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.
5118 Introduction Efficient methods for the reliable in vitro expansion of tumor-reactive T cells will surely broaden the applicability of adoptive T cell therapy in cancer. In this study we investigated the antigen-independent stimulation and expansion of human T cells in peripheral blood mononuclear cells (PBMC) and in long-term cultured tumor-reactive CD8+ T cell lines using superparamagnetic beads coated with antibodies to CD3 and the costimulatory molecules CD28 and CD137. Methods T cell numbers were measured in healthy donor PBMC after in vitro stimulation with Dynabeads® coated with CD3/CD28/CD137 versus Dynabeads® coated with CD3/CD28 (all beads +/- 100 U/mL IL-2) versus IL-2 alone at different bead/cell ratios (3:1, 1:1). Expansion was also analyzed in human renal cell carcinoma-reactive CD8+ T cell lines after restimulation with tumor cells (weekly), CD3/CD28 beads and CD3/CD28/CD137 beads, respectively (bead/cell ratio of 1:5, 100 U/mL IL-2 added). Expanded T cell lines were phenotyped for expression of activation, differentiation and homing molecules (i.e. CD27, CD28, CD45RA, CD45RO, CD57, CD62L, CD137, CCR7) and were also tested for function. Results T cells in PBMC showed an increased expansion rate of up to 17-fold during a 2-week culture period using beads with IL-2 added versus IL-2 alone (p<0.0001 for CD3/CD28/CD137; p<0.0001 for CD3/CD28). The difference between CD3/CD28/CD137 beads and CD3/CD28 beads was not significant (p=0.4). Bead/cell ratios of 1:1 and 3:1 expanded T cells in PBMC with similar efficiency. In addition, IL-2 was essential to obtain maximum T cell proliferation. Peripheral blood CD4+ and CD8+ T cells showed a strong increase of CD137 surface expression starting 12-24 hours upon stimulation, regardless which beads were used. In contrast to PBMC, tumor-reactive CD8+ T cell lines expanded more rapidly using CD3/CD28/CD137 beads versus CD3/CD28 beads (p=0.03). Stimulation with CD3/CD28/CD137 beads was comparably efficient versus the control arm using weekly addition of tumor cells and IL-2. Simultaneous addition of beads and tumor cells did not have a synergistic effect. CD8+ T cell lines analyzed 12 days after bead-induced in vitro expansion versus weekly tumor stimulation showed a comparable level of tumor reactivity in IFN-g ELISPOT assay. Phenotypically, expression of CD137 on CD8+ T cell lines showed maximum up-regulation 24 hours after beads stimulation and persisted for at least 72 hours. In contrast, cultures stimulated solely with tumor cells showed a much shorter and transient CD137 expression with an earlier peak level after 12 hours. Other phenotypic markers were similar on tumor-reactive T cell cultures, except for increased CD62L expression after bead-induced stimulation. Conclusion Antigen-independent in vitro expansion of T cells in PBMC was equally efficient using CD3/CD28 beads or CD3/CD28/CD137 beads, respectively. In contrast, we observed an increased growth rate for tumor-reactive CD8+ T cell lines when activated with CD3/CD28/CD137 beads compared to CD3/CD28 beads. Antitumor reactivity of T cell lines was maintained during the antigen-independent stimulation step. Bead activation was associated with increased expression of the lymph node homing receptor CD62L on antitumor CD8+ T cell lines, which indicates a central memory phenotype. Our data suggest that the conjugation of anti-CD137 antibodies to the traditionally used CD3/CD28 beads improves their expansion capacity for antitumor CD8+ T cell lines. Disclosures No relevant conflicts of interest to declare.
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