Combined megaplex TCR isolation and SMART-based real-time quantitation methods for quantitating antigen-specific T cell clones in mycobacterial infection

Abstract

Despite recent advances in measuring cellular immune responses, the quantitation of antigen-specific T cell clones in infections or diseases remains challenging. Here, we employed combined megaplex TCR isolation and SMART-based real-time quantitation methods to quantitate numerous antigenspecific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vβ families and 13 Jβ segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNγ-producing T cells that were purified by flow cytometry sorting. The SMART amplification technique was then validated for its capacity to proportionally enrich cellular TCR mRNA/cDNA for real-time quantitation of large numbers of T cell clones. SMART amplified cDNA was shown to maintain relative expression levels of TCR genes when compared to unamplified cDNA. While the SMART-based real-time quantitative PCR conferred a detection limit of 10 −5 to 10 −6 antigen-specific T cells, the clonotypic primers specifically amplified and quantitated the target clone TCR but discriminated other clones that differed by ≥2 bases in the DJ regions. Furthermore, the combined megaplex TCR isolation and SMART-based real-time quantiation methods allowed us to quantitate large numbers of PPD-specific IFNγ-producing T cell clones using as few as 2×10 6 PBMC collected weekly after mycobacterial infection. This assay system may be useful for studies of antigen-specific T cell clones in tumors, autoimmune and infectious diseases.

TCR Repertoire, Clonal Dominance, and Pulmonary Trafficking of Mycobacterium-Specific CD4+ and CD8+ T Effector Cells in Immunity Against Tuberculosis

“…Intracellular cytokine staining (ICS) for IFN-γ + cells in PBMCs was done as previously described (11, 15, 18, 22). Stained cells were fixed in 2% formaldehyde.…”

“…Stained cells were fixed in 2% formaldehyde. IFN-γ + CD4 + CD3 + T cells and IFN-γ + CD8 + CD3 + T cells were then purified and enumerated by flow cytometry sorting, essentially as we previously described (15, 23). The formaldehyde-fixed PPD-specific CD4 T cells were subjected to TCRVDJ DNA isolation using the megaplex PCR.…”

“…We have recently used our decade-long TCR expertise and developed a useful method to isolate TCR VDJ clonotypic sequences from a limited number of purified protein deritative (PPD)-specific IFN-γ–producing T cells and to specifically measure clonotypic TCR frequencies in the T cell pool using the PCR-based real-time quantitation (15). The real-time quantitation technique using clonotypic primers specifically quantitates a target clonotypic TCR clone at a detection limit of 10 –5 Ag-specific T cells, and it discriminates other clones that differ by two or more bases in DJ regions (15).…”

“…The real-time quantitation technique using clonotypic primers specifically quantitates a target clonotypic TCR clone at a detection limit of 10 –5 Ag-specific T cells, and it discriminates other clones that differ by two or more bases in DJ regions (15). Employing this useful method, we investigated TCR Vβ repertoires/CDR3 usage, clonal expansion or dominance, and pulmonary trafficking or accumulation for PPD-specific T effector cells producing anti-TB cytokine IFN-γ during bacillus Calmette-Guérin (BCG) vaccination and subsequent M. tuberculosis challenge of macaques.…”

Missing: A diagnostic technique to enumerate antigen-specific T cells

“…An alternative strategy has been to analyse the genes of the T cell receptor via spectratyping or other methods [29–32]. Spectratyping (Immunoscope®) is based on PCR of the T cell receptor variable gene in T cells from peripheral blood and electrophoretic separation of the PCR products to produce a Gaussian shaped distribution of fragment lengths [33,34].…”