The 4-1BBL is a type II surface glycoprotein of the tumor necrosis factor (TNF) superfamily, and is generally expressed in APCs, such as dendritic cells, macrophages, and activated B cells . 4-1BBL binds to 4-1BB (also called CD137), a member of the TNFR superfamily, and enhances T cell activation . Emerging lines of evidence shows that activation of 4-1BB by agonistic monoclonal antibodies (Abs) or over-expressing 4-1BBL enhances T cell proliferation and cytokine production, up-regulates anti-apoptotic gene expression and prevents activation-induced cell death [15–17]. However, systemic administration of anti-4-1BB agonistic Abs is unsuccessful in correcting the defect in response to severe influenza in 4-1BBL-deficient mice, which is possibly because anti-4-1BB can target many cell types and lead to high levels of cytokine production and immunopathology [18, 19]. A HIV-1 effective vaccine should induce both antigen-specific cellular and humoral immunity and DNA vaccine for inducing 4-1BBL expression is a superior adjuvant to enhance HIV-specific immunity than anti-4-1BB agonistic antibody . Based on these findings, our previous study has shown that recombinant attenuated Salmonella harboring the 4-1BBL gene can induces high levels of 4-1BBL expression in dendritic and other immune cells . In the current study, we explored the effect of oral treatment with a recombinant Salmonella-based 4-1BBL vaccine on the development of DMH-induced colorectal cancers in rats and the potential immune mechanisms.
A previous study indicated that abnormal morphology characterized by accumulation of undifferentiated cells with pleiomorphic and conspicuous nucleoli in a small cluster of neighboring crypts was found at 12 weeks post DMH administration. Microscopic carcinomatous foci were observed at 15 weeks post DMH treatment . Therefore, in this present study, the vaccine administration was started 14 weeks after the DMH treatment.
Inoculation of recombinant Salmonella appeared to induce more 4-1BBL expression in the spleens and colorectal tumor tissues. Evidentially, we detected the reporter GFP mRNA transcripts and protein expression only in the spleens from the SL3261C and SL3261R groups of rats. Similarly, there was more 4-1BBL expression in the spleens and colorectal tissues than the other groups. In this study, we found that SL3261R suppressed tumor growth as compared with PBS group (P < 0.05). However, no significant difference was found between SL3261R group and SL3261 or SL3261C group (P > 0.05), although slightly decreased number of colorectal tumors was observed in SL3261R group. As Salmonella can localize to transplantable murine tumors and partially inhibit tumor growth , it is possible that the attenuated Salmonella (SL3261) itself may have anti-tumor effect. We further staged the colon cancer and found that most of the tumors in PBS groups were in late stage (Dukes’ stage D). In SL3261 and SL3261C groups, tumors were found in middle and late stages (Dukes’ stage C D), whereas tumors in SL3261R group were in early and middle stages (Dukes’ stage A B C). Together, our data extended previous findings and indicated that recombinant bacteria-based 4-1BBL vaccines effectively inhibited the development of colorectal cancer .
CD3+CD8+ cells are major players of T cell immunity against tumors . CD25 is the α-chain of IL-2R, which is absent on naive resting T cells, except for T regulatory cells. Following stimulation by “signal two”, activated T cells initiate IL-2 expression and secretion, which acts as an autocrine growth factor because activated T cells express high-affinity IL-2R. IL-2 exerts its pleiotropic activities through binding to either dimeric receptor composed of β-chain IL-2R (CD122) and common cytokine receptor γ-chain (CD132) or the receptor composed of trimeric IL-2R. alpha; IL-2Rβ, and common cytokine receptor γ-chain. CD25 has been considered as a marker of activated lymphocytes [24, 25]. Consistent with our previous results , the number of CD3 + CD8+ and CD3 + CD25+ T cells in peripheral blood obtained from SL3261R group was increased following induction of colorectal tumors for 16 weeks, when animals were given drug administration for once (P < 0.05 compared with PBS group). Nevertheless, no statistical difference was found between SL3261R group and SL3261 or SL3261C group (P > 0.05), which might be due to the short time period of drug administration and only once vaccine administration. Significant elevated number of CD3 + CD25+ T cells in peripheral blood was detected 21 weeks following tumor induction, when animals were given 3 times of drug administration in SL3261R group (P < 0.05 compared with all groups), suggesting the recombinant bacteria-based 4-1BBL vaccines can enhance the immune function of tumor-bearing mice. We did not observe significant difference in the frequency of peripheral blood and splenic CD3+CD8+ T cells between the SL3261R and other groups of rats at 21 weeks post DMH treatment, It is possible that CD3+CD8+ T cells may migrate into the tumors with the progression of tumorigenesis. More importantly, we detected significantly higher levels of splenic IFN-γ responses in the SL3261R group of rats. Given that IFN-γ is predominantly secreted by Th1 and cytotoxic CD8+ T cells (the stronger IFN-γ responses), together with a higher frequency of activated T cells suggest that the recombinant vaccine may enhance T cell immunity that inhibits the development of colorectal cancers in rats .