Vaccination with ΔCD40L or Flagellin Gene-Modified T Cells Activates Dendritic Cells In Vivo and Induces a Potent Anti-Tumor Immune Response

Cancer vaccines have shown promise in small animal models of cancer, but have thus far been disappointing in clinical settings. Successful induction of a systemic and long-term anti-tumor immune response following vaccination is dependent on delivery of tumor-associated antigens to lymphoid tissues, in combination with the activation of professional antigen presenting cells (APCs). Here we describe a novel live T cell vaccine (TCV) that delivers antigenic peptides to secondary lymph nodes while simultaneously activating endogenous dendritic cells (DCs) through transgenic expression of CD40L or bacterial flagellin (fliC).

To generate TCVs, murine splenocytes were isolated from wild-type type C57BL/6 mice. Following activation with anti-CD3/anti-CD28 microbeads, splenocytes were transduced with pRV2011-luciferase-IRES-Thy1.1, pRV2011-CD40L-IRES-Thy1.1 or pRV2011-fliC-IRES-Thy1.1 retrovirus. Analysis of TCV migration to lymphoid organs was performed by bioluminescence imaging for firefly luciferase. Following transduction with CD40L and fliC molecules, TCVs were measured for transduction efficiency (Thy1.1) and transgene expression using FACS analysis of CD40L or by Western blot, respectively. TCVs were subsequently pulsed with MHC class I-restricted epitopes for ovalbumin_257-264 (SIINFEKL) or Trp2_180-188 (SVYDFFVWL) peptides and injected intravenously at a dose of 1×10⁷ TCVs per vaccination. To test the protective effects of TCVs, C57BL/6 mice were immunized at days 0 and 14 and then challenged with either 5×10⁵ B16-OVA (for TCV-SIIN) or parental B16.F10 (for TCV-SVYD) melanoma tumor cells. To examine the ability of TCVs to eliminate established tumors, mice received B16-OVA or B16.F10 tumor cells followed by vaccination with TCVs on days 3, 9 and 15. Immunological studies were performed on a subset of mice (n=5 per group) to analyze induction of tumor-specific T cells using tetramer and IFN-g ELIspot assays. In vivo activation of lymph node DCs was performed by FACS analysis for CD11c+ DC co-expressing CD86 and I-A/I-E mouse MHC class II antibodies.

Following activation, TCVs were efficiently transduced with retrovirus (>85% CD40L) or expressed high levels of fliC. Bioluminescent imaging showed that luciferase-expressing TCVs rapidly migrated to lymphoid organs including the spleen and cervical and inguinal lymph nodes demonstrating the capacity of TCVs to co-localize with professional APCs. Importantly, irradiation (30 Gy) of TCVs completely abrogated migration and persistence highlighting the requirement for live TCVs. Next we examined whether TCV-CD40L or TCV-fliC could induce a protective immune response against B16 tumors. Administration of TCV-fliC-SIIN (OVA) and TCV-CD40L-SIIN primed peptide-specific CD8+ T cells, and led to decreased tumor growth and increased survival in mice subsequently challenged with B16-Ova (p<0.05). This response corresponded with a statistically significant (p<.05) increase in SIIN-specific CD8+ T cells as measured by tetramer FACS analysis and IFN-g ELIspot assays. Vaccination of mice with established tumors showed similar tumor suppression with both TCV designs (p<05). As OVA is a xenogenic antigen, we next examined whether TCVs pulsed with Trp2 peptide (SVYD) could induce similar protective effects. While vaccination with SVYD-pulsed T cells alone (no gene modification) did not inhibit tumor growth, expression of CD40L or fliC by TCV pulsed with Trp2 peptide suppressed B16.F10 tumor proliferation and increased survival in mice with pre-established tumors (p<.05). As found in the B16.OVA experiments, immunological protection correlated with a dramatic increase in SVYD-specific CD8+ T cells in the spleen, tumor draining lymph nodes and tumor.

The efficient delivery of tumor-associated antigens to lymphoid tissues by TCVs overcomes a major limitation of alternative vaccine strategies. Vaccination with peptide-pulsed TCVs primes antigen-specific T cell responses with anti-tumor capability, and endogenous DC maturation leads to the inhibition of established B16-Ova and B16-F10 tumors. This illustrates the role of endogenous DC as mediators of the vaccine response and demonstrates the effectiveness of using TCVs to deliver antigen in the context of DC activating molecules.

No relevant conflicts of interest to declare.