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.

Figures

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 Kb/Ova tetramer-positive cells in the total CD8+ T cell population (left) and the absolute number of Kb/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 Kb/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) Kb/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 Kb/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.