Estimating the precursor frequency of naive antigen-specific CD8 T cells

Abstract

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.

Figure 1.

Adoptive transfer of increasing numbers of D^bGP33-specific TCR Tg CD8⁺ T cells (Vα2⁺Vβ8.1⁺), followed by challenge with LCMV, results in an increasing proportion of the total epitope-specific response being contributed by donor cells. (A) Analysis of recipient mice after viral challenge. Contour plots are gated on CD8⁺ T cells and percentages indicate the relative proportions of D^bGP33⁺ cells that are Vα2⁺ or Vα2⁻. Numbers above each plot indicate the number of TCR-Tg CD8⁺ T cells transferred. (B) Analysis of Vβ8.1 usage by epitope-specific cells. Histograms are gated on CD8⁺D^bGP33⁺Vα2⁺ cells. Percentages under each histogram indicate the total Vα2⁺Vβ8.1⁺ portion of the D^bGP33-specific CD8⁺ T cell response. (C) The donor TCR-Tg fraction of the epitope-specific response is plotted vs. the number of TCR-Tg cells engrafted. Open symbols circles represent mice that were given only TCR-Tg cells, while filled circles represent mice which were given TCR-Tg cells diluted in 2 × 10⁶ naive polyclonal B6 splenocytes. The solid line is the best fit to the equation F = T/(T + P) while the dashed line indicates the dose of donor cells at which 50% of the D^bGP33-specific response is endogenous.

Figure 2.

Engraftment of TCR-Tg and polyclonal B6 CD8⁺ T cells are similar. (A) Splenocytes from either TCR-Tg or B6 (Thy1.1⁺) mice were injected into naive, nonirradiated B6 (Thy1.2⁺) recipients. Percentages indicate the fraction of naive TCR-Tg or B6 donor CD8⁺ T cells recoverable from spleens of recipient mice 2 d after transfer. Each bar represents >10 mice and error bars indicate standard deviation from the mean. (B) To discount differences in survival of TCR-Tg vs. B6 cells, 10⁸ purified polyclonal CD8⁺ cells from C57Bl/6 Thy1.1⁺ mice (which, given our estimates, contains ∼500 precursor cells) were mixed with 500 purified TCR-Tg Thy1.1⁺/Thy1.2⁺ CD8⁺ cells. These were then injected into naive B6 Thy1.2⁺ recipients. After challenge with LCMV, epitope-specific donor cells (Thy1.1⁺) from recipient mice were analyzed for Thy1.2 (TCR Tg only) expression. Percentages indicate the proportion of the CD8⁺D^bGP33⁺Thy1.1⁺ response due to TCR-Tg (Thy1.2⁺) or polyclonal B6 (Thy1.2⁻) donor cells.

Figure 3.

TCR-Tg and polyclonal B6 epitope-specific CD8⁺ T cells have similar proliferative properties. LCMV immune mice containing a mixture of TCR-Tg and endogenous D^bGP33-specific CD8⁺ T cells were rechallenged with either LCMV (clone-13) (A) or a vaccinia virus expressing the GP33 epitope (VV-GP33) (B). PBMCs were analyzed for the frequency of D^bGP33-specific cells (bar graphs, left axis) as well as the fraction of the D^bGP33⁺ response from individual mice due to donor TCR-Tg cells (right axis) before (memory) and after (secondary) rechallenge. Graphs indicate mean frequency of antigen specific cells (>6 mice per group) and error bars indicate standard deviation from the mean.

Figure 5.

Transfer of increasing numbers of donor cells does not affect the TCR affinity or diversity of the endogenous D^bGP33 response. TCR affinity. (A) Relative TCR affinity of P14 TCR transgenic (filled circles) versus polyclonal (open circles) D^bGP33-specific CD8 T cells. For quantitative comparison of tetramer fall-off kinetics, data are shown as the (−) change in normalized total fluorescence (NTF, as described in Materials and Methods) over time. The mean (± SD) slope of each interval is equivalent to (NTF_i − NTF_i-1)/t where t is the length of the time interval. (B) Relative TCR affinity of endogenous D^bGP33-specific CD8 T cells from mice whose D^bGP33 response was completely due to endogenous cells (circles), 25–33% donor cells (triangles), or 66–75% donor cells (squares) were analyzed by tetramer fall-off assay as described. (C) TCR diversity. Endogenous D^bGP33-specific CD8 T cells from mice containing either completely endogenous (open circles) or 50% transgenic D^bGP33 cells (filled circles) were analyzed for TCR-Vβ usage using a panel of monoclonal antibodies. Shown is the percentage of the endogenous (Vα2⁻) D^bGP33 response using each Vβ gene segment.

Figure 4.

No suppression of the endogenous D^bGP33-specific CD8⁺ T cell response due to limited antigen availability. (A) No suppression of the endogenous response was observed in the range of precursor frequency. Mice receiving different numbers of D^bGP33-specific TCR Tg CD8⁺ T cells were challenged with LCMV and 7 d after infection, splenocytes were analyzed by flow cytometry. The magnitude of the endogenous polyclonal (open circles) and donor transgenic (filled circles) D^bGP33-specific CD8⁺ response is shown per spleen versus the number of engrafted donor cells. (B) Increased antigen load results in precisely the same dose dependence of donor cells. Mice receiving different numbers of donor TCR-Tg cells were challenged with LCMV (ARM) (circles) or the more virulent LCMV (clone-13) (triangles). Plotted is the fraction of the response due to donor cells versus the number of donor cells engrafted.