Article Figures & Data
Figures
- FIGURE 1.
rLM-OVA induces an OVA-specific CD4 T cell response. A, Schematic diagram of the Ag cassette in rLM-OVA. The cassette is integrated into the genome between two convergent transcriptional units, the lecithinase and lactate dehydrogenase (LDH) operons. It contains truncated OVA (aa 134–387) fused to the ha mAb epitope, the signal sequence (SS) and promoter (Phly) of the hly gene, and an erythromycin-resistance gene (Emr). B, Western blot analysis of supernatants from rLM-OVA and wtLM cultures. The OVA fusion protein (35 kDa) was detected using mAb to the ha epitope. C, Proliferation of OVA-specific CD4 T cells in rLM-OVA-infected mice. CFSE-labeled splenocytes from DO11.10 mice were adoptively transferred into BALB/c-Thy1.1 recipients, which were then infected with 5 × 104 CFU (0.1 LD50) of rLM-OVA or 5 × 103 CFU (0.1 LD50) of wtLM, or mock-infected with PBS. On day 8 postinfection, splenocytes were harvested and stained with mAb to Thy1.2, CD4, and KJ1-26. DO11.10 cells were identified by gating on Thy1.2+CD4+ cells and analyzed for CFSE fluorescence intensity. Data are representative of three experiments with at least two mice per group.
- FIGURE 2.
CD4 T cells undergo limited division and clonal expansion in comparison to CD8 T cells. CFSE-labeled splenocytes from DO11.10 and P14 mice were adoptively transferred into congenic Thy1.1 recipients, which were then infected with 5 × 104 CFU (0.1 LD50) of rLM-OVA or 5 × 105 CFU (0.1 LD50) of rLM-gp33, respectively. On days 0, 3, and 8 postinfection, splenocytes were harvested and stained with Thy1.2, CD4, and KJ1-26 mAb to identify DO11.10 cells or with Thy1.2, CD8 mAb, and/or Dbgp33 tetramer to identify P14 cells. A, Proliferation of OVA-specific CD4 T cells and gp33-specific CD8 T cells was analyzed by measuring their CFSE fluorescence intensity (nonrecruited cells are separated from recruited cells by a dashed line). B, IFN-γ production by responding OVA-specific CD4 T cells and gp33-specific CD8 T cells on day 8 postinfection was measured by intracellular cytokine staining (dotted lines, nonrecruited cells; solid lines, recruited cells). Data are representative of three experiments with at least two mice per group.
- FIGURE 3.
Responding CD4 T cells exhibit proliferative arrest in early divisions. A, CFSE profiles of OVA-specific cells following rLM-OVA infection. Experiments were performed as described in Fig. 1. A, On days 1, 14, and 21 postinfection, CFSE profiles of DO11.10 cells were analyzed. B, On days 3 and 8 postinfection, the forward scatters of DO11.10 cells were analyzed (dotted lines, recruited cells; solid lines, naive cells).
- FIGURE 4.
Infectious dose has little effect on the proliferative patterns of CD4 and CD8 T cells. Experiments were conducted as described in Fig. 2. CFSE profiles of DO11.10 and P14 cells were analyzed on day 8 postinfection with 0, 5 × 102, 5 × 103, or 5 × 104 CFU of rLM-OVA or rLM-gp33.
- FIGURE 5.
CD4 and CD8 T cells are inherently different in their proliferative responses. A, CFSE-labeled splenocytes from OT-II and OT-I mice were adoptively transferred into congenic Thy1.2 recipients, which were then infected with 5 × 105 CFU (0.1 LD50) of rLM-OVA. On days 0, 3, and 8 postinfection, proliferation of OVA-specific CD4 and CD8 T cells was analyzed by measuring their CFSE fluorescence intensity. B, Same as in A, except that enriched T cells from OT-II and OT-I mice were pooled, CFSE-labeled, and adoptively transferred into the same congenic Thy1.2 recipients. OT-I and OT-II cells were identified by first gating on the Thy1.1+Vα2+ subset and then separated by CD8+ staining. C, CFSE-labeled splenocytes from C57BL/6 mice were stimulated with anti-CD3 mAb and CFSE profiles of CD4 and CD8 T cells were analyzed at different time points to measure their proliferation. Similar results were obtained when splenocytes from BALB/c mice were used.
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