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.

Systems integration of innate and adaptive immunity

“…Several of the identified genes had demonstrated functional relationships to cytotoxic responses, including the inhibitory killer cell immunoglobulinlike receptor KIR2DL1, the NK cell activating receptor CLEC2D [54], and the NK-cell signaling adaptor EAT-2 [55], and many more were protein-protein interaction network neighbors of genes that comprise "Cytotoxicity", "T cells", and "Lymphoid lineage" transcriptional modules. Consistent with the identified association between EAT-2 expression and CD8+ T-cell responses, it was recently reported that adenoviral expression of EAT-2 as part of a vaccine strategy enhanced vaccine-induced T-cell responses [56,57]. Furthermore, integration of these genes with published innate and immunogenicity data for the yellow fever vaccine [18] revealed that two of them (CRIP3 and NPB) exhibited expression responses that were consistently associated with impaired CD8+ T-cell immunogenicity.…”

“…It is also possible to directly include host modulatory components within the vaccines themselves in order to effectively reprogram the host. As mentioned above, expressing an NK-cell signaling adaptor [56,57] as part of an adenoviral vaccine strategy enhanced vaccineinduced CD8+ T-cell responses. Collectively, these studies suggest that it will be possible to employ systems vaccinology to identify critical nodes in the host response, and then construct improved vaccines that specifically activate or suppress those nodes.…”

Strengthened tumor antigen immune recognition by inclusion of a recombinant Eimeria antigen in therapeutic cancer vaccination

“…We and others have verified that rAd5-CEA vaccination of murine and human subjects induces significant CEA-specific CMI [7, 8, 27]. To examine if the addition of rAd5-rEA to a TAA-expressing vaccine would further promote anti-TAA CMI responses, we treated mice with rAd5-CEA and an equivalent dose of either rAd5-rEA or a GFP-expressing control adenovirus vector (rAd5-GFP).…”

“…ELISpot mouse IFNγ and IL-2 kits (eBioscience, San Diego, CA), as previously described [27]. In brief, splenocytes (5 × 10 6 cells/well) from individual mice were incubated on capture antibody pre-treated plates with individual CEA peptides or a CEA peptide pool (0.4 μg/well).…”

“…In vivo CTL analysis was performed similarly to previously described studies [27]. Briefly, mice were vaccinated with 5 × 10 9 vp rAd5-CEA and 5 × 10 9 vp of either rAd5-GFP or rAd5-rEA.…”