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. 1997 Jan 20;185(2):251-62.
doi: 10.1084/jem.185.2.251.

Distinct costimulatory molecules are required for the induction of effector and memory cytotoxic T lymphocytes

Y Liu  1 , R H Wenger, M Zhao, P J Nielsen
Affiliations

Distinct costimulatory molecules are required for the induction of effector and memory cytotoxic T lymphocytes

Y Liu et al. J Exp Med. .

Abstract

A successful T cell immune response has two major products: effector T cells which directly or indirectly remove the antigens, and memory T cells, which allow a faster and more efficient recall response when challenged by related antigens. An important issue is whether costimulatory molecules on the antigen-presenting cells are involved in determining whether T cells will differentiate into effector or memory cells after antigenic stimulation. To address this issue, we have produced mice with targeted mutations of either the heat-stable antigen (HSA), or both HSA and CD28. We show that CD28/B7 and HSA provide two alternative costimulatory pathways for induction of immunological memory to influenza virus. Furthermore, our results revealed that B7 is essential for the generation of effector T cells from either naive or memory T cells, while HSA is not necessary for the generation of effector T cells. Our results demonstrate that the induction of memory T cells and effector T cells can utilize distinct costimulatory molecules. These results have important implications on lineage relationship between effector and memory T cells.

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Figures

Figure 1
Figure 1
Production of mice homozygous for a disrupted HSA gene. (a) The disruption of the HSA gene in C57BL/6 ES line BL/6-III was achieved by replacement of the HSA promotor and the first exon with a neomycin-resistance expression cassette. The coding portions of both HSA exons are depicted by filled boxes. (b) Southern blot analysis of HSA genotypes using a Pvu II fragment located between nucleotides −710, and −1790 relative to the transcriptional start site of the HSA gene as a hybridization probe.
Figure 2
Figure 2
Mice with targeted mutations of HSA and/or CD28 produce normal numbers of T cells, B cells and APC. Mouse spleen cells were analyzed by flow cytometry after staining with either anti-HSA (20C9), anti-CD28 (37N), anti-Mac-1 (TIB128), anti-dendritic cell (HB224), FITC-labeled anti-CD4, and phycoerythrin-labeled anti-CD8 mAbs. CD4, CD8, CD28, B220, and HSA typing was done with freshly isolated spleen cells in pools from three mice. Expression of CD28 on gated T cells is presented. Macrophage and dendritic cells were determined using low-density spleen cells, enriched by centrifugation over a 55% Percoll medium. The genotypes and antibodies used are marked on the panels.
Figure 3
Figure 3
Primary in vivo CTL response against influenza virus in WT mice (a), and mice with a targeted mutation of HSA (b), CD28 (c), or of both HSA and CD28 (d). Spleen cells were harvested from mice on day 7 after A/JAP-infection. The CTL activities were tested on either peptide (AA365-380 of the nucleoprotein from A/JAP virus)- pulsed (EL4-NP), or unpulsed EL4 cells (EL4). Representatives of three independent experiments using pooled spleen cells from 2–4 mice in each group are shown.
Figure 4
Figure 4
Anti-B7 mAbs completely block the production of effector CTL from naive T cells in both WT mice (a) or HSA-KO (b) mice. WT and mutant mice were infected with 1,000 HAU/mouse of influenza virus A/JAP by intraperitoneal injection on day 0. The mice were injected with either a mixture of normal rat and hamster IgG, or anti-B7-1 + anti-B7-2 mAbs (3A12+GL1) on days −1, 0, +1, at a dose of 100 μg/ mouse/injection. Data presented are representative of three experiments using pooled spleen cells from 2–3 mice per group.
Figure 5
Figure 5
Requirement of costimulatory molecules for the generation of primed T cells assayed at 8 d after priming: microplate culture in the absence of exogenous cytokines. (a) Anti-viral CTL of in vitro-stimulated spleen cells from either naive or A/JAPprimed mice. (b) Requirement of B7 family members for the generation of primed T cells. WT and HSA-KO mice were infected with 1,000 HAU/mouse of influenza virus A/JAP by intraperitoneal injection on day 0. On days −1, 0, +1, these mice were injected with either a mixture of normal rat and hamster IgG, or anti-B7-1 + antiB7-2 mAbs (3A12+GL1) at a dose of 100 μg/mouse/injection. Spleen cells were harvested 8 d after viral infection and were restimulated with irradiated, A/JAP-infected syngeneic spleen cells for 6 d in vitro, and CTL activity determined. Responder cells used were from pools of two spleens. Data in b were lysis of NP peptide (AA365-380)-pulsed EL4 target (104/well) only. The lysis of unpulsed EL4 cells is not shown but was always less than 10%. Representative of three experiments, with two mice per group are shown.
Figure 6
Figure 6
Either HSA or CD28-mediated costimulation is sufficient for generation of primed T cells: bulk cultures. (a and b) Anti-HSA mAb 20C9 blocks T cell memory in CD28KO (b) but not WT mice (a), as measured by recall CTL responses in vitro. CD28-deficient mice and syngeneic WT mice were treated with either normal hamster Ig or anti-HSA mAb (300 μg/mouse/injection) on day −1, day 0, and day 1. On day 0, these mice were injected intraperitoneally with influenza virus A/JAP (1,000 HAU/mouse), spleen cells were harvested on day 7; and were restimulated with A/JAP-infected (1,000 HAU/12 × 106 cells, 37°C,1 h), irradiated syngeneic spleen cells for 5 d (responder: stimulator ratio 5:1, with responder cells at a density of 106/ml). At the end of culturing, viable cells were harvested and the cytotoxicity of these cells was determined by a 6 h 51Cr-release assay. (c) Recall CTL responses in mice with different targeted mutations. The pooled spleen cells from groups of 2–3 mice were harvested on day 7 after i.v. injection of A/JAP (300 HAU), recall CTL activity determined as in a and b. Representatives of 2–3 independent experiments are shown. Note overall CTL responses in c are stronger than in a and b due to a stronger priming via i.v. injection. Dashed lines are the lysis of EL-4 cells in the absence of NP peptide.
Figure 7
Figure 7
Requirement for costimulatory molecules in the generation of memory T cells assayed at 100 d after priming: microplate culture in the absence of exogenous cytokines. Both WT and HSA-KO mice were injected with influenza virus A/JAP (1,000 HAU/mouse) (day 0) and treated with either a mixture of hamster and rat Ig or a mixture of anti-B7-1 plus B7-2 on days −1, 1, 3, 7, 10, 13, 21 (200 μg/ mAb/injection). At 100 d after the viral infection, pooled spleen cells (three mice per group) were stimulated in vitro, CTL activity was determined 5 d after stimulation. Similar results were obtained when memory CTL activity was measured at 30 d after infection.
Figure 8
Figure 8
B7, but not HSA, plays an important role in the induction of effector T cells from memory T cells, regardless of the priming conditions: microplate culture in the absence of exogenous cytokines. Spleen cells from either WT (a and b) or HSA-deficient mice (c and d) pretreated with either normal Ig (a and c) or a mixture of anti-B7-1 and anti-B7-2 (b and d) were infected with A/JAP virus as detailed in the legend to Fig. 3. Increasing numbers of spleen cells were stimulated in vitro for 5 d with A/JAP-infected spleen cells, in the presence of either medium, or a mixture of anti-B7-1 and anti-B7-2, or anti-HSA mAb (final concentration at 5 μg/ml), and CTL activity was determined in a 6-h 51Cr-release assay. Representative of three independent experiments are shown. Similar results were obtained when memory activity is measured in bulk cultures.
Figure 9
Figure 9
A model for the involvement of costimulatory signals in the generation of effector and memory T cells. Two major products, effector and memory T cells, are produced from naive T cells after viral infection. The production of memory T cells requires costimulation by either HSA or B7, while the production of effector T cells utilizes B7 but not HSA. By definition, memory T cells give rise to effector T cells after further stimulation by antigen. However, effector T cells are unlikely to be mandatory precursors for memory T cells and distinct costimulatory molecules could be used at different phases of the immune response.
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