CR) and antibody-mediated rejection (AMR) by mean of microarray analyses. By a retrospective study conducted on a historical EMB collection, we first showed a strong immunologic modulation during CR. For the same CR histological grading, two transcriptional profiles were identified according to the inflammation level. Moreover, myocardial GEP modifications were observed one month before the occurrence of CR, while histological characteristics showed no abnormality. A second study conducted on a prospective collection of both EMB and peripheral blood samples confirmed the results obtained on EMB and showed peripheral blood GEP modulations during both CR and even one month earlier. Finally, we have also shown for the first time in heart transplantation, myocardial and peripheral GEP modulations in AMR. Identification of modulated pathways in both types of rejection should allow for the determination of rejection biomarkers.

La survenue de la maladie du greffon contre l’hôte aiguë (GVHa) après l’allogreffe de cellules souches hématopoïétiques (CSH) reste une source majeure de mortalité et morbidité pour laquelle des avancées thérapeutiques restent indispensables. Les lymphocytes NKT invariants (iNKT) possèdent de multiples propriétés à potentiel immuno-régulateur et anti-tumoral, mais peu de données existent chez l’homme dans le cadre de l’allogreffe de CSH. Nous avons récemment montré qu’une bonne reconstitution en iNKT post greffe ainsi qu’une bonne richesse et capacité d’expansion des iNKT du greffon, notamment du sous type CD4-, étaient corrélés à une moindre survenue de GVHa chez les patients. Ce travail avait pour objectif de déterminer quel sous type de iNKT humain pouvait réguler la réponse allo-immune et ses mécanismes d’action. Dans le modèle murin humanisé de xéno-GVH, chez la souris NSG, nous avons mis en évidence que le sous-type iNKT CD4- humain ,contrairement au sous type CD4+, permettait de diminuer la survenue de GVH et d’améliorer la survie des souris NSG greffées avec des PBMC humains enrichis ou non en lymphocytes iNKT. Nous avons montré de façon concordante, in vivo et in vitro, que les iNKT CD4- humains diminuent le profil Th1 et Th17 des lymphocytes T conventionnels et leur marqueurs d’activation après stimulation allo- et xéno-génique. Par ailleurs, les iNKT CD4- humains induisent la mortalité des cellules dendritiques in vitro et in vivo, contrairement au sous-type iNKT CD4+ qui induit leur maturation. Par contre, les iNKT CD4- n’ont pas d’impact sur les lymphocytes T régulateurs, ni sur la prolifération des T conventionnels. Ils n’altèrent pas non plus la qualité de la prise de greffe des cellules humaines dans la souris NSG. Nous concluons, que contrairement aux iNKT murins, le sous-type iNKT CD4- humain, et non pas CD4+, est capable de diminuer la survenue de xéno-GVH dans le modèle murin NSG par une régulation directe de la réponse immune allogénique. Ces résultats obtenus dans ce modèle préclinique ouvrent la possibilité d’une nouvelle thérapie cellulaire utilisant ces cellules chez l’homme pour prévenir la survenue de GVH aiguë. / The occurrence of acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (allo-SCT) is a major source of mortality and morbidity and new therapeutic advances are urgently needed. Despite the numerous immunomodulatory properties depicted for the iNKT lymphocytes, there are few studies of these lymphocytes in human allo-SCT and GVHD setting. We previously reported that a higher iNKT reconstitution in patients after allo-SCT and higher graft CD4- iNKT expansion rates, were associated with reduced risk of aGVHD in patients. In this study we aimed to assess a direct immune regulatory role of CD4+ or CD4- iNKT subsets and their mechanisms of action during the allo-immune response. We demonstrated that the human iNKT CD4- subset, but not the CD4+ subset, was associated with reduced xeno-GVHD and prolonged survival of humanized NSG mice infused with human PBSC with or without iNKT cells. We also concomitantly showed, both in vitro and in vivo, that human iNKT CD4- could dampen Th1 and Th17 conventional T cell allo and xeno-responses as well as T cell activation markers. In addition, whereas iNKT CD4+ could stimulate DC maturation, iNKT CD4- induced their apoptosis, in vitro and in vivo. We did not observe any impact on regulatory T cells and conventional T cell proliferation. iNKT CD4- did not impact the engraftment of human cells in NSG mice. We conclude that, by contrast with mice, in humans, the CD4- subset and not their CD4+ counterpart can directly down-regulate the allo-immune response. These results obtained in a robust humanized preclinical xeno-GVH mice model could open new strategies of cellular therapy in the prevention of acute GVHD.

Purpose: In an Orthopedic office in Northeast Tennessee clinical decision making about injection options for non-surgical candidates with knee osteoarthritis is unclear. Aims: This quality improvement project will develop a clinical guideline so that providers know criteria for choosing optimal knee alternative treatments for non-surgical knee patients. Outcome measures: An expert panel gave feedback and advice on the information presented for the injection types of Platelet Rich Plasma, Corticosteroids, Amniotic Allograft and Hyaluronic Acid. They edited the guideline then sent the edits back to be complied and edited using the Delphi method. Process and Methods: The expert feedback was then be collected in a non-identifiable fashion and the guideline was rewritten based on the panel advice. Then the guideline was then presented to the practice site and the site reviewed and rated the guideline on clarity, accuracy and ease of use. Results: The guideline was rated by the practice site as able to be adopted into practice and used at the site. Findings and Limitations: Limitations include the practice site is constantly changing and new implementations could be overlooked. The expert panel are all busy professionals and finding time to review and critique a guideline is extensive. Conclusions and Implications: This novel guideline will improve healthcare by eliciting an expert panel of orthopedics that perform injections to assist in compiling the most accurate up to date guideline through which will create enhanced decision making and overall better patient care when choosing knee injections for non surgical patients.

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.

Dans des modèles expérimentaux, une tolérance d'allogreffe a pu être induite en transférant des lymphocytes T CD4+ régulateurs (Treg) spécifiques d'antigènes (Ag) du donneur expandus ex vivo. Les données ont démontré l'importance des Treg d'allospécificité indirecte (Treg indirects) dans l'induction d'une tolérance à long terme. L'expansion de Treg indirects ex vivo est problématique, principalement à cause de la difficulté d'obtenir en grand nombre des cellules présentatrices d'antigène autologues (CPA)pour stimuler les Treg. Les lymphocytes B (LB) sont des APC accessibles, présentes en grand nombre et ont un fort potentiel régulateur. Cependant, l'utilisation de LB autologues comme APC est rendue problématique par leur incapacité à présenter les Ag dont ils ne sont pas spécifiques.Dans ce travail de thèse, nous avons développé une approche nanobiotechnologique permettant de transformer des LB polyclonaux autologues en puissants stimulateurs de Treg spécifiques de l'Ag.Des Ag particulaires synthétiques (SPAg) ont été générés en fixant sur des nanosphères fluorescentes de 400 nm de diamètre : (i) des anticorps monoclonaux dirigés contre un domaine constant de la chaine légère kappa du récepteur des LB, et (ii) des Ag modèles.Les SPAg se comportent comme des Ag particulaires naturels lorsqu'ils sont incubés in vitro avec des LB murins ou humains. Les SPAg se lient à la surface des LB kappa+, déclenchent un signal d'activation et sont internalisés dans leur endosomes. Les LB chargés en SPAg induisent l'activation et la prolifération des lymphocytes T CD4+ spécifiques de l'Ag in vitro.Des propriétés régulatrices peuvent être conférées aux LB chargés en SPAg en les stimulant avec du CpG. Les LB régulateurs générés n'induisent pas de prolifération des T CD4+ effecteurs mais, au contraire, entrainent une prolifération importante des Treg.Cette approche apparait comme une alternative innovante pour expandre des Treg spécifiques de l'Ag ex vivo / Allograft tolerance has been obtained in experimental models with adoptive transfer of ex vivo-expanded regulatory T cells (Treg) specific for donor antigens. Preclinical data have shown that Treg specific for indirectly presented alloantigens (indirect Treg) are mandatory for long-term tolerance. However, the ex vivo expansion of indirect Treg faces limitations,related essentially to the source of autologous antigen-presenting cells (APCs) used to stimulate T cells in vitro. B cells are (i) potent regulatory cells and (ii) APCs able to establish a privileged crosstalk with CD4+ T cells. However, the use of B cells as APCs is made problematic due to their inability to internalize and present non-cognate antigens. We have developed a novel nanobiotechnology-based approach to turn autologous polyclonal B cells into potent stimulators of antigen-specific T reg.Synthetic particulate antigens (SPAg) were generated by immobilizing (i) monoclonal antibodies directed against a framework region of B cell receptor (BCR) kappa-light chains and (ii) model antigens on fluorescent nanospheres of 400 nm in diameter.SPAg behaved like genuine particulate antigens when incubated in vitro with polyclonal murine B cells. SPAg bound to surface BCR of any kappa-positive B cells, triggered activation signal and were internalized in late endosomal compartment of B cells. SPAgloaded B cells induced activation and proliferation of antigen-specific T cells. This approach was transposable to humans’ cells. Importantly, regulatory properties could be conferred toSPAg-loaded B cells by CpG stimulation. SPAg-loaded regulatory B cells prevented proliferation of effector CD4+ T cells and induced proliferation of antigen-specific Treg in vitro.Autologous polyclonal B cells loaded with SPAg appear as an innovative platform to expand Treg ex vivo. This approach may improve the efficiency and costs of current procedures