Pancreatic cancer is one of the most aggressive human malignancies and still represents a major therapeutic challenge. Despite the infiltration of large numbers of immune cells into pancreatic cancer, the immune system fails to prevent disease development and progression. Recently, dysregulation of immune responses by aberrant expression of negative costimulatory molecules by tumor cells has been suggested to play a potential role in the evasion of tumors from the immune system [
4,
5]. For example, tumor B7-H1 expression has been shown to significantly correlate with negative outcome in several human malignancies, including pancreatic cancer [
11,
13‐
16]. B7-H3 is another member of the B7 family of costimulatory molecules which serves as an accessory costimulatory regulator of T cell responses following initial T cell priming. The exact physiological function of B7-H3 and especially its role in the development and progression of human cancers are still elusive.
In this study, we demonstrated that the cultured pancreatic cancer cell lines Panc-1, MiaPaCa-2, and SU86.86 constitutively express B7-H3. Moreover, we demonstrated that B7-H3 gene expression is upregulated in pancreatic cancer tissues in comparison to normal pancreatic tissues. Immunohistochemical analysis revealed B7-H3 protein expression by pancreatic cancer cells. Tumor cell-associated B7-H3 expression significantly correlated with better postoperative survival. Moreover, we showed that B7-H3 gene expression in pancreatic cancer tissues significantly correlated with the levels of CD4 and CD8. Further immunohistochemical analyses on the distribution of CD4+ and CD8+ T cells revealed the infiltration of CD4+ and CD8+ T cells into areas of B7-H3 positive pancreatic cancer cells. More importantly, the number of tumor-infiltrating CD8+ T cells significantly correlated with the levels of B7-H3 on pancreatic cancer cells. Therefore, one could speculate that tumor-associated B7-H3 expression might act as a positive regulator of antitumor response in pancreatic cancer. B7-H3 might trigger the infiltration of CD8+ T cells into cancerous tissues and might also enhance tumor immunogenicity by stimulation of tumor-infiltrating CD8+ T cells. These findings are in accordance with previous reports on the function of B7-H3. Chapoval and colleagues reported that human B7-H3 costimulates proliferation of CD8+ T cells, enhances the induction of cytotoxic T cells, and selectively stimulates IFN-γ production by T cells in the presence of T cell receptor signaling in vitro [
17]. Further data in support of a positive costimulatory role of B7-H3 in the regulation of immune response come from an experimental study showing that acute and chronic cardiac allograft rejection can be reduced in B7-H3 knock-out mice [
26]. Recently, B7-H3 has also been implicated as a potential stimulator of antitumor immunity. In vivo, transfection of B7-H3 into P815 mouse tumors led to tumor regression and amplification of tumor-specific CD8+ CTL response in syngeneic mice suggesting enhancement of tumor immunogenicity by preferential stimulation of CD8+ T cell responses [
19]. Further evidence in favor of a possible tumor-protective effect of B7-H3 expression in cancers comes from a clinical study investigating the expression of B7-H3 in human gastric carcinoma. The majority of tumors showed B7-H3 expression in tumor cells. Moreover, the presence of B7-H3 positive tumor cells was associated with improved patient survival [
27]. Although these results clearly implicate a tumor-protective effect of B7-H3, the exact physiologic/pathologic role of B7-H3 remains ambiguous, because B7-H3 has also been shown to inhibit CD4+ and CD8+ T cell proliferation and IFN-γ production mediated by anti-CD3 in mice [
28]. Recently, single or duplicate constructs of the immunoglobulin-V-like and immunoglobulin-C-like domains of B7-H3 have been shown to downregulate both T cell proliferation and cytokine production in response to CD3/CD28-mediated costimulatory activation [
29]. Further evidence that B7-H3 may serve as an inhibitor of antitumor immunity includes several clinical studies. In prostate cancer, Zang et al. evaluated the expression of B7-H3 in over eight hundred prostate cancer patients and reported that 93% of the examined tumors displayed aberrant B7-H3 expression [
23]. High tumor B7-H3 expression was associated with disease progression and spread as well as with poor patient survival [
23]. Accordingly, tumor cell and tumor vasculature B7-H3 expression significantly correlated with an increased risk of death from clear cell renal cell carcinoma [
30]. In these studies, however, the functional aspect of B7-H3 expression was not tested in detail. For example, correlation of B7-H3 expression by cancer cells with tumor-infiltrating immune cells was either not tested or not significant. The reason for the contrasting effects of B7-H3 in cancer might also be related to varying counter-receptors involved in different tumor entities. So far, one receptor of B7-H3 has been identified [
31]. Hashiguchi and co-workers have recently reported that murine B7-H3 specifically binds to triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2, TREML2), which is expressed on both CD4+ and CD8+ T cells [
31]. Moreover, the authors demonstrated that stimulation with B7-H3 transfectants preferentially upregulated the proliferation and IFN-γ production of CD8
+ T cells [
31]. In humans, however, a receptor for B7-H3 has not been identified yet.
Another possible mechanism involved in antitumor immunity includes the induction of tumor-suppressive cytokines. Here, we found that B7-H3 was significantly associated with IFN-γ levels. IFN-γ is an important pro-inflammatory Th1 cytokine and has been implicated as a mediator of an extrinsic tumor-suppressor mechanism in immunocompetent hosts. IFN-γ is usually produced by activated cytotoxic T lymphocytes, macrophages, and natural killer (NK) cells [
32]. In mice, treatment with neutralizing monoclonal antibodies to IFN-γ resulted in a faster growth of immunogenic sarcomas that were transplanted into mice [
33]. Furthermore, endogenously produced IFN-γ has been shown to be protective against transplanted, chemically induced, and spontaneous tumors [
34]. Therefore, B7-H3 might act as an immunostimulatory agent through the induction of IFN-γ.
Mechanisms regulating B7-H3 expression are still unclear. The local cytokine milieu in the tumor microenvironment represents a potential regulator of B7-H3. Indeed, IFN-γ has previously been shown to enhance surface expression of B7-H3 on bone marrow derived dendritic cells and on monocytes [
18]. Therefore, we investigated the potential effects of the Th1 cytokine IFN-γ and Th2 cytokine IL-4 on the expression of B7-H3 on cultured pancreatic cancer cells. We found that both IFN-γ and IL-4 treatment resulted in a marked increase of B7-H3 on SU86.86 but not on Panc-1 pancreatic cancer cells. Furthermore, we found that B7-H3 positively correlated with IFN-γ levels in vivo. Taken together, these findings suggest a potential involvement of IFN-γ (and IL-4) in the regulation of B7-H3 expression in pancreatic cancer.