Longitudinal requirement for CD4+ T cell help for adenovirus vector-elicited CD8+ T cell responses

Abstract

Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8(+) T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8(+) T cell responses by Ad vector immunization is longitudinally dependent on CD4(+) T cell help for a prolonged period. Depletion of CD4(+) T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8(+) T cells, decreased T-bet and eomesodermin expression, impaired KLRG1(+) effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8(+) T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4(+) T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8(+) T cells. These data demonstrate a prolonged temporal requirement for CD4(+) T cell help for vaccine-elicited CD8(+) T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8(+) T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4(+) T cell immune deficiencies.

FIGURE 1.

CD4⁺ T cell help is required for the development of CD8⁺ T cell responses following i.m. Ad vaccination. (A and B) C57BL/6, CD4 KO, or MHC class II KO mice were immunized i.m. with 10⁹ vp of Ad26-Gag or were unimmunized (naive C57BL/6). Animals were bled longitudinally, and SIV Gag-specific CD8⁺ T cells were quantified by tetramer staining. Representative plots (A) and group averages (B) for D^b/AL11⁺ cells as a percent of CD8⁺ T cells are shown. Data are from n = 8 per group (pooled from two independent experiments). (C and D) C57BL/6, CD4 KO, or MHC class II KO mice were coimmunized i.m. with 10⁹ vp each of Ad26-Gag and Ad26-Env. Splenocytes were harvested on day 28 after immunization and stimulated with the indicated peptide pool. Representative plots of IFN-γ⁺ CD8⁺ T cells (C) and group averages (D) are shown. Data are from n = 8 per group (pooled from two independent experiments). (E) C57BL/6, CD4 KO, or MHC class II KO mice were coimmunized i.m. with 10⁹ vp each of Ad5HVR48-Gag and Ad5HVR48-Env. Splenocytes were harvested on day 28 after immunization and stimulated with the indicated peptide pool. Percent of IFN-γ⁺ cells as a fraction of CD8⁺ T cells are shown. Data are from n = 8 per group (pooled from two independent experiments). Mean ± SEM are shown. *p < 0.01.

FIGURE 2.

Temporal requirement for CD4⁺ T cell help for the induction and expansion of CD8⁺ T cell responses following Ad vaccination. (A) C57BL/6 mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) beginning on days −1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 and immunized i.m. with 10⁹ vp of Ad26-Gag on day 0. Untreated controls are designated by a circle with a diagonal line though it. (B) Mice from each treatment group were bled at the indicated time points, and AL11-specific CD8⁺ T cells were assessed by D^b/AL11 staining. Inset, the fold-decrease in the number of AL11-specific CD8⁺ T cells in blood from day 14 to 28 after immunization is shown. Groups of mice administered with anti-CD4 beginning on day 9–13 were pooled for analysis. (C and D) On day 28 after immunization, the total number of AL11 peptide-specific CD8⁺ T cells per 10⁶ PBMCs (C) or per tissue (D) were quantified by D^b/AL11 staining. (E) On day 28 after immunization, the total number of AL11 peptide-specific CD8⁺ T cells was assessed by ex vivo restimulation with AL11 peptide followed by intracellular cytokine staining for IFN-γ. Data are from n = 7–22 per group (pooled from two to six independent experiments). Mean ± SEM are shown. *p < 0.001.

FIGURE 3.

Effector differentiation of CD8⁺ T cells is dependent on CD4⁺ T cell help following Ad vaccination. C57BL/6 mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) beginning on days 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 or were left untreated, and were immunized i.m. with 10⁹ vp of Ad26-Gag on day 0. On day 28 after immunization, animals were sacrificed and tissues were harvested. KLRG1 and CD127 expression on D^b/AL11⁺ CD8⁺ T cells was assessed. (A and B) Representative flow plots (A) and group averages (B) of KLRG1 and CD127 expression on D^b/AL11⁺ CD8⁺ T cells in the spleen. (C and D) Group summary of KLRG1 and CD127 expression of D^b/AL11⁺ CD8⁺ T cells in blood (C) and liver (D). (E) Absolute number of KLRG1⁺CD127⁻ or KLRG1⁻CD127⁺ D^b/AL11⁺ CD8⁺ T cells in the spleen. Anti-CD4 treatment on days −1, 1, 2, and 3 after immunization had insufficient numbers of D^b/AL11⁺ cells for analysis. Data are from n = 8–16 per group (pooled from two to four independent experiments), except for PBMC anti-CD4 day 9, 10, and 12 groups, which are n = 3–4 per group (one experiment). Mean ± SEM are shown. *p < 0.05.

FIGURE 4.

CD8⁺ T cells in mice depleted of CD4⁺ T cells exhibit an abnormal phenotype after i.m. Ad vaccination. C57BL/6 mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) on days 5, 8, or 11 or left untreated and immunized i.m. with 10⁹ vp of Ad26-Gag. On day 28 after immunization, animals were sacrificed and splenocytes were isolated. (A and B) Expression level of the indicated protein as a percentage (A) or mean fluorescence intensity (B) on gated D^b/AL11⁺ cells. Dashed lines indicate CD44^lo CD8⁺ (naive) T cells, and solid-shaded histograms indicate D^b/AL11⁺ CD8⁺ T cells. Data are from n = 8–16 per group (pooled from two to four independent experiments). (C) Gene expression of sorted D^b/AL11⁺ CD8⁺ T cells from untreated animals or animals treated with GK1.5 (anti-CD4) on days 5 or 8 after immunization was determined by quantitative real-time PCR. Gene expression was normalized to Gapdh. Data are from n = 4–6 per group (pooled from two independent experiments). (D) Annexin V expression and uptake of vital dye by D^b/AL11⁺ cells was assessed. Data are from n = 8 per group (pooled from two independent experiments). Mean ± SEM are shown. ^#p = 0.067, *p < 0.05.

FIGURE 5.

CD8⁺ T cells primed with reduced CD4⁺ T cell help fail to protect against lethal recombinant L. monocytogenes challenge. (A) C57BL/6 mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) on day 8 or left untreated, and immunized with 10⁹ vp of Ad26-GP. On day 30 after immunization, mice were challenged with 1.75 × 10⁵ to 2.5 × 10⁵ CFU Lm-GP33. (B) Frequency of D^b/GP33⁺ CD8⁺ T cells in the blood on day 30 after immunization. (C) KLRG1 and CD127 expression on D^b/GP33⁺ CD8⁺ T cells in the blood on day 30 after immunization. (D) Survival of mice after Lm-GP33 challenge. Data are from n = 16–32 per group (A–C) or n = 7–19 per group (D) pooled from two to four independent experiments. Mean ± SEM are shown. ^#p = 0.08, *p < 0.05.

FIGURE 6.

Normal contraction of the CD8⁺ T cell response requires prolonged CD4⁺ T cell help. (A) C57BL/6 mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) on days −1, 7, 14, 21, or 28 or left untreated, and immunized i.m. with 10⁹ vp of Ad26-Gag. (B) Animals were bled longitudinally, and Gag-specific CD8⁺ T cells were quantified by D^b/AL11 tetramer staining. (C) Frequency of KLRG1 and CD127 expression on D^b/AL11⁺ CD8⁺ T cells in blood. Data are from n = 8–12 per group (pooled from two to three independent experiments). Mean ± SEM are shown. *p < 0.05.

FIGURE 7.

CD8⁺ T cells primed with at least 8 d of CD4⁺ T cell help proliferate upon secondary Ag exposure. (A) CD45.1⁺ mice were depleted of CD4⁺ T cells by i.p. administration of mAb GK1.5 (anti-CD4) on days 8, 11, or 14 or left untreated, and immunized i.m. with 10⁹ vp of Ad26-Gag (day 0). On day 28 postimmunization, CD8⁺ T splenocytes were pooled, enriched by negative selection, and labeled with CFSE; 1.5 × 10⁴ D^b/AL11⁺ CD8⁺ T cells were transferred by tail vein injection into naive CD45.2⁺ recipients. One day after transfer, animals were immunized i.m. with 10⁹ vp of Ad5HVR48-Gag. (B) Representative plots of D^b/AL11⁺ CD45.1⁺ donor cells as a fraction of CD8⁺ T cells in the spleen on days 8 or 14. (C) Representative histograms of CFSE dilution in the donor D^b/AL11⁺ CD45.1⁺ CD8⁺ T cell population on day 8 after boost (open histogram) or the donor CD45.1⁺ CD8⁺ T cell population with no boosting immunization (solid histogram). (D) Absolute number of D^b/AL11⁺ CD45.1⁺ CD8⁺ T cells in the spleen on days 8 or 14 after immunization. Frequency of donor D^b/AL11⁺ CD45.1⁺ CD8⁺ T cells at the time of immunization (day 0) is based on 10% engraftment. Data are from n = 6–13 recipient mice per group on day 8 (pooled from three independent experiments) and n = 5 recipient mice per group on day 14 (from one experiment). Mean ± SEM are shown.

FIGURE 8.

CD40 signaling is required for programming CD8⁺ T cells with anamnestic potential. (A–C) C57BL/6, CD40L KO, and CD40 KO animals were immunized i.m. with 10⁹ vp of Ad26-Gag (n = 8 per group pooled from two independent experiments). (A) The number of D^b/AL11⁺ CD8⁺ T cells in the spleen of immunized animals on day 28 after immunization. (B) Splenocytes were harvested on day 28 after immunization and the number of IFN-γ⁺ CD8⁺ T cells was quantified following stimulation with an overlapping SIV Gag peptide pool. (C) KLRG1 and CD127 expression were assessed on splenic D^b/AL11⁺ CD8⁺ T cells on day 28 after immunization. (D and E) C57BL/6, CD40L KO, and CD40 KO animals were immunized i.m. with 10⁹ vp of Ad26-Gag and on day 56 after priming were boosted with 10⁹ vp of Ad5HVR48-Gag (n = 4 per group from one experiment). (D) The frequency of D^b/AL11⁺ CD8⁺ T cells per 10⁶ PBMCs after the boost was assessed. (E) The fold-change in frequency of D^b/AL11⁺ CD8⁺ T cells per 10⁶ PBMCs from 1 d before boost to day 7 after boost was assessed. (F) C57BL/6 or CD40L KO animals were immunized i.m. with 10⁹ vp of Ad26-Gag. On day 50 after immunization, 5 × 10⁴ D^b/AL11⁺ CD8⁺ T cells were enriched by negative selection and transferred i.v. to congenically marked (CD45.1⁺) naive recipients (n = 8–9 per group from two independent experiments). One day after transfer, recipient mice were immunized i.m. with 10⁹ vp of Ad5HVR48-Gag. On day 12 after secondary immunization, the number of donor D^b/AL11⁺ CD45.2⁺ CD8⁺ T cells in PBMCs, spleen, and liver was assessed. Mean ± SEM are shown. ^#p = 0.06, *p < 0.05.

FIGURE 9.

Schematic representation of longitudinal requirement for CD4⁺ T cell help. Summary of longitudinal requirement for CD4⁺ T cell help for the induction of CD8⁺ T cell responses, effector differentiation, and normal contraction following Ad vector vaccination. The requirement for CD4⁺ T cell help was prolonged but waned over time.