Massive expansion of antigen-specific CD8+ T cells during an acute virus infection

Abstract

During LCMV infection, CD8+ T cells expand greatly. Bystander activation has been thought to play a role because few cells score as LCMV specific in limiting dilution analysis. In contrast, we find that at least a quarter of the CD8+ cells secrete IFNgamma specifically in response to LCMV peptides at the peak of the response. Moreover, by analyzing the expansion of adoptively transferred LCMV-specific, TCR-transgenic CD8+ T cells in congenic hosts, we have determined that most of the CD8+ cell expansion is virus specific. Analysis of the effect of the monospecific TCR-transgenic T cells on the host response to three LCMV epitopes suggests that CTL precursors compete for sites on the APC in an epitope-specific fashion and that this competition determines the specificity of the response.

Figure 1. LCMV Infection of C57Bl/6 Mice Induces Massive Expansion of CD8⁺ T Cells

Representative CD4 versus CD8 flow cytometry profiles of splenocytes from uninfected (A) or day 8 LCMV-infected B6 mice (B). The numbers beside the gating boxes indicates the percentage of live-gated cells. d8, day 8.

Figure 2. Antigen-Specific Cytolytic Activity Is Proportional to the Frequency of IFNγ-Secreting Cells

(A) Erythrocyte-depleted spleen cells were prepared from a pair of day 8 LCMV-infected B6 mice and were used to determine primary ex vivo CTL activity in an 8 hr ⁵¹Cr-release assay against EL4 target cells uncoated or coated with the indicated LCMV peptides. (B) The same pool of effector cells was used in an ELISPOT assay to determine the frequency of cells secreting IFNγ in response to LCMV epitope peptides. The number of CD8⁺ cells was determined by FACS analysis. Error bars indicate the standard deviation of triplicate samples. Three additional experiments yielded similar results. d8, day 8. (C) The titration of spots per well versus number of LCMV day 8 splenocytes per well is shown for ELISPOT data from a separate experiment. Splenocytes were stimulated in triplicate samples with either control EL4 cells (open circles) or with gp33-coated EL4 cells (filled squares). Stimulation with gp276- or np396-coated EL4 cells resulted in similarly linear responses (data not shown).

Figure 3. Adoptively Transferred TCR-Transgenic CD8⁺ T Cells Do Not Expand Nonspecifically during VV or LCMV Infection

Flow cytometry profiles of pooled spleen cells from pairs of B6.PL control mice (A) and B6.PL host mice that received 10⁷ purified OT-1 CD8⁺ T cells and were injected the next day with PBS (B), VVflu-np (C), LCMV (D), or VVova (E). Splenocytes were analyzed on day 6 (A–C and E) or day 8 (D) of infection. The numbers beside the gating boxes indicate the number of donor CD8⁺ cells as a percentage of total CD8⁺ cells. A repetition of the experiment yielded similar results.

Figure 4. GP33-Specific TCR-Transgenic CD8⁺ T Cells Expand Dramatically during LCMV Infection

Control B6.PL mice (A and C) or B6.PL mice that had received 10⁵ P14 spleen cells (~3 × 10⁴ transgenic CD8⁺ cells) 1 day earlier (B and D) were infected with LCMV. After 8 days spleens were removed and assayed for CD4 versus CD8 staining (A and B) and donor (CD8⁺Thy1.2⁺) CTL content (C and D). The numbers beside the gating boxes in (A) and (B) indicate the percentage of live gated cells; in (C) and (D) the numbers indicate the number of Thy1.2⁺ donor cells as a percentage of CD8⁺ cells. Results are from representative individual mice from groups of three. In uninfected chime-ric mice the donor cells were less than 0.5% of CD8⁺ cells (data not shown). d8, day 8.

Figure 5. Adoptive Transfer of a Limited Number of gp33-Specific CD8⁺ T Cells Does Not Significantly Alter the Overall Response to gp33, gp276, or np396 during the Immune Response to LCMV

Primary CTL activity of splenocytes from uninfected B6.PL mice that received 10⁵ P14 cells (open circles) and day 8 LCMV-infected mice (filled symbols) that received no P14 cells (squares), 10³ P14 cells (diamonds), 10⁵ P14 cells (triangles), or 10⁶ P14 cells (crosses) 1 day prior to infection. Lysis was determined on day 8 of infection by ⁵¹Cr-release assay using EL4 targets without additional peptide (A) and EL4 pulsed with gp33 (B), gp276 (C), or np396 (D) peptides. Data are presented as the average specific lysis of targets by effectors from three individual mice per group, with standard deviation indicated by error bars (some of which fall within the symbols). Two repetitions of the experiment yielded similar results.

Figure 6. Both Adoptively Transferred P14 and Host CD8⁺ T Cells Are Functionally Active in LCMV-Infected PL/P14 Chimeras

A group of three B6.PL mice received 10⁴ P14 spleen cells. The next day the PL/P14 chimeras (C, F, and I) and groups of three B6 (A, D, and G) and B6.PL (B, E, and H) mice were infected with LCMV. On day 8 of infection, splenocytes pooled for each group were prepared and treated with rabbit complement alone (A–C), with anti-Thy1.1 (anti-host) plus complement (D–F), or with anti-Thy1.2 (anti-donor) plus complement (G–I), and the ability of the surviving cells to lyse uncoated EL4 targets and EL4 cells coated with the indicated LCMV peptides was determined in a ⁵¹Cr-release assay.

Figure 7. Competition for MHC Class I–Peptide Complexes during CTL Priming

(Left) A virus-infected APC displays a large number of MHC class I–peptide complexes, indicated by the different numbers on the surface of the cell. (Center) When CTL precursors specific for epitopes 1 and 2 (T₁ and T₂) interact with the APC, they aggregate their respective target MHC–peptide complexes at the zones of contact. (Right) When enough T₁ interact with the APC, they reduce the available MHC–peptide 1 complexes to the point where other T₁ cells cannot be primed, although cells of other specificities (T₂) can still interact productively with the APC.