Using nanoparticles as artificial antigen presenting cells to activate human CD4 T cells for immunotherapy

Introduction: CD4 T cells participate in anti-tumor immune responses, recruit innate immune cells, directly lyse tumor cells, and assist in CD8 T cell activation. CD4 T cell-based adoptive cell transfer (ACT) therapies have seen clinical success across multiple cancer types, including metastatic melanoma and epithelial cancer. One of the challenges of CD4 T cell-based ACT therapy is finding methods to expand tumor antigen-specific T cells ex vivo. Therefore, engineering artificial platforms for antigen-specific T cell expansion is critical since it provides greater control over studying T cell biology and has significant translational relevance. We developed a nanoscale platform that expands human antigen-specific T cells in vitro, allowing us to study the effector and helper function of CD4 T cells through precise tuning of material parameters, including shape, size, stiffness, and ligand patterning.

Materials and

Methods: Our lab utilizes artificial antigen-presenting presenting cells (aAPCs) – iron dextran particles conjugated with MHC class II proteins and the co-stimulatory molecule anti-CD28 to activate T cells. This aAPC platform provides T cells with two critical signals for T cell activation, which permits the activation of cognate antigen-specific CD4 T cells ex vivo. We isolate human CD4 T cells from peripheral blood mononuclear cells (PBMC) of healthy donors who are HLA DR4+ and DP4+. We then stimulate CD4 T cells with HLA II aAPCs in media with inflammatory cytokine and access the specificity, effector function, and memory profiles through tetramer staining, cytokine release assays, and peptide-pulsed lymphoblastoid cell line (LCL) killing assays.

Results, Conclusions, and Discussions: Co-culturing class II aAPCs with human CD4 T cells significantly increases the frequency of cognate antigen-specific CD4 T cells. Specifically, we successfully expanded tetanus toxoid (TT)-specific and herpes simplex virus (HSV)-specific HLA- DP4+ CD4 T cells from healthy human PBMCs with aAPC. By the end of the 21-day expansion period, the antigen-specific cells showed substantial proliferation, escalating from under 1% to approximately 10% for HSV and exceeding 30% for TT (Figure 1A). The resulting antigen-specific CD4 T cells display high levels of effector cytokine production, including TNF-a, IFN-g, IL-2, and granzyme B (Figure 1B).
Furthermore, these CD4 T cells demonstrated antigen-specific cytotoxic capabilities (Figure 1C) and are predominantly effector memory T cells (Figure 1D). Upon examination of the activation mechanism, we found that inhibition of PI3K resulted in a complete reduction of the antigen-specific population, indicating a dependence on the PI3K pathway. Preliminary data shows that inhibition of NFAT increased the percentage and cell count (3-fold) of antigen-specific CD4 T cells compared to control untreated cells (data not shown).
Our findings demonstrate effective expansion of human antigen-specific CD4+ T cells using the artificial antigen-presenting cell (aAPC) platform. The expanded antigen-specific CD4+ T cells exhibited effector functions, including high levels of cytokine production, antigen-specific cytotoxicity, and an effector memory phenotype. Mechanistic studies revealed the dependence on the PI3K pathway for aAPC-mediated expansion, while NFAT inhibition enhanced the frequency of antigen-specific CD4+ T cells. These findings lay the groundwork for further exploration and refinement of our aAPC platform, aiming to improve the efficacy of rare neoantigen-specific CD4+ T-cell-based cancer immunotherapies for the benefit of patients with CD4 T cell-enriched cancers.

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