Background
CD4 T cells participate in anti-tumor immune responses, recruiting innate immune cells, direct lysis of tumor cells, and assisting CD8 activation. CD4 T cell-based adoptive cell transfer (ACT) therapies have clinical success across multiple cancer types, including metastatic melanoma and epithelial cancer.1 2 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 murine and human antigen-specific T cells in vitro. 3 4 This platform allows 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.
Methods
Our lab utilizes artificial antigen-presenting presenting cells (aAPCs) – iron dextran particles conjugated with MHC class II proteins and the co-stimulatory molecule 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.
Result
Co-culturing Class II aAPCs and human CD4 T cells significantly increase the frequency of cognate antigen-specific CD4 T cells. Specifically, we successfully expanded tetanus toxin (TT)-specific and herpes simplex virus (HSV)-specific HLA-DP4-positive 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).
Conclusions
These data show that aAPC platform can expand antigen-specific CD4 T cells from a rare precursor population. Therefore, this technology is beneficial to expand rare neoantigen-specific cells for ACT for CD4 T cell-enriched cancers.
Acknowledgements
SK would like to thank everyone in the Schneck labs for their help. Special thank you to Ariel Isser for his work, training and support. Finally, SK recognizes support from these funding sources: Ministry of Education, Taiwan and NIH.
References
Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, Jungbluth A, Gnjatic S, Thompson JA, Yee C. New England Journal of Medicine. 2008 Jun 19;358(25):2698–703.
Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, Wunderlich JR, Somerville RP, Hogan K, Hinrichs CS, Parkhurst MR. Science. 2014 May 9;344(6184):641–5.
Perica K, Bieler JG, Schutz C, Varela JC, Douglass J, Skora A, Chiu YL, Oelke M, Kinzler K, Zhou S, Vogelstein B. ACS nano. 2015 Jul 28;9(7):6861–71.
Isser A, Silver AB, Pruitt HC, Mass M, Elias EH, Aihara G, Kang SS, Bachmann N, Chen YY, Leonard EK, Bieler JG. Nature communications. 2022 Oct 14;13(1):6086.
Ethics Approval
All uses of human material in this study have been approved by the ethical committee of Johns Hopkins University; approval number NA-00027947.
Consent
All recruited volunteers provided written informed consent. A copy of the written consent is available for review by the Editor of this journal.
Abstract 1215 Figure 1
Expansion of antigen-specific CD4 T cells from DPB1*04:01 healthy donor peripheral blood mononuclear cells (PBMC). CD4 T cells treated with DP4-restricted herpes simplex virus (HSV) peptide-loaded aAPC (first row) or tetanus toxin (TT) peptide-loaded aAPCs (second row) in medica supplemented with cytokine mixes: IL-2, IL-4, IL-1β, and IFN-. (A) Representative tetramer staining of stimulated CD4 T cells on days 0, 7, 14, and 21. Cells are gated on the CD4+ population and stained for PD-conjugated HSV, TT, or CLIP tetramers. (B) Intracellular cytokine production of HSV peptide-specific CD4 T cells on day 21. (C) Killing assay activated CD4 T cells isolated from donors 169 and 174. Target cells are lymphoblastoid cell lines (LCLs) pulsed with HSV peptide at 30mM for an hour. (D) memory subset composition.