In Vitro Stimulation and Expansion of Human Tumour‐Reactive CD8⁺ Cytotoxic T Lymphocytes by Anti‐CD3/CD28/CD137 Magnetic Beads

Abstract

Adoptive immunotherapy with tumour‐reactive CD8+ cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen‐independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour‐reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4‐1BB). T cells in PBMCs showed an increased expansion rate of 15‐ to 17‐fold during a 2‐week culture period using antibody‐conjugated beads with interleukin‐2 (IL‐2) added versus IL‐2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour‐reactive CD8+ CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4‐fold ±1.2) compared with CD3/CD28 beads (10.6‐fold ±0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead‐activated cultures expressed consistently higher levels than tumour‐stimulated cultures. CTLs analysed after bead‐induced expansion versus weekly tumour stimulation showed equal IFN‐γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen‐independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti‐CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour‐reactive CD8+ CTLs.

PD-1 blockade restores impaired function of ex vivo expanded CD8⁺ T cells and enhances apoptosis in mismatch repair deficient EpCAM⁺PD-L1⁺ cancer cells

“…The median expansion of the T cells was ~1,000-fold, and the majority of the cells were CD3 + CD8 + T cells (range 64%–87%, mean =79%) after expansion 31,32. The presence of other cytotoxic cell types, including CD3 − CD56 + NK cells (range 0.1%–5.4%; mean =1.72%) and CD3 + CD56 + NKT cells (range 0.3%–15.1%; mean =6.34%) was also determined (Figure S3A) and found to be unchanged.…”

“…The CD3/TCR complex stimulation of tumor reactive T cells mediated by co-stimulatory signals to retain proliferation and functional properties of antigen specific T cells 32. This procedure is similar to that used to expand tumor-specific T cells, non-specific cytotoxic cells (eg, NK, NKT, CD8 + T), and MHC-independent CAR T cells.…”

The Immune System in the Elderly: A Fair Fight Against Diseases?

“…This is of major importance to retain T-cell functionality for adoptive cell transfer therapy. A study showed that taken as a whole, T cells expand similarly following stimulation with CD3/CD28/4-1BB, but tumor-reactive T-cell expansion was significantly higher compared with CD3/CD28 stim ulation alone without loss of functionality [87]. Cytokines were also shown to play a pivotal role in T-cell survival, homeostasis and activation.…”

Efficacy of ex vivo activated and expanded natural killer cells and T lymphocytes for colorectal cancer patients

“…A number of approaches have been investigated for the growth and expansion of NK (12)(13)(14) and T cells (15,16), in order to obtain a maximum-fold expansion. Takada et al (17) and Dewan et al (18) successfully expanded NK and T cells by severalfold from a low number of peripheral blood mononuclear cells (PBMCs) in order to develop a multipronged approach to cancer management.…”