Antigen delivered by anthrax lethal toxin induces the development of memory CD8+ T cells that can be rapidly boosted and display effector functions

Abstract

Memory CD8+ T cells are essential for protective immunity against many intracellular pathogens; therefore, stimulation of this population of cells is an important goal of vaccination. We have previously shown that a detoxified derivative of Bacillus anthracis anthrax lethal toxin (LT) can deliver heterologous CD8+ T-cell epitopes to the major histocompatibility complex class I processing and presentation pathway of murine host cells and that immunization of mice with these LT-antigen fusion proteins leads to the induction of antigen-specific CD8+ T cells. In this report we extend these findings to include a detailed characterization of the phenotypic and functional properties of the T cells stimulated by the LT-based system. We found that after an initial period of expansion and contraction, antigen-specific CD8+ T cells differentiated into a pool of memory cells that produced gamma interferon and displayed in vivo cytotoxic activity. The transition to memory cells appeared to be quite rapid based on an analysis of the phenotypic marker CD127 and the effectiveness of a booster immunization administered early after the initial immunization. We also investigated the composition of the memory T-cell pool induced by this system and found that while one immunization induced a mixture of effector memory T cells (CD62Llow) and central memory T cells (CD62Lhigh), a second immunization preferentially elevated the effector memory T-cell frequency. Finally, we demonstrated that mice that received prime-boost immunizations of LT-antigen proteins were more protected in a Listeria monocytogenes challenge model than mice that received only one immunization.

FIG. 1.

NP-specific CD8⁺ T cells expand and contract following LT-NP immunization. Groups of BALB/c mice were immunized i.p. with 30 pmol of LT-NP, 30 pmol of PA, or 30 pmol of LFn-NP_118-126 in the absence of PA or were not immunized. The frequencies of splenic NP-specific T cells on the indicated days after immunization were determined by (A) IFN-γ ELISPOT analysis or (B) flow cytometry using L^dNP tetramers. Data were compiled from several independent experiments, and the values are the results for 3 to 25 mice (A) and 7 to 10 mice (B). The dotted line indicates the limit of detection for the ELISPOT assay. (C) Expression of the T-cell activation marker CD44 on CD8⁺ L^dNP⁺ splenocytes isolated at the peak of the response to either LT-NP (day 6) or L. monocytogenes-NP (LM-NP) (day 7) or on naïve CD8⁺ cells from unimmunized mice.

FIG. 2.

CD8⁺ T cells induced by LT-NP rapidly display a CD127^high phenotype. Groups of BALB/c mice were immunized with 30 pmol of LT-NP i.p. or 0.1 LD₅₀ of L. monocytogenes-NP (LM-NP) i.v. At the indicated days after immunization (days 6, 9, 12, and 28 for LT-NP and days 7, 9, 12, and 28 for L. monocytogenes-NP) splenocytes were enriched for T cells and analyzed by flow cytometry. NP-specific T cells (CD8⁺ L^dNP⁺, as defined by the gate shown in the dot plots on the left, where the numbers indicate the percentages of CD8⁺ cells that are L^dNP⁺ for the representative mice shown) were analyzed for CD127 expression. Representative dot plots indicate the percentages of CD127^high and CD127^low cells from individual mice. The graph shows the average percentage of CD127^high cells in the total CD8⁺ L^dNP⁺ cells from 3 to 10 mice per time point.

FIG. 3.

Booster LT-NP immunizations increase the frequency of NP-specific memory T cells. BALB/c mice were immunized i.p. either once or twice (with the second immunization 21 [D21] or 9 [D9] days after the first immunization, as indicated) with 30 pmol of LT-NP. Twenty-eight days after the final immunization the frequency of splenic, NP-specific CD8⁺ T cells was determined using L^dNP tetramers and flow cytometry. The data represent 4 to 16 mice per group, pooled from several independent experiments. For comparisons with mice immunized once, one asterisk indicates that the P value is <0.05, and two asterisks indicate that the P value is <0.01.

FIG. 4.

Reduced CD62L expression on secondary memory cells compared to primary memory cells. BALB/c mice were immunized i.p. either once or twice (21 days after the first immunization [D21]) with 30 pmol of LT-NP. Splenocytes were harvested 28 days later, enriched for T cells, and analyzed by flow cytometry. (A) Representative dot plots from individual mice demonstrating the distribution of CD62L^high and CD62L^low cells. Plots were gated on CD8⁺ L^dNP⁺ cells. (B) Percentages of CD62L^high and CD62L^low cells among CD8⁺ L^dNP⁺ cells from 10 to 16 mice per group compiled from three independent experiments. CD62L^high and CD62L^low were defined based on the gates indicated in panel A. (C) Calculated percent increases in the numbers of CD62L^high and CD62L^low cells 28 days after a second LT-NP immunization compared to the numbers 28 days after a first immunization.

FIG. 5.

LT-NP induces IFN-γ-producing, cytotoxic memory T cells that can be boosted by a second immunization. Groups of mice were immunized i.p. with 30 pmol of LT-NP either once or twice (9 [D9] or 21 [D21] days after the first immunization, as indicated) or were not immunized. (A) Splenocytes were harvested 28 days later, and the percentage of CD8⁺ T cells that produced IFN-γ in response to the NP peptide was determined by intracellular cytokine staining. (B) Twenty-eight days after the final immunization, mice were injected i.v. with a 1:1 mixture of CFSE^low NP peptide-pulsed splenocytes and CFSE^high unpulsed splenocytes. The percentage of in vivo specific lysis of the peptide-pulsed splenocytes was determined by flow cytometry 18 h later. The data are the means and standard deviations for 4 to 16 mice (A) or 10 to 15 mice (B) from several independent experiments. An asterisk indicates that the P value is <0.01 for a comparison with mice immunized once (A) or for a comparison with either unimmunized mice or mice immunized once (B).

FIG. 6.

LT-NP immunizations protect mice against L. monocytogenes-NP challenge. Groups of BALB/c mice were immunized with 30 pmol of LT-NP once or twice (21 days after the first immunization [D21]) or were not immunized. After 28 days all mice were injected i.v. with 0.73 LD₅₀ of L. monocytogenes-NP. The number of bacteria per spleen was determined 72 h later. The lines indicate the mean numbers of CFU/spleen for the groups. The results are representative of three independent experiments. The asterisk indicates that the P value is <0.01 for a comparison with unimmunized mice.