Improved cytotoxic T-lymphocyte immune responses to a tumor antigen by vaccines co-expressing the SLAM-associated adaptor EAT-2

Abstract

The signaling lymphocytic activation molecule-associated adaptor Ewing's sarcoma's-activated transcript 2 (EAT-2) is primarily expressed in dendritic cells, macrophages and natural killer cells. Including EAT-2 in a vaccination regimen enhanced innate and adaptive immune responses toward pathogen-derived antigens, even in the face of pre-existing vaccine immunity. Herein, we investigate whether co-vaccinations with two recombinant Ad5 (rAd5) vectors, one expressing the carcinoembryonic antigen (CEA) and one expressing EAT-2, can induce more potent CEA-specific cytotoxic T lymphocyte (CTL) and antitumor activity in the therapeutic CEA-expressing MC-38 tumor model. Our results suggest that inclusion of EAT-2 significantly alters the kinetics of Th1-biasing proinflammatory cytokine and chemokine responses, and enhances anti-CEA-specific CTL responses. As a result, rAd5-EAT2-augmented rAd5-CEA vaccinations are more efficient in eliminating CEA-expressing target cells as measured by an in vivo CTL assay. Administration of rAd5-EAT2 vaccines also reduced the rate of growth of MC-38 tumor growth in vivo. Also, an increase in MC-38 tumor cell apoptosis (as measured by hematoxylin and eosin staining, active caspase-3 and granzyme B levels within the tumors) was observed. These data provide evidence that more efficient, CEA-specific effector T cells are generated by rAd5 vaccines expressing CEA, when augmented by rAd5 vaccines expressing EAT-2, and this regimen may be a promising approach for cancer immunotherapy in general.

Figure 1

Kinetics of proinflammatory cytokine and chemokine production following recombinant Ad5 vector expressing EAT-2 (rAd5-EAT2) administration. Wild-type (WT) C57BL/6 mice (n = 8) were either mock injected or intravenously injected with 7.5 × 10¹⁰ vps of either rAd5-EAT2 or rAd5-Null control vectors. Plasma was collected at 3, 6, 10, 24 and 48 h after virus injection. Cytokine induction was evaluated using a 23-plex multiplexed bead array-based quantitative system. Increases (a) or decreases (b) in cytokines and chemokines responses following EAT-2 overexpression are shown. The bars represent mean±_s.d. Statistical analysis was completed using a two-way analysis of variance (ANOVA) with a Student–Newman–Keuls post-hoc test, P<0.05 was deemed a statistically significant difference. * Denotes P<0.05, ** denotes P<0. 01, # denotes P<0.001, statistically significant difference from mock-injected animals.

Figure 2

Dose curve analysis for carcinoembryonic antigen (CEA)-specific CD8⁺ T cells induced by recombinant Ad5 vector (rAd5)-CEA vaccine. Wild-type (WT) C57BL/6 mice (n = 3) were co-immunized intramuscularly in the tibialis anterior with escalating viral particles (VPs) of rAd5-CEA mixed with rAd5-GFP (total of 1 × 10⁸, 1 × 10⁹ and 1 × 10¹⁰ vps mixed before injection). At 14 dpi, splenocytes were collected and stimulated ex vivo with the CEA-specific peptide mix, followed by interferon (IFN)-γ ELISPOT (a) or FACS intracellular staining analysis for IFNγ and TNFα (b), performed as described in Materials and methods. Spot-forming cells (SFCs) were quantified using an ELISPOT reader. Data are presented as mean±_s.d. Statistical analysis was completed using a one-way analysis of variance (ANOVA) with a Student–Newman–Keuls post-hoc test, P<0.05 was deemed as a statistically significant difference. * Denotes P<0.05, ** denotes P<0. 01, *** denotes P<0.001, statistically significant difference from naive animals. # denotes P<0.05, statistically significant difference from animals vaccinated with 1 × 10⁸ and 1 × 10⁹ of rAd5-CEA + rAd5-GFP vaccines.

Figure 3

Carcinoembryonic antigen (CEA)-specific T-cell immune responses elicited by recombinant Ad5 vector (rAd5)-CEA and rAd5-Ewing's sarcoma's-activated transcript 2 (EAT2) co-immunization. Wild-type (WT) C57BL/6 mice (n = 4) were co-immunized intramuscularly in the tibialis anterior with equivalent viral particles (VPs) of rAd5-CEA mixed with either rAd5-GFP=or rAd5-EAT2 (total of 1 × 10⁹ vps mixed before injection). At 14 dpi, splenocytes were collected and stimulated ex vivo with the CEA-specific peptide mix, followed by interferon (IFN)-γ (a) and interleukin (IL)-2 or IL-4 (b) ELISPOT performed as described in Materials and methods. Spot-forming cells (SFCs) were quantified using an ELISPOT reader. Data are presented as mean±_s.d. Data are representative of two independent experiments with similar results. Statistical analysis was completed using two-way analysis of variance (ANOVA) with a Bonferroni post-hoc test, P<0.05 was deemed as a statistically significant difference. Representative data from two independent experiments are shown.

Figure 4

Inclusion of Ewing's sarcoma's-activated transcript 2 (EAT-2) adaptor in the vaccine formulation enhances carcinoembryonic antigen (CEA)-specific CD8⁺ T cells. C57BL/6 (n = 4) mice were co-immunized with equivalent viral particles (VPs) of Ad-CEA mixed with either Ad5- GFP or Ad5-EAT2 (1 × 10⁹ total vps). At 14 dpi, the mice were killed and lymphocytes were isolated from spleen. Multiparameter flow cytometry was used to enumerate the frequency of cytokine-producing CD8⁺ T cells. Gate were set based on negative control (naive) and placed consistently across samples. The total frequency of CD8⁺ T cells derived from C57BL/6 mice expressing interferon (IFN)-γ (a), granzyme B (b) or TNFα (c) is shown. The bars represent mean±_s.d. Data are representative of two independent experiments with similar results. Statistical analysis was completed using one-way analysis of variance (ANOVA) with a Student–Newman–Keuls post-hoc test, P<0.05 was deemed as a statistically significant difference. * Denotes P<0.05, statistically different from naive animals.

Figure 5

Inclusion of Ewing's sarcoma's-activated transcript 2 (EAT-2) adaptor in the vaccine formulation enhances carcinoembryonic antigen (CEA)-specific CD8⁺ T cells. C57BL/6 (n = 4) mice were co-immunized with equivalent viral particles (VPs) of Ad-CEA mixed with either Ad5-GFP or Ad5-EAT2 (1 × 10⁹ total vps). At 14 dpi, the mice were killed and lymphocytes were isolated from spleen. Multiparameter flow cytometry was used to enumerate the frequency of cytokine-producing CD8⁺ T cells. The total frequency of CD8⁺ T cells derived from C57BL/6 mice expressing interferon (IFN)-γ and granzyme B is shown. The bars represent mean±_s.d. Data are representative of two independent experiments with similar results. Statistical analysis was completed using one-way analysis of variance (ANOVA) with a Student– Newman–Keuls post-hoc test, P<0.05 was deemed a statistically significant difference. * Denotes P<0. 05, ** denotes P<0.01, statistically different from naive animals.

Figure 6

Increased in vivo cytolytic activity of the carcinoembryonic antigen (CEA)-specific T cells in recombinant Ad5 vector (rAd5)-CEA and rAd5-Ewing's sarcoma's-activated transcript 2 (EAT2) co-immunized mice. C57BL/6 (n = 6) mice were co-immunized with equivalent viral particles (VPs) of rAd5-CEA mixed with either rAd5-EAT2 or rAd5-GFP (1 × 10⁹ total vps). At 14 dpi, syngeneic splenocytes were pulsed with either an irrelevant peptide (NYD-pep) and stained with 1 μm CFSE^Low or with CEA-specific peptides, and labeled with 10 μm (CFSE^High). Twenty hours after adoptive transfer into either naive or co-immunized mice, splenocytes were collected using a LSRII flow cytometer. (a) Representative figures of the CTL analysis from naive or vaccinated mice are shown. (b) Analysis for percent CEA-specific killing is shown. % carboxyfluorescein succinimidyl ester (CFSE)-positive cells were quantified using FlowJo software. % specific killing = 1 – ((% CFSE^High /% CFSE^Low)_immunized/(%CFSE^High /% CFSE^Low)_{non-immunized}). * Denotes P<0.05, *** denotes P<0.001 statistically different from naive animals. Representative figure of two independent experiments is shown.

Figure 7

Recombinant Ad5 vector (rAd5)-Ewing's sarcoma's-activated transcript 2 (EAT2) treatment of established carcinoembryonic antigen (CEA)-expressing tumors. C57BL/6 mice (n = 16 per group) were inoculated with 10⁶ MC-38-CEA adenocarcinomas cells SQ into the left flank at day 0. Five days following tumor implant, mice were either unvaccinated or vaccinated with the indicated viral vectors. Tumors were measured by two opposing directions and volumes were calculated according to the formula: volume = ((a × b)²/2). (a) Expression of CEA on MC-38 cells prior to tumor implantation. (b) Percent tumor volume increase at day 8. (c) Kinetics of tumor growth in naive or vaccinated mice. Error bars represent the s.d. of the tumor volume. Data are representative of two independent experiments with similar results.

Figure 8

Tumor tissues derived from recombinant Ad5 vector expressing EAT-2 (rAd5-EAT2) and rAd5-EAT2-co-vaccinated mice have increased caspase-3 activation and granzyme B expression. Tumor tissues derived from rAd5-carcinoembryonic antigen (CEA)/rAd5-EAT2 co-injected animals (n = 6 per group) have numerous necrotic foci and pyknotic nuclei. (a-e) Hematoxylin and eosin (H&E). Immunohistochemistry for active caspase-3 (f–j) and granzyme B (k–o) (red) was performed on tumor tissue (18 dpi) shows limited cell death in naive and rAd5-CEA-immunized animals. In contrast, rAd5-CEA/rAd5-EAT2-co-injected mice have highly pyknotic nuclei and cellular debris marking more widely dispersed necrotic tissue. Immunohistochemistry for active caspase-3 (b) and granzyme (c) (red) was performed on tumor tissues at the end of the study (18 dpi). Tumor tissues derived from rAd5-EAT2 co-injected mice show a strong signal for active caspase-3 and granzyme B. Quantification was performed using the ImageJ software, (NIH, Bethesda, MD, USA).