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Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 151-168). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Abstract Food hypersensitivities are becoming increasingly common worldwide. Previous studies indicate that cell mediated immunity has a role in delayed paediatric gastrointestinal food hypersensitivities, but the exact pathogenetic mechanisms are unknown. Cytotoxic activation of T-lymphocytes is known to play an important role in the pathogenesis of celiac disease (CD). The pathogenetic mechanisms of cow's milk protein sensitive enteropathy (CMSE) are largely unknown. CMSE is a non-IgE related type of food hypersensitivity with variable gastrointestinal symptoms but no visible mucosal abnormalities on light microscopy. The diagnosis is based on an open or blinded elimination/challenge test, as the endoscopic, histological and laboratory findings are generally non-specific. This thesis aims to characterize the role of lymphocyte cytotoxicity in the pathogenesis and diagnosis of CMSE in preschool and school aged children, including comparison with CD where the pathogenetic significance of cytotoxicity is well established. The study cohort consisted of 151 children, including 57 with untreated CMSE, 18 with treated CMSE, 24 with CD, and 52 controls. Using immunohistochemistry, the mucosal expressions of cytotoxic T cell-restricted intracellular antigen type 1 (TIA-1), perforin, granzyme A and B were analysed in the duodenal bulb and descending duodenum. Intraepithelial T-lymphocytes were labelled with CD3, alpha/beta and gamma/delta T cell receptor antigens. To determine the rates of overall and epithelial apoptosis as well as proliferation, the immunohistochemical TUNEL technique, M30 and Ki-67 antibodies were used. Serum levels of granzymes, CD30 and soluble Fas were studied using ELISA method. The number of intraepithelial lymphocytes with TIA-1, perforin and granzyme A containing granules was increased in CMSE. This increase was related to antigen challenge and not a constitutional abnormality. The cytotoxic reaction in CMSE differed from that in CD by being of lesser magnitude, concerning predominantly the descending duodenum and not showing signs of cytotoxicity related epithelial destruction. The serum levels of GrA, GrB and CD30 were increased in both CMSE and CD, correlating with the number of duodenal CD3+, alpha/beta and gamma/delta+ intraepithelial lymphocytes. The results strongly support the role of cell-mediated immunity in the pathogenesis of CMSE. Mucosal cytotoxic activation seems to be manifested by the release of cytoxicity related proteins in serum. This provides a new approach to the monitoring of intestinal immune activation which could help in diagnosis and in objectively monitored treatment response.

Modelos murinos de infecção revelaram que as células T CD8+ (LTCs) são essenciais no controle de Trypanosoma cruzi, agente etiológico da Doença de Chagas. No entanto, durante a doença, os LTCs são notavelmente alterados e sub-ótimos, estabelecendo a fase crônica. Nos últimos anos, vários trabalhos sob a Doença de Chagas visam melhorar o desempenho dos LTCs. Apesar que os mecanismos citotóxicos, como perforina/granzima e interações Fas/FasL, pelos LTCs são bem caraterizados, a regulação de ambos não são completamente entendidos. Porém, nós investigamos se alguns parâmetros podem estar envolvidos na regulação in vivo das respostas efetoras dos LTCs pela vacina terapêutica de Trypanosoma cruzi baseado em Adenovírus humano tipo 5. Nós encontramos que a carga viral e o tempo após imunização têm um impacto na comutação do fenótipo citotóxico dos LTCs. Nossa descoberta evidencia os papeis não redundantes da perforina e FasL e a importância destes parâmetros na citotoxicidade FasL-independente, eventos envolvidos na busca uma efetiva vacina contra a Doença de Chagas. / Models of infection revealed that CD8+ T cells (CTLs) are essential for control of Trypanosoma cruzi, etiologic agent of Chagas disease. However, during disease, the CTLs are remarkably delayed and suboptimal establishing the chronic phase. In the latest years, several works about Chagas disease aim to enhance the performance of CTLs. Although the effector mechanisms of target cells elimination as perforin/granzymes and Fas/FasL interactions by CTLs are well characterized, the regulation of both are non-completed understood. Here, we investigated if some parameters can be involved in the in vivo regulation of CTL effector responses by a human type 5 Adenovirus-based Trypanosoma cruzi therapeutic vaccine. We found that, the viral load and time post-immunization have an impact in the CTL switching killing phenotype. Our findings shows the evidence of the nonredundant roles of the perforin and FasL and the importance of those parameters in the FasL-independent cytotoxicity, events related with the searching for an effective vaccine against Chagas disease.

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-18T21:46:22Z No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) / Made available in DSpace on 2012-07-18T21:46:22Z (GMT). No. of bitstreams: 1 Fernanda Oliveira Novais Avaliacao do papel....pdf: 2271339 bytes, checksum: eee5aa084065678cf55651296f356919 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Linfócitos T CD8+ são células do sistema imune adaptativo capazes de induzir a morte de células infectadas através de mecanismos citotóxicos. No modelo de infecção intradérmica por Leishmania revelou-se que os linfócitos T CD8+ são responsáveis tanto pela indução de patogênese bem como pela imunidade contra a infecção primária por L. major. Até o momento, o papel dos linfócitos T CD8+ não foi estudado na infecção experimental por L. braziliensis. Neste estudo, investigamos o recrutamento dos linfócitos T CD8+ para o sítio de infecção e determinamos a sua função. Cinco semanas após a infecção intradérmica por L. braziliensis, camundongos BALB/c apresentaram um aumento na porcentagem de linfócitos T CD8+ presentes na orelha infectada e estes produziram, principalmente, IFN-g e Granzima B. Já no linfonodo de drenagem, estas células não produzem granzima mas, sim, IFN-g e TNF-a. Utilizando o mesmo modelo de infecção, camundongos BALB/c ou C57BL/6 depletados de linfócitos T CD8+ ou camundongos deficientes em b2-microglobulina ou em CD8 apresentaram redução no tamanho da lesão ao longo da infecção e menor carga parasitária cinco semanas após a infecção. A depleção de linfócitos T CD8+ não induziu qualquer alteração no recrutamento e produção de IFN-g, TNF-a e IL-10 pelos linfócitos T CD4+ no sítio de infecção ou no linfonodo de drenagem. Além disso, a capacidade proliferativa ou a produção de citocinas específicas in vitro após estímulo com células dendríticas infectadas por L. braziliensis não sofreram alteração. A ausência de linfócitos T CD8+ após a 8 infecção por L. braziliensis também não alterou o recrutamento de monócitos inflamatórios nem a sua diferenciação em células dendríticas. Por último, a análise histológica e de citometria de fluxo mostrou aumento no recrutamento de neutrófilos para o sítio de infecção e este resultado pode ser correlacionado com o controle da doença. Para confirmar o envolvimento dos linfócitos T CD8+ no desenvolvimento de lesão por L. braziliensis, transferimos linfócitos T CD8+ de camundongos naïve ou imunes, bem como linfócitos T CD4+ somente ou linfócitos T CD8+ e T CD4+ para camundongos RAG-/-. Neste contexto, a transferência de linfócitos T CD8+ naïve ou imunes induziu uma intensa patologia no sítio de infecção bem como a disseminação de parasitas para outros sítios, como a orelha não infectada, pata e nariz. Camundongos RAG-/- controle e aqueles que receberam linfócitos T CD8+ naïve ou imunes apresentam a mesma quantidade de parasitas no sítio de infecção, embora o aspecto da lesão tenha sido muito diferente. A transferência de linfócitos T CD4+ foi capaz de controlar a carga parasitária nestes animais e o mesmo foi observado após transferência de linfócitos T CD4+ e T CD8+ em conjunto. Nestes animais não observamos lesões em outros sítios, indicando que os linfócitos T CD8+ contribuem para a disseminação dos parasitas. Por último, transferimos linfócitos T CD8+ provenientes de camundongos selvagens ou deficientes de IFN-g e perforina e observamos que, na ausência de perforina, a patologia e a disseminação parasitária são controladas. Portanto, este estudo sugere envolvimento dos linfócitos T CD8+ na patogênese induzida por L. braziliensis devido ao seu potencial citotóxico e, em paralelo, inibindo o recrutamento de neutrófilos para o sítio de infecção. / CD8+ T lymphocytes are part of the adaptive immune system and are considered cytotoxic because of their ability to induce death in infected cells. Using the intradermal model of Leishmania infection, it has been shown that CD8+ T cells play an essential role in both pathogenesis and immunity to primary infection with L. major in the skin. So far, the role of these lymphocytes in the experimental model of infection using L. braziliensis has not been evaluated. In this study we determined the recruitment and function of these cells upon infection with L. braziliensis. Five weeks after infection, the frequency of CD8+ T cells was increased in the dermal site and these cells produced mainly IFN-g and granzyme B in infected mice. In the draining lymph nodes, these cells produced high levels of IFN-g and TNF-a, but not granzyme B. Using the same intradermal model of infection, we analysed the outcome of infection in the absence of CD8+ T lymphocytes using both antibody depletion in BALB/c and C57BL/6 mice and mice deficient in b2- microglobulin and CD8. In all groups, the absence of CD8+ T cells was correlated with better control of lesion development and parasite load in both depleted BALB/c and in b2-microglobulin deficient mice. In the absence of CD8+ T cells, CD4+ T lymphocytes were recruited to the same extension and produced same levels of IFN-g, TNF-a and IL-10 both in the infected ear and in draining lymph nodes when compared to infected mice that were not depleted. Also, there was no change in the proliferative potential and in IFN-g production by these cells after re-stimulation with infected dendritic cells. Analysis of inflammatory monocyte recruitment and differentiation of these cells into dendritic cells were similar in both depleted and non-depleted mice. On the other hand, histological and flow cytometric analyses showed increased neutrophil recruitment to the site of infection and this can be correlated with disease control. To confirm the role of CD8+ T cells in the lesion development of L. braziliensis infected mice, we then transferred CD8+ T cells from naïve or immune mice, as well as CD4+ T cells alone or together with T CD8+ to RAG deficient mice. The transfer of CD8+ T cells from immune or naïve mice into RAG recipients induced an intense pathology upon infection with L. braziliensis in the infection site, but also in uninfected tissues such as the uninfected ear, nose and footpad. Evaluation of parasite numbers in the infected ear showed that RAG deficient mice without T cells and those transferred with CD8+ T cells from naïve or immune mice had similar number of parasites although the pathology was very different. The transfer of CD4+ T cells alone or in association with CD8+ T cells induced parasite control in the infection site. In these mice, we could not detect lesions in other sites and we concluded that the transfer of CD8+ T cells alone induces parasite dissemination in RAG deficient mice. Finally, the transfer of CD8+ T cells from perforin deficient mice led to control in lesion development and in parasite dissemination. In this study we can conclude that CD8+ T cells are involved in the pathogenesis of L. braziliensis due to their cytotoxic potential and by inhibiting neutrophil recruitment to the infection site.

Les cellules dendritiques plasmacytoïdes (pDC) sont spécialisées dans la lutte antivirale, notamment grâce à leur capacité à sécréter des IFN de type I. Néanmoins, elles sont aussi impliquées dans Pactivation des réponses immunitaires adaptatives, et des lymphocytes T (LT) en particulier. C'est pourquoi, lors d'épisodes inflammatoires chroniques ou incontrôlés, les pDC sont à l'origine de l'initiation ou du maintien de syndromes inflammatoires et du développement de pathologies auto-immunes. Il doit donc exister des mécanismes permettant de contrôler l'activité de ces cellules. À l'aide d'un modèle in vivo d'inflammation non virale induite par l'injection d'un anticorps anti-CD3 (Ac aCD3), nous avons observé une apoptose des pDC dans différents organes lymphoïdes, et ce de façon dépendante de l'activation des lymphocytes T. De plus, nous avons pu observer que la diminution de la survie des pDC dans ce contexte inflammatoire n'était pas associée à l'orage cytokinique induit par l'efièt mitogénique de l'Ac aCD3. En revanche nos résultats montrent que les LT CD8* et la voie cytotoxique de la perforine dans ce contexte inflammatoire aigu sont responsables de la déplétion des pDC. Nous avons également étendu ces résultats à d'autres situations inflammatoires stériles comme lors de la maladie du greffon contre l'hôte. Ces données suggèrent que cette voie de régulation pourrait être utilisée à des fins thérapeutiques, afin de contrôler la survie des pDC impliquées dans la physiopathologie de syndromes auto-immuns comme le lupus érythémateux disséminé, le psoriasis, la sclérose en plaques ou encore le diabète de type I. / Plasmacytoid dendritic cells (pDC) are specialized in type I interferons (IFN-I) secretion to control viral infections. However, these cells can also activate adaptive immune responses, and polarize T cells. Indeed, during chronic or uncontrolled inflammatory episodes, pDC can induce or maintain inflammatory syndromes and autoimmune diseases. So some mechanisms should exist to control the fonction of these cells. In an in vivo modcl of non viral inflammation induced by the injection a CD3-specific antibody (aCD3 Ab), we could observed pDC's apoptosis dependent of T cell activation in different lymphoid organs. Moreover, we could observe that this depletion of pDC was not associated with the cytokinic storm induced by the mitogenic effect after aCD3 Ab treatment. On the other hand our data shovved that CD8+ T cells and the perforin pathway in this acute inflammatory context are responsible for pDC depletion We also obtained the same results in other non viral inflammation settings such as graft versus host disease. Overall, these data suggesi that this regulation pathway could be used for therapeutic purposes, to control pDC survival and avoid their involvement in the physiopathology of autoimmune disorders like systemic lupus erythematosus, psoriasis, multiple sclerosis or type I diabetes.

Orientador: Maria Letícia Cintra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T06:34:44Z (GMT). No. of bitstreams: 1 Lage_Denise_D.pdf: 12121159 bytes, checksum: f93ebe72b9dc8acf40f7ff2245de6a21 (MD5) Previous issue date: 2014 / Resumo: O líquen plano (LP) pode afetar a pele e/ou as mucosas. Histologicamente apresenta infiltrado linfo-histiocitário na junção epitélio-tecido conjuntivo e apoptose de células epiteliais basais. No LP oral (LPO), ocorre erosão frequente pela maior intensidade da necrose. O LP cutâneo (LPC) e o LPO apresentam características histopatológicas similares, mas o curso clínico é diverso. O LPC costuma ter seu curso limitado, enquanto o LPO é frequentemente recidivante. A erupção liquenoide a droga (ELD) desenvolve-se após semanas da ingestão do medicamento e a resolução do quadro é lenta após a interrupção, dificultando o diagnóstico diferencial com o LP idiopático. Os achados clínicos e histológicos podem ser indistinguíveis daqueles do LP, mas a patogênese da ELD não é conhecida. Diferenças locais no sistema imune da mucosa oral e pele poderiam explicar a diversidade no comportamento clínico do LP. Quanto à ELD, há poucas publicações sobre as alterações imunes que atuam no seu desenvolvimento. A citotoxidade celular é mediada, dentre outros mecanismos, por grânulos contendo granzima B e perforina, produzidos por linfócitos T citotóxicos e células natural killers. Com o objetivo de estudar a citotoxicidade celular na patogênese destas doenças, foram analisadas 29 amostras de LPO, 16 de LPC e 6 de ELD. Os cortes foram corados pela H&E e técnica de imuno-histoquímica, para a demonstração de linfócitos CD4 e CD8, macrófagos HAM 56+ e MAC 387+, granzima B, perforina e ICAM-1. As amostras de LPO apresentaram maior densidade de células granzima B+ e perforina+, em comparação às do LPC. Nos dois grupos de doenças, quanto maior era o número de células perforina+, maior era o de células granzima B+. Maior número de células CD4-positivas foi encontrado nas lesões ativas, quando comparado com o das regressivas, no LPO, mas não no LPC. À comparação entre o LPC e a ELD, quanto maior o número de células CD8-positivas, maior era o número de células que expressavam a perforina no grupo LPC. Quanto maiores eram os valores da granzima B, maiores os da perforina, no grupo LPC. Quanto maiores eram os valores da granzima B, maiores os de células apoptóticas agregadas, no grupo da ELD. Nas amostras do LPC, quanto maiores os valores das células T, maiores os dos macrófagos HAM56-positivos e vice-versa. Nas amostras da ELD, foi encontrada correlação negativa entre o número de células T e o de histiocitos jovens (MAC 387+). Havia correlação positiva entre o número de células T e o de células CD8, no grupo da ELD. O mesmo não ocorreu, no grupo do LPC. Concluindo, a expressão aumentada dos grânulos citotóxicos no LPO pode estar associada à maior gravidade da doença na mucosa. Os resultados favorecem um papel mais importante da granzima B e linfócitos TCD8+, no mecanismo patogenético da ELD, comparativamente com o da perforina, de maior importância no LPC. É possível que a ação da granzima B esteja ligada ao número abundante de clusters encontrado na ELD. Embora o LPC e a ELD apresentem semelhanças clínicas e histológicas, a etiopatogênese parece ser distinta / Abstract: Lichen planus (LP) can affect the skin and/or mucous membranes. Histologically it presents lymphohistiocytic infiltrate in the epithelium-connective tissue junction and apoptosis of basal epithelial cells. In oral LP (OLP), erosion occurs frequently by higher intensity of necrosis. Cutaneous LP (CLP) and OLP present similar histopathological features, but the clinical course is diverse. Spontaneous remission is common in CLP, but OLP follows a prolonged course, with periods of remission and relapse. Lichenoid drug eruption (LDE) develops after weeks of drug intake and the resolution of lesions is slow after drug discontinuation, hampering the differential diagnosis with (idiopathic) LP. Clinical and histological findings of LDE may be indistinguishable from those of LP, but LDE pathogenesis is poorly understood Local differences in the immune system of the skin and oral mucosa could explain the diversity in the clinical behavior of CLP and OLP. Regarding LDE, there are few publications on the immune changes that act in its development. Cellular cytotoxicity is mediated, among other mechanisms, by granules containing perforin and granzyme B, produced by cytotoxic T lymphocytes and NK cells. In order to study cellular cytotoxicity in the pathogenesis of these diseases, we analyzed 29 samples of OLP, 16 of CLP and 6 of LDE. The sections were stained for H&E and immunohistochemically targeted with CD4, CD8, HAM 56, MAC387, granzyme B, perforin and ICAM-1. OLP specimens exhibited higher density of cytotoxic granules (perforin and granzyme B) when compared with CLP. In both groups of diseases, the greater the number of perforin+ cells, the greater was the number of granzyme B+ cells. Increased number of CD4+ cells was found in active lesions as compared with the regressive ones in OLP but not in the CLP. The comparison between CLP and LDE revealed that the greater the number of CD8+ cells, the greater the number of cells expressing perforin in CLP group. The higher were the values of granzyme B, the higher the perforin values in the CLP group; the higher were the values of granzyme B, the higher the number of clusters of apoptotic cells in the LDE group. Within CLP group, the higher were the values of T cells, the greater the number of HAM56+ macrophages and vice versa. In LDE samples, negative correlation was found between the number of T cells and young histiocytes (MAC 387+). There was a positive correlation between the number of T cells and CD8 cells in LDE group, but not in CLP group. Concluding, increased expression of cytotoxic granules in OLP may be associated with greater mucosa severity. The results favor a greater role of granzyme B and CD8+ lymphocytes in the pathogenic mechanism of LDE, when compared with perforin, of greater importance in CLP. It is possible that the action of granzyme B is connected to the abundant number of clusters found in LDE. Although CLP and LDE present clinical and histological similarities, the etiopathogenesis appears to be distinct / Doutorado / Anatomia Patologica / Doutora em Ciências Médicas

Toxoplasma gondii, an obligate intracellular protozoan parasite, establishes a latent, chronic infection by forming cysts preferentially in the brain after replication of tachyzoites in various organs during the acute stage of infection. Chronic infection with T. gondii is one of the most common parasitic diseases in humans. The immune system is required for maintaining the latency of chronic infection. Reactivation of infection can occur in immunocompromised individuals, such as AIDS patients, which results in the development of life-threatening toxoplasmic encephalitis (TE). IFN-gamma-dependent, cell mediated immune responses play an essential role in preventing the reactivation of chronic infection of T. gondii in the brain. In my dissertation study, we examined the mechanisms of IFN-gamma-mediated prevention of TE by using models of reactivation of chronic infection in BALB/c mice. This strain of mouse is genetically resistant to T. gondii infection and establishes a latent chronic infection as do immunocompetent humans, and therefore provides an excellet model for this purpose. Our laboratory previously demonstrated that both T cells and IFN-gamma-producing non-T cells are required for genetic resistance of BALB/c mice against development of TE. However, the function of T cells required for the resistance is still unclear. Therefore, in the present study, we examined whether IFN-gamma production or perforin-mediated cytotoxicity of T cells play an important role in their protective activity against TE. Immune T cells were obtained from infected IFN-gamma-knockout (IFN-g-/-), perforin-knockout (PO), and wild-type (WT) BALB/c mice, and transferred into infected, sulfadiazine-treated athymic nude mice which lack T cells but have IFN-gamma-producing non-T cells. Control nude mice that had not received any T cells developed severe TE due to reactivation of infection and died after discontinuation of sulfadiazine treatment. Animals that had received immune T cells from either PO or WT mice did not develop TE and survived. In contrast, nude mice that had received immune T cells from IFN-gamma-/- mice developed severe TE and died as early as control nude mice. T cells obtained from spleens of the animals that had received either PO or WT T cells both produced large amounts of IFN-gamma following stimulation with T. gondii antigens in vitro. In addition, the amounts of IFN-gamma mRNA expressed in the brains of PO T-cell recipients did not differ from those of WT T-cell recipients. These results indicate that IFN-gamma production, but not perforin-mediated cytotoxic activity, by T cells is required for prevention of TE in genetically resistant BALB/c mice. In our attempt to identify a T cell population(s) that produces IFN-gamma in the brain and plays an important role for prevention of TE, we analyzed T cell receptor (TCR) Vb chain usage in T cells expressing IFN-gamma in the brains of infected BALB/c mice. We found T cells bearing TCR V beta8 chain to be the most frequent IFN-g-producing population in the brains of infected animals. To examine the role of IFN-gamma production by this T cell population for prevention of TE, V beta8+ immune T cells purified from spleens of infected BALB/c and IFN-g-/- mice were transferred into infected, sulfadiazine-treated athymic nude mice. After discontinuation of sulfadiazine treatment, control nude mice that had not received any T cells and animals that had received Vb8+ T cells from IFN-g-/- mice all died due to reactivation of infection (TE). In contrast, animals that had received the cells from WT mice survived. These results indicate that IFN-gamma production by Vb8+ T cells in the absence of any other T cell population can prevent reactivation of infection. Thus, V beta8+ T cells play a crucial role in genetic resistance of BALB/c mice to TE through their production of IFN-gamma. When V beta8+ immune T cells were divided into CD4+ and CD8+ subsets, a potent protective activity was observed only in the CD8+ subset whereas a combination of both subsets provided greater protection than did the CD8+Vb8+ population alone. These results indicate that CD8+ subset of V beta8+ T cells is a major afferent limb of IFN-gamma-mediated resistance of BALB/c mice against TE, although the CD4+ subset of the T cell population works additively or synergistically with the CD8+V beta8+ population. T cells need to enter into the brains of infected mice to demonstrate their protective activity against TE. This migration is mediated, in part, by endothelial adhesion molecules. Since IFN-gamma is essential for preventing reactivation of chronic infection with this parasite in the brain, we examined whether this cytokine plays an important role in expression of lymphocyte and endothelial adhesion molecules and recruitment of T cells into the brain during chronic infection with T. gondii using IFN-g-/- and WT BALB/c mice. Although the number of cerebral vessels expressing intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) increased in both WT and IFN-g-/- mice following infection, there were more VCAM-1+ vessels in brains of infected WT than infected IFN-g-/- mice; in contrast, numbers of ICAM-1+ vessels did not differ between strains. We did not detect endothelial E-selectin, P-selectin, MAdCAM-1 or PNAd in any of the brains. Significantly fewer CD8+ T cells were recruited into brains of infected IFN-g-/- than WT mice. Treatment of infected IFN-g-/- mice with recombinant IFN-gamma restored the expression of VCAM-1 on their cerebral vessels and recruitment of CD8+ T cells into their brains, confirming an importance of this cytokine for up-regulation of VCAM-1 expression and CD8+ T cell trafficking. In infected WT and IFN-g-/- animals, almost all cerebral CD8+ T cells had an effector/memory phenotype (LFA-1high, CD44high and CD62Lneg) and approximately 38% were positive for a4b1 integrin (the ligand for VCAM-1). In adoptive transfer of immune spleen cells, pre-treatment of the cells with a monoclonal antibody against a4 integrin markedly inhibited recruitment of CD8+ T cells into the brain of chronically infected wild-type mice. These results indicate that IFN-g-induced expression of endothelial VCAM-1 and its binding to a4b1 integrin on CD8+ T cells is important for recruitment of the T cells into the brain during the chronic stage of T. gondii infection. Since we found strong expression of ICAM-1 on endothelia and LFA-1 on T cells in the brains of infected mice, LFA-1/ICAM-1 interaction, in addition to a4b1 integrin/VCAM-1 interaction, may also be involved in this process. As mentioned earlier, CD8+ T cells are crucial for prevention of TE in BALB/c mice. Therefore, IFN-gamma-mediated expression of VCAM-1 and its binding to a4b1 integrin for recruitment of CD8+ T cells may play a critical role in genetic resistance of BALB/c mice to development of TE. / Ph. D.