The genes for perforin, granzymes A–C and IFN‐γ are differentially expressed in single CD8+ T cells during primary activation

Abstract

Here we show that the genes for perforin, the three major T cell granzymes (A-C) and IFN-gamma are differentially expressed during primary activation of naive CD8(+) T cells, kinetically and at the single-cell level. When CD44(low)CD62L(high)CD8(+) lymph node T cells were activated with IL-2 and immobilized antibodies to CD3, CD8 and CD11a, expression of perforin, granzyme B and IFN-gamma mRNAs was induced by day 2, and increased in parallel with perforin-dependent cytolytic activity. Granzyme C and A transcripts were not detected until 1 and 3 days later respectively. Single-cell PCR showed that expression frequencies rose in parallel with total levels of each mRNA, but that individual cells expressed diverse combinations of perforin, granzyme A-C and IFN-gamma mRNAs. These expression patterns indicated that the delayed expression of granzymes A and C was not due to late activation of distinct cell subpopulations. Statistical analysis of the data suggested that each gene was differentially regulated at the single-cell level. Individual naive CD8(+) T cells gave rise over 7 days to clones that expressed all five products at the clonal level, but also expressed diverse combinations at the single-cell level. We conclude that, during primary activation, CD8(+) T cells progressively acquired the ability to express most or all of these genes, and that the variable expression patterns observed among single cells within clones and populations reflected transient rather than heritable differences in expression profile.

Reduced antigen concentration and costimulatory blockade increase IFN‐γ secretion in naive CD8+ T cells

“…Expression of perforin and IFN-c/TNF-a is also largely segregated. The genes for perforin, granzymes and IFN-c are also differentially expressed in naive CD8 + T cells during primary activation [29]. One explanation for this heterogeneity is a stochastic and independent regulation of individual effector molecules determined by the intensities of the stimulation during primary activation, as suggested by our study.…”

Granzyme A expression reveals distinct cytolytic CTL subsets following influenza A virus infection

“…These data support the notion that the different grz family members are regulated independently. Previous analysis of effector mRNA expression has demonstrated that there is heterogeneity of grz/pfp expression within virus-specific CTL, with little evidence of effector mRNA co-expression [7,8,12,25]. To date, this heterogeneity has not been examined at the protein level for murine virus-specific CTL.…”

Heterogeneity of Effector Phenotype for Acute Phase and Memory Influenza A Virus-Specific CTL

“…The PCR utilized 1 U of Taq polymerase (Invitrogen Life Technologies), 1.5 mM MgCl 2 , 0.2 mM dNTP, and 20 pmol each of the sense and antisense primers. The majority of the primers used to amplify each region have been described previously (26). Otherwise, the primers were: GzmK, EX sense 5Ј-GGCCATTTATGGCGTCC ATCCAG-3Ј, antisense 5Ј-TCACCTGGCATTTGGTCCCA-3Ј and IN sense 5Ј-GCAAGCATATTTGTGGAGGA-3Ј, antisense 5Ј-GTCGTGAG AATGGGATGAAC-3Ј; CD8␣, EX sense 5Ј-ATGGACGCCGAACTTGG TCA-3Ј, antisense 5Ј-GTTCGCAGCACTGGCTTGGT-3Ј, IN sense 5Ј-GTC AGAAGGTGGACCTGGTATG-3Ј, antisense 5Ј-GAACTTGTTCAGGGT GAGAACG-3Ј.…”

“…After polyclonal in vitro stimulation of naive CD8 ϩ T cells, early transcription of Pfp and GzmB mRNA is followed by GzmA mRNA expression 3-4 days later (26). The single-cell effector mRNA profiles were very heterogeneous, suggesting that Gzm and Pfp transcription may not be coregulated (26,27).…”

“…After polyclonal in vitro stimulation of naive CD8 ϩ T cells, early transcription of Pfp and GzmB mRNA is followed by GzmA mRNA expression 3-4 days later (26). The single-cell effector mRNA profiles were very heterogeneous, suggesting that Gzm and Pfp transcription may not be coregulated (26,27). An indication that this functional diversity may also apply in vivo was found for influenza A virus-specific CTL isolated from both lung and mesenteric lymph nodes 7 days after primary challenge (27).…”