Vaccines with enhanced costimulation maintain high avidity memory CTL

Abstract

The avidity of Ag-specific CTL is a critical determinant for clearing viral infection and eliminating tumor. Although previous studies have demonstrated that vaccines using enhanced costimulation will enhance the level and avidity of Ag-specific T cells from naive mice, there are conflicting data about the effects of vaccines using enhanced costimulation (vector or dendritic cell based) on the survival of memory T cells. In this study we have first extended previous observations that primary vaccination with a recombinant vaccinia virus (rV-) expressing a model Ag (LacZ) and a triad of T cell costimulatory molecules (B7-1, ICAM-1, and LFA-3 (designated TRICOM)) enhances the level and avidity of T cells from naive vaccinated C57BL/6 (Thy1.2) mice. Adoptive transfer of Thy1.1 memory CD8(+) T cells into naive Thy1.2 C57BL/6 mice was followed by booster vaccinations with a recombinant fowlpox (rF-)-expressing LacZ (rF-LacZ) or booster vaccinations with rF-LacZ/TRICOM. Analysis of levels of beta-galactosidase tetramer-positive T cells and functional assays (IFN-gamma expression and lytic activity) determined that booster vaccinations with rF-LacZ/TRICOM were superior to booster vaccinations with rF-LacZ in terms of both maintenance and enhanced avidity of memory CD8(+) T cells. Antitumor experiments using a self-Ag (carcinoembryonic Ag (CEA) vaccines in CEA transgenic mice bearing CEA-expressing tumors) also demonstrated that the use of booster vaccinations with vaccines bearing enhanced costimulatory capacity had superior antitumor effects. These studies thus have implications in the design of more effective vaccine strategies.

Figure 1

Immunization with TRICOM-based vaccines in naive mice induced high avidity antigen-specific CTL. Panel A: C57BL/6 mice were vaccinated once with buffer (HBSS, inverted open triangles), rV-LacZ (open squares), rV-LacZ/B7-1 (closed circles) or rV-LacZ/TRICOM (closed triangles). After 30 days, splenocytes were harvested. β-gal-specific CD8⁺ T-cell precursor frequency and avidity were determined by intracellular IFN-β staining. Panel B: β-gal-specific CD8⁺ T-cell avidity as determined by cytolytic assay. Splenic T cells from rV-LacZ and rV-LacZ/TRICOM immunized mice were stimulated with irradiated B cells pulsed with 1μg/ml β-gal peptide for 5 days. CTL avidity was determined using lytic assay as described in Materials and Methods. rV-LacZ (open squares), rV-LacZ/TRICOM (closed triangles).

Figure 2

Phenotypic analysis of memory T cells for adoptive transfer. Thy1.1 mice were vaccinated with rV-LacZ/TRICOM vaccine. Four weeks later, Pan T cells were isolated using Pan T isolation kit by AutoMACS (Miltenyi Bitotech). Memory T cells were purified by depleting CD62L^high T cells, using CD62L-labeled beads, from the isolated Pan T cells. The purified memory T cells were stained with indicated flourochrome labeled antibodies and analyzed by flow cytometry. Top line, FSC vs. SSC, CD4 vs. CD8 and β-gal tetramer vs. CD8 were graphed as dot plot. For the histogram, isotype control (gray curves) was overlaid with specific antibody staining (solid silhouettes).

Figure 3

Functional analysis of antigen-specific memory T cells after booster with rF-LacZ or rF-LacZ/TRICOM vaccines. Memory T cells were prepared and adoptively transferred as described in Materials and Methods and in the legend to Fig. 2. Four weeks after one booster vaccination with the indicated vaccine, T cells were isolated for functional assay. For IFN-γ production, purified memory Thy1.1 T cells (A, left panel) and endogenous Thy1.2 T cells (A, right panel) were stimulated with irradiated B cells pulsed with β-gal peptide (1μg/ml) for 24 h. IFN-γ production was detected using a Cytometric Bead Array as described in Materials and Methods. Note the difference in scale between the panels of A. Panel B: For intracellular IFN-γ staining, purified Pan T cells were stimulated with irradiated B cells pulsed with control OVA peptide or β-gal peptide (1μg/ml) for 6 h. At the end of incubation, cells were stained with anti-CD8, Thy1.1, Thy1.2 and anti-IFN-γ antibodies. IFN-γ producing CD8⁺ T cells in gated Thy1.1⁺ and Thy1.2⁺ T cell populations were analyzed using CellQuest software. These data are representative of two similar experiments.

Figure 4

Effect of multiple booster vaccinations with rF-LacZ versus rF-LacZ/TRICOM on the expansion of antigen-specific memory T cells. Memory T cells were purified from rV-LacZ/TRICOM vaccinated Thy1.1 mice as described in Materials and Methods and in the legend to Fig. 2, and were adoptively transferred to C57BL/6 mice (Thy1.2). One week after adoptive transfer, mice were vaccinated three times with the indicated vaccines at 2-week intervals. Five days after second and third vaccinations, three mice from each group were sacrificed and splenocytes were prepared for monitoring expansion of CD8⁺/β-gal tetramer ⁺ T cells in the gated Thy1.1 T cell population.

Figure 5

The effect of repeated booster vaccinations on the expansion of antigen-specific memory CD8⁺ T cells and avidity maturation. Memory T cells (Thy1.1) were prepared and adoptively transferred to Thy1.2 mice as described in Materials and Methods and in the legend to Fig. 2. Four weeks after three booster vaccinations at 2-week intervals with either rF-LacZ or rF-LacZ/TRICOM, splenocytes were purified and stained with indicated antibodies for antigen-specific memory T-cell expansion. Thy1.1⁺ T cells were purified, stimulated in vitro with β-gal peptide for 5 days and CTL avidity was titrated using a cytolytic assay. Panel A: Thy1.1 and β-gal tetramer staining of splenocytes from mice boosted with rF-LacZ or rF-LacZ/TRICOM. Panel B: Peptide titration of CTL avidity. Panel C: Normalization of data in B for avidity calculation. These data are representative of two similar experiments.

Figure 6

CTL avidity titration in freshly isolated memory T cells following booster vaccinations with rF-LacZ or rF-LacZ/TRICOM using intracellular IFN-γ staining. Thy1.1 mice were vaccinated with rV-LacZ/TRICOM. Thy1.1⁺ memory T cells were prepared and adoptively transferred to Thy1.2 mice as described in Materials and Methods and in the legend to Fig. 2. Four weeks after three booster vaccinations with either rF-LacZ or rF-LacZ/TRICOM at 2-week intervals, Thy1.1⁺ T cells were purified as described in Materials and Methods and stimulated with irradiated B cells pulsed with graded concentrations of β-gal peptide for 6 h. Cells were then stained with anti-Thy1.1, CD8 and anti-IFN-γ antibodies. Panel A: Phenotypic analysis of intracellular IFN-γ staining using graded concentrations of β-gal peptide. Panel B: Dose response curve of data collected from Panel A. Panel C: Avidity titration curves via normalization of data (see Refs. 20, 21, 25) from Panel B. These experiments were repeated twice with similar results.

Figure 7

Induction of anti-tumor responses in CEA transgenic (CEA-Tg) mice by recombinant poxviral vectors. CEA-Tg mice bearing 14-day established peripancreatic metastases were divided into three treatment groups. Tumors were transplanted by intrasplenic injection of MC-38 colon carcinoma cells that were transduced with CEA (day 0). Group 1 (n = 10, closed squares) received an rV-CEA/TRICOM prime vaccination (T) followed by three weekly boosts with rF-CEA/TRICOM (T). Group 2 (n = 10, open circles) received an rV-CEA/TRICOM prime vaccination followed by three weekly boosts with rF-CEA (C). Group 3 (n = 10, open squares) received an HBSS prime vaccination followed by three weekly boosts with HBSS. Groups 1-2 prime vaccinations were administered with recombinant GM-CSF and low-dose IL-2, and all booster vaccinations were admixed with rF-GM-CSF and low-dose IL-2. Mice in each group were monitored weekly for survival.