Defective CD8 T cell memory following acute infection without CD4 T cell help

Abstract

The CD8+ cytotoxic T cell response to pathogens is thought to be CD4+ helper T cell independent because infectious agents provide their own inflammatory signals. Mice that lack CD4+ T cells mount a primary CD8 response to Listeria monocytogenes equal to that of wild-type mice and rapidly clear the infection. However, protective memory to a challenge is gradually lost in the former animals. Memory CD8+ T cells from normal mice can respond rapidly, but memory CD8+ T cells that are generated without CD4 help are defective in their ability to respond to secondary encounters with antigen. The results highlight a previously undescribed role for CD4 help in promoting protective CD8 memory development.

Fig. 1

Mice deficientin CD4⁺ T cells show a progressive loss in protective immunity. (A) Wild-type C57BL/6 and major histocompatibility complex (MHC) class II–deficient (class II^−/−) mice were injected intravenously with 2 × 10³ rLmOva and, on the indicated number of days postinfection (PI), the number of live bacteria per spleen was determined (7). (B) Previously immunized mice were challenged intravenously with 10⁵ rLmOva at14, 28, and 60 days PI. Protective immunity was measured by determining the number of colony-forming units (CFU) in spleen 3 days after challenge. An unimmunized set of mice was also given 10⁵ rLmOva as a control. The values shown represent the mean CFU ± SE. The limit of detection in all experiments was 20 CFU per spleen (designated by the dotted line and arrow). (C) Mice were injected intravenously with 2 × 10³ rLmOva; 7, 14, 28, 60, and 120 days after immunization, memory CD8⁺ T cell numbers were measured. The percentage of K^b/Ova tetramer-positive cells in the total CD8⁺ T cell population (left) and the absolute number of K^b/Ova tetramer-positive cells in the spleen (right) are plotted. Numbers on the graphs represent the percentage of memory CD8⁺ T cells from class II^−/− compared with wild-type mice. The data shown are representative of two independent studies, with two to three mice per group at each time point.

Fig. 2

Equivalent primary CD8⁺ T cell response, but weak secondary response, in MHC class II– deficient mice compared with wild-type mice. Naïve and 60-day PI mice were given 2 × 10³ or 10⁵ rLmOva, respectively. Primary CD8⁺ T cell responses were measured 7 days PI by K^b/Ova-specific tetramer staining, intracellular IFN-γ staining, and ⁵¹Cr-release cytotoxicity assays (7). To examine secondary CD8⁺ T cell responses in previously immunized mice, we performed similar assays 3 days after challenge. (A and C) K^b/Ova tetramer and intracellular IFN-γ staining were done to determine the percentage of antigen-specific cells in the CD8⁺ T cell population (top numbers), as well as in the whole splenocyte pool (bottom numbers in parentheses). (A) CD44⁺, tetramer⁺ cells in the CD8⁺ T cell population. (C) CD8⁺, IFN-γ–producing cells in total splenocytes. (B) Total numbers of K^b/Ova-specific cells in spleens were determined. (D) Four-hour ⁵¹Cr-release assays were performed to measure specific killing of OVA peptide–loaded EL-4 cells by wild-type (filled squares) and MHC class II^−/− (open circles) splenocytes taken directly ex vivo. (E) At80 days PI, wild-type mice were given phosphate-buffered saline (PBS) or CD4-depleting antibody, GK1.5, before challenge with bacteria. The data shown are representative of two independent studies, with two mice per group at each time point.

Fig. 3

Memory CD8⁺ T cells from MHC class II^−/− mice show reduced ability to proliferate after transfer into wild-type mice. CD8⁺ T cells (Thy1.2) from wild-type and class II^−/− mice 80 days after priming were enriched with magnetic anti-CD4 bead depletion (7) and 10⁴ Ova-specific cells adoptively transferred into separate wild-type F₁ (C57BL/6 × B6.PL) (Thy1.1 × Thy1.2) hosts. The next day, all mice were injected intravenously with 2 × 10³ rLmOva, and CD8⁺ T cell responses were assayed 7 days later. As a control (to monitor the expansion levels of endogenous CD8⁺ T cells), F₁ hostmice that had not received memory cells were similarly infected. (A) Before adoptive transfer, CD4-depleted cell populations were stained for CD8 and intracellular IFN-γ. The percentages of Ova-specific cells within the CD8⁺ population (top numbers) and total pool (bottom numbers in parentheses) are shown. (B) On day 7 after infection, intracellular IFN-γ staining of splenocytes was performed. All plots show IFN-γ–producing transferred (Thy1.1⁻) and host (Thy1.1⁺) cells within the total cell population. The percentages of antigen-specific cells within the splenic CD8⁺ (top numbers) and total cell (bottom numbers in parentheses) population are shown.