Subsets of memory cytotoxic T lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo

Abstract

Vaccine-elicited cytotoxic T lymphocytes (CTL) should be long-lived memory cells that can rapidly expand in number following re-exposure to antigen. The present studies were initiated to analyze the ability of plasmid interleukin-12 (IL-12) to augment CTL responses in mice when delivered during the peak phase of an immune response elicited by a plasmid human immunodeficiency virus type 1 gp120 DNA vaccine. Delivery of plasmid IL-12 on day 10 postimmunization resulted in a robust expansion of gp120-specific CD8+ T cells, as measured by tetramer, gamma interferon ELISPOT, and functional-killing assays. Interestingly, this delayed administration of plasmid IL-12 had no significant effect on antigen-specific CD4(+)-T-cell and antibody responses. Phenotypic analyses suggested that administration of plasmid IL-12 near the time of the peak CTL response activated and expanded antigen-specific effector cells, preventing their loss through apoptosis. However, this IL-12-augmented population of gp120-specific CD8+ T cells did not efficiently expand following gp120 boost immunization, suggesting that these effector cells would be of little utility in expanding to contain a viral infection. Analyses of the phenotypic profile and anatomic distribution of the plasmid IL-12-augmented CTL population indicated that these lymphocytes were primarily effector memory rather than central memory T cells. These observations suggest that CTL-based vaccines should elicit central memory rather than effector memory T cells and illustrate the importance of monitoring the phenotype and functionality of vaccine-induced, antigen-specific CTL.

FIG. 1.

p18-specific CD8⁺-T-cell responses elicited by DNA vaccination with delayed plasmid IL-12 administration. Groups of BALB/c mice were immunized with 50 μg of sham PMV plasmid, 50 μg of PMV-gp120 alone, or 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 given concurrently or on day (d.) 4, 7, 10, or 14 following vaccine administration. Peripheral blood was obtained from individual mice at the indicated times, and antigen-specific CD8⁺ T cells were detected with an H-2D^d/p18 tetramer. Data are presented as the percentage of gated CD8⁺ T cells that bound tetramer, as measured by flow cytometry, and are the means of six mice per group ± SEM.

FIG. 2.

Functional analysis of CD8⁺ T cells from mice immunized with DNA vaccine with or without delayed plasmid IL-12 administration. Groups of BALB/c mice were immunized with 50 μg of sham PMV plasmid, 50 μg of PMV-gp120 alone, or 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 on day (d.) 10 following vaccine administration. Spleen cells were harvested from individual mice on day 18 postimmunization for functional analysis. (A) Total splenocytes or splenocytes depleted (Depl.) of CD8⁺ or CD4⁺ T cells were evaluated for IFN-γ production in an ELISPOT assay following stimulation with the gp120-derived p18 epitope peptide or a pool of 47 overlapping peptides spanning the HIV-1 IIIB gp120 protein. Data are presented as the mean number of antigen-specific spots per 10⁶ spleen cells ± SEM with four mice per group. (B) Spleen cells from mice immunized with sham PMV plasmid (triangles), PMV-gp120 alone (circles), or PMV-gp120 plus plasmid IL-12 DNA (squares) were cultured for 7 days in the presence of 10 ng of p18 peptide per ml. Cells were then harvested and used as effectors in a ⁵¹Cr release assay to assess lysis of P815 tumor cell targets incubated overnight in the presence of medium alone (open symbols) or p18 peptide (filled symbols). Results are expressed as the percent specific ⁵¹Cr release and are the means ± SEM of four mice per group. E:T ratio, effector-to-target ratio.

FIG. 3.

Splenocyte gp120-specific proliferative responses and serum anti-gp120 antibody titers elicited by DNA vaccination with or without delayed plasmid IL-12 administration. (A) Groups of BALB/c mice were immunized with 50 μg of sham PMV plasmid, 50 μg of PMV-gp120 alone, or 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 on day (d.) 10 following vaccine administration. Spleen cells from individual mice were harvested on day 18 postimmunization for functional analysis. Total splenocytes or splenocytes depleted of CD4⁺ or CD8⁺ T cells were stimulated with the indicated concentrations of recombinant HIV-1 IIIB gp120 protein, and proliferation responses were measured by [³H]thymidine incorporation. Results are presented as the mean SI ± SEM of four mice per group. (B) Groups of BALB/c mice were immunized with 50 μg of sham PMV plasmid, 50 μg of PMV-gp120 alone, or 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 given on the indicated days. On day 28 postimmunization, serum was collected from individual mice and the titer of gp120-specific antibody was determined by ELISA. Data are presented as the geometric mean titer ± SEM of eight mice per group.

FIG. 4.

Phenotypic analysis of p18-specific CD8⁺ T cells elicited by DNA vaccination with or without delayed administration of plasmid IL-12. BALB/c mice were immunized with 50 μg of PMV-gp120 alone (black bars) or with 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 on day 10 following vaccine administration (checkered bars). Spleen cells were harvested from individual mice at the indicated times, and the expression of CD11a (A) and CD54 (B) on gated H-2D^d/p18 tetramer-positive CD8⁺ T cells was measured by flow cytometry. Expression of these molecules on CD8⁺ T cells from naive animals is also indicated (hatched bars). Data are presented as the mean fluorescence intensity (MFI) and represent the means of four mice per group ± SEM.

FIG. 5.

Annexin V staining of p18-specific CD8⁺ T cells elicited by DNA vaccination with or without delayed administration of plasmid IL-12. Groups of BALB/c mice were immunized with 50 μg of PMV-gp120 alone (closed circles) or with 50 μg of PMV-gp120 plus 200 μg of PMV-IL-12 on day 10 following vaccine administration (open circles). Spleen cells from individual mice were harvested at the indicated times and stained with anti-CD8 MAb, H-2D^d/p18 tetramer, and annexin V. Data are presented as the percentage of gated CD8⁺ T cells that bound tetramer (A) and the percentage of gated tetramer-positive CD8⁺ T cells that bound high levels of annexin V (B), as measured by flow cytometry. The background level of annexin V binding to gated CD8⁺ T cells from naive animals is also indicated. Data are the mean of four mice per group ± SEM.

FIG. 6.

p18-specific memory CD8⁺-T-cell expansion elicited in mice primed by DNA vaccination with or without delayed plasmid IL-12 administration. Groups of BALB/c mice were immunized with 50 μg of PMV-gp120 plus either 200 μg of sham PMV plasmid or 200 μg of PMV-IL-12 given on day (d.) 10 following vaccine administration. On day 130 postimmunization, mice were boosted with 50 μg of PMV-gp120 plasmid alone. Peripheral blood was obtained from individual mice at the indicated times, and antigen-specific CD8⁺ T cells were detected with an H-2D^d/p18 tetramer. Data are presented as the percentage of gated CD8⁺ T cells that bound tetramer, as measured by flow cytometry, and are the means of eight mice per group ± SEM.

FIG. 7.

Analysis of p18-specific CD8⁺-T-cell central memory and effector memory subsets elicited by DNA vaccination with or without delayed administration of plasmid IL-12. Groups of BALB/c mice were immunized as described in the legend to Fig. 6. Peripheral blood was obtained from individual mice at the indicated times, and expression of CD62L on gated H-2D^d/p18 tetramer-positive CD8⁺ T cells was measured by flow cytometry. Data are presented as the percentage of CD8⁺ T cells that were either tetramer positive and CD62L⁺ or tetramer positive and CD62L⁻ and are the means of eight mice per group ± SEM. d., day.