Previous reports have established that immortalized NHAs do not differ in their expression levels of B7-H1 protein from their normal counterparts [11
]. Here, NHAs transfected with E6/E7 did not show a significant increase in level of B7-H1 protein expression when treated with IFN-γ, while both Ras+ NHAs and Akt+ NHAs showed a significant increase in B7-H1 protein expression levels with IFN-γ treatment (). Exposure to IFN-γ resulted in a dramatic increase in B7-H1 protein levels in Ras+/Akt+ NHAs ().
Figure 1 B7-H1 protein levels are elevated by interferon-γ on astrocytes with Akt and Ras activation. Standardized fluorescence intensity of B7-H1 protein expression on the surface of human astrocytes immortalized by hTERT, E6, and E7 (E6/E7), immortalized (more ...)
In vivo, glioblastoma multiforme (GBM) cells show a range of functional PTEN expression (), and loss of PTEN is correlated with activation of the PI3K pathway and increased B7-H1 expression [11
]. Based on immunohistochemical findings of PTEN levels, established glioma cell lines were categorized as PTEN deficient (PTEN-), if positive staining was found in 0–25% of cells (2/6 cell lines) or in 25–50% of cells (1/6). Tumors were considered PTEN intact (PTEN+), if positive staining was found in 50–75% of cells (1/6) or in 75–100% of cells (2/6).
Figure 2 Variation in PTEN expression in human malignant glioma. (A) Patient A, Hematoxylin and Eosin, 100×, showing glioblastoma. (B) Patient A, PTEN stain, 100×, shows little to no positivity to stain using Rabbit anti-human PTEN antibody. Positive (more ...)
Transcript levels of B7-H1 were not significantly different in PTEN- tumors compared to PTEN+ tumors at baseline (). In contrast, expression levels of B7-H1 protein were significantly higher in PTEN- tumors compared to PTEN+ tumors at baseline (). Treatment with IFN-γ resulted in increased B7-H1 transcript and protein expression in both PTEN- and PTEN+ cell lines (). In addition, IFN-γ induced a significantly larger degree of increase in levels of B7-H1 protein and transcript in PTEN- tumor cells than in PTEN+ tumors ().
Figure 3 PTEN loss leads to superinduction of B7-H1 in response to interferon-γ through transcriptional and translation regulation. (A) Representative B7-H1 transcript levels relative to HPRT in PTEN deficient (PTEN -) and PTEN wildtype (PTEN +) primary (more ...)
To investigate the functional consequences of the observed superinduction of B7-H1 in PTEN- tumors by IFN-γ, the degree of apoptosis of autologous lymphocytes and T-cells were analyzed in co-culture experiments. Degree of apoptosis was measured by positivity of Annexin V staining using flow cytometry. Proportions of viable cells were determined by those with normal size and granularity, negativity of Annexin V stain and positivity for CD3 +/− CD45. As illustrated in , co-culture of autologous lymphocytes with PTEN- tumor cells resulted in a significantly higher degree of lymphocyte apoptosis than co-culture of lymphocytes with autologous PTEN+ tumor cells (mean % apoptosis: 45% and 12%, respectively; p<0.001). Co-culture with autologous PTEN- tumor cells treated with IFN-γ demonstrated a significantly increased degree of lymphocyte apoptosis (mean % apoptosis from 45% to 72%; p<0.001), along with decreased subsets of viable lymphocytes. For samples from patients with PTEN+ tumor, co-culture experiments after treatment of tumors with IFN-γ did not demonstrate a significantly higher degree of lymphocyte apoptosis.
After co-culture with autologous tumor cells, T-cells of patients with PTEN- tumors had equivalent levels of tumor-induced apoptosis as T-cells derived from patients with PTEN+ tumors (). Compared to co-culture with tumors without treatment of IFN-γ, T-cells after co-culture with autologous PTEN- tumor cells treated with IFN-γ resulted in a significantly increased level of apoptosis (23% to 64%; p<0.001). After IFN-γ treatment there was also a decrease in proportion of viable T-cells (data not shown). This increase in apoptosis was not observed for co-culture experiments utilizing samples from PTEN+ glioma patients.
To determine if the effects of IFN-γ seen above on levels of B7-H1 protein and T-cell apoptosis are mediated by activation of the PI3K pathway, B7-H1 protein measurements using flow cytometry and T-cell killing assays were repeated with tumors after treatment with inhibitors of the PI3K pathway, AKT III, LY294, Rapa, and HNK. For PTEN- samples, inhibition of the PI3K pathway decreased the B7-H1 levels of IFN-γ treated tumor cells to levels prior to treatment with IFN-γ (). The percentage of T-cells undergoing apoptosis after co-culture with autologous PTEN- glioma cells treated with IFN-γ was greatly reduced by treatment of any of the four PI3K inhibitors prior to co-culture ().
Figure 4 Inhibition of PI3K pathway inhibits induction of B7-H1 and enhanced T-cell killing by interferon-γ. (A) Representative standardized fluorescence intensity of B7-H1 protein levels in PTEN deficient glioma cells treated with PI3K pathway inhibitors. (more ...)