RESUMO
Tracking antigen-specific T cell responses over time within individuals is difficult because of lack of knowledge of antigen-specific TCR sequences, limitations in sample size, and assay sensitivities. We hypothesized that analyses of high-throughput sequencing of TCR clonotypes could provide functional readouts of individuals' immunological histories. Using high-throughput TCR sequencing, we develop a database of TCRß sequences from large cohorts of mice before (naive) and after smallpox vaccination. We computationally identify 315 vaccine-associated TCR sequences (VATS) that are used to train a diagnostic classifier that distinguishes naive from vaccinated samples in mice up to 9 months post-vaccination with >99% accuracy. We determine that the VATS library contains virus-responsive TCRs by in vitro expansion assays and virus-specific tetramer sorting. These data outline a platform for advancing our capabilities to identify pathogen-specific TCR sequences, which can be used to identify and quantitate low-frequency pathogen-specific TCR sequences in circulation over time with exceptional sensitivity.
Assuntos
Rastreamento de Células , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Vírus/metabolismo , Sequência de Aminoácidos , Animais , Células Clonais , Feminino , Biblioteca Gênica , Masculino , Camundongos Endogâmicos C57BL , Orthopoxvirus , Peptídeos/química , Infecções por Poxviridae/virologia , Receptores de Antígenos de Linfócitos T/química , VacinaçãoRESUMO
In a previous study, a new set of Y-chromosome short tandem repeats, the OSU 10-locus set (MPM1 and MPM2), was shown to have a higher discrimination power when evaluated against the 10 SWGDAM loci on a common population panel. Here, we describe the optimization of the multiplex reactions using dye-labeled primers followed by performance evaluations. The loci exhibited high precision, human male specificity, reliability in different body fluids, high sensitivity, stability, and the ability to amplify nonprobative casework and mixture samples. Stutter for the all of the loci, with the exception of the highly polymorphic locus DYS688, was similar to that observed for autosomal loci. The results of the performance evaluations reinforced the utility of these loci.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , Loci Gênicos , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex , Animais , Sangue , DNA/isolamento & purificação , Primers do DNA , Eletroforese , Feminino , Corantes Fluorescentes , Haplótipos , Humanos , Masculino , Saliva , SêmenRESUMO
A comparative analysis of two Y-STR loci sets was conducted on a population sample of 224 individuals, 114 Caucasians and 110 African Americans. One set of loci, designated the OSU 10-locus set, comprises variable, single copy, male-specific loci that are dispersed across the Y-chromosome. Parallel evaluations were performed using the 10 Y-chromosome loci most frequently used for forensic analysis, the loci chosen as the SWGDAM Y-STR loci. The OSU 10-locus set had a greater average number of alleles per locus and higher average gene diversity than the SWGDAM loci. The OSU 10-locus set found 220 unique haplotypes in 224 individuals. In approximately 6000 pairwise haplotype comparisons for each population with each set of loci, the OSU 10-locus set also yielded a greater average number of allelic differences per pair than the SWGDAM loci. Finally, the overall linkage disequilibrium levels were lower for the OSU 10-locus set in the Caucasian population. In general, the OSU 10-locus set revealed a higher power of discrimination than the SWGDAM set.
