RESUMO
There are well-characterized age-related changes in the peripheral repertoire of CD8 T cells characterized by reductions in the ratio of naive:memory T cells and the development of large clonal expansions in the memory pool. In addition, the TCR repertoire of naive T cells is reduced with aging. Because a diverse repertoire of naive T cells is essential for a vigorous response to new infections and vaccinations, there is much interest in understanding the mechanisms responsible for declining repertoire diversity. It has been proposed that one reason for declining repertoire diversity in the naive T cell pool is an increasing dependence on homeostatic proliferation in the absence of new thymic emigrants for maintenance of the naive peripheral pool. In this study, we have analyzed the naive CD8 T cell repertoire in young and aged mice by DNA spectratype and sequence analysis. Our data show that naive T cells from aged mice have perturbed spectratype profiles compared with the normally Gaussian spectratype profiles characteristic of naive CD8 T cells from young mice. In addition, DNA sequence analysis formally demonstrated a loss of diversity associated with skewed spectratype profiles. Unexpectedly, we found multiple repeats of the same sequence in naive T cells from aged but not young mice, consistent with clonal expansions previously described only in the memory T cell pool. Clonal expansions among naive T cells suggests dysregulation in the normal homeostatic proliferative mechanisms that operate in young mice to maintain diversity in the naive T cell repertoire.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Receptores de Antígenos de Linfócitos T/biossíntese , Envelhecimento/genética , Animais , Linfócitos T CD8-Positivos/citologia , Separação Celular , Células Clonais , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/genética , Feminino , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Homeostase/imunologia , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/imunologiaRESUMO
Two periplasmic binding proteins of E. coli, the leucine specific-binding protein (LS) and leucine-isoleucine-valine binding protein (LIV), have high similarity in their structure and function, but show different substrate specificity. A key difference between these proteins is residue 18 in the binding pocket, a tryptophan residue in the LS and a tyrosine residue in the LIV. To examine the role of this residue in binding specificity, we used fluorescence and (19)F NMR to monitor ligand binding to three mutants: LSW18Y, LSW18F and LIVY18W. We observed leucine binding to all proteins. LS binds L-phenylalanine but the mutation from Trp to Tyr or Phe disallows this ligand and expands the binding repertoire to L-isoleucine and L-valine. The LIVY18W mutant still retains the ability to bind L-isoleucine and also binds L-phenylalanine.
Assuntos
Escherichia coli/metabolismo , Leucina/metabolismo , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Genes Bacterianos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Conformação Proteica , Desnaturação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Triptofano/metabolismo , UreiaRESUMO
The identification of T cell epitopes is crucial for the understanding of the host immune response during infection. While much is known about the MHC class I-restricted response following influenza virus infection of C57BL/6 mice, with over 16 CD8 epitopes identified to date, less is known about the MHC class II-restricted response. Currently, only a few I-A(b)-restricted T helper epitopes have been identified. Therefore, several important questions remain about how many class II epitopes exist in this system and whether these epitopes are evenly distributed within the most abundant viral proteins. In order to address these questions, we analyzed the repertoire of epitopes that drive the CD4+ approximately 20-30 epitopes drive the CD4 T cell response and that the majority of these peptides are derived from the NP and HA proteins. We were also able to demonstrate that vaccination with one of the newly identified epitopes, HA(211-225)/A(b), resulted in increased epitope-specific T cell numbers and a significant reduction in viral titers following influenza virus challenge.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Genes MHC da Classe II/imunologia , Hemaglutininas Virais/imunologia , Vírus da Influenza A/imunologia , Animais , Epitopos , Vírus da Influenza A/química , Vírus da Influenza A/enzimologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Recent studies have shown that CD4(+) memory T cells persist in nonlymphoid organs following infections. However, the development and phenotype of these peripheral memory cells are poorly defined. In this study, multimerized MHC-Ig fusion proteins, with a covalently attached peptide sequence from the Sendai virus hemagglutinin/neuraminidase gene, have been used to identify virus-specific CD4(+) T cells during Sendai virus infection and the establishment of peripheral CD4(+) memory populations in the lungs. We show declining frequencies of virus-specific CD4(+) T cells in the lungs over the course of approximately 3 mo after infection. Like peripheral CD8(+) T cells, the CD4(+) have an acutely activated phenotype, suggesting that a high level of differentiation is required to reach the airways and persist as memory cells. Differences in CD25 and CD11a expression indicate that the CD4(+) cells from the lung airways and parenchyma are distinct memory populations.