Duration of antigen availability influences the expansion and memory differentiation of T cells

Abstract

The initial engagement of the TCR through interaction with cognate peptide-MHC is a requisite for T cell activation and confers Ag specificity. Although this is a key event in T cell activation, the duration of these interactions may affect the proliferative capacity and differentiation of the activated cells. In this study, we developed a system to evaluate the temporal requirements for antigenic stimulation during an immune response in vivo. Using Abs that target specific Ags in the context of MHC, we were able to manipulate the duration of Ag availability to both CD4 and CD8 T cells during an active infection. During the primary immune response, the magnitude of the CD4 and CD8 T cell response was dependent on the duration of Ag availability. Both CD4 and CD8 T cells required sustained antigenic stimulation for maximal expansion. Memory cell differentiation was also dependent on the duration of Ag exposure, albeit to a lesser extent. However, memory development did not correlate with the magnitude of the primary response, suggesting that the requirements for continued expansion of T cells and memory differentiation are distinct. Finally, a shortened period of Ag exposure was sufficient to achieve optimal expansion of both CD4 and CD8 T cells during a recall response. It was also revealed that limiting exposure to Ag late during the response may enhance the CD4 T cell memory pool. Collectively, these data indicated that Ag remains a critical component of the T cell response after the initial APC-T cell interaction.

Figure 1. Antigen is required for several days for optimal CD8 T cell response to virus infection

Mice were infected with 10⁵ pfu of VSV-Ova-NJ and treated with 500ug of the blocking 25-D1.16 mAb at the indicated times. Control mice received mouse IgG1 isotype mAb. Mice were then bled at days 7, 9, 11, and 15 post-infection. OVA-specific CD8 T cells were quantitated by flow cytometry using an OVA:K^b tetramer. (A) Blood analysis at day 7; (B) Kinetics of the OVA-specific CD8 T cell response; (C) Analysis of ova-specific and VSV-nucleoprotein (N) -specific CD8 T cells after antibody treatment at day 0; (D) Total numbers of ova-specific splenic CD8 T cells seven days after infection and treatment with the indicated mAbs. The x-axis in A&B indicates the time post-infection that mice were treated with control or blocking antibody. Graphs represent the mean +/− SEM of Ova-tetramer positive cells as a percentage of the entire CD8 T cell population from 3–4 mice per group. These data are representative of three similar experiments. *, p< 0.01.

Figure 2. Blocking antigen presentation with 25-D1.16mAb after influenza virus infection inhibits the expansion of CD8 T cells

Mice were infected with 1000 pfu of WSN-OVA1 and treated with 500ug of the blocking 25-D1.16 mAb or IgG at the indicated times. On day 10 post infection cells from the lung, mediastinal lymph node (MLN) and spleen were stained with OVA:K^b tetramer and antibodies to CD8 and CD11a then analyzed by flow cytometry. A. Representative plots of gated MLN CD8+ lymphocytes showing staining for CD11a and OVA:K^b tetramer. Values indicate the percentages of tetramer+ cells among CD8+ T cells. B. Graph represents the total number of OVA-specific CD8+ cells in the indicated tissues; n=3. This experiment was performed 2 additional times with similar results. Days indicate the day on which mice were treated with mAb.

Figure 3. Inhibition of the CD8 T cell response by 25-D1.16mAb is through the abrogation of expansion of previously primed cells

Mice were infected with 1000 pfu WSN-OVA and treated with PBS or 500ug of the blocking 25-D1.16 mAb at the indicated times post infection. Mice from the different groups were sacrificed at the indicated times and lymphocytes isolated from the lung, MLN and spleen, stained with OVA:K^b tetramer and antibodies to CD8 and CD11a then analyzed by flow cytometry. Graphs depict mean +/− SEM of the total number of CD11^hitet⁺ CD8 cells in the indicated tissues of mice from the various groups; n=4.

Figure 4. Antigen specific CD4 T cells require sustained antigenic stimulation for optimal expansion and cytokine production

(A) Kinetics of TEa TCR transgenic CD4 T cells in the blood in response to VSV-GSE at different times during the primary immune response under conditions of varying antigen availability. 1×10⁴ TEa T cells were transferred one day before infection with 1×10⁵ pfu of VSV-GSE. Separate groups of mice received 500 μg of the Y -Ae antibody on different days of the response. At the time of infection, or at 24 hour intervals after infection for up to five days, mice were treated with Y-Ae. The populations of TEa cells were analyzed at the indicated times by flow cytometry using a congenic marker. Data represents the mean +/− SEM of four mice/group and are representative of several independent experiments. (B) Individual time points from A. The x-axis indicates the time after infection when mice were treated with the Y-Ae mAb. *, p <0.05. (C) Magnitude of the TEa response in spleen and lung at day 6 post-infection following Y-Ae treatment at day 1,2,3, or 4 post-infection. Mice were treated as in A, with the exception that they received 100ug of Ab. *, p <0.05. **, p<0.01. Data for A-C is derived from 3–4 mice per time point and treatment group and is representative of 3–4 experiments. (D) Limiting the duration of antigen availability results in reduced cytokine production by CD4 T cells. IL-2 and IFN-γ expression by TEa cells in the spleen 5 days after infection. Following adoptive transfer and infection as in (A), mice were injected with Y-Ae or mouse IgG2b control antibody at the indicated times after infection. 5 days after infection gated TEa cells were analyzed for IL-2 and IFN-γ production by intracellular staining after Eα peptide stimulation in vitro. Values represent the mean of 2 mice per group.

Figure 5. The duration of antigen availability during the primary immune response influences the generation of antigen-specific memory CD8 T cells

Mice were infected with VSV-OVA-NJ (A) or influenza virus (B) and treated with the 25-D1.16 mAb at the times indicated after infection. Control mice were treated with a mouse IgG1 mAb at the time of infection. At 10 weeks (A) or day 31 (B) post infection the indicated tissues were harvested and analyzed by flow cytometry for the presence of Ova-specific CD8 T cells. Data represents the percentage of Ova-tetramer positive CD8 T cells relative to the total CD8 T cell population from the indicated tissue. The x-axis represents the time after infection that mice were treated with mAb. Data is representative of two independent experiments with 3–4 mice per group. *, p<0.05.

Figure 6. Duration of antigen availability during the primary immune response influences the generation of antigen-specific memory CD4 T cells

1×10⁴ TEa CD4 T cells were adoptively transferred into congenic B6 mice. One day later mice were infected with VSV-GSE. At the times indicated after infection mice were treated with 100ug of Y-Ae mAb. Control mice were treated with 100ug of mouse IgG2b antibody. Four weeks later, cells from the spleen and lung were analyzed for the presence of donor TEa cells by flow cytometry. Graph indicates the mean +/− SEM from 5 mice per group (Day 1, n=3) and is representative of two independent experiments. *, p< 0.05.

Figure 7. Memory CD8 T cells require a reduced period of antigenic stimulation for optimal expansion

Mice were immunized with 1×10⁵ VSV-OVA-NJ 60 days prior to secondary challenge with 1×10⁵ VSV-OVA-Ind and treated with 500ug of 25-D1.16 mAb at the time of infection (D0) or at 24 hour intervals after the secondary challenge (D1-D4). Control mice (Ig Control) were treated with 500ug of mouse IgG1 mAb. Memory indicates the population of Ova-tet+ cells from mice that only received primary infection. Five days after secondary infection, Ova-tetramer positive CD8 T cells from the spleen and lung were assessed via flow cytometry. Graphs represent the mean +/− SEM from 4–5 mice per group of Ova-tetramer positive cells as a percentage of the entire CD8 T cell population. Representative data from two independent experiments. *, p < 0.01.

Figure 8. Memory CD4 T cells require a reduced period of antigenic stimulation during secondary encounter with antigen

1×10⁴ TEa CD4 T cells were adoptively transferred into B6 congenic mice that were subsequently infected with 1×10⁵ PFU of VSV-GSE-Ind. Seven months later mice were recalled with 1×10⁶ PFU of VSV-GSE-NJ. At the indicated times after recall infection, mice were treated with 500ug of Y-Ae mAb, or mouse IgG2b at the time of infection (Control). Upper left panel: memory TEa cells in the blood prior to recall infection. Cells were identified by flow cytometric analysis of CD4+CD45.2+ T cells. The x-axis denotes the mice assigned to each group for the recall response. Upper right panel: analysis of TEa cells in the blood 4 days after the recall response. Bottom panels: Five days after the recall response, the TEa CD4 T cell response in the spleen and lung was analyzed by flow cytometry. *, p < 0.05.

Figure 9. Duration of antigen availability and the recall response of antigen-specific memory CD4 T cells generated by peptide immunization

1×10⁴ naïve TEa CD4 T cells were adoptively transferred into congenic B6 mice. Memory TEa cells were generated as described in the materials and methods. Four weeks after immunization, memory TEa cells were recalled by infection with VSV-GSE-Ind. Five days after infection, cells from the spleen, lung, and peripheral lymph nodes (PLN) were analyzed for the presence of TEa cells (gated on CD4+CD45.2+ cells). Graphs represent the mean +/− SEM of TEa CD4 T cells relative to the total CD4 population from 4 mice per group. Data is representative of two independent experiments. *, p<0.05. **, p<0.01.