Contribution of T-cell receptor repertoire breadth to the dominance of epitope-specific CD8+ T-lymphocyte responses

Abstract

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (Vbeta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse Vbeta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused Vbeta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.

FIG. 1.

Immunodominant and subdominant SIV/SHIV epitopes have similar peptide binding affinities for MHC class I. (A) Peak tetramer responses for the dominant p11C and subdominant p68A epitopes in SIV-infected Mamu-A*01-positive rhesus monkeys. (B) Peak tetramer responses for the dominant p199RY and subdominant p56 epitopes in SIV-infected Mamu-A*02-positive monkeys. (C) Binding affinities of p11C and p68A for Mamu-A*01. (D) Binding affinities of p199RY and p56 for Mamu-A*02.

FIG. 2.

Two-step amplification of cellular RNA does not bias the detected TCR repertoire. (A and B) Total cellular RNA extracted from CD8⁺ T lymphocytes was evenly divided among eight tubes, and 10² to 10⁹ copies of kanamycin (KAN) RNA were added to the tubes. Separate cDNA synthesis reactions were performed on each sample, and all samples were individually preamplified, with the number of cycles determined by the previously described SYBR green test run. These preamplified samples were then analyzed using the TCR α-chain and kanamycin probes. C_T, cycle threshold. (C and D) Various amounts of cellular RNA were used in five separate preamplification reactions, while the amount of exogenously added kanamycin was kept constant in each reaction. The number of cycles was again determined by a SYBR green test run. R² values calculated from the linear regression of panels A and C are 0.9889 and 0.9637, respectively. CREq, cell RNA equivalent.

FIG. 3.

p11C-stimulated tetramer-sorted PBMCs are a distinct subset of the total CD8⁺ T-lymphocyte population. (A) Real-time PCR Vβ quantitation of total peripheral blood cDNA generated from fresh p11C tetramer-sorted PBMCs and from 12-day p11C-stimulated, p11C tetramer-sorted PBMCs. (B and C), Vβ repertoire analysis from two monkeys (CJ2P and 134, respectively) of fresh sorted CD8⁺ T lymphocytes and 12-day p11C-stimulated, p11C tetramer-sorted PBMCs.

FIG. 4.

CD8⁺ T-lymphocyte populations recognizing dominant Mamu-A*01- or Mamu-A*02-restricted epitopes consist of a diverse Vβ repertoire, while those recognizing subdominant epitopes have a more restricted Vβ repertoire. (A) Mamu-A*01-restricted p11C-specific T-lymphocyte Vβ repertoires. (B) Mamu-A*01-restricted p68A-specific T-lymphocyte Vβ repertoires. (C) Mamu-A*02-restricted p199RY-specific T-lymphocyte Vβ repertoires. (D) Mamu-A*02-restricted p56-specific T-lymphocyte Vβ repertoires.

FIG. 5.

Spectratyping analysis reveals CDR3 diversity within represented Vβ families of CD8⁺ T-lymphocyte populations specific for dominant Mamu-A*01- and Mamu-A*02-restricted CD8⁺ T-lymphocyte epitopes and a single CDR3 length for Vβ families of CD8⁺ T lymphocytes specific for subdominant epitopes. Shown are spectratyping results for representative Vβ families found in (A) p11C-specific, (B) p68A-specific, (C) p199RY-specific, and (D) p56-specific CD8⁺ lymphocyte populations.

FIG. 6.

TCR Vβ CDR3 sequences of CD8⁺ T lymphocytes specific for the Mamu-A*01-restricted p11C and p68A epitopes. CDR3 sequences are arranged in order of corresponding Jβ families. a.a, amino acids.

FIG. 7.

TCR Vβ CDR3 sequences of CD8⁺ T lymphocytes specific for the Mamu-A*02-restricted p199RY and p56 epitopes. CDR3 sequences are arranged in order of corresponding Jβ families. a.a, amino acids.