Immunophenotyping of circulating T cells in a mucosal leishmaniasis patient coinfected with HIV

HIV coinfection modifies the clinical course of leishmaniasis by promoting a Th2 pattern of cytokine production. However, little information is available regarding the lymphocytic response in untreated coinfected patients. This work presents the immunophenotyping of Leishmania-stimulated T cells from a treatment-na��ve HIV+ patient with ML. Leishmania braziliensis antigens induced CD69 expression on CD3+CD4+ and CD3+CD8+ cells. It also increased IL-4 intracellular staining on CD3+CD4+GATA3- population and decreased the percentage of CD3+CD4+IL-17+ cells. This suggests that modulations in the IL-4R/STAT6 pathway and the Th17 population may serve as parasitic evasion mechanisms in HIV/ML. Further studies are required to confirm these results.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ, under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Senescencia de celulas T humanas inducida por tumores: un nuevo mecanismo de evasi��n de la respuesta inmune. Senescence of human T cells iduced by tumors. A new mechanism of evasion of immune response.

La respuesta antitumoral en individuos con c��ncer depende, en gran medida, de c��lulas del sistema inmune capaces de reconocer y eliminar las c��lulas tumorales. Sin embargo, los tumores tienen la capacidad de evadir la respuesta inmune a trav��s de diversos mecanismos como por ejemplo inducir la muerte de c��lulas claves del sistema inmune [1-3]. Previamente nosotros demostramos mediante un modelo in vitro que dependiendo de las condiciones de interacci��n entre tumor y linfocitos (tiempo y relaci��n num��rica), los tumores pueden inducir apoptosis o senescencia de linfocitos T provenientes de donantes sanos. Nuestros resultados son los primeros en demostrar que c��lulas tumorales de diversos or��genes pueden inducir senescencia de c��lulas T a trav��s de factor/s solubles. Tambi��n demostramos que a diferencia con los que ocurre in vivo, tanto las c��lulas CD4 como las CD8 son susceptibles a adquirir fenotipo de senescencia. Estudiando las implicancias que puede tener la senescencia sobre la funcionalidad de la c��lula T, observamos que las c��lulas T CD4+ y CD8+ senescentes pueden suprimir una respuesta linfoproliferativa. Si bien las c��lulas CD8+CD28- han sido identificadas in vivo, nosotros demostramos que c��lulas CD4+ CD28- tiene capacidad supresora [4]. En base a estos resultados, nuestra hip��tesis es que las c��lulas T senescentes inducida por tumores pueden regular nuestro sistema de defensa actuando sobre la respuesta inmune adaptativa y posiblemente sobre la innata y, por consiguiente, postulamos que la senescencia de c��lulas T puede ser considerada como otro de los mecanismos de evasi��n de la respuesta inmunePlanteamos as�� los siguientes objetivos espec��ficos: -Evaluar como las c��lulas T senescentes inducidas por tumores pueden regular la respuesta inmune.-Evaluar la participaci��n de mediadores solubles capaces de regular la senescencia de c��lulas T inducida por tumores. En la actualidad existen estrategias inmuno-terap��uticas que avizoran resultados promisorios. Sin embargo, el control de c��lulas T inmunosupresoras permanece como unos de los grandes desaf��os. Nuestro proyecto proveer�� conocimientos sobre un fen��meno muy poco estudiado y por consiguiente muy poco valorado a la hora de dise��ar estrategias terap��uticas para la cura del c��ncer.

C��lulas dendr��ticas y su funcionamiento durante el envejecimiento. Efecto of aging on the function of dendritic cells.

La inmunosenescencia es definida como el estado de desregulaci��n de la funci��n inmune, que contribuye a la morbilidad y mortalidad debida a una mayor incidencia o reactivaci��n de enfermedades infecciosas y de fen��menos autoinmunes y c��ncer. Durante el envejecimiento hay un decaimiento de la funci��n del sistema inmune. Aunque est�� bien documentada la declinaci��n de la funci��n de las c��lulas T en individuos envejecidos, es escasa la informaci��n disponible acerca de c��mo el envejecimiento afecta a las c��lulas dendr��ticas (DCs) y en particular a su rol en la activaci��n de linfocitos T CD4+ y CD8+. Nuestra hip��tesis es que las c��lulas dendr��ticas juegan un rol importante en la desregulaci��n de la funci��n inmune observada durante el envejecimiento. Por ello, el Objetivo General de este proyecto es caracterizar el estado funcional de las c��lulas dendriticas en ratones envejecidos y su contribuci��n a las alteraciones del sistema inmune durante el envejecimiento. Para ello, estudiaremos la composici��n y estado de activaci��n de las DCs de los ��rganos linf��ticos y tejidos perif��ricos de ratones envejecidos, y su capacidad para ser activadas in vitro e in vivo por diferentes ligandos de los receptores tipo Toll (TLR). Adem��s, estudiaremos la capacidad in vitro, ex vivo e in vivo de las DCs de ratones envejecidas para capturar, procesar y presentar ant��genos a linfocitos T CD4+ y CD8+ y finalmente la capacidad de las DCs de ratones envejecidos para montar una respuesta mediada por linfocitos T CD4+ y CD8+. Para ello, luego de transferir DCs de ratones envejecidos cargadas con ant��geno a animales j��venes v��rgenes, evaluaremos la respuesta T CD4 y CD8 en los animales receptores frente a dicho ant��geno. Basado en nuestra trayectoria en la inmunogerontolog��a experimental, creemos que con este proyecto podremos obtener informaci��n que permitir�� abordar el estudio del efecto del envejecimiento sobre el sistema inmune desde una nueva perspectiva, para en un futuro poder extender el mismo estudio en seres humanos y as�� desarrollar modelos m��s eficientes de inmunoterapia en individuos envejecidos.

Modulaci��n de la activaci��n de c��lulas dendr��ticas por ant��genos de Fasciola hepatica y ligandos para receptores Toll: Potencial terap��utico en respuestas anti-inflamatorias. Fasciola hepatica antigens and TLR ligands modulate dendritic cells activation: therapeutic potential in anti-inflammatory responses.

Antecedentes: En nuestro laboratorio hemos demostrado que ant��genos (Ags) de Fasciola hepatica inducen en c��lulas dendr��ticas murinas (CD), diferentes propiedades tolerog��nicas como la incapacidad por si mismos de inducir la maduraci��n de las c��lulas, la resistencia a la maduraci��n por ligandos de TLR, el incremento en la producci��n de IDO y tambi��n la capacidad de esta estas c��lulas de dirigir la respuesta inmune hacia un perfil Th2 y T reg. Por otra parte ha sido bien documentado que CD con caracter��sticas tolerog��nicas, ya sea inmaduras o semimaduras, son ��tiles para reducir respuestas inflamatorias excesivas tales como las que ocurren en enfermedades autoinmunes. Adem��s hemos demostrado que CD tratadas con Ags del par��sito en conjunto con un ligando Toll (CpG-ODN) producen altos niveles de citoquinas anti-inflamatorias (IL-10 y TGF-���) bajos de citoquinas proinflamatorias (TNF, IL-6, IL-12). Hip��tesis: El fenotipo semimaduro alcanzado en las CDpodr��a ser utilizado para reducir la inflamaci��n en un modelo de enfermedad autoinmune en donde existe una exacerbada respuesta Th1 y Th17, ya que la producci��n elevada de IL-10 y TGF-��� podr��a inhibir o controlar estas respuestas de manera directa o a trav��s de la inducci��n de c��lulas T regulatorias. Objetivos: En este proyecto nosotros proponemos la inmunizaci��n de animales susceptibles (ratones DBA1/j), al desarrollo de artritis inducida por col��geno (AIC) con CD tratadas con Ags de F. hepatica en conjunto con CpG-ODN para reducir los s��ntomas cl��nicos de la enfermedad. Materiales a utilizar: En nuestro laboratorio hemos desarrollado un modelo de artritis inducida por col��geno (AIC) mediante dos inmunizaciones de ratones DBA1/j con col��geno tipo II bovino y adyuvante de Freund. El modelo permiti�� establecer un ��ndice cl��nico mediante la hinchaz��n en las patas de los animales. Doce d��as posteriores a la primera inmunizaci��n los animales ser��n inyectados con CD tratadas con: 1. PBS, 2.Extracto total de F.hepatica (TE) + CII, 3. CpG + CII, 4. TE+CpG+CII Se realizar�� la observaci��n macrosc��pica diaria, a partir de los 7 d��as de la 2a inmunizaci��n Luego del sacrificio las articulaciones de las patas se preparar��n para realizar un an��lisis histol��gico. Se detectar�� en suero los niveles de anticuerpos IgG1 (perfil Th2) y de IgG2a (perfil Th1) mediante la t��cnica de ELISA. Se detectar�� tambi��n el perfil de citoquinas en los n��dulos drenantes por la t��cnica de ELISA y adicionalmente la poblaci��nes celulares de c��lulas T regulatorias (Treg) CD4+CD25+Foxp3 o c��lulas Tr1. Resultados esperados: Pensamos que el tratamiento de los animales que desarrollan AIC con CD semimaduras (por el tratamiento con TE y CpG), ser��n capaces de migrar a los ��rganos linfaticos y secretar TGF-be(inductora de c��lulas T reg), IL-10 (inductoras de c��lulas Tr1), IDO inhibitoria de la respuesta de Li T y promotor de c��lulas T reg, tambi��n podr��a generarse una respuesta Th2 (por la presencia de ant��genos del par��sito), y estas respuestas aisladas o en forma sin��rgica podr��an inhibir las respuestas de tipo Th17 y Th1 asociadas a la patolog��a en esta enfermedad. Importancia del proyecto: En el desarrollo de la artritis existe un aumento de la inmunidad mediada por c��lulas, asi como de la respuesta inmune humoral hacia componentes de la matriz del cart��lago. El tratamiento convencional de la artritis recae en general en el uso de inmunosupresores no-espec��ficos, los cuales poseen una variedad de efectos adversos y la inhibici��n de la respuesta inflamatoria no es espec��fica. En este proyecto proponemos el uso de CD tratadas con ant��genos del helminto F. hepatica y CpG ligando Tol que capacita a estas c��lulas para generar una respuesta adaptativa de tipo regulatoria, ��til en la inhibici��n de las respuestas inflamatorias como la que ocurre durante la progresi��n de artritis reumatoidea en un modelo experimental en ratones. We have shown that F. hepatica Ags-treated dendritic cells (DC) together with a TLRl ligand (CpG-ODN) produce high levels of anti-inflammatory cytokines (IL-10 and TGF-Beta) and low of proinflammatory cytokines (TNF, IL-6, IL -12). Hypothesis: The semimature phenotype achieved by DC, could be used to reduce inflammation in a model of autoimmune disease. The high production of IL-10 and TGF-Beta by these cells could directly or through the induction of T reg cells inhibit the inflammatory response. Objective: In this project we propose the immunization of DBA1 / j mice, susceptible to the development of collagen-induced arthritis (CIA) with F. hepatica-treated DC in conjunction with CpG-ODN to reduce clinical signs of disease. Materials: In our laboratory, we developed the CIA model by two immunizations of DBA1 / j mice with bovine type II collagen and Freund's adjuvant. The model allowed to stablish a clinical index by swelling in the legs of animals. Twelve days after the first immunization the animals are injected with DC treated with: 1. PBS 2. F.hepatica Extract (TE) + CII, 3. CpG + CII, 4. TE + CpG + CII Macroscopic observation will take place daily from 7 days of the 2nd immunization. After sacrifice the joints of the legs will be prepared for histological analysis. Serum levels of IgG1 antibodies (Th2 profile) and IgG2a (Th1 profile) will be detected by ELISA. It will also detected the cytokine profile in draining lymph nodes by ELISA and additionally the cell populations of regulatory T cells (Treg) CD4 + CD25 + Foxp3 or Tr1 cells. Expected results: We believe that the treatment of animals that had developed CIA with DC will be able to migrate to lymphatic organs and secrete TGF-B (T reg cell-inducing), IL-10 (inducing Tr1 cells), IDO (inhibitory of T cells and inducing of T reg cells) could alone or in synergy inhibit Th17-type responses and Th1 associated with the pathology in this disease.

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.

Transplantation tolerance induced by regulatory T cells: in vivo mechanisms and sites of action.

The mechanisms by which CD4(+)CD25(+)Foxp3(+) T (Treg) cells regulate effector T cells in a transplantation setting and their in vivo homeostasis still remain to be clarified. Using a mouse adoptive transfer model, we analyzed the in vivo expansion, trafficking, and effector function of alloreactive T cells and donor-specific Treg cells, in response to a full-thickness skin allograft. Fluorescent-labeled CD4(+)CD25(-) and antigen-specific Treg cells were transferred alone or co-injected into syngeneic BALB/c-Nude recipients transplanted with skins from (C57BL/6 x BALB/c) F1 donors. Treg cells divided in vivo, migrated and accumulated in the allograft draining lymph nodes as well as within the graft. The co-transfer of Treg cells did not modify the early activation and homing of CD4(+)CD25(-) T cells in secondary lymphoid organs. However, in the presence of Treg cells, alloreactive CD4(+)CD25(-) T cells produced significantly less IFN-gamma and were present in reduced numbers in the secondary lymphoid organs. Furthermore, time-course studies showed that Treg cells were recruited into the allograft at a very early stage after transplantation and effectively prevented the infiltration of effector T cells. In conclusion, suppression of rejection requires the early recruitment to the site of antigenic challenge of donor-specific Treg cells, which then mainly regulate the effector arm of T cell alloresponses.

Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects.

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-β. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.

CD4+, CD8+ and CD4- CD8- T cell-subsets can confer protection against Leishmania m. mexicana infection

We studied the role of CD4+, CD8+, CD4- CD8- T cells and IgG anti-Leishmania after infection or vaccination in the CBA/ca mouse. Mice were either infected with L. m. mexicana promastigotes or vaccinated with parasite-membrane antigens incorporated into liposomes. Successfully vaccinated mice were used as cell-donors in adoptive transfer experiments. Naive, syngeneic recipients received highly-enriched CD4+, CD8+ or CD4- CD8- T cells from those two set of donors and challenged with live parasites. Our results showed that, both CD4+ and CD8+ T cells from infected or vaccinated donors conferred significant disease-resistance to naive recipients. In addition, adoptive transfer of CD4- CD8- T cells from vaccinated donors significantly delayed lesion growth in recipient mice. We concluded that vaccination of CBA mice correlates with the induction of protective CD4+, CD8+ and CD4- CD8- T cells and the synthesis of IgG anti-Leishmania.