Invasion and destruction of a murine fibrosarcoma by Salmonella-induced effector CD8 T cells as a therapeutic intervention against cancer

Abstract

We have developed a new vaccination strategy by using the Salmonella type III secretion system (T3SS) to translocate heterologous antigens into the cytosol of host cells. This leads to an efficient antigen-specific CD8 T cell induction. Recently, we have demonstrated the use of Salmonella's T3SS for the immunoprophylaxis of a solid tumor. The murine fibrosarcoma WEHI 164 was transfected with the DNA sequence encoding the MHC class I-peptide p60(217-225) from Listeria monocytogenes. In the present study, we used this tumor model to investigate the potential of vaccination with recombinant Salmonella in a therapeutic setting. BALB/c mice were subcutaneously challenged with WEHI-p60 cells. Simultaneously or 4 days later, these mice received either an orogastric or intravenous immunization with Salmonella translocating p60. Interestingly, 71-80% of the intravenously and 50-52% of the orogastrically immunized mice showed a complete tumor regression after 14 days. In addition, the distribution of tetramer-positive p60(217-225)-specific CD8 T cell subpopulations in blood and tumor tissue was analyzed. Co-staining with CD62L and CD127 revealed that the frequencies of p60(217-225)-specific effector and effector memory CD8 T cells in blood and in fibrosarcoma tissue were related to the kinetics of tumor regression. In summary, our study demonstrates that therapeutic vaccination with Salmonella leads to efficient induction of tumor-invading effector CD8 T cells that may result in significant tumor regression.

Fig. 1

Regression of fibrosarcoma growth after therapeutic vaccination with recombinant Salmonella. BALB/c mice received a subcutaneous injection of 5 × 10⁶ WEHI-p60 cells into the flank on day 0. Mice given the fibrosarcoma cells were inspected on days 4, 5, 7, 10 and 14 for tumor growth and size. a Immunization group A: application of Salmonella vaccine strain SB824 (pHR241) expressing translocated YopE/p60 via the intravenous route on day 0. b Immunization group B: application of Salmonella vaccine strain SB824 (pHR241) via the intravenous route on day 4. c Immunization group C: application of Salmonella vaccine strain SB824 (pHR241) via the orogastric route on day 0. d Immunization group D: application of Salmonella vaccine strain SB824 (pHR241) via the orogastric route on day 4. Negative control mice were immunized with plasmidless SB824 (S) or remained non-immunized (NI). Means and standard deviations of ten mice per vaccination group are indicated. Asterisks indicate values that differ significantly from the corresponding values obtained from mice of the control groups S and NI (*p < 0.01, **p < 0.005)

Fig. 2

Percentages of tumor-free mice after intravenous or orogastric therapeutic vaccination with recombinant Salmonella. Determination of tumor size was performed 14 days after WEHI-p60 inoculation. Complete tumor regression was defined by a calliper detection threshold of 3 mm × 3 mm, successful protection against tumor rechallenge after 1 month and no cancer relapse during the observation period of 3 months. Immunization group A: application of Salmonella vaccine strain SB824 (pHR241) via the intravenous route on day 0. Immunization group B: application of SB824 (pHR241) via the intravenous route on day 4. Immunization group C: application of SB824 (pHR241) via the orogastric route on day 0. Immunization group D: application of SB824 (pHR241) via the orogastric route on day 4. Negative control mice were immunized with plasmidless SB824 (S) or remained non-immunized (NI). Means and standard deviations of ten mice per vaccination group are indicated. Asterisks indicate values that differ significantly from the corresponding values obtained from mice of control groups S and NI (***p < 0.001) or indicate values of groups A and B that differ significantly from those of groups C and D (**p < 0.005)

Fig. 3

Representative dot plots of p60_217–225-tetramer-positive CD8 T cells in blood samples (panels A, C, and S) or tumor tissue (panel F) from BALB/c mice (n = 5 per group) determined 2 weeks after WEHI-p60 fibrosarcoma inoculation. Panels A and F show representative dot plots of mice immunized intravenously with SB824 (pHR241); panel C shows a representative dot plot of mice vaccinated with SB824 (pHR241) via the orogastric route. Panel S depicts a representative dot plot of mice vaccinated intravenously with plasmidless SB824

Fig. 4

Differentiation patterns of p60-specific CD8 T cells into phenotypically distinct subsets in blood samples (panel A) or tumor tissue (panel F) from BALB/c mice (n = 5 per group) determined 2 weeks after WEHI-p60 fibrosarcoma inoculation and intravenous vaccination with SB824 (pHR241). Cells were stained for expression of CD127 (x-axis) and CD62L (y-axis). The percentage of cells in each quadrant is indicated. CD127^high/CD62L^high central memory CD8 T cells (TCMC); CD127^high/CD62L^low effector memory CD8 T cells (TEMC); CD127^low/CD62L^low effector CD8 T cells (TEC)

Fig. 5

Absolute numbers of p60-specific CD8 T cells per milliliter blood and differentiation patterns of phenotypically distinct subsets (TEMC, TEC and TCMC) on day 14 after tumor inoculation. Cells were stained for expression of CD127 and CD62L. Immunization group A: application of Salmonella vaccine strain SB824 (pHR241) via the intravenous route on day 0. Immunization group B: application of SB824 (pHR241) via the intravenous route on day 4. Immunization group C: application of SB824 (pHR241) via the orogastric route on day 0. Immunization group D: application of SB824 (pHR241) via the orogastric route on day 4. Immunization group S: application of SB824 via the intravenous route on day 0. NI: non-immunized mice received phosphate-buffered saline on day 0. Asterisks indicate values that differ significantly from the corresponding values obtained from mice of the control groups S and NI (***p < 0,001) or indicate values of groups A and B that differ significantly from those of group C and D (**p < 0.005)

Fig. 6

Detection of tumor-invading p60-specific CD8 T cells in fibrosarcoma tissue. Regression of tumor growth in mice intravenously vaccinated on day 0 with SB824 (pHR241) (see Fig. 1a) was plotted against the numbers of CD8 T cells (light gray area) and p60_217-225 tetramer-positive TEC and TEMC (dark gray area), respectively, per 10³ tumor cells. Means and standard deviations of ten mice per vaccination group are indicated