Genotoxic effects in occupational exposure to formaldehyde: A study in anatomy and pathology laboratories and formaldehyde-resins production

ABSTRACT – Background: According to the Report on Carcinogens, formaldehyde ranks 25th in the overall U.S. chemical production, with more than 5 million tons produced each year. Given its economic importance and widespread use, many people are exposed to formaldehyde environmentally and/or occupationally. Presently, the International Agency for Research on Cancer classifies formaldehyde as carcinogenic to humans (Group 1), based on sufficient evidence in humans and in experimental animals. Manyfold in vitro studies clearly indicated that formaldehyde can induce genotoxic effects in proliferating cultured mammalian cells. Furthermore, some in vivo studies have found changes in epithelial cells and in peripheral blood lymphocytes related to formaldehyde exposure. Methods: A study was carried out in Portugal, using 80 workers occupationally exposed to formaldehyde vapours: 30 workers from formaldehyde and formaldehyde-based resins production factory and 50 from 10 pathology and anatomy laboratories. A control group of 85 non-exposed subjects was considered. Exposure assessment was performed by applying simultaneously two techniques of air monitoring: NIOSH Method 2541 and Photo Ionization Detection equipment with simultaneously video recording. Evaluation of genotoxic effects was performed by application of micronucleus test in exfoliated epithelial cells from buccal mucosa and peripheral blood lymphocytes. Results: Time-weighted average concentrations not exceeded the reference value (0.75 ppm) in the two occupational settings studied. Ceiling concentrations, on the other hand, were higher than reference value (0.3 ppm) in both. The frequency of micronucleus in peripheral blood lymphocytes and in epithelial cells was significantly higher in both exposed groups than in the control group (p < 0.001). Moreover, the frequency of micronucleus in peripheral blood lymphocytes was significantly higher in the laboratories group than in the factory workers (p < 0.05). A moderate positive correlation was found between duration of occupational exposure to formaldehyde (years of exposure) and micronucleus frequency in peripheral blood lymphocytes (r = 0.401; p < 0.001) and in epithelial cells (r = 0.209; p < 0.01). Conclusions: The population studied is exposed to high peak concentrations of formaldehyde with a long-term exposure. These two aspects, cumulatively, can be the cause of the observed genotoxic endpoint effects. The association of these cytogenetic effects with formaldehyde exposure gives important information to risk assessment process and may also be used to assess health risks for exposed worker

Effect of sialic acid loss on dendritic cell maturation

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.

T cell Maturation and Regulatory T Cell Differentiation:From the Thymus to the Periphery

Immunological tolerance, that is, the failure to mount an immune response to an otherwise immunogenic molecule, is one of the fundamental questions in immunology. The fact that lymphocytes express antigen receptors that are generated randomly and have the potential to recognize any conceivable antigen, adds another puzzle to the physiology of immunological tolerance. The other side of the coin, the general absence of immune responses to self antigens, is ensured by a tight regulation and several selection steps during T and B cell differentiation. One of these processes is the differentiation of regulatory T cells (Treg). While developing in the thymus, T cell clones bearing receptors with high affinity/avidity to antigens present at the time of differentiation may be eliminated by apoptosis or, alternatively, express Foxp3 and become Treg. Treg are key players in the regulation of immunological tolerance since humans and mice with complete loss of function variants of this gene develop fatal autoimmune conditions early in life.(...)

The role of α2,3- and α2,6-sialylation

RESUMO: Os glicoconjugados que decoram a superfície celular e os lípidos e proteínas secretados ocupam o ponto de encontro onde normalmente ocorrem interacções críticas homólogas (hospedeiro-hospedeiro) e heterólogas (hospedeiro-patogénio). Apesar de ser largamente aceite que os glicanos são parte integrante do processo de imunidade, continua a não ser claro qual o papel que os glicanos, em toda a sua diversidade, tomam no quadro geral da imunidade. Os glicanos, que são frequentemente terminados por resíduos de ácido siálico, podem ser alterados por factores externos, tais como patogénios, ou por acontecimentos fisiológicos celulares específicos. Normalmente em posição terminal, as glico-estruturas que contêm ácido siálico assumem um papel fundamental numa quantidade substancial de receptores imunes envolvidos na adesividade e tráfico celular, tal como as Selectinas e as Siglecs, das quais se sabe apresentarem uma relevante função imune. À altura do início desta tese, era sabido que os ácidos siálicos expressos à superfície das células poderiam modular mecanismos importantes nas respostas imunes adaptativas. Considerando a posição de charneira que as células dendríticas (DCs) ocupam na transição da resposta imune inata para a adaptativa, antecipámos que os ácidos siálicos poderiam também modular mecanismos relevantes nas DCs humanas. As DCs têm uma função muito relevante na verificação e captura antigénica, migração para os gânglios linfáticos e apresentação antigénica aos linfócitos, uma sequência de funções que conduz, em ultima instância, à indução da resposta inata adaptativa. Considerando estas premissas, a nossa hipótese principal foi que os ácidos siálicos podem influenciar funções relevantes das DCs, tais como captura de antigénios, maturação, migração para os gânglios linfáticos e apresentação antigénica às células Para testar esta hipótese, dividimos o trabalho em quatro partes: 1) Analisámos os glicanos sialilados de superfície, expressos durante a diferenciação de monócitos humanos em DCs (moDCs). Os nossos dados mostraram que a expressão dos glicanos com ligações em O (O-glicanos) e sialilados em α2,3, assim como glicanos com ligações em N (N-glicanos) sialilados em α2,6 e α2,3 aumentou durante o processo de diferenciação das moDCs. Contribuindo para esta nova configuração glicosídica, três sialiltransferases (STs) poderão estar envolvidas: a ST6Gal-1 correlaciona-se com a expressão aumentada de N-glicanos sialilados em α2,6; a ST3Gal-1 contribui para a sialilação em α2,3 de O-glicanos, em especial de antigénios T; e a ST3Gal-4 poderá ser responsável pelo aumento de N-glicanos sialilados em α2,3. Após estímulo e consequente maturação das moDCs, ambos os níveis de expressão génica de ST6Gal-1 e ST3Gal-4 são negativamente modificados sendo, também, que a expressão de ST3Gal-1 varia consoante o estímulo. 2) Estudámos posteriormente as consequências da modulação dos ácidos siálicos de superfície nas funções das DCs. Observámos que a remoção dos ácidos siálicos de superfície diminui significativamente a capacidade de macropinocitose e endocitose mediada por receptores nas moDCs. Em contrapartida, o tratamento com sialidase aumentou significativamente a capacidade das moDCs para fagocitar Escherichia coli. Determinou-se também que este mecanismo requer a existência de ácido siálico presente nas E. coli indicando um mecanismo de interacção hospedeiro-patogénio dependente de ácido siálico em ambas as partes envolvidas. As moDCs tratadas com sialidase também apresentam um nível superior de expressão de moléculas de MHC e moléculas co-estimulatórias, sugerindo um fenótipo celular mais maduro. Recorrendo ao modelo de ratinho, utilizaram-se DCs derivadas de células da medula (BMDCs) de ratinhos deficientes em ST3Gal-1 e ST6Gal-1. Estes ensaios revelaram que quer a endocitose quer a maturação são influenciadas por modificações 37 nos glicanos sialilados em α2,3 ou α2,6. A detecção e quantificação de proteínas Nglicosiladas e sialiladas em α2,6 apontou para um potencial envolvimento de integrinas β2 nestes mecanismos. 3) O efeito da sialilação em α2,6 na migração das DCs para os gânglios linfáticos foi também analisado. Observámos que BMDCs deficientes para ST6Gal-1 apresentam uma redução de cerca de 50% nos níveis de migração das DCs para os gânglios linfáticos, tal como aferido em ensaios de inflamação in situ e estudos de transferência adoptiva de células. Uma redução dos níveis deste tipo de migração foi também observada quando BMDCs nativas foram transferidas para ratinhos receptores deficientes em ST6Gal-1. São, contudo, necessários mais ensaios de forma a identificar as moléculas envolvidas neste processo. 4) Por último, analisámos o impacto da sialilação na estimulação antigénica das DCs às células T. Assim, concluiu-se que moDCs tratadas com sialidase apresentam um nível de expressão superior de IL-12, TNF-ɑ, IL-6 e IL-10, e activação do factor de transcrição nuclear kappa B (NF-κB). As DCs tratadas com sialidase induziram uma maior proliferação nas células T, com expressão correspondente de interferão-γ. Este dado sugere que a remoção de ácidos siálicos de superfície contribui para o desenvolvimento de uma resposta pro-inflamatória do tipo 1 por células T auxiliares (resposta Th1). Considerando estes dados no seu todo, concluímos que o ácido siálico tem um papel marcante nas funções imunes das DCs. Alterações à concentração de ácido siálico à superfície das células podem alterar a endocitose/fagocitose, maturação, migração para os tecidos e gânglios linfáticos e capacidade estimulatória para com as células T. Complementando estes dados, as ligações glicosídicas de ácidos siálicos criados por ST6Gal-1 e ST3Gal-1 são funcionalmente relevantes. A modulação programada da sialilação do glicocálice, mediada por sialidases individuais ou sialiltransferases é uma possibilidade aceitável para a melhoria da fagocitose por DCs e da sua potência imunológica. Este facto tem um significado particular para imunoterapias baseadas em DCs, podendo provar-se decisivo para a sua eficiência e aplicabilidade num futuro muito próximo.-------------------------------ABSTRACT: Glycans decorating cell surface and secreted proteins and lipids occupy the junction where critical host–host and host-pathogen interactions occur. In spite of the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their variety and variability contribute to the overall immune response remains poorly defined. Glycans, frequently terminated by sialic acid residues, may be modified by external factors such as pathogens or upon specific physiological cellular events. The terminal, privileged positions of sialic acid-modified structures makes them key, fundamental determinants for a number of immune receptors with known involvement in cellular adhesiveness and cell trafficking, such as Selectins and Siglecs, with known relevant immune functions. At the time this thesis was initiated, it was established that sialic acids expressed at cell surface could modulate important mechanisms of the adaptive immune responses. Given the key role of dendritic cells (DCs) in the transition from innate to the adaptive immune responses, we anticipated that sialic acids could also modulate important mechanisms of human DCs. DCs have a relevant role in antigen screening and uptake, migration to lymph nodes and antigen presentation to lymphocytes, ultimately triggering the adaptive immune response. Therefore, our primary hypothesis was that sialic acids may modulate DC functions, such as antigen uptake, maturation, homing to lymph nodes and antigen presentation to T cells. To test this hypothesis, we divided our work in four parts. 1) Surface sialylated glycans expressed during differentiation from human monocytes to DCs (moDCs) were analyzed. Our data showed that α2,3-sialylated O-glycans and α2,6- and α2,3-sialylated N-glycans expression increased during moDC differentiation. Three main sialyltransferases (STs) are committed with this new glycan configuration: ST6Gal- 1 correlates with the increased expression of α2,6-sialylated N-glycans; ST3Gal-1 32 contributes for the α2,3-sialylation of O-glycans, especially T antigens; and ST3Gal-4 may contribute for the increased α2,3-sialylated N-glycans. Upon moDC maturation, ST6Gal-1 and ST3Gal-4 are downregulated and ST3Gal-1 is altered in a stimulus dependent manner. 2) We subsequently analyzed the consequences of the modulation of cell surface sialic acids in DC functions. We observed that removing surface sialic acid by sialidase significantly decreased the capacity of moDCs to micropinocytose and receptormediated endocytose. In contrast, treatment with a sialidase significantly improved the capacity of moDCs to phagocytose Escherichia coli. The improved phagocytosis mechanism required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. Sialidase-treated moDCs have increased expression of MHC and co-stimulatory molecules, suggesting a more mature phenotype. Experiments using mouse bone marrow-derived DCs (BMDCs) from ST3Gal-1-/- and ST6Gal-1-/- strains indicated that endocytosis and maturation are influenced by changes in either α2,3 or α2,6-sialylated glycans. The analysis of α2,6-sialylated, N-glycosylated proteins, strongly suggested the potential involvement of β2 integrins, underlying these mechanisms. 3) The effect of α2,6-sialylation in DC homing to lymph nodes was also analyzed. We observed that BMDCs deficient for ST6Gal-1 have an almost 50% reduction in DC homing, as assayed by in situ inflammation and adoptive transfer studies. A reduction in DC homing was also observed when wild type BMDCs were transferred into ST6Gal-1-/- recipient mice. Further investigations are necessary to identify the molecules involved in this process. 4) Finally, we also analyzed the impact of sialylation on DCs ability to prime T cells. Sialidase-treated moDCs show increased gene expression of IL-12, TNF-α, IL-6 and IL- 10 cytokines, and activation of the transcription factor nuclear factor-κB. Sialidase33 treated DCs induced a higher proliferative response of T cells with concomitant higher expression of interferon-γ, suggesting that the clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. Together, our data strongly support sialic acid’s relevance in DC immune functions. Alterations of cell surface sialic acid content can alter the endocytosis/phagocytosis, maturation, migration/homing and the ability for T cell priming in human DCs. Moreover, sialic acid linkages created by ST6Gal-1 and ST3Gal-1 are functionally relevant. The engineering of cell surface sialylation, mediated by individual sialidases or sialyltransferases is a likely possibility to fine tune DC phagocytosis and immunological potency, with particular significance to DC-based therapies.

Nanomateriais manufaturados : avaliação de segurança através da caracterização dos seus efeitos genéticos

RESUMO - Os nanomateriais manufaturados (NMs), isto é, fabricados deliberadamente para fins específicos, apresentam propriedades físico-químicas únicas como a dimensão, área superficial ou funcionalização, que lhes conferem caraterísticas mecânicas, óticas, elétricas e magnéticas muito vantajosas para aplicações industriais e biomédicas. Efetivamente, a tecnologia baseada nos NMs, ou nanotecnologia, foi identificada como uma key enabling technology, impulsionadora do crescimento económico dos países industrializados, devido ao seu potencial para melhorar a qualidade e desempenho de muitos tipos de produtos e de processos. Contudo, a expansão da utilização de NMs contrasta com a insuficiente avaliação de risco para a saúde humana e para o ambiente, sendo considerados como um risco emergente para a saúde pública. As incertezas sobre a segurança dos NMs para a saúde pública advêm sobretudo de estudos epidemiológicos em humanos expostos a nanomateriais produzidos como consequência dos processos e atividades humanas e da poluição. Uma das principais preocupações relativamente aos efeitos adversos dos NMs na saúde humana é o seu potencial efeito carcinogénico, que é sugerido por alguns estudos experimentais, como no caso dos nanomateriais de dióxido de titânio ou dos nanotubos de carbono. Para avaliar em curto termo as propriedades carcinogénicas de um composto, utilizam-se frequentemente ensaios de genotoxicidade em linhas celulares de mamífero ou ensaios em modelos animais, em que se analisa uma variedade de lesões genéticas potencialmente relacionados com o processo de carcinogénese. No entanto, a investigação sobre as propriedades genotóxicas dos NMs não foi, até hoje, conclusiva. O presente estudo tem por objectivo principal caracterizar os efeitos genotóxicos associados à exposição a nanomateriais manufaturados, de forma a contribuir para a avaliação da sua segurança. Constituíram objectivos específicos deste estudo: i) avaliar a genotoxicidade dos NMs em três tipos de células humanas expostas in vitro: linfócitos humanos primários, linha celular de epitélio brônquico humano (BEAS-2B) e linha celular de adenocarcinoma epitelial de pulmão humano (A549); ii) avaliar a sua genotoxicidade num modelo de ratinho transgénico; iii) investigar alguns mecanismos de acção que poderão contribuir para a genotoxicidade dos nanomateriais, como a contribuição de lesões oxidativas para a genotoxicidade induzida pelos NMs in vitro, e a investigação da sua bioacumulação e localização celular in vivo. Foram analisados os efeitos genotóxicos associados à exposição a duas classes de NMs, dióxido de titânio e nanotubos de carbono de parede múltipla, bem como a um NM de óxido de zinco, candidato a ser utlilizado como controlo positivo de dimensão nanométrica. Os xx NMs utilizados foram previamente caracterizados com detalhe relativamente às suas características físico-químicas e também relativamente à sua dispersão em meio aquoso e no meio de cultura. A metodologia incluiu ensaios de citotoxicidade e de genotoxicidade in vitro, designadamente, ensaios de quebras no DNA (ensaio do cometa) e nos cromossomas (ensaio do micronúcleo) em células humanas expostas a várias concentrações de NMs, por comparação com células não expostas. Também foram realizados ensaios in vivo de quebras no DNA, quebras cromossómicas e ainda um ensaio de mutações em vários órgãos de grupos de ratinhos transgénicos LacZ, expostos por via intravenosa a duas doses de dióxido de titânio. Foi investigada a existência de uma relação dose-resposta após exposição das células humanas ou dos animais a NMs. A contribuição de lesões oxidativas para a genotoxicidade após exposição das células aos NMs in vitro foi explorada através do ensaio do cometa modificado com enzima. Realizaram-se estudos histológicos e citológicos para deteção e localização celular dos NMs nos órgãos-alvo dos ratinhos expostos in vivo. Os resultados demonstraram efeitos genotóxicos em alguns dos NMs analisados em células humanas. No entanto, os efeitos genotóxicos, quando positivos, foram em níveis reduzidos, ainda que superiores aos valores dos controlos, e a sua reprodutibilidade era dependente do sistema experimental utilizado. Para outros NMs, a evidência de genotoxicidade revelou-se equívoca, conduzindo à necessidade de esclarecimento através de ensaios in vivo. Para esse fim, recorreu-se a uma análise integrada de múltiplos parâmetros num modelo animal, o ratinho transgénico baseado em plasmídeo contendo o gene LacZ exposto a um NM de dióxido de titânio, NM-102. Embora tenha sido demonstrada a exposição e a acumulação do NM no fígado, não se observaram efeitos genotóxicos nem no fígado, nem no baço nem no sangue dos ratinhos expostos a esse NM. Neste estudo concluiu-se que algumas formas de dióxido de titânio e nanotubos de carbono de parede múltipla produzem efeitos genotóxicos em células humanas, contribuindo para o conjunto de evidências sobre o efeito genotóxico desses NMs. As diferenças observadas relativamente à genotoxicidade entre NMs do mesmo tipo, mas distintos em algumas das suas características físico-quimicas, aparentemente não são negligenciáveis, pelo que os resultados obtidos para um NM não devem ser generalizados ao grupo correspondente. Para além disso, a genotoxicidade equívoca verificada para o NM-102 em células humanas expostas in vitro, não foi confirmada no modelo in vivo, pelo que o valor preditivo da utilização dos ensaios in vitro para a identificação de NMs com efeitos genotóxicos (e portanto potencialmente carcinogénicos) ainda tem de ser esclarecido antes de ser possível extrapolar as conclusões para a saúde humana. Por sua vez, como a informação aqui produzida pelas metodologias in vitro e in vivo não reflete os efeitos de exposição continua ou prolongada, que poderá conduzir a efeitos genotóxicos distintos, esta xxi deverá ser complementada com outras linhas de evidência relativamente à segurança dos NMs. Perante a incerteza dos níveis de exposição real do organismo humano e do ambiente, a segurança da utilização dos NMs não pode ser garantida a longo prazo e, tendo em conta a elevada produção e utilização destes NMs, são prementes futuros estudos de monitorização ambiental e humana.

T-cell receptor V beta use predicts reactivity and tolerance to Mlsa-encoded antigens.

T lymphocytes reactive with the product of the Mlsa-allele of the minor lymphocyte stimulating (Mls) locus use a predominant T-cell receptor beta-chain variable gene segment (V beta 6). Such V beta 6-bearing T cells are selectively eliminated in the thymus of Mlsa-bearing mice, consistent with a model in which tolerance to self antigens is achieved by clonal deletion.

Constitutive activation of Wnt signaling favors generation of memory CD8 T cells.

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

Role of cytokines in secondary lymphoid organ development

Summary Secondary lymphoid organs are sites of antigen presentation, clonal expansion of B and lymphocytes, and affinity maturation of B lymphocytes. In the intestine, these immune functions occur mainly in Peyer's patches (PP). PP develop through the interplay of two main cell types, haematopoietic cells and meserichyrnal cells. One particular haematopoietic cell type was identified as the inductive cell type in the formation of both PP and lymph nodes and was therefore designated as lymphoid tissue inducer cell. For a successful PP organogenesis, the crucial molecular components involved in the crosstalk of inducer cells and their mesenchymal target cells are adhesion molecules, lymphotoxin (LT) family members, and cytokines. In particular, the interleukin 7 receptor (IL-7R) expressed on inducer cells is absolutely required. To investigate the contribution of the ligand for the IL-7R. the cytokine IL-7, in the process of PP formation, we analyzed double transgenic (TG) mice. These mice resulted from an interbreeding of an IL-7TG mouse strain where the transgene is under the control of the MHC class II promoter with a second transgenic mouse strain, which overexpresses a transactivator for MHC class II genes. Double TG offsprings revealed higher levels of IL-7 mRNA occuring earlier in embryogenesis. Consequently, double TG mice showed a striking phenotype with a 3- to 5-fold increase in PP numbers compared to single IL-7TG or control littermates. Analysis of embryonic double TG intestines demonstrated that the process of PP development was already elevated during development as early as the embryonic day 16.5. Importantly, inducer cells were significantly increased in numbers in these embryonic intestines. Furthermore, the expression of LT? mRNA, which at this early time point is exclusively expressed by inducer cells, was also increased in double TG animals. These data clearly indicate a direct influence of IL-7 on the expansion of lymphoid tissue inducer cells and on the availability of LT? leading to a higher frequency of developing PP in fetal life. Interestingly, in addition to an enhanced frequency of PP development, in double TG mice, three additional phenotypic differences were observed. i) Lymphocyte infiltration in various non-lymphoid organs, such as stomach, salivary gland, and liver. Subsequent analysis demonstrated that B lymphocytes were predominant within these tertiary lymphoid structures. ii) Ectopic lymph node-like structures containing both B and T lymphocytes were found near the inguinal lymph node. iii) Double TG mice had a severe bone resorption syndrome most likely as a consequence of the pro-osteoclastic effect of IL-7. Taken together, these results show that IL-7 plays a key role in the homeostasis of inducer cells, in the generation of PP in the gut, in the formation of ectopic lymphoid tissue, and in bone resorption. Résumé Les organes lymphoïdes secondaires sont les lieux de présentation des antigènes aux lymphocytes, permettant l'expansion des lymphocytes B et T et la maturation d'affinité des lymphocytes B. Dans l'intestin, ces fonctions immunitaires se déroulent dans les plaques de Peyer (PP). Ces plaques se développent grâce à l'interaction des cellules hématopoïétiques avec des cellules mésenchymales. Un type particulier de cellules hématopoïétiques a été identifié comme cellule inductrice dans la formation des PP et des ganglions lymphatiques et de ce fait a été désigné cellule inductrice des tissus lymphoïdes. Durant l'organogénèse des PP, les composants moléculaires cruciaux impliqués dans l'interaction des cellules inductrices et des cellules mésenchymales sont les molécules d'adhésion, les membres de la famille des lymphotoxines (LT) et les cytokines. En particulier, le récepteur de l'interleukine 7 (IL-7R) exprimé par les cellules inductrices est absolument nécessaire. Pour étudier le rôle du ligand de l'IL-7R, l'interleukine IL-7, dans la formation des PP, nous avons croisé une lignée de souris transgénique (TG) surexprimant IL-7 sous contrôle du promoteur MHC class Il avec une lignée de souris transgénique surexprimant un transactivateur des genes MHC class II. Les souris doubles TG présentent une concentration élevée d'ARNm de l'IL-7 durant l'embryogénèse, ce qui résulte en une augmentation du nombre de PP de 3 à 5 fois en comparaison aux souris ayant seul le transgène IL-7 et aux souris contrôles. L'analyse des intestins des souris doubles TG démontre que le processus de développement des PP était élevé dès le jour 16.5 du développement embryonnaire. L'augmentation du nombre des cellules inductrices dans ces intestins embryonnaires est signilicative. De plus l'expression de l'ARNm LT?, qui à ce stade précoce est exclusivement exprimé dans les cellules inductrices, est également augmenté dans les doubles TG. Ces résultats indiquent clairement une influence directe d'IL-7 sur l'expansion des cellules inductrices des tissues lymphoïdes et sur la synthèse de LT? induisant une augmentation des PP se développant durant la vie foetale. En plus du développement accru des PP dans les souris doubles TG, trois différences phénotypiques ont été observées. i) L'infiltration lymphocytaire dans différents organes non-lymphoïdes, comme l'estomac, les glandes salivaires et le foie. Des analyses complémentaires ont demontré que les lymphocytes B étaient prédominants dans ces structures lymphoïdes tertiaires. ii) Des structures de ganglions lymphatiques ectopiques contenant des lymphocytes B et T ont été trouvées près des ganglions lymphatiques inguinaux. iii) Les souris doubles TG présentent un syndrome de résorption osseuse sévère probablement dû à l'effet pro-osteoclaste d'IL-7. Globalement, ces résultats montrent que IL-7 joue un rôle clé dans l'homéostasie des cellules inductrices dans la génèse de PP de l'intestin, dans la formation des tissus lymphoïdes ectopiques et dans la résorption osseuse.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Notch1 and T-cell development: insights from conditional knockout mice.

Notch proteins influence cell-fate decisions in many developmental systems. Gain-of-function studies have suggested a crucial role for Notch1 signaling at several stages during lymphocyte development, including the B/T, alphabeta/gammadelta and CD4/CD8 lineage choices. Here, we critically re-evaluate these conclusions in the light of recent studies that describe inducible and tissue-specific targeting of the Notch1 gene.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

B and T lymphocyte attenuator regulates CD8+ T cell-intrinsic homeostasis and memory cell generation.

B and T lymphocyte attenuator (BTLA) is a negative regulator of T cell activation, but its function in vivo is not well characterized. Here we show that mice deficient in full-length BTLA or its ligand, herpesvirus entry mediator, had increased number of memory CD8(+) T cells. The memory CD8(+) T cell phenotype resulted from a T cell-intrinsic perturbation of the CD8(+) T cell pool. Naive BTLA-deficient CD8(+) T cells were more efficient than wild-type cells at generating memory in a competitive antigen-specific system. This effect was independent of the initial expansion of the responding antigen-specific T cell population. In addition, BTLA negatively regulated antigen-independent homeostatic expansion of CD4(+) and CD8(+) T cells. These results emphasize two central functions of BTLA in limiting T cell activity in vivo.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.