Contribution of T cell receptor affinity to overall avidity for virus-specific CD8+ T cell responses

Abstract

Prior analysis has characterized the clonal characteristics of effector CD8(+) T cells specific for the prominent influenza A virus nucleoprotein (NP) and acid polymerase (PA) peptides presented by H2D(b). Using a single-cell approach and determination of CDR3beta profiles, a limited, predominantly "public" repertoire was found for CD8(+)D(b)NP(366)(+)Vbeta8.3+ cells, whereas diverse and "private" T cell antigen receptor (TCR)beta clonotypes were typical of the CD8(+)D(b)PA(224)(+)Vbeta7+ response. This single-cell approach has now been used to relate the contributions of particular clonotypes (or affinities) to high-avidity TCRs, as defined by binding under conditions of limiting tetramer availability. At least by the measure of CDR3beta usage, no difference could be found between total and high-avidity populations in the spectrum of TCR-pMHC affinities throughout the limited, and relatively public, CD8(+)D(b)NP(366)(+)Vbeta8.3+ populations. Conversely, the more even (by clone size), diverse, and private CD8(+)D(b)PA(224)(+)Vbeta7+ response was characterized by the clear partitioning of the largest T cell clones in the high-avidity compartment. These results suggest that the relatively constrained CD8(+)D(b)NP(366)(+)Vbeta8.3+ set utilizes a relatively narrow range of affinities, whereas the broader CD8(+)D(b)PA(224)(+)Vbeta7+ response is induced at a range of TCR-pMHC affinities. Thus, whereas TCR sequence (or affinity) appears to contribute substantially to the avidity profile of diverse virus-specific CD8+ populations, other mechanisms may be prominent where the TCR spectrum is more limited.

Fig. 1.

Measurement of avidity in CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ and CD8⁺D^bPA₂₂₄⁺Vβ7⁺ cells by tetramer dilution analysis. Lymphocytes were obtained from spleens of individual mice at day 8 after intranasal immunization with HKx31 of PR8-primed mouse, enriched for CD8⁺ T cells, and stained with D^bNP₃₆₆ (A) or D^bPA₂₂₄ (B) tetramers conjugated to Streptavidin-PE at concentrations ranging from 1:100 to 1:20,000. All of the cells were subsequently stained with anti-CD8α-APC, and either anti-Vβ8.3 for D^bNP₃₆₆⁺CD8⁺ (A) cells or anti-Vβ7 antibodies for D^bPA₂₂₄⁺CD8⁺ (B) cells. Lymphocytes stained with tetramers at the indicated dilutions (i.e., 1:100 for the total epitope-specific population and 1:10,000 for the limiting tetramer⁺ cells) were single-cell sorted into the cDNA buffer by using the MoFlo sorter. Shown is the percentage of tetramer⁺ Vβ⁺ CD8⁺ T cells.

Fig. 2.

The frequency of singletons and repeated clonotypes in epitope-specific cells stained with tetramers at 1:100 and 1:10,000 dilutions. Clonotypes from a pool of CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ (A) and CD8⁺D^bPA₂₂₄⁺Vβ7⁺ (B) sets were analyzed according to the number of repeats found in individual mice. The sequences were divided into singletons (found only once in the particular repertoire), “2 to 4,” “5 to 9,” “10 to 14,” and “15+” groups. The frequency at which each group of repeats was found within Vβ8.3⁺ (A) and Vβ7⁺ (B) sequences is shown. The tetramer⁺CD8⁺ populations stained at 1:100 dilution are represented in filled bars, whereas cells stained at 1:10,000 dilution are shown in open bars.

Fig. 3.

Increased frequency of the prominent clonotypes in CD8⁺D^bPA₂₂₄⁺Vβ7⁺ cells stained at the limiting tetramer dilution. The frequencies of the two predominant clonotypes in CD8⁺D^bPA₂₂₄⁺Vβ7⁺ populations stained with the tetramer at 1:10,000 dilution were analyzed for their respective frequencies in the total pool of CD8⁺D^bPA₂₂₄⁺Vβ7⁺ cells stained under saturating conditions (1:10,000). A graphic representation of increased frequencies of clonotypes in the high-avidity PA₂₂₄⁺Vβ7⁺CD8⁺ cells is compared with the total PA₂₂₄⁺Vβ7⁺CD8⁺ cells (P < 0.004, Wilcoxon).

Fig. 4.

Prominent clonotypes in CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ cells stained with the limiting tetramer dilution (1:10,000) and their respective frequencies in the total tetramer⁺ population determined under saturating conditions (1:100). A graphic representation of increased frequencies of clonotypes in the high-avidity CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ cells when compared with the total CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ cells is shown (P = 0.91, Wilcoxon).

Fig. 5.

Clonotype distribution of CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ and CD8⁺PA₂₂₄Vβ7⁺ cells determined by saturating (1:100) or limiting (1:10,000) tetramer dilutions. The clonotypes obtained from individual mice for CD8⁺D^bPA₂₂₄⁺Vβ7⁺ cells stained with 1:100 (filled squares) or 1:10,000 tetramer dilution (open squares), and CD8⁺D^bNP₃₆₆⁺Vβ8.3⁺ cells stained at 1:100 (filled circles) or 1:10,000 tetramer dilution (open circles) were ranked according to their size and cumulative proportion of total clonotypes plotted against a cumulative percentage of total sequences. Shown are results obtained from all of the clonotypes found for the five mice tested. A mouse with <20 sequences obtained for the CD8⁺D^bPA₂₂₄⁺Vβ7⁺ response was excluded from the analysis.