Boosting functional avidity of CD8+ T cells by vaccinia virus vaccination depends on intrinsic T-cell MyD88 expression but not the inflammatory milieu

Abstract

T-cell functional avidity is a crucial determinant for efficient pathogen clearance. Although recombinant DNA priming coupled with a vaccinia-vectored vaccine (VACV) boost has been widely used to mount robust CD8+ T-cell responses, how VACV boost shapes the properties of memory CD8+ T cells remains poorly defined. Here, we characterize the memory CD8+ T cells boosted by VACV and demonstrate that the intrinsic expression of MyD88 is critical for their high functional avidity. Independent of selection of clones with high-affinity T-cell receptor (TCR) or of enhanced proximal TCR signaling, the VACV boost significantly increased T-cell functional avidity through a decrease in the activation threshold. VACV-induced inflammatory milieu is not sufficient for this improvement, as simultaneous administration of the DNA vaccine and mock VACV had no effects on the functional avidity of memory CD8+ T cells. Furthermore, reciprocal adoptive transfer models revealed that the intrinsic MyD88 pathway is required for instructing the functional avidity of CD8+ T cells boosted by VACV. Taking these results together, the intrinsic MyD88 pathway is required for the high functional avidity of VACV-boosted CD8+ T cells independent of TCR selection or the VACV infection-induced MyD88-mediated inflammatory milieu.

Importance: Functional avidity is one of the crucial determinants of T-cell functionality. Interestingly, although it has been demonstrated that a DNA prime-VACV boost regimen elicits high levels of T-cell functional avidity, how VACV changes the low avidity of CD8+ T cells primed by DNA into higher ones in vivo is less defined. Here, we proved that the enhancement of CD8+ T cell avidity induced by VACV boost is mediated by the intrinsic MyD88 pathway but not the MyD88-mediated inflammatory milieu, which might provide prompts in vaccine design.

FIG 1

VACV boosts CD8⁺ T-cell functional avidity by decreasing the CD8⁺ T-cell activation threshold. (A) Vaccination schedule. Three vaccination regimens were included in these studies. Vaccine was administered intramuscularly (i.m.) to BALB/c mice at weeks 0, 2, 4, and 6. All assays for characterization of T-cell immunity were carried out 4 weeks after the final inoculation. The vaccines express HIV-1 CN54-Gag. (B to D) Magnitude of Gag-specific CD8⁺ T-cell responses induced by different regimens. Representative flow-cytometric plots of tetramer (tet) staining (B) and intracellular staining (C) are shown on the left. Summary data are shown on the right. The ELISpot data are shown in panel D. SFCs were counted for 10⁶ cells. (E to G) CD8⁺ T-cell functional avidity was enhanced by VACV boost. The functional avidity of a dominant epitope (E) and a subdominant epitope (F) are shown. The EC₅₀ data are shown in panel G. (H) The T-cell activation threshold was determined as the sensitivity of CD8⁺ T cells to anti-CD3ζ antibody stimulation. The immediate responses after stimulation were monitored by Ca²⁺ influx in antigen-specific CD8⁺ T cells by flow cytometry for 5 min. Examples of flow-cytometric plots are on the left, and the concentrations of anti-CD3ζ antibodies for activation of Ca²⁺ influx in tetramer-positive CD8⁺ T cells from each mouse are displayed on the right. Data are representative of at least three independent experiments with at least 4 mice per group. Statistical analysis was performed by t test using SPSS16.0 software, and the P values of the comparisons between any two groups are labeled.

FIG 2

Both VACV boost and DNA boost increase the functional avidity of CD8⁺ T cells by selecting a higher-affinity TCR Vβ repertoire. (A) T-cell affinity was determined with the tetramer dissociation assay. The percentages of MFI of T cells stained with diluted tetramer in the maximal MFI were calculated and graphed. (B and C) TCR Vβ preferential usages were determined with a panel of 15 anti-Vβ antibodies and are displayed for each mouse (B) and as grouped data (C). These experiments were performed as described in the legend to Fig. 1.

FIG 3

Enhanced functional avidity induced by VACV boost is independent of TCR selection and enhanced TCR proximal signaling. (A) Wild-type C57BL/6 mice were adoptively transferred with purified monoclonal TCR OT-I CD8⁺ T cells and immunized with vaccines that expressed OVA. Two vaccination regimens were administered at weeks 0 and 2, and assays were carried out 4 weeks after the last inoculation. (B) Numbers of antigen-specific CD8⁺ T cells from mice receiving a DNA boost and from mice receiving a VACV boost. (C) Functional avidity as measured by response to decreasing amounts of peptide antigen was assessed in CD8⁺ T cells from animals receiving either the VACV boost or the DNA boost. (D) EC₅₀ data were calculated for peptide concentrations in cells from animals receiving the VACV boost or the DNA boost. (E) The difference in the functional avidity was confirmed by using 2-fold dilutions of peptides. (F to H) The activation of proximal TCR signaling in the OVA-specific OT-I CD8⁺ T cells following DNA boost and VACV boost was assessed. (F) Splenocytes were stained for surface markers and stimulated with OVA peptide at 5 μg/ml, and then the levels of phospho-ZAP-70 and phospho-Lck were assessed by intracellular staining. Representative plots of gated CD8⁺ CD45.1⁺ cells stained with Phosflow antibodies are shown for the MFI of the activated forms of ZAP-70 (G) and Lck (H). Data are representative of three independent experiments with at least three mice per group. Statistical analysis was performed by t test using SPSS16.0 software, and the P values for the comparisons between any two groups are labeled. ns, not significant.

FIG 4

Boost in avidity in CD8⁺ T cells following VACV vaccination is independent of the inflammatory milieu. The bulk of CD8⁺ T cells from each immunized mouse group was mixed with equivalent amounts of splenocytes from naive mice (A) or mock VACV-infected mice (B). The functional avidity of CD8⁺ T cells was measured in response to decreasing peptide concentration. (C) CD8⁺ and CD8⁻ T cells from DNA vaccine (DNA-prime and DNA-boost) and VACV boost groups were cross-mixed before assessment of functional avidity of the CD8⁺ T cells. (D to F) Mixed mock VACV and DNA vaccines were administered to concurrently induce the inflammatory milieu with a DNA boost. The magnitude (D) and functional avidity (E and F) were measured. These experiments were performed as described in the legend to Fig. 1, and the dominant epitope, Gag49, was employed for stimulation. Data represent three independent experiments with at least three mice per group. Statistical analysis was performed by t test using SPSS16.0 software, and the P values of comparisons between any two groups are labeled.

FIG 5

MyD88 is essential for eliciting high-avidity CD8⁺ T cells. (A) MyD88^−/− mice were immunized i.m. with vaccines that express OVA. Two vaccination regimens were performed. The magnitude (B) and the functional avidity (C and D) of OVA-specific CD8⁺ T cells from MyD88^−/− and wild-type mice that were immunized were quantified by ELISpot assay. Statistical analysis was performed by t test using SPSS16.0 software, and the P values of comparisons between any two groups are labeled.

FIG 6

Attenuated cytokine production and normal maturation of MyD88^−/− DCs in response to VACV infection. (A) Secretion of inflammatory cytokines by splenocytes from WT mice or MyD88^−/− mice stimulated with DNA, mock VACV, and UV-inactivated mock VACV (UV-VACV) for 20 h in 96-well plates at 37°C. Medium was included as a negative control, and LPS was used as a positive control. (B) Comparison of the production of cytokines following treatment with mock VACV and UV-VACV in cells from MyD88^−/− and WT mice. LPS was used as a positive control. (C) Splenocytes from MyD88^−/− or C57BL/6 mice were harvested and incubated with mock VACV (MOI of 1), LPS (100 ng/ml), or medium in 96-well plates for 9 h and 20 h and then stained with the indicated antibodies. DCs were gated on CD11c⁺ CD14⁻ cells. The levels of CD80 and CD86 are presented as histograms. Data are representative of three independent experiments with at least three mice per group.

FIG 7

Transferring of WT CD8⁺ T cells into MyD88^−/− mice rescues the enhanced functional avidity of OVA-specific CD8⁺ T cells boosted by VACV. Purified OT-I CD8⁺ T cells were adoptively transferred into MyD88^−/− mice. The recipients were then immunized according to the schedule shown in panel A. The magnitude (B) and the functional avidity (C and D) of OVA-specific CD8⁺ T cells were quantified by ELISpot assay. Statistical analysis was performed by t test using SPSS16.0 software, and the P values of comparisons between any two groups are labeled.

FIG 8

T-cell-intrinsic MyD88 pathway is required for eliciting high-avidity CD8⁺ T cells following VACV boost. The OT-I mice were crossed with MyD88^−/− mice to generate MyD88-deficient T cells carrying TCR recognizing OVA (MyD88^−/− OT-I). Purified CD8⁺ T cells from MyD88^−/− OT-I mice were adoptively transferred into WT mice, which were immunized as depicted in panel A. The magnitude (B) and the functional avidity (C and D) of OVA-specific CD8⁺ T cells induced by VACV boost or DNA boost in both MyD88^−/− and WT transferred cells was analyzed. Statistical analysis was performed by t test using SPSS16.0 software, and the P values of comparisons between any two groups are labeled.