The impact of a boosting immunogen on the differentiation of secondary memory CD8+ T cells

Abstract

While recent studies have demonstrated that secondary CD8+ T cells develop into effector-memory cells, the impact of particular vaccine regimens on the elicitation of these cells remains poorly defined. In the present study we evaluated the effect of three different immunogens--recombinant vaccinia, recombinant adenovirus, and plasmid DNA--on the generation of memory cellular immune responses. We found that vectors that induce the rapid movement of CD8+ T cells into the memory compartment during a primary immune response also drive a rapid differentiation of these cells into effector-memory CD8+ T cells following a secondary immunization. In contrast, the functional profiles of both CD8+ and CD4+ T cells, assessed by measuring antigen-stimulated gamma interferon and interleukin-2 production, were not predominantly shaped by the boosting immunogen. We also demonstrated that the in vivo expression of antigen by recombinant vectors was brief following boosting immunization, suggesting that antigen persistence has a minimal impact on the differentiation of secondary CD8+ T cells. When used in heterologous or in homologous prime-boost combinations, these three vectors generated antigen-specific CD8+ T cells with different phenotypic profiles. Expression of the memory-associated molecule CD27 on effector CD8+ T cells decreased following heterologous but not homologous boosting, resulting in a phenotypic profile similar to that seen on primary CD8+ T cells. These data therefore suggest that the phenotype of secondary CD8+ T cells is determined predominantly by the boosting immunogen whereas the cytokine profile of these cells is shaped by both the priming and boosting immunogens.

FIG. 1.

Kinetics of HIV-1 Env-specific CD8⁺ T cells elicited after prime-boost immunization with different recombinant vectors. BALB/c mice were primed with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), or plasmid DNA (50 μg) expressing the HIV-1 envelope protein. Ten weeks after the first immunization, mice were boosted with homologous or heterologous combinations of immunogens using the previously mentioned vectors and the same quantity and route of administration used for the priming immunizations; p18-specific CD8⁺ T cells in the peripheral blood of individual mice were quantitated with an H-2D^d/p18 tetramer. Data are presented as the percentages of CD8⁺ T cells that bind tetramer and represent the means of five mice per group ± SE.

FIG. 2.

Gating strategies used for the phenotypic analysis. p18-specific CD8⁺ T cells were identified by gating on the CD8⁺ T cells in the lymphocyte population that bind the H-2D^d/p18 tetramer. These cells were divided into effector CD62L^lo CD127^lo (E), effector-memory CD62L^lo CD127^hi (EM), and central-memory CD62L^hi CD127^hi (CM) cell subsets using gates based on the relevant “fluorescence minus one” (FMO) controls (A). We then measured the expression of the CD27 surface molecule on these E, EM, and CM cell populations (B).

FIG. 3.

Differentiation of p18-specific CD8⁺ T cells into effector, effector-memory, and central-memory subsets. Mice were primed with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), or plasmid DNA (50 μg) expressing the HIV-1 envelope protein. Ten weeks after the first immunization, mice were boosted with homologous or heterologous combinations of immunogens using the previously mentioned vectors and the same quantity and route of administration used for the priming immunizations. Data are presented as the percentage of effector (E), effector-memory (EM), or central-memory (CM) p18-specific CD8⁺ T cells and represent the means of five mice per group ± SE.

FIG. 4.

Expression of CD27 on effector, effector-memory, and central-memory subsets of p18-specific CD8⁺ T cells. Mice were primed with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), or plasmid DNA (50 μg) expressing the HIV-1 envelope protein. Ten weeks after the first immunization, mice were boosted with homologous or heterologous combinations of immunogens using the previously mentioned vectors and the same quantity and route of administration used for the priming immunizations. Data are presented as the percentage of CD27 expression on effector (E), effector-memory (EM), or central-memory (CM) p18-specific CD8⁺ T cells and represent the means of five mice per group ± SE.

FIG. 5.

In vivo expression of the luciferase protein by rVac, rAd, and DNA plasmid. Mice were immunized with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), and DNA (50 μg) expressing the luciferase protein, and ten weeks later they were boosted with homologous or heterologous combinations of immunogens using the noted vectors and the same quantity and route of administration used for the priming immunizations. The levels of luciferase expression were measured over time in the immunized mice using IVIS. (A) Representative images of luciferase expression in the mice following priming immunization. (B) The mean values of the amount of luciferase expressed by groups of four or five mice ± SE following the priming immunization or following homologous or heterologous prime-boost immunizations using the different vectors. The dotted line represents the level of background luminescence. RLU, relative light units.

FIG. 6.

Functional analysis of p18-specific CD8⁺ T cells elicited after priming by different recombinant vaccine vectors. Mice were immunized with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), or plasmid DNA (50 μg) expressing the HIV-1 envelope protein. Splenocytes were harvested on day 7 (rVac), day 12 (rAd), or day 14 (plasmid DNA) after the priming immunization or 10 weeks later (preboost). The splenocytes were then cultured for 6 h in the presence of medium alone (Med.) or p18 peptide (2 μg/ml) (A) or cultured for 6 h in the presence of a pool of 47 overlapping peptides spanning the HIV-1 IIIB gp120 protein (2 μg/ml) (B). Data are presented as the percentages of tetramer-positive CD8⁺ T cells or Env peptide-reacting CD4⁺ T cells staining positively for IFN-γ, IL-2, or CD107a and CD107b and represent the means of five mice per group ± SE. *, P < 0.001 (production of cytokines by splenocytes of rVac- and plasmid DNA-immunized mice compared to cytokines produced after immunization with rAd); #, P < 0.001 (production of cytokines by splenocytes collected 10 weeks postimmunization compared to cytokines measured at the time of the peak immune response).

FIG. 7.

Analysis of Env-specific CD8⁺ and CD4⁺ T-cell function after homologous or heterologous boosting immunization. Groups of mice were primed with rVac (2 × 10⁷ PFU), rAd (2 × 10⁷ particles), or DNA (50 μg) (V, A, and D, respectively) and 10 weeks later were boosted with both homologous and heterologous combinations of immunogens using the noted vectors and the same quantity and route of administration used for the priming immunization. Splenocytes were harvested after the boosting immunization and cultured for 6 h in the presence of medium alone (Med.) or p18 peptide (2 μg/ml) (A), or cultured for 6 h in the presence of a pool of 47 overlapping peptides spanning the HIV-1 IIIB gp120 protein (2 μg/ml) (B). Data are presented as the percentages of tetramer-positive CD8⁺ or Env peptide-reacting CD4⁺ T cells staining positively for IFN-γ, IL-2, or CD107a and CD107b and represent the means of five mice per group ± SE. *, P < 0.001 (production of cytokines by CD4⁺ T cells following heterologous versus homologous prime-boost with rVac or rAd); #, P < 0.01 (production of cytokines by CD8⁺ T cells generated after rVac/rAd immunization compared to the other groups).