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 antigen-specific T cell clones using limited amounts of specimens. The megaplex TCR isolation covered the repertoire comprised of recombinants from 24 Vbeta families and 13 Jbeta segments, and allowed us to isolate TCR VDJ clonotypic sequences from one or many PPD-specific IFNgamma-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 >or=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 IFNgamma-producing T cell clones using as few as 2 x 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.

Fig. 1

Combined megaplex TCR isolation and SMART-based real-time quantitative PCR methods for quantitating antigen-specific T cell clones. (A) Megaplex TCR isolation and SMART-based real-time quantitative methods. (B) Complementary regions of each of the 25 Vβ family-specific primers and 13 Jβ-specific primers. Three sets of nested Vβ family-specific primers were designated as external (ext.Vβ), middle (mid.Vβ), and internal (int.Vβ) Vβ family-specific primers. Likewise, three sets of Jβ family-specific primers were designated as ext.Jβ, mid.Jβ and int.Jβ. Note that ext.Jβ-specific primers were designed based on intron sequences close to Jβ segments.

Fig. 2

Three-round nested megaplex PCR were able to amplify TCR VDJ DNA from small numbers of formaldehyde-fixed T cells. Lane M on the 2% agarose gel was DNA size markers. Lanes 1 and 2 were PCR products amplified from sample DNA of 200 T cells; Lanes 3 and 4 were PCR products each starting from 20 T cells. Lanes 5 and 6 were PCR products each starting from 4 cells; Lanes 7, 8, 9 and 10 were PCR products each amplified from single cell DNA. (A) The 2% agarose gel showed TCR VDJ DNA amplified from primary megaplex PCR using 25 ext.Vβ and 13 ext.Jβ primers (Tables 1A–1C). The expected size ranged from 280 to 350 bp. Only primer dimers/multimers were seen. (B) VDJ DNA amplified from the 2nd megaplex PCR using 1 μl of the 1st round PCR product as template, 25 mid.Vβ and 13 mid.Jβ primers. The expected VDJ DNA sizes ranged from 150 to 280 bp. (C) VDJ DNA amplified from the 3rd megaplex PCR using 1 μl of 500-fold diluted 2nd PCR product as template, 25 int.Vβ, and 13 int.Jβ primers. The expected sizes ranged from 120 to 250 bp. The VDJ DNA were confirmed by direct sequencing.

Fig. 3

SMART amplification of cDNA enriched cellular mRNA/cDNA without altering TCR β gene profiles. Shown are scattergrams and regression plots comparing sixteen gene expression ratios of individual VβJβ-bearing genes versus Cβ genes or the house-keeping β-actin or GAPDH gene between cDNA (pre-amplification) and SMART-amplified cDNA (post-amplification) derived after different PCR cycles. Data are shown as log10 scales, and presented as mean ratios of genes (transcripts of a VβJβ gene/transcripts of Cβ or β-actin or GAPDH gene), which were calculated from the results in three different assays. SMART-amplified cDNA was generated from the same cDNA derived from a BCG-infected monkey.

Fig. 4

Immune responses of PPD-specific IFNβ-producing CD4 T cell clones after BCG infection of monkeys. Clonotypic TCR sequences were derived from PPD-specific IFNβ-producing CD4 T cells. These antigen-specific T cells were selected by intracellular cytokine staining of PPD-stimulated PBMC obtained 3 and 4 weeks after BCG infection, fixed by 2% formaldehyde and purified by flow cytometry sorting. Real time quantitation of PPD-specific IFNβ-producing CD4 T cell clones was done using SMART cDNA (18 cycles) derived from frozen PBMC collected before and after BCG infection. Shown are only the clones that were detectable in real-time PCR. Twenty-four, thirty and fifteen VβJβ clones isolated from monkeys 124, 227 and 336, respectively, were undetectable due to their low frequencies (<1×10⁵) in PBMC. Similar expression levels for dominant clones 124Vβ21Jβ2.4, 124Vβ20Jβ1.5, 336Vβ7Jβ1.6 and 336Vβ9Jβ2.1 were also confirmed by real-time PCR using conventional cDNA derived from pre- and week 4 PBMC.