Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

Abstract

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.

Figure 1

Mice inoculated with GPVNPV lack robust CD8⁺ T cell responses to LCMV-specific dominant epitopes. Total number (±SD, n = 3) of CD8⁺ T cells producing IFN-γ from H-2^b mice 8 days following inoculation with either LCMV Armstrong or GPVNPV in response to the NP396–404, GP33–41, GP34–41, and GP276–286 wild-type peptides and variant peptides (containing the single amino acid substitution). Each mouse is analyzed individually, and data are representative or at least two independent experiments. *, p < 0.05 (Mann-Whitney t test).

Figure 2

Epitope loss does not lead to significant changes in the CD8⁺ T cell immunodominance hierarchy. H-2^b and H-2K^b−/− mice were inoculated with 1 × 10⁵ PFU LCMV Armstrong or GPVNPV i.p. Eight days later, splenocytes were analyzed. Total number (±SD, n = 3–9) of CD8⁺ T cells producing IFN-γ from immunized H-2^b mice (A) as well as H-2K^b−/− mice (B) in response to the known 28 LCMV-specific H-2^b-restricted epitopes. Splenocytes from immunized mice were stimulated with the indicated peptides and BFA for 5 h, surface stained for CD8, permeabilized, and then stained for intracellular IFN-γ. “Major” refers to dominant epitopes that induce the most robust CD8⁺ responses, while “intermediate” and “minor” refer to dominant epitopes that generate moderate and weak CD8⁺ responses, respectively, following acute LCMV infection. NP396–404, GP33–41, GP34–41, and GP276–286 wild-type peptides and variant peptides were used to stimulate splenocytes from LCMV-immunized and GPVNPV-immunized mice, respectively. A “v” indicates variant peptides and an arrow indicates D^b-restricted epitopes. Each mouse is analyzed individually, and results are averages of at least two independent experiments.

Figure 3

Augmenting epitope complexity does not change immunodominance of LCMV-specific CD8⁺ T cell responses. H-2^b wild-type and F₁ hybrid mice were inoculated i.p. with 1 × 10⁵ PFU of LCMV Armstrong. Eight days later, splenocytes from immunized mice were stimulated with the known 28 LCMV-specific H-2^b-restricted epitopes in the presence of BFA for 5 h. Total number (±SD, n = 6) of CD8⁺IFN-γ⁺ T cells were enumerated by staining for CD8 and intracellular IFN-γ. Each mouse is analyzed individually, and data are the averages of two independent experiments.

Figure 4

CD8⁺ T cell immunodominance is not altered by changing the context of viral infection. H-2^b mice were inoculated with either 1 × 10⁷ PFU rVACV-LCMV GPC, rVACV-LCMV NP, or rVACV-LCMV L. Seven days after rVACV infection, splenocytes were stimulated with indicated peptides and stained for CD8 and intracellular IFN-γ. Total number (±SD, n = 6) of CD8⁺IFN-γ⁺ T cells responding to the known 28 LCMV-specific epitopes. Each mouse is analyzed individually, and data are averages of two independent experiments.

Figure 5

Comparison between MHC binding affinity and the total number of epitope-specific CD8⁺ T cells producing IFN-γ after acute LCMV infection. Each filled circle represents the IC₅₀ value and total number CD8⁺IFN-γ⁺ T cells for the wild-type 28 LCMV-specific epitopes, while the open circles represent corresponding values for the variant epitopes. The horizontal lines correspond to IC₅₀ values of 25 and 500 nM, while the vertical lines are thresholds for either the major, intermediate, or minor epitopes.

Figure 6

Peptide-specific CD8⁺ T cell responses following peptide pool immunization. H-2^b mice were immunized s.c. with a peptide pool containing all 28 LCMV-specific peptides. Eleven to 12 days after immunization, splenocytes were stimulated with the indicated peptides and stained for CD8 and intracellular IFN-γ. Total number (±SD, n = 6) of CD8⁺IFN-γ⁺ T cells responding to each of the known LCMV-specific epitopes was measured. Each mouse is analyzed individually, and data represent average values from two independent experiments.

Figure 7

Frequencies of LCMV-specific T cell precursor populations found in naive H-2^b mice. Total number of tetramer-specific CD8⁺ T cells in spleen and lymph nodes from naive H-2^b mice (filled symbols; n = 15–19) and from OT-1 Rag^−/− TCR transgenic mice (open symbols; n = 3–5) following tetramer-based enrichment. Each symbol represents an individual mouse, whereas horizontal bars represent average precursor values. When no tetramer-specific CD8⁺ T cells were detected in OT-1 Rag^−/− mice, precursor values were set at a detection threshold of 10 cells.

Figure 8

Immunogenicity and naive T cell precursor frequencies specific for the GP33–41/D^b wild type and variant epitopes. A, Total number (±SD, n = 6) of CD8⁺IFN-γ⁺ T cells responding to the GP33–41/D^b wild-type and variant epitopes following individual peptide immunization of H-2^b mice. Each mouse is analyzed individually, and data represent average values from two independent experiments. B, Total number of GP33–41/D^b wild-type and GP33–41/D^b variant-specific CD8⁺ T cells in spleen and lymph nodes from naive H-2^b mice (filled symbols; n = 15–19) and from OT-1 Rag^−/− TCR transgenic mice (open symbols; n = 3–5) following tetramer-based enrichment. Each symbol represents an individual mouse, whereas horizontal bars represent average precursor values. When no tetramer-specific CD8⁺ T cells were detected in OT-1 Rag^−/− mice, precursor values were set at a detection threshold of 10 cells.