Mimicking homeostatic proliferation in vitro generates T cells with high anti-tumor function in non-lymphopenic hosts

Abstract

CD8(+) T cells undergoing homeostatic proliferation (HP) in a lymphopenic environment acquire a central memory-like phenotype (CD44(+) CD62L(+) Ly6c(+)). Such cells are readily functional in vitro, with a strong capacity to secrete IFNγ and IL-2 and to lyse target cells upon antigen recognition. In vivo, these memory-like T cells display potent anti-tumor reactivity. When addressing whether these remarkable properties were "acquired" or dependent on sustained HP, we observed, for the first time, that memory-like T cells retained full anti-tumor functions even when removed from their lymphopenic environment and retransferred into non-lymphopenic P14/Rag2(-/-) recipients (where HP is prevented). Moreover, memory-like T cells were superior to in vitro expanded effector T cells. We next sought to determine the conditions required to reproduce such a potent phenotype in vitro, in order to obtain optimal cells for adoptive cell transfer therapy. Assessing ex vivo lymph node cultures, dendritic cells, fibroblastic reticular cells, and HP-associated cytokines, we found that stimulation of naïve T cells with anti-CD3/CD28 beads and IL-15 (IL-7 was dispensable) led to the generation of memory-like T cell with a similar phenotype. Both in vitro and in vivo memory-like T cells retained the capacity to efficiently control tumor growth in non-lymphopenic hosts upon adoptive cell transfer. A similar phenotype could be imparted to human peripheral blood leukocytes with comparable culture conditions. Our data reinforce the idea that in vitro-generated memory-like T cells could benefit adoptive cell transfer therapies.

Fig. 1

Naïve 2C T cells transferred into Rag2^−/− mice acquire a memory-like phenotype and become readily functional. Naive 2C T cells (3–5 × 10⁶) were transferred i.v. in Rag2^−/− lymphopenic mice to undergo homeostatic proliferation and generate the “HP” T cells. In parallel, effector T cells were generated in vitro by stimulating naïve 2C T cells, on day 0 and day 7, with 40 Gy-irradiated P815.B7.1 mastocytoma tumor cells (at a 1:5 ratio). The phenotype and function of the indicated cell types were analyzed 14 days later from pooled spleens and LNs. a Flow cytometry analysis of naïve, effector, and HP T cell at day 14. b 1 × 10⁵ 2C T cells from the indicated groups were stimulated for 18 h with P815 tumor cells at a 1:1 ratio. IFNγ production was measured by ELISA (error bars are SD of triplicates). c Lytic capacity was analyzed in a standard 4-h chromium release assay of 2.5 × 10³ radiolabeled target cells and the indicated increasing ratios of T cells. EL4 targets were used as negative controls. d P14/Rag2^−/− received 1 × 10⁶ P1.HTR.C tumor cell subcutaneous on day 0. On day 1, the mice received either, no cells (mock), 1 × 10⁵ in vitro-stimulated T cells (effector) or 1 × 10⁵ T cell having undergone 14 days of homeostatic proliferation in Rag2^−/− mice (HP). Results are shown for 6 mice/group (mean ± SEM). Differences between tumor curves of effector and HP groups are significant (p = 0.037) as calculated by Wilcoxon rank sum test. Numbers between brackets represent the number of mice alive at completion of the experiment. Data shown are representative of 2 independent experiments

Fig. 2

Assessing the factors necessary to reproduce HP-like proliferation in vitro. CFSE-labeled naïve 2C T cells (5 × 10⁶) were transferred into a Rag2^−/−. After 15 h, inguinal LNs were removed and either placed a intact in culture or b as single cell suspension after homogenization for an additional 4 days. Cells were harvested and analyzed by flow cytometry for CD62L, CD11c, MHCII, and CFSE expression. Represented are lymphoid-gated CD8⁺, 1B2⁺ (anti-2C TCR) cells (left) and total gated CD3⁻, CD11c⁺, MHC-II⁺ dendritic cells (right). c FRCs or DCs were loaded on collagen-coated ceramic structures on day-2 and day-1, respectively. Naïve CFSE-labeled 2C T cells were added on day 0 and cultured in the presence of IL-15 and IL-7. CFSE dilution, and CD62L expression was analyzed 7 days later. Alternatively, DCs and T cells were cultured in flat bottom 24-well tissue culture plates. d, e CFSE-labeled T cells were co-cultured with the indicated ratio of FRC or DC in the presence of the indicated cytokines for 7 days. CD62L and CFSE expression of CD8⁺, 1B2⁺ lymphocytes is shown. Each panel is representative of 2–3 individual experiments

Fig. 3

Anti-CD3/CD28 bead stimulation with IL-15 in vitro leads to memory-like phenotype acquisition. The phenotype of 2C T cells at day 14 after in vivo HP, or in vitro stimulation is represented. HP and effector cells were generated as described in Fig. 1. Naïve T cells were stimulated with anti-CD3/CD28 beads at a ratio of 1:3 in the presence of 200 ng/ml IL-15 + 400 ng/ml IL15RFc-Ig and when indicated 50 ng/ml IL-7. Depicted surface stains are gated on CD8⁺ 1B2⁺ lymphoid cells. Data are representative of 3 individual experiments using pooled T cells from 3 donor mice each

Fig. 4

In vitro-generated memory-like T cells are readily functional upon antigen recognition. Naive 2C T cells (3–5 × 10⁶) were transferred i.v. in Rag2^−/− lymphopenic mice to undergo homeostatic proliferation and generate the “HP” T cells. In parallel, effector T cells were generated in vitro by stimulating naïve 2C T cells, on day 0 and day 7, with 40 Gy-irradiated P815.B7.1 mastocytoma tumor cells (at a 1:5 ratio). Bead-stimulated T cells were obtained from naive CD8⁺ 2C T cells stimulated with anti-CD3/CD28 bead (ratio T:bead = 1:3) and the indicated cytokines. a The effector T cells and bead-stimulated T cells were harvested after 14 days of culture and viable T cells were determined by trypan blue exclusion. The average fold expansion ± SD of three independent experiments is represented. b IFNγ and c IL-2 intracellular cytokine production was determined after a 4-h co-culture in vitro with P1.HTR.C or P815.B7.1 tumor cells at an E:T ratio of 1:2. Shown are the mean percentages ± SD of CD8⁺, 1B2⁺ lymphoid cells producing cytokines corrected for background (using irrelevant EL4 target cells). Representative of 2 independent experiments

Fig. 5

Beads + IL-15 stimulation generates T cells with potent anti-tumor activity in non-lymphopenic hosts. P14/Rag2^−/− received 1 × 10⁶ P1.HTR.C tumor cell subcutaneous on day 0. On day 1, the mice received either no cells (mock), 1 × 10⁵ in vitro-stimulated T cells (effector or bead-stimulated), or 1 × 10⁵ T cell having undergone 14 days of homeostatic proliferation in Rag2^−/− mice. Results are the mean of 6 mice per group (±SEM). Numbers between brackets represent the number of mice alive at completion of the experiment. Data shown are representative of 2 independent experiments

Fig. 6

Beads + IL-15 stimulation of human PBL generates memory-like T cells that are readily functional. PBL were stimulated with soluble anti-CD3 antibody and IL-2 or anti-CD3/CD28 beads + IL-15 ± IL-7 at a bead to PBL ratio of 3:1. After 2 days of stimulation, the T cells were retrovirally transduced to express the 1D3 TCR specific for MART-1_26–35. a Surface expression of CD62L, CD45RA, and CD45RO among CD3⁺, CD8⁺, lymphocytes was determined after 14 days of culture. b Mean fold expansion of T cells from 5 different healthy donors after 14 days of culture. c intracellular IFNg d IL-2 (on fixed cells) and e CD107a surface expression (on live cells), was measured after a 4-h in vitro co-culture with the melanoma cell lines mel 526, mel 624 (both HLA-A2⁺, MART-1⁺), and mel 888 (HLA-A2⁻, negative control). T cells alone are shown as control. The graphs represent the mean percentage of CD8⁺ CD3⁺ lymphocytes normalized for MART-1-tetramer⁺ T cells. f Depicts the fraction of CD8⁺ MART-1-Tetramer⁺ lymphocytes that have undergone proliferation as measured by CFSE dilution after 4 days of co-culture. Data represent mean ± SD. For a–d and f, 5 different healthy donors were analyzed. Data in e are determined in two healthy donors