28% of CTLs inside the tumor were Ag specific and exhibited higher levels of PD-1 expression compared to the same cells type found in the blood. (Figures 3D and 3E). These data show that Ag fusion with trafficking signals from the MHC class I molecule significantly increases the potency of the AAV6-based vaccine by targeting both CD4+ and CD8+ T?cells and by creating a higher number of memory cells. Open in a separate window Figure?2 663-optOva Has Superior Capacity to Generate an Ag-Specific Immune Response (A) Analysis of IFN secretion by spleen CD4+ T?cells stimulated with MHC class II Ova-immunodominant peptide Ova323C339 2?weeks after immunization measured by ELISPOT assay. (B) Levels of Ova257C264- tet+ CD8+ T?cells in the blood 2?weeks after vaccination. Verteporfin (C) The representative analysis of changes in activation markers on cell surface of Ova257C264- tet+ CD8+ T?cells compared to naive cells in the blood 3?weeks after vaccination. OD, optical density. (D) Levels of terminally activated and memory precursor effector T?cells after vaccination, which were calculated after analysis of data. Cell populations were defined as follows: T effector (TE) cells: Ova257C264- tet+/CD44+/CD62L?; terminally differentiated T effector (terminal TE): Ova257C264- tet+/CD44+/CD62L?/KLRG1+/CD127?; T effector memory (TEM): Ova257C264- tet+/CD44+/CD62L?/KLRG1?/CD127+; and T central memory (TCM): Ova257C264-tet+/CD44+/CD62L+/KLRG1?/CD127+. ? p 0.05. Open in a separate window Figure?3 Comparison of Functional Activity of Ova-Specific CD8+ T Cells after 663-Ova and 663-optOva Vaccination (ACC) Functional analysis 4?weeks after vaccination. (A) IFN production Slc7a7 by splenocytes re-stimulated with Ova257C264 peptide (SIINFEKL) 4?weeks after immunization. (B) cytotoxicity assay 4?weeks after immunization. Syngeneic naive splenocytes were divided into two equal parts and labeled with low and high concentrations of CFSE. The fraction labeled with low CFSE concentration was also pulsed with Ova257C264 peptide before splenocytes were mixed and adoptively transferred to immunized mice. 16?h later, CFSE-labeled populations were analyzed in the spleen. (C) Representative results for each group. (DCF) Functional analysis 9?weeks after vaccination. (D) IFN production by splenocytes re-stimulated with Ova257C264 peptide (SIINFEKL) 9?weeks after immunization. (E and F) killing capacity of mice immunized 9?weeks before analysis. 16?h after adoptive transfer of labeled splenocytes. *p? 0.05; **p? 0.01. Induction of Anti-tumor Protection after Vaccination The vaccines anti-cancer potential was tested against a highly aggressive syngeneic B16F10 melanoma tumor stably expressing Ova (B16/Ova). First, the vaccines ability to prevent the spread of metastasis was investigated in a metastatic model after intravenous (i.v.) injection of tumor cells. Vaccination with intramuscular injections of 663-optOva demonstrated excellent protection against lung metastasis compared with non-relevant control. Vaccinated mice developed very few or no tumor nodules on lungs compared to non-vaccinated mice, which had more than 200 nodules 19?days after the injection of 5? 105 tumor cells (Figure?4A). Second, in prophylactic solid tumor models, mice were immunized by intramuscular injection with 663-Ova or 663-optOva and then challenged with 5? 105 B16/Ova cells inoculated intradermally into the right flank to mimic melanoma development. In this model, tumors formed in a distal site that was less exposed to circulating immune cells. Vaccination induced a significant delay in tumor growth (Figure?4B); on average, the tumor become noticeable 7?days later for 663-Ova and 10? days later for Verteporfin 663-optOva compared to non-immunized mice, although tumors eventually Verteporfin recapitulate in all mice (Figure?4C). To understand why the vaccination was only partially effective, we tested how well tumor cells retain Ova expression under the treatment. We analyzed tumor lysates by western blotting for Ova expression at day 25 after inoculation (Figure?4D). At this time point, B16/Ova cells continued to express Ova in tumors of non-immunized mice; however, in immunized mice, tumors completely lost the expression of Ova. Hence, the limited success of our vaccination in a solid tumor model can be explained in part by the loss of Ag expression, allowing tumors to escape elimination by vaccine-induced CTLs. Open in a separate window Figure?4 Analysis of Vaccine Effectiveness against B16/Ova Tumor (A) Prophylactic 663-optOva vaccination gives protection against metastatic tumor spread. B16/Ova metastatic nodules are visible as black dots. Vaccinated mice had 0C13 B16/Ova nodules on the lungs. Non-vaccinated mice all had more than 200 B16/Ova nodules on lungs, so all values were marked as 200 for the purpose of this graph (right panel). Left panel: images of lungs. **p? 0.01. (B) Growth of intradermal B16/Ova tumor implanted on mouse flanks.
28% of CTLs inside the tumor were Ag specific and exhibited higher levels of PD-1 expression compared to the same cells type found in the blood