We observed DiO signals largely localized to the periphery of P14 T cells incubated at 4C. conventional protocols using non-specific CD3 and CD28 antibodies without the need for costimulation signals. T cells treated with synthetic APCs produce effector cytokines and demonstrate cytotoxic activity when co-cultured with tumor cells presenting target antigen TIL activation include both cell-based and biomaterials-based approaches. K562 cells engineered to display CD3 and CD28 antibodies or scFvs on their surface have previously been shown to activate and expand T cells by greater than 50-fold over the course of 9 days.[6C8] For clinical use, GMP-grade magnetic beads (e.g., Dynabeads) or polymeric matrices (e.g., Miltenyi T Cell TransAct?) coupled with CD3 and CD28 antibodies are routinely used to activate T cells prior to ACT.[9C10] These approaches are adequate for therapies involving genetically engineered transgenic T cells with T cell receptors (TCR) or chimeric antigen receptors (CAR) in which the cell product consists of a single antigen-specific population. However, for TIL therapy, nonspecific stimulation by CD3/CD28 results in the activation of both tumor-specific and non-specific clones, leading to an overall ALS-8112 decrease in antigen-specificity and limiting the ability of tumor-specific T cells to persist post-transfusion.[11C13] More sophisticated strategies for selective activation and expansion of antigen-specific T cells involve the use of magnetic nanoparticles[13C16] or 3D-scaffolds[17C18] decorated with major histocompatibility complexes (MHCs) presenting disease-specific peptides. These pMHC-based materials have been shown to ALS-8112 be able to rapidly activate and expand low-frequency antigen-specific T cell populations directly from mouse splenocytes or human PBMCs, producing cells capable of potent cytotoxic activity in pre-clinical models.[13C14, 17C18] Recombinant pMHC molecules are typically used as multimeric complexes to analyze or sort antigen-specific T cells by flow-, mass-, or DNA-based cytometry.[19C23] Here we develop synthetic antigen-presenting cells (synAPCs) comprising liposomes surface-conjugated with pMHC molecules for antigen-specific activation of T cells. We optimize pMHC density on liposomes for T cell binding and demonstrate that synAPCs activate antigen-specific T cells ALS-8112 activation of T cells prior to viral transduction and adoptive cell transfer in CAR T cell therapies.[31] Compared to na?ve cells, we found that P14 CD8+ T cells incubated ALS-8112 with Db-GP33 synAPCs showed increased expression of the activation markers CD44 and CD69 to levels that were equivalent to cells treated with CD3/CD28 antibodies (Figure 3B). Upregulation of activation markers was seen even in the absence of co-stimulatory signaling with synAPCs, demonstrating that potent stimulation of the TCR through multivalent presentation of pMHC molecules on the surface of a lipid membrane surface is sufficient to induce T cell activation. To verify that activated cells exhibited cytotoxic activity, we treated P14 CD8+ T cells with bare liposomes (lacking pMHC), Db-GP33 synAPCs, or CD3/CD28 and then co-cultured these T cells with MC38 tumor cells alone or MC38 tumor cells pulsed with GP33 peptide. We observed no significant difference in the expression of IFN by intracellular staining when T cells treated with bare liposomes were co-cultured with MC38 WT or MC38-GP33 cells (Figure 3C). By contrast, T cells that were stimulated with Db-GP33 synAPCs showed a significant increase in IFN expression when cultured with GP33-pulsed MC38 cells (****p 0.001 by two-way ANOVA). To verify that activation by synAPCs leads to target cell killing, we used a second TCR/pMHC combination and primed CD8+ T cells from OT1 TCR transgenic mice, which recognize a Kb-restricted ovalbumin antigen (OVA), with bare liposomes,, Db-GP33 synAPCs, or Kb-OVA synAPCs before co-culturing each population with B16-OVA tumor cells. We found that only OT1 T cells that had been stimulated with Kb-OVA synAPCs killed B16-OVA cells with increasing effector:target Rabbit Polyclonal to GHITM cell ratios (Figure 3D). These results show that pMHC-conjugated liposomes activate antigen-specific T cell populations and that activated cells retain cytotoxic activity. Open in a separate window Figure 3. T cells activated by synAPCs demonstrate cytotoxic activity. (A) pMHC liposomes stimulate T cells through pMHC-TCR interactions. Activated T cells then exert cytotoxic effects on target cells. (B) Histograms of CD44 and CD69 expression on T cells stimulated with synAPCs or anti-CD3/anti-CD28 antibodies. (C) P14 CD8+ T cells activated with Db-GP33 synAPCs have higher IFN expression when incubated with target cells pulsed with GP33 peptide than when incubated with control cells. Data shown as mean s.d. n=3 ****p 0.0001 by two-way ANOVA with Tukeys multiple comparison test. (D) OT1 CD8+ T cells stimulated with Kb-OVA synAPCs show cytotoxic activity against B16-OVA target cells. Data shown as mean s.d. n=4 ***p 0.001, ****p 0.0001 by two-way ANOVA with Tukeys multiple.