Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells

Abstract

Memory T lymphocytes proliferate in vivo in the absence of antigen maintaining a pool of central memory T cells (T(CM)) and effector memory T cells (T(EM)) with distinct effector function and homing capacity. We compared human CD4(+) naive T, T(CM), and T(EM) cells for their capacity to proliferate in response to cytokines, that have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T(EM), while T(CM) were less responsive and naive T cells failed to respond. Dendritic cells (DCs) and DC-derived cytokines allowed naive T cells to proliferate selectively in response to IL-4, and potently boosted the response of T(CM) to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15Rbeta and the common gamma chain (gamma(c)). The extracellular signal regulated kinase and the p38 mitogen-activated protein (MAP) kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures, some of the proliferating T(CM) differentiated to T(EM)-like cells acquiring effector function and switching chemokine receptor expression from CCR7 to CCR5. The sustained antigen-independent generation of T(EM) from a pool of T(CM) cells provides a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4(+) memory T cells.

Figure 1.

Kinetics of TCR- and cytokine-induced T cell proliferation. CFSE-labeled naive CD4⁺ T cells were stimulated with either TSST-loaded autologous DCs (A) or with a cytokine mixture (IL-7, IL-15, TNF-α, IL-6, and IL-10) (B). Cell division was measured by flow cytometry at the time indicated.

Figure 1.

Kinetics of TCR- and cytokine-induced T cell proliferation. CFSE-labeled naive CD4⁺ T cells were stimulated with either TSST-loaded autologous DCs (A) or with a cytokine mixture (IL-7, IL-15, TNF-α, IL-6, and IL-10) (B). Cell division was measured by flow cytometry at the time indicated.

Figure 2.

Differential requirements for cytokine-induced proliferation of naive T, T_CM, and T_EM cell subsets. (A) CFSE-labeled naive and memory CD4⁺ T cells were cultured for 7 d with IL-7 and IL-15 alone or in combination with autologous DCs, in the absence or presence of a blocking anti-MHC class II antibody, or with the supernatant of 24-h LPS-activated DCs. (B) CD4⁺ naive, T_CM, and T_EM cells were isolated by cell sorting from peripheral blood according to their expression of CD45RA and CCR7. CFSE-labeled cells were cultured in the presence of different cytokines as indicated. Cell division was assessed by flow cytometry after 7 d. One representative experiment out of five and eight, respectively.

Figure 2.

Differential requirements for cytokine-induced proliferation of naive T, T_CM, and T_EM cell subsets. (A) CFSE-labeled naive and memory CD4⁺ T cells were cultured for 7 d with IL-7 and IL-15 alone or in combination with autologous DCs, in the absence or presence of a blocking anti-MHC class II antibody, or with the supernatant of 24-h LPS-activated DCs. (B) CD4⁺ naive, T_CM, and T_EM cells were isolated by cell sorting from peripheral blood according to their expression of CD45RA and CCR7. CFSE-labeled cells were cultured in the presence of different cytokines as indicated. Cell division was assessed by flow cytometry after 7 d. One representative experiment out of five and eight, respectively.

Figure 3.

Modulation of cytokine receptors on T cell subsets by DC-derived cytokines or IL-4. Naive, T_CM, and T_EM cells were cultured for 16 h in the absence (red) or in the presence of TNF-α, IL-6, and IL-10 (blue) or IL-4 (green) and stained with antibodies to IL-2/IL-15Rβ, γc and IL-4Rα. The black lines show staining with isotype-matched controls. One representative experiment out of five.

Figure 4.

Differential requirements for signal transduction pathways in TCR- versus cytokine-driven proliferation. (A) CFSE-labeled CD4⁺ T cells were stimulated with plate-bound anti-CD3 or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d in the absence (control) or in the presence of optimal concentrations of the indicated inhibitors. (B and C) CD4⁺ T cells were stimulated as above for 20 min in the presence or absence of inhibitors. ERK (B) and p38 (C) were assessed by immunoblotting with antibodies specific for the active kinases. Protein amount was controlled by reblotting with an antibody specific for total p38 (data not shown). One experiment out of four and three, respectively.

Figure 4.

Differential requirements for signal transduction pathways in TCR- versus cytokine-driven proliferation. (A) CFSE-labeled CD4⁺ T cells were stimulated with plate-bound anti-CD3 or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d in the absence (control) or in the presence of optimal concentrations of the indicated inhibitors. (B and C) CD4⁺ T cells were stimulated as above for 20 min in the presence or absence of inhibitors. ERK (B) and p38 (C) were assessed by immunoblotting with antibodies specific for the active kinases. Protein amount was controlled by reblotting with an antibody specific for total p38 (data not shown). One experiment out of four and three, respectively.

Figure 4.

Differential requirements for signal transduction pathways in TCR- versus cytokine-driven proliferation. (A) CFSE-labeled CD4⁺ T cells were stimulated with plate-bound anti-CD3 or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d in the absence (control) or in the presence of optimal concentrations of the indicated inhibitors. (B and C) CD4⁺ T cells were stimulated as above for 20 min in the presence or absence of inhibitors. ERK (B) and p38 (C) were assessed by immunoblotting with antibodies specific for the active kinases. Protein amount was controlled by reblotting with an antibody specific for total p38 (data not shown). One experiment out of four and three, respectively.

Figure 5.

Cytokine expanded T_CM differentiate and switch chemokine receptor expression. CFSE-labeled CD4⁺ naive T and T_CM cells were stimulated with plate-bound anti-CD3 and anti-CD28 or with cytokines (IL-7, IL-15, TNF-α, IL-6, IL-10) and stained after 7 d with antibodies specific for the indicated surface receptors (y axis, PE, or APC staining). One experiment out of five.

Figure 6.

Cytokine-driven differentiation of naive T and T_CM cells. (A) CFSE-labeled CD4⁺ naive T and T_CM cells were stimulated with TSST-pulsed DCs or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d. Intracellular IFN-γ and IL-4 were measured 6 h after stimulation with PMA and ionomycin. Numbers indicate the percentage of cytokine-producing cells. (B) CFSE-labeled CD4⁺ naive T cells were stimulated with allogeneic DCs or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d in the absence of IL-4 and IL-12 or in the presence of either IL-4 and anti-IL-12 or IL-12 and anti-IL4. IFN-γ and IL-4 production were measured 6 h after stimulation with PMA and ionomycin. The dot plots display cells, which had undergone the same number of divisions. One representative experiment out of six.

Figure 6.

Cytokine-driven differentiation of naive T and T_CM cells. (A) CFSE-labeled CD4⁺ naive T and T_CM cells were stimulated with TSST-pulsed DCs or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d. Intracellular IFN-γ and IL-4 were measured 6 h after stimulation with PMA and ionomycin. Numbers indicate the percentage of cytokine-producing cells. (B) CFSE-labeled CD4⁺ naive T cells were stimulated with allogeneic DCs or with cytokines (IL-7, IL-15, TNF-α, IL-6, and IL-10) for 7 d in the absence of IL-4 and IL-12 or in the presence of either IL-4 and anti-IL-12 or IL-12 and anti-IL4. IFN-γ and IL-4 production were measured 6 h after stimulation with PMA and ionomycin. The dot plots display cells, which had undergone the same number of divisions. One representative experiment out of six.