The Timing of Stimulation and IL-2 Signaling Regulate Secondary CD8 T Cell Responses

<div><p>Abstract</p><p>Memory CD8 T cells provide protection to immune hosts by eliminating pathogen-infected cells during re-infection. While parameters influencing the generation of primary (1°) CD8 T cells are well established, the factors controlling the development of secondary (2°) CD8 T cell responses remain largely unknown. Here, we address the mechanisms involved in the generation and development of 2° memory (M) CD8 T cells. We observed that the time at which 1° M CD8 T cells enter into immune response impacts their fate and differentiation into 2° M CD8 T cells. Late-entry of 1° M CD8 T cells into an immune response (relative to the onset of infection) not only facilitated the expression of transcription factors associated with memory formation in 2° effector CD8 T cells, but also influenced the ability of 2° M CD8 T cells to localize within the lymph nodes, produce IL-2, and undergo Ag-driven proliferation. The timing of stimulation of 1° M CD8 T cells also impacted the duration of expression of the high-affinity IL-2 receptor (CD25) on 2° effector CD8 T cells and their sensitivity to IL-2 signaling. Importantly, by blocking or enhancing IL-2 signaling in developing 2° CD8 T cells, we provide direct evidence for the role of IL-2 in controlling the differentiation of Ag-driven 2° CD8 T cell responses. Thus, our data suggest that the process of 1° M to 2° M CD8 T cell differentiation is not fixed and can be manipulated, a notion with relevance for the design of future prime-boost vaccination approaches.</p></div

The timing of stimulation modulates the function of 2° M CD8 T cells.

<p><b>A)</b> Total numbers of 2° M Thy1.1 P14 CD8 T cells in mL of PBL, and in the spleen, lung, and LN of individual mice from ‘early’ and ‘late’ group of mice 7 months after the initiation of the experiment. <b>B)</b> Representative dot plots showing cytokine production by 2° M P14 CD8 T cells, isolated from the spleen of individual mice after short <i>ex vivo</i> incubation in the presence of GP33 peptide. Numbers represent the percentage of 2° M P14 CD8 T cells that were positive for IFNγ and IL-2. <b>C)</b> Percentage of IFNγ producing 2° M P14 CD8 T cells that co-produce IL-2. Dots represent individual mice and the line represents the mean. <b>D)</b> Experimental design, 2° M Thy1.1 P14 CD8 T cells from ‘early’ and ‘late’ groups of mice were isolated on day 260 after transfer by positive selection and transferred in equal numbers (1.5x10⁴ cells/mouse, i.v.) into naïve B6 Thy1.2/1.2 recipients 1 day before Att LM-GP33 (1x10⁷ CFU/mouse i.v.) infection. <b>E)</b> Representative histograms showing the expression of the molecules CD27, CD62L, and KLRG1 on transferred 2° M P14 CD8 T cells from ‘early’ and late’ groups of mice. Shaded graphs represent isotype control staining and open graphs represent specific Ab staining on gated 2° M Thy1.1 P14 CD8 T cells. <b>F)</b> Representative dot plots showing 3° expansion of P14 CD8 T cells on day 6 after infection with Att LM-GP33. Numbers indicate the percentage of P14 CD8 T cells in the PBL. <b>G)</b> Percentage of P14 CD8 T cells in the PBL of individual mice on day 6 is shown. Dots represent individual mice and the line represents the mean. Data are of 5 mice per group and representative of 2–3 independent experiments. The p values are indicated; ns- not significant.</p

1° M CD8 T cells are not recruited simultaneously after infection.

<p><b>A)</b> Experimental design. CFSE-labeled 1° M Thy1.1 P14 CD8 T cells (2x10⁶ cells/mouse, i.v.) were adoptively transferred into naïve B6 Thy1.2 recipient mice before LCMV infection. A non-infected group of recipient mice served as a control. The expression of <b>B)</b> CD25 and <b>C)</b> CD69 on CFSE-labeled 1° M P14 CD8 T cells isolated from the spleen of mice 24 and 48 hours after infection was determined. Numbers indicate the percentage of cells positive for the indicated molecule. Representative profiles of 3 mice per group are shown. Experiments in panels A-C are representative of 3 independent and similar experiments.</p

The timing of stimulation regulates IL-2 sensitivity and expression of cell-cycle related proteins.

<p><b>A)</b> Representative histograms showing the expression of the CD25 molecule on 2° effector Thy1.1 P14 CD8 T cells isolated from the spleens of ‘early’ and ‘late’ groups of mice on indicated days after transfer. Shaded graphs represent isotype control staining and open graphs represent specific Ab staining on gated 2° effector Thy1.1 P14 CD8 T cells. Histograms are concatenated using FlowJo software from 1 of 2 independent experiments displaying equal representation from 3 individual mice. <b>B)</b> Percentage of 2° effector P14 CD8 T cells positive for CD25 on various days after transfer. Data are presented as mean+ SEM of 3 mice per group. <b>C)</b> Log geometric mean fluorescence intensity (gMFI) of CD25⁺ 2° effector P14 CD8 T cells. Data are presented as mean+ SEM of 3 mice per group. <b>D)</b> Histograms showing the expression of CD25 on 2° effector P14 CD8 T cells isolated from the spleen 5 days after transfer. Shaded graphs represent isotype control staining and open graphs represent staining on gated 2° effector Thy1.1 P14 CD8 T cells. Black numbers indicate the percentage of P14 CD8 T cells positive for CD25 and grey numbers indicate the gMFI of CD25⁺ P14 CD8 T cells. Histograms are concatenated using FlowJo software from one of two independent experiments displaying equal representation from 4–5 individual mice. <b>E)</b> STAT5-pY694 was measured in 2° effector P14 CD8 T cells in the absence (shaded histograms) or after 15 minutes of IL-2 stimulation (open histograms). <b>F)</b> Protein expression of Foxm1, Cyclin A, and Cyclin B1 in 2° effector (day 7) and 1° M P14 CD8 T cells. All experiments are representative of 2–3 independent experiments. The p values are indicated; ns-not significant.</p

Timing of stimulation impacts proliferative expansion and transcriptional program of 2° effector CD8 T cells.

<p><b>A)</b> Experimental design. Naïve B6 Thy1.2/1.2 mice received a transfer of 1° M Thy1.1 P14 CD8 T cells (2x10⁴ cells/mouse, i.v.) on the day of (‘early’ group) or 3 days after (‘late’ group) infection with LCMV (2x10⁵ PFU/mouse i.p.). <b>B)</b> The percentage of 2° effector P14 CD8 T cells in the PBL at day 7 after transfer. Dots represent individual mice and the line represents the mean. <b>C)</b> Representative dot plots showing the expression of KLRG1 and CD127 molecules on 2° effector P14 CD8 T cells isolated from the spleen at day 7 after transfer. The percentage of 2° effector P14 CD8 T cells expressing a <b>D)</b> KLRG1^hi CD127^lo or <b>E)</b> KLRG1^lo CD127^hi phenotype. <b>F)</b> Total RNA was extracted from 2° effector P14 CD8 T cells and analyzed for the expression of indicated transcripts using quantitative RT-PCR. Relative expression to Hprt is shown. The data are mean + SD of triplicate measurements of a total of three samples from each group. G) Representative histograms showing the expression of the molecules Bcl2, Eomes, Tcf1, and Tbet on 2° effector P14 CD8 T cells from spleens of mice from ‘early’ and ‘late’ groups. Shaded graphs represent isotype control staining and open graphs represent specific Ab staining on gated 2° effector Thy1.1 P14 CD8 T cells. Black numbers indicate the percentage of P14 CD8 T cells positive for indicated markers and grey numbers indicate gMFI of P14 CD8 T cells. Data are of 3–5 mice per group and experiments are representative of 2–3 independent experiments. The p values are indicated.</p