Exogenous IL-2 delays memory precursors generation and is essential for enhancing memory cells effector functions

Abstract

To investigate the impact of paracrine IL-2 signals on memory precursor (MP) cell differentiation, we activated CD8 T cell in vitro in the presence or absence of exogenous IL-2 (ex-IL-2). We assessed memory differentiation by transferring these cells into virus-infected mice. Both conditions generated CD8 T cells that participate in the ongoing response and gave rise to similar memory cells. Nevertheless, when transferred into a naive host, T cells activated with ex-IL-2 generated a higher frequency of memory cells displaying increased functional memory traits. Single-cell RNA-seq analysis indicated that without ex-IL-2, cells rapidly acquire an MP signature, while in its presence they adopted an effector signature. This was confirmed at the protein level and in a functional assay. Overall, ex-IL-2 delays the transition into MP cells, allowing the acquisition of effector functions that become imprinted in their progeny. These findings may help to optimize the generation of therapeutic T cells.

Keywords: Biological sciences; Immune system evolution; Immunology; Molecular biology.

Graphical abstract

Figure 1

Ex-IL-2 impacts the phenotype but not the proliferation of in vitro activated CD8 T cells See also Figure S1. (A–F) 1.5 × 10⁵ magnetically purified naive F5 CD8 T cells labeled with CTV (CellTrace Violet) were cultured with CpG-matured, peptide-loaded cDC at a ratio of cDC:CD8 = 1:10, in the presence or absence of ex-IL-2 (11,5 ng/mL). The strategy to gate divided CD8 T cells is described in Figure S1A. (A) Left: The number of divided CD8 T cells (that has undergone at least one division) was determined on days 3, 4 and 5. Right: The cell number ratio between cells cultured in the absence or presence of ex-IL-2 was calculated. (B) CD8 proliferation in the presence (red) or absence (black) of ex-IL-2 was analyzed after 4 days by CTV dilution and is represented as overlay histogram. (C) Median fluorescence intensity (MFI) of Ki67 was measured on divided cells after 4 days of activation and the MFI-ratio between cells cultured in the absence or presence of ex-IL-2 was calculated. (D) Expression of CD25, EOMES and Bcl-2 by divided CD8 T cells was analyzed 4 days after activation. Representative histograms of cells cultured in the presence (red) or absence (black) of ex-IL-2 is shown. (E) Kinetics of the percentage of EOMES⁺ and CD25⁺ cells, as well as the level of Bcl-2 expression by divided CD8 T cells. (F) Expression of pAkt and pSTAT5 by divided CD8 T cells were analyzed 4 days after activation. Representative histograms (left panel) and MFI (right panel) of cells cultured in the presence (red) or absence (black) of ex-IL-2 are shown. The mean ± SEM of triplicate cultures from one representative experiment out of at least five independent experiments is shown in panel A (right)-B-D-E, and one out of two in panel F. The mean ± SD of five and six experiments is shown in panel A (Left) and C, respectively. The statistical significance of the difference between the mean value of ratios and the hypothetical value of 1 was determined by the one sample t-test.

Figure 2

Lower CD8 cellular concentration strongly increases the dependency on ex-IL-2 See also Figure S2. (A, B, D, E) 1.5 × 10⁵, 5 × 10⁴ or 1.5 × 10⁴ purified naive F5 CD8 T cells labeled with CTV were cultured with CpG-matured, peptide-loaded cDC at a ratio of cDC:CD8 = 1:10, in the presence or absence of ex-IL-2 (11,5 ng/mL) for 4 days. (A) Left: The number of divided CD8 T cells (that has undergone at least one division) was determined for each cell concentration. Right: The cell number ratio between cells cultured in the absence or presence of ex-IL-2 was calculated for each cell concentration. (B) CD8 proliferation in the presence (red) or absence (black) of ex-IL-2 was analyzed after 4 days by CTV dilution and represented as overlay histograms. (C) 5 × 10⁴ purified naive F5 CD8 T cells labeled with CTV were activated as in (A), in the presence or absence of 3 × 10⁵ C57BL/6J splenocytes, and their proliferation was analyzed by CTV dilution 4 days later. (D) Median fluorescence intensity (MFI) of CD25 and Bcl-2 was measured on divided cells for each cell concentration. (E) Expression of CD62L by CD8 T cells activated at different concentrations with ex-IL-2. Representative histograms (Left) and individual percentages of positive cells (Right) are shown. The mean ± SEM of triplicate cultures from one representative experiment out of three independent experiments is presented in panel A (right) and D. The mean ± SD of three independent experiments is shown in panel A (Left) and E. In A (Left), the statistical significance of the difference between the mean value of ratios and the hypothetical value of 1 was determined by the one sample t-test (A). In E, the statistical significance of differences was determined by one-way ANOVA followed by Tukey’s post-hoc test (ns = p > 0.05, ∗ = p ≤ 0.05).

Figure 3

Cells activated with and without ex-IL-2 have a similar potential to participate in an ongoing immune response See also Figure S3. (A–F) CTV-labelled purified naive F5 CD8 T cells, at a concentration of 6 × 10⁵/mL (B and D-F) or 1.2 × 10⁵/mL (3 × 10⁴/well) and 6 × 10⁵/mL (1.5 × 10⁵/well) (C), were cultured with CpG-matured, peptide-loaded cDC at a ratio of cDC:CD8 = 1:10 for 4 days. Divided CD8 cells were sorted by flow cytometry and 1 × 10⁶ (B; D-F) or 2 × 10⁴ (C) cells were adoptively transferred into vaccinia virus-infected C57BL/6J mice (4 days post-infection). (A) Outline of the experimental scheme. (B and C) The number of TCR Vβ11⁺ F5 CD8 T cell was determined in the blood on day 8 (B) and in the spleen on day 32 after activation (C). (D) The expression of CD62L, NKG2D, CD29, CD49d, and CD49a was analyzed on TCR Vβ11⁺ F5 CD8 T cells from spleen on day 32 and represented as histogram. Representative histograms (top) and individual percentages of positive cells (bottom) are shown. (E) The expression of CD43 and CD27 was analyzed on TCR Vβ11⁺ F5 CD8 T cells from spleen on day 32. Representative histograms (left) and individual percentages of CD27⁺CD43⁻ and CD27⁺CD43⁺ cells (right) are shown. (F) On day 32, 3 × 10⁶ splenocytes were stimulated with NP68 (10 nM) for 4 h. The expression of IFN-γ and CCL5 by F5 CD8 T was analyzed by flow cytometry. One representative out of four independent experiments is presented. The results are expressed as the mean ± SD (n = 4 mice per group). The statistical significance of differences was determined by the Student’s t test (ns = p > 0.05, ∗ = p ≤ 0.05).

Figure 4

Ex-IL-2 promotes direct in vivo memory differentiation of in vitro activated cells See also Figure S3. (A–E) CTV-labelled purified naive F5 CD8 T cells at a concentration of 6x10⁵/mL (C-E) or 1.2 × 10⁵/mL (3 × 10⁴/well) and 6 × 10⁵/mL (1.5 × 10⁵/well) (B) were cultured with CpG-matured, peptide-loaded cDC at a ratio of cDC:CD8 = 1:10 for 4 days. Divided CD8 cells were sorted by flow cytometry and 1 × 10⁶ (C-E) or 2 × 10⁴ (B) cells were adoptively transferred into naive C57BL/6J mice. (A) Outline of the experimental scheme. (B) The number of TCR Vβ11⁺ F5 CD8 T cell was determined in the spleen on day 32 after activation. (C) The expression of CD43 and CD27 was analyzed on TCR Vβ11⁺ F5 CD8 T cells from spleen on day 32. Representative histograms and individual percentages of CD27⁺CD43⁻ and CD27⁺CD43⁺ cells are depicted. (D) The expression of CD62L, NKG2D, CD29, CD49d and CD49a was analyzed on TCR Vβ11⁺ F5 CD8 T cells from spleen on day 32. Representative histograms and individual values for each mouse are shown. (E) On day 32, 3 × 10⁶ splenocytes were stimulated with NP68 (10 nM) for 4 h. The expression of IFN-γ and CCL5 by F5 CD8 T was analyzed by flow cytometry. One representative out of four independent experiments is presented. The results are expressed as the mean ± SD (n = 4 mice per group). The statistical significance of differences was determined by the Student’s t test (ns = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.0001).

Figure 5

Single cell RNA seq analysis reveals that ex-IL-2 delays the differentiation of activated CD8 T cells in memory precursor, while driving the acquisition of effector function See also Figures S4 and S5. (A) UMAP projection of cells sorted on day 0, 3, 4 and 5 after activation with or without ex-IL-2 and colored according to cluster identification by Seurat package. (B) Left: UMAP projection of cells as in A, colored according to the experimental time points and conditions of culture (+/− ex-IL-2). Right: Percentages of cells from each experimental condition in each cluster. (C and E) Memory precursor (C) and terminal effector (TE) (E) signature enrichment per cluster. The dotted line represents the threshold above which cells are considered positive for the gene expression signature. The legend indicates the percentage of cells positive for a given signature in each cluster. AUC: area under the curve. (D and F) The cells positive for the memory precursor (D) or the terminal effector (TE) (F) signature are colored on the UMAP. (G and H) RNA velocities of cells activated without ex-IL-2 (G) or with ex-IL-2 (H) for 3, 4 or 5 days are projected onto the UMAP. Colors are the same as in B.

Figure 6

CD8⁺ T cells activated in the presence of ex-IL-2 display stronger effector functions See also Figure S6. 1.5 × 10⁵ magnetically purified naive F5 CD8⁺ T cells labeled with CTV were activated with CpG-matured, NP68-loaded cDC at a ratio of cDC:CD8 = 1:10, in the presence or absence of ex-IL-2 (11,5 ng/mL). (A) Median Fluorescence Intensity (MFI) of TCF1 was measured on divided cells after 4 days of activation and the ratio between cells cultured in the absence and cells cultured in the presence of ex-IL-2 was calculated. (B and C) After 3, 4, or 5 days of activation, CD8⁺ T cells were restimulated for 2h with NP68 and the percentages of IFN-γ- (B) and GzmB- (C Left) expressing CD8⁺ T cells were measured on divided cells. Representative dot plots and individual percentages of GzmB⁺ cells are also depicted (C Right). (D) Percentages of live EL4 target cells, loaded or not with NP68 peptide, after 4h of co-culture with effector CD8⁺ T cells activated for 5 days. Values from three independent experiments are presented. The results are expressed as the mean +SD. E:T ratio = effector:target ratio. The mean ± SD of six independent experiments is shown in panel A. The mean ± SEM of triplicate cultures from one representative experiment out of three independent experiments is shown in panel B and C (right). In A, the statistical significance of the difference between the mean value of ratios and the hypothetical value of 1 was determined by the one sample t-test. In D, the statistical significance of differences between F5 CD8⁺ cultured with or without ex-IL-2 at each E:T ratio was assessed by a two-way ANOVA followed by Sidak’s multiple comparison test (ns = p > 0.05, ∗∗ = p ≤ 0.01 and ∗∗∗ = p ≤ 0.001).