RESUMO
BACKGROUND: Non Obese Diabetic mice lacking B cells (NOD.Igµ(null) mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age. METHODS: We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igµ(null) mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes. RESULTS: We found that B cell reconstitution of NOD.Igµ(null) mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igµ(null) mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro. CONCLUSIONS: Diabetes in NOD.Igµ(null) mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.
Assuntos
Linfócitos B/imunologia , Diabetes Mellitus Experimental/imunologia , Cadeias mu de Imunoglobulina/imunologia , Imunofenotipagem/métodos , Ilhotas Pancreáticas/imunologia , Linfócitos T/imunologia , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Proliferação de Células , Células Clonais , Ciclofosfamida , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Rejeição de Enxerto/complicações , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Memória Imunológica/imunologia , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos NOD , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Baço/patologia , Linfócitos T/citologiaRESUMO
Monkeypox virus (MPV) is a virulent human pathogen that has gained increased attention because of its potential use as a bioterrorism agent and inadvertent introduction into North America in 2003. The US outbreak also provided an important opportunity to study MPV-specific T cell immunity. Although MPV-specific CD4(+) and CD8(+) T cells could recognize vaccinia virus (VV)-infected monocytes and produce inflammatory cytokines such as IFNgamma and TNFalpha, they were largely incapable of responding to autologous MPV-infected cells. Further analysis revealed that, unlike cowpox virus (CPV), MPV did not interfere with MHC expression or intracellular transport of MHC molecules. Instead, MPV-infected cells were capable of preventing T cell receptor (TcR)-mediated T cell activation in trans. The ability to trigger a state of nonresponsiveness represents a unique MHC-independent mechanism for blocking antiviral T cell activation and inflammatory cytokine production and is likely an important attribute involved with viral dissemination in the infected host.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Monkeypox virus/imunologia , Mpox/imunologia , Apresentação de Antígeno/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Infecções por Vírus Epstein-Barr/imunologia , Regulação da Expressão Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Herpesvirus Humano 4/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Vacínia/imunologia , Vaccinia virus/imunologia , Replicação ViralRESUMO
The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a "T cell-driven" methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.