Investigating Cytotoxic Function in CD4+ T-cells for Cancer Immunotherapy 2260082

Abstract Introduction T-cell-based immunotherapies like ACT have mostly focused on CD8 T cells, but cytotoxic CD4 T cells (CD4 CTLs) are increasingly recognized as contributors to anti-tumor immunity. As such, we created a CD4 T-cell activating nanoparticle platform, termed the artificial antigen presenting cell (aAPC). Under Th1-skewing conditions, aAPC-stimulated CD4 T cells upregulate granzyme B and mediate cell lysis. We hypothesize that aAPCs can provide a tunable system to probe how environmental cues influence CD4 T-cell cytotoxicity. Methods aAPCs are formed by covalently conjugating signal 1 to amine-coated iron-oxide nanoparticles. CD4 T-cells from transgenic OT-II mice were isolated from organs and cultured in vitro with aAPCs and soluble signals 2 and 3 for 7 days. Cells were expanded in the presence of CD4 subset-polarizing mixes (Th1, Th2, or Treg), using aAPCs of varying diameters (70 to 4000 nm), or with aAPCs presenting different signal 1 molecules (I-ab OVA or anti-CD3). CD4 CTL and CD4 phenotypic markers (CD44, CD62L, and granzyme B) were assessed with flow cytometry. Results aAPCs were used to activate OT-II CD4 T cells in vitro. Th1-skewing conditions promoted expression of cytotoxic-associated transcription factors. Th2- and Treg-skewing conditions failed to induce granzyme B. Smaller aAPCs (70 and 250 nm) supported greater cytotoxic function than larger particles (4 um). Antigen-specific stimulation elicited stronger cytotoxicity than non-specific stimulation. Higher IL-2 concentrations increased both frequency and expression of granzyme B, and favored an effector over memory phenotype. Conclusion We have shown that aAPCs can drive differentiation of CD4 CTL in vitro. Furthermore, we show that CD4 CTL phenotype and function are tunable by cytokine concentration and milieu, particle size, and antigen specificity. These findings lay the groundwork for further investigation of this non-canonical T-cell subset, with the ultimate goal of improving the efficacy of T-cell-based cancer immunotherapies. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)