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Background T-cell-based cancer immunotherapies like adoptive cell transfer (ACT) have largely focused on CD8 + T cells, but cytotoxic CD4+ T cells (CD4 CTLs) are increasingly recognized as contributors to anti-tumor immunity. To explore this, we created a CD4+ T-cell activating nanoparticle platform, termed the artificial antigen presenting cell (aAPC), using conjugated signal 1 (pMHC class II), along with soluble signals 2 (anti-CD28), and 3 (cytokines). Under Th1-skewing conditions, aAPC-stimulated CD4+ T cells upregulated granzyme B and mediated antigen-specific lysis. Given the limited understanding of CD4 CTL differentiation and function, we hypothesized that aAPCs could provide a tunable system to probe how environmental cues influence CD4+ T-cell cytotoxicity.Methods CD4+ T-cells from transgenic OT-II were isolated from spleens and lymph nodes using a negative selection kit and cultured in vitro with aAPCs and soluble signals 2 and 3 for 7 days. aAPCs are formulated by covalently conjugating signal 1 to amine-coated iron-oxide nanoparticles functionalized with Sulfo-SMCC. 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-a b OVA or anti-CD3). Markers previously associated with CD4+ CTLs and phenotypic markers (CD44, CD62L, and granzyme B) were assessed with flow cytometry.Results aAPCs were used to activate CD4+ T-cells from OT-II mice in vitro and assess conditions influencing cytotoxicity ( figure 1a). Th1-skewing conditions promoted expression of cytotoxic-associated transcription factors, including Blimp1 and ThPok, consistent with prior reports (figure 1b). In contrast, Th2- and Treg-skewing conditions failed to induce granzyme B, highlighting the importance of skewing signals in CD4 CTL differentiation (figure 1c). We also observed that smaller aAPCs (70 and 250 nm) supported greater cytotoxic function than larger particles (4 um) (figure 2a) and antigen-specific stimulation via I-ab OVA-loaded aAPCs elicited stronger cytotoxicity than non-specific anti-CD3-conjugated particles (figure 2b). Finally higher IL-2 concentrations in Th1-skewing conditions increased both the frequency and expression of granzyme B (figure 2c), and favored an effector (CD44+CD62L–) over memory (CD44+CD62L+) phenotype (figure 2d).Conclusions In conclusion, we have demonstrated 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.Acknowledgements Mara Lanis would like to thank everyone in the Schneck lab for their help. Special thank you to Sujin Kim, Joseph Choy, and Si-Sim Kim for their help and endless support.Ethics Approval All procedures for animal experiments were approved by the Johns Hopkins University’s Animal Care and Use Committee and compliant with the National Institutes of Health guidelines. The approved protocol number is M022M199.Abstract 1012 Figure 1Examination of molecular mechanisms in CD4 CTL. a) Signal 1 is conjugated to nanoparticle while signals 2 and 3 are added to the media. b) Presence of cytotoxicity-associated transcription factors in CD4s activated by aAPC or dynabeads. c) Measurement of cytotoxicity in multiple CD4 T-cell subsetsAbstract 1012 Figure 2Investigating environmental effects on CD4 cytotoxicity. a) Measurement of granzyme B positive cells activated with different sized aAPCs or b) different signal 1. c) Comparison of amount of granzyme B and d) percentage of effector vs memory T-cells for Th1-skewing medias with different concentrations of IL-2