Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses

Abstract

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Figure 1.

LLO_91–99–specific CD8⁺ T cell response during primary and secondary infection with L. monocytogenes (A) Primary infection: mice infected with L. monocytogenes were left untreated (control) or received anti-CD4 mAb (anti-CD4). (B) Secondary infection: mice were infected and after 60 d challenged with L. monocytogenes. During challenge infection, mice were left untreated or received anti-CD4 mAb. At the indicated days, spleen cells were stained with Cy5-conjugated anti-CD8α mAb, FITC-conjugated anti-CD62L mAb, and PE-labeled LLO_91–99-tetramers, and analyzed by flow cytometry after the addition of propidium iodide. Figures show percent values of live CD62L^lowtetramer⁺ cells of total CD8⁺ cells. Data represent mean ± SD of three mice per group and time point. Experiments in A and B are representative of two or three experiments, respectively.

Figure 2.

Response against peptide and DNA boost-immunization of L. monocytogenes–primed mice after CD4⁺ T cell depletion. Mice were infected with L. monocytogenes. After 100 d, mice were immunized either with the peptide LLO_91–99 in incomplete Freund's adjuvant subcutaneously or with pChly DNA using the gene gun. Groups of mice were left untreated (control) or were injected with anti-CD4 mAb during the boost immunization. On day 7, spleen cells were stained and analyzed by flow cytometry. Dot plots depict CD62L and LLO_91–99-tetramer staining of viable CD8α gated cells. The experiment shown is representative of two independent experiments with three individually analyzed mice per group.

Figure 3.

Effector functions of LLO_91–99–specific CD8⁺ T cells after prime/boost DNA immunization. Mice were immunized with pChly using the gene gun. After 50 d, a boost immunization with the same DNA was performed. Mice were left untreated (control) or were treated with anti-CD4 mAb during the boost immunization. At day 7, mice were killed. (A) Spleen cells were stained and analyzed by flow cytometry. Dot plots depict CD62L and LLO_91–99-tetramer staining of viable CD8-gated T cells and figures represent percent values calculated for CD8⁺ T cells only. (B) Spleen cells were cultured for 5 h with or without the peptide LLO_91–99 and stained extracellularly for CD8 and intracellularly for IFN-γ and TNF-α or with isotype control mAbs. Dot blots show CD8-gated cells, and figures give percent values calculated for CD8⁺ T cells only. Values for isotype controls were below 0.05% (data not depicted). Dot blots in A and B show corresponding results from the same mice. (C) Spleen cells from untreated (diamonds) or anti-CD4 mAb-treated mice (triangles) were incubated for 4 h with target cells with (filled symbols) or without LLO_91–99 (open symbols). After 4 h, lysis of target cells was determined. C shows results from three individually analyzed mice per group. Experiments in A–C are representative for at least two independent experiments with three individually analyzed mice per experimental group in each experiment.

Figure 4.

Inhibition of memory CD8⁺ T cell responses by CD4⁺ and CD25⁺ cells BALB/c mice were immunized with pChly using the gene gun, and after 35 d, a boost immunization with the same DNA was performed. During boost immunization, mice were treated with purified rat Ig or the mAb indicated. On day 7, spleen cells were analyzed with LLO_91–99-tetramers (A) and for IFN-γ production after incubation with LLO_91–99 (B), as described in Fig. 3. Without peptide stimulation, we observed <4,000 IFN-γ⁺CD8⁺ T cells/spleen in all samples analyzed (data not depicted). The experiment shown is representative of three similar experiments and represents mean ± SD of three individually analyzed mice per group. Differences to rat Ig treatment: *P < 0.05; **P < 0.01; NS, P > 0.05.

Figure 5.

Inhibition of memory CD8⁺ T cell responses by CD4⁺CD25⁺ cells in a transfer model SCID mice received CD4-depleted cells from mice previously immunized by DNA vaccination. Groups of mice received in addition purified CD4⁺CD25⁻ and CD4⁺CD25⁺ cells from either naive or L. monocytogenes–infected mice. Immediately after cell transfer, mice were DNA immunized with pChly. On day 7, spleen cells were analyzed with LLO_91–99-tetramers (A) and for IFN-γ production after incubation with LLO_91–99 (B), as described in Fig. 3. Without peptide stimulation we observed <1,500 IFN-γ⁺CD8⁺ T cells/spleen in all samples analyzed (data not depicted). The experiment shown represents mean ± SD of three individually analyzed mice per group and is representative for three independent experiments. Differences to gene gun treated mice that received only CD4⁺ T cell–depleted cells: *P < 0.05; **P < 0.01; NS, P > 0.05.