Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells

Abstract

Two competing theories have been put forward to explain the role of CD4(+) T cells in priming CD8(+) memory T cells: one proposes paracrine secretion of interleukin 2 (IL-2); the other proposes the activation of antigen-presenting cells (APCs) via the costimulatory molecule CD40 and its ligand CD40L. We investigated the requirement for IL-2 by the relevant three cell types in vivo and found that CD8(+) T cells, rather than CD4(+) T cells or dendritic cells (DCs), produced the IL-2 necessary for CD8(+) T cell memory. Il2(-/-) CD4(+) T cells were able to provide help only if their ability to transmit signals via CD40L was intact. Our findings reconcile contradictory elements implicit in each model noted above by showing that CD4(+) T cells activate APCs through a CD40L-dependent mechanism to enable autocrine production of IL-2 in CD8(+) memory T cells.

Figure 1

CD4⁺ T cells require CD40L, not IL-2, to provide help to CD8⁺ T cells. (a) Flow cytometry of splenic CD8⁺ T cells from C57BL/6J mice that had undergone thymectomy and were first depleted of CD4⁺ T cells, then given injection of PBS alone (No OT-II) or 5 × 10⁴ OT-II cells (OT-II) or OT-II Il2^−/− cells (OT-II Il2^−/−), then primed 1 d ater with 5 × 10⁶ Act-mOVA H-2K^b-deficient splenocytes and challenged 30 d later with 5 × 10⁶ plaque-forming units (PFU) of vaccinia virus–OVA. Numbers adjacent to outlined areas ndicate percent H-2K^b–OVA tetramer–positive CD8⁺ T cells (mean ± s.e.m. of six mice). (b,c) Frequency (b) and absolute number (c) of FN-γ⁺ CD8+ T cells among total splenocytes from the mice in a (n = 6 per group), assessed by intracellular staining at day 7 (primary) or at day 35 in mice that had (secondary) or had not (memory) been challenged with vaccinia virus–OVA 5 d earlier. Each symbol represents an individual mouse; small horizontal lines indicate the mean (b). (d) Flow cytometry of CD8⁺ T cells in blood from the mice in a that were given no antibody treatment (far left) or treated with control immunoglobulin (control Ig) or blocking antibody to CD40L (α-CD40L), assessed at day 7 (primary), day 30 (memory) or day 35 (secondary) after mice were challenged with vaccinia virus–OVA 5 d earlier. Numbers adjacent to outlined areas indicate percent OVA(257–264)–H-2K^b tetramer-specific CD8+ T cells (mean ± s.e.m. of six mice). NS, not significant; *P < 0.05 (two-tailed unpaired t-test). Data are representative of three (a,b) or two (c,d) independent experiments (mean ± s.e.m.).

Figure 2

DC-derived IL-2 is not required for the secondary population expansion of CD8⁺ T cells. (a) Flow cytometry of splenocytes from H-2K^bm1 mice given injection of 5 × 10⁴ OT-I H-2K^bm1 CD45.1⁺ cells, then primed 1 d later with 7 × 10⁴ wild-type (WT) or Il2^−/− Act-mOVA DCs, followed by analysis of the primary CD8⁺ T cell response after 7 d (top row) or boosted 139 d later with 7 × 10⁵ wild-type or Il2^−/− Act-mOVA DCs, followed by analysis of the OT-I response in both primed and boosted groups 5 d later. Numbers adjacent to outlined areas indicate percent OT-I CD8⁺ T cells among total splenocytes (mean ± s.e.m.). (b) Absolute number of OT-I CD8⁺ T cells among total splenocytes during the memory and secondary response in a. Data are representative of two experiments (mean ± s.e.m. of three to four mice in b).

Figure 3

CD8⁺T cells require autocrine IL-2 for a memory response to a replicating immunogen. (a,b) Frequency (a) and absolute number (b) of OT-I (CD45.1⁺) CD8⁺ T cells among total splenocytes from intact C57BL/6J (CD45.2⁺) mice (+ help) or C57BL/6J (CD45.2⁺) mice depleted of CD4⁺ T cells (– help; n = 3–4 per group) given 50 wild-type or Il2^−/− (CD45.1⁺) OT-I CD8⁺ T cells, then infected 1 d later with 1 × 10⁶ PFU vaccinia virus–OVA and, for some groups (secondary), challenged 40 d later with 0.6 half-maximal lethal dose of L. monocytogenes–OVA, followed by analysis at day 7 (primary), or at day 45 for mice that had (secondary) or had not (memory) been challenged with L. monocytogenes–OVA 5 d earlier. Each symbol represents an individual mouse; small horizontal lines indicate the average (a). *P < 0.05 (two-tailed unpaired t-test). Data are representative of three experiments (error bars (b), s.e.m.). (c) IFN-γ production by the endogenous (CD45.1⁻) and OT-I (CD45.1⁺) CD8⁺ T cells during the primary, memory and secondary responses in a,b. Numbers in quadrants indicate percent cells in each (mean ± s.e.m.). Data are representative of three experiments with 8–12 mice. (d) Population expansion of the OT-I CD8⁺ T cells in a,b. Data are representative of three experiments.

Figure 4

CD8⁺T cells require autocrine IL-2 for a memory response to a nonreplicating immunogen. (a,b) Frequency (a) and absolute number (b) of OT-I (CD45.1⁺) CD8⁺ T cells among total splenocytes from C57BL/6 (CD45.2⁺) mice (n = 3–4 per group) given 50 wild-type or Il2^−/− (CD45.1⁺) OT-I CD8⁺ T cells, then immunized 1 d later with 5 × 10⁶ Act-mOVA H-2K^b-deficient splenocytes and, for some groups (secondary), challenged 30 d later with 5 × 10⁶ PFU vaccinia virus–OVA, followed by analysis at day 7 (primary), or at day 45 in mice that had (secondary) or had not (memory) been challenged with vaccinia virus–OVA 5 d (presented as in Fig. 3a,b). (c) IFN-γ production by endogenous (CD45.1^-) and OT-I (CD45.1⁺) CD8⁺ T cells during the primary, memory and secondary responses in a (presented as in Fig. 3c; n = 8–12 mice). Data are representative of two experiments (mean ± s.e.m. in b,c).

Figure 5

Functional and phenotypic comparison of wild-type and IL-2-deficient OT-I CD8⁺ T cells. (a) Frequency of OT-I CD8⁺ T cells producing IFN-γ alone (IFN-γ⁺) or both IFN-γ and tumor necrosis factor (IFN-γ⁺TNF⁺) among total splenocytes from C57BL/6J (CD45.2⁺) mice (n = 3–4 per group) given wild-type (OT-I) or Il2^−/− (OT-I Il2^−/−) CD45.1⁺ OT-I CD8⁺ T cells and infected 1 d later with 1 × 10⁶ PFU vaccinia virus–OVA, analyzed by intracellular staining of CD45.1⁺ cells at day 7 (primary), or at day 45 in mice that had (secondary) or had not (memory) been challenged with 0.6 half-maximal lethal dose of L. monocytogenes–OVA 5 d earlier. *P < 0.05 and **P < 0.005 (two-tailed unpaired t-test). (b) Frequency of OT-I cells with positive or negative expression of the activation and memory markers KLRG-1 and CD127 (IL-7 receptor α-chain) on CD45.1⁺ cells in the blood from the mice in a at day 7 (primary). (c) Frequency of OT-I CD8⁺ T cells with the surface phenotype of central memory cells (CD127⁺CD62L⁺) or effector memory cells (CD127⁺CD62L⁻) at day 40 (memory) in the blood of the mice in a. (d) In vivo cytotoxicity of wild-type and Il2^−/− OT-I cells 6 d after immunization with vaccinia virus–OVA, presented as the frequency of tetramer-positive OT-I cells in wild-type mice (numbers above outlined areas; left) and frequency of target cells loaded with various concentrations of the cytosolic dye CSFE and pulsed with the peptide epitope of adenovirus early 1B protein amino acids 192–200 (E1B) or OVA(257–264) (OVA) 16 h after adoptive transfer into the immunized mice (right; numbers in plots indicate percent CSFE⁺ cells). (e) Expression of granzyme B by wild-type and Il2^−/− OT-I cells at days 0–5 after immunization of the mice in a with vaccinia virus–OVA. IgG (key), staining with isotype-matched control antibody. Data are representative of three (a,c) or two (b,d,e) experiments (error bars (a–c), s.e.m.).