We consistently observed constitutive expression of TLT-2 in LN CD8+ T cells from naive mice, and its expression was comparable in RLN CD8+ T cells from tumour-bearing Selleckchem beta-catenin inhibitor mice. Here, we examined TLT-2 expression in TIL for the first time. We found a marked lymphocyte infiltration within the B7-H3/SCCVII tumour mass, indicating active anti-tumour immune responses in the B7-H3+ tumour sites. Surprisingly, the majority of CD8+ TIL in the B7-H3/SCCVII-inoculated mice
lost TLT-2 expression, and the cells expressing activation marker down-regulated TLT-2 expression. These findings suggest that activation signals to CD8+ T cells induce down-regulation of TLT-2. Although we tried to detect TLT-2 expression by immunofluorescence histostaining, TLT-2 expression was undetectable so we could not examine the distribution of TLT-2+ versus TLT-2− CD8 TIL in the tissues. We also found that TGF-β, which is often secreted from solid tumour cells like squamous cell carcinomas or tumour-associated cells, down-regulated TLT-2 expression. It is therefore possible that some tumour-related environmental factor(s) may have caused
TLT-2 down-regulation. TLT-2 down-regulation occurred at the local tumour sites and this may have contributed to the limited efficacy of B7-H3-transduced tumours. Our results from the TLT-2-transduced CD8+ T-cell study suggest that the TLT-2 expression level is more critical than that of B7-H3 to deciding whether there is a contribution of the B7-H3–TLT-2 pathway. Over-expression of B7-H3 is no longer required selleck compound when sufficient TLT-2 expression is provided on the surface of CD8+ T cells (Fig. 6d). In
contrast to broad and abundant B7-H3 expression, TLT-2 expression levels in T cells are tightly regulated. Additional approaches for preventing TLT-2 down-regulation or enhancing TLT-2 expression at tumour sites may be needed. We performed experiments to block the B7-H3–TLT-2 pathway, using anti-B7-H3 and anti-TLT-2 mAbs, to confirm the functional contribution of B7-H3 and TLT-2 in B7-H3-introduced tumour-mediated immunity. Unfortunately, mafosfamide there was no effect on the tumour regression induced by B7-H3-introduced tumours that expressed high levels of B7-H3. Interestingly, growth of the parental tumour, which expressed endogenously low levels of B7-H3, was accelerated by treatment with either anti-B7-H3 or anti-TLT-2 mAb. This suggests the immunoenhancing effects of the B7-H3–TLT-2 pathway in tumour immunity against parental tumours. We have previously attempted and failed to reverse the enhanced responses induced by B7-H3- or TLT-2-transduced cells using the same anti-B7-H3 and TLT-2 mAbs in vitro, although these mAbs could inhibit B7-H3 immunoglobulin binding, assessed by flow cytometry, and the functional endogenous TLT-2 and B7-H3 interaction in contact hypersensitivity in vivo.28 The low affinity of our blocking mAbs may explain the failure.