RESUMO
The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.
Assuntos
Diversidade de Anticorpos/genética , Regiões Determinantes de Complementaridade/genética , Cadeias Pesadas de Imunoglobulinas/genética , Adulto , Sequência de Aminoácidos , Subpopulações de Linfócitos B/metabolismo , Sequência de Bases , Códon de Terminação , Regiões Determinantes de Complementaridade/química , Feminino , Expressão Gênica , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina A/genética , Imunoglobulina A/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Região de Junção de Imunoglobulinas/química , Região de Junção de Imunoglobulinas/genética , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Masculino , Pessoa de Meia-Idade , Fases de Leitura , Reprodutibilidade dos Testes , Recombinação V(D)J , Adulto JovemRESUMO
Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.