Tumor-Expressed B7-H1 and B7-DC in Relation to PD-1+ T-Cell Infiltration and Survival of Patients with Cervical Carcinoma

Cervical cancer is the second most common cancer in women worldwide (1). It develops as a result of an uncontrolled persistent infection with a high-risk type of human papilloma virus (HPV), in particular, types HPV16 and HPV18 (2). The occurrence of HPV-induced cancer is strongly associated with failure to mount a strong HPV-specific type 1 T-helper and cytotoxic T-lymphocyte response (3–5), the lack of CD8+ T cells migrating into the tumor cell nests, the induction of HPV16-specific regulatory T cells, and the influx of regulatory T cells into the tumor (6, 7). Moreover, the ratio between the tumor-infiltrating CD8+ T cells and coinfiltrating CD4+Foxp3+ regulatory T cells is an independent prognostic factor for overall survival (8), indicating the key role of these different types of T cells in cervical cancer.

Activated T cells can express the programmed cell death 1 (PD-1) receptor, which can bind B7-H1 (PD-L1) and B7-DC (PD-L2). B7-H1 could be induced to express by a wide variety of immune cells and nonhematopoetic cell types, whereas B7-DC is expressed mainly on activated macrophages and dendritic cells (9). Upon simultaneous engagement of both, the T-cell receptor and PD-1–negative immunoregulatory signals are transferred to the T cells, resulting in a decreased effector response and T-cell tolerance (10). PD-1/B7-H1 interactions have been shown to inhibit a wide range of immune responses against pathogen, tumor, and self-antigens (11, 12).

More recently, it has been reported that B7-H1 and B7-DC are exploited by tumors to evade immune responses. B7-H1 is found to express on cell surface in most human cancers, and this expression was correlated with poor clinical prognosis in renal, gastric, ovarian, breast, and esophageal carcinomas (13–17). The role of B7-DC in the suppression of immune responses remains controversial (18). Because of the strong association between tumor-infiltrating lymphocytes (TIL) and the prognosis of cervical cancer (6, 8) and the fact that PD-1 has been reported to be expressed by tumor-infiltrating CD8+ T cells, CD4+ T cells, and regulatory T cells (10), we studied the expression and function of B7-H1, B7-DC, and PD-1 in cervical cancer. Here, we show that PD-1 is expressed on a substantial number of tumor-infiltrating CD8+, CD4+, and regulatory T cells. B7-H1, however, is expressed in only a small group of cervical cancer patients and does not confer a survival disadvantage. Interestingly, when the tumors of this group of patients are infiltrated with a high number of tumor-infiltrating CD4+Foxp3+ regulatory T cells, the expression of B7-H1 may bestow a survival benefit.

A previously described tissue array containing 115 cervical cancer samples of patients, all of whom underwent a radical hysterectomy, was used for the B7-H1 and B7-DC staining (8). Standard immunohistochemical staining was done using antibodies against human B7-H1 (clone 5H1) and B7-DC (R&D Systems). The tissue array sections were deparaffinized, and antigen retrieval was done using EDTA. To reduce nonspecific binding, sections were incubated overnight at 4°C with 10% rabbit serum. The B7-H1 and B7-DC antibodies were used in 1:200 and 1:800 dilutions, respectively. The Powervision detection system was applied (DAKO). The tissue array was evaluated and scored by two experienced researchers (R. Karim; E.S. Jordanova) independently. Expression groups were defined based on the presence or absence of membranous staining.

Eight cases with B7-H1–positive and eight cases with B7-H1–negative cervical cancer specimens were selected from the tissue array based on a comparable amount of tumor-infiltrating T cells to avoid a potential bias between the two groups. The simultaneous immunohistochemical staining of three different epitopes applied to 4-μm, formalin-fixed, paraffin-embedded tissue sections has been reported by us before (6, 8). Briefly, the sections were incubated overnight with a mix of anti-CD8 (4B11; mouse IgG2b; Novo Castra), anti-Foxp3 (clone 236A/E7; Abcam), and anti–PD-1 (R&D Systems) after antigen retrieval with EDTA. Slides were washed and incubated with a combination of the fluorescent antibody conjugates goat anti-mouse IgG2b–Alexa-546, goat anti-rabbit IgG1–Alexa-488, and donkey anti-goat–Alexa-647. Alexa Fluor conjugates were obtained from Molecular Probes. The images were captured with a confocal Laser Scanning Microscope (Zeiss LSM510, Zeiss). Ten images were scanned per slide. For each case, one successive negative control slide was included. The intraepithelial TIL count was presented as the number of cells per square millimeter.

A previously isolated HPV16-specific CD4+FoxP3+ regulatory T-cell clone (7) C148.31 and an influenza-specific CD4+Foxp3- T-helper clone (B1.50) were stained with goat anti–PD-1 (R&D Systems), followed by anti-goat biotin (Dako) and streptavidin-allophycocyanin (APC) (eBioscience). PD-1 expression was analyzed by flow cytometry. The effect of B7-H1 on the proliferative response of these two clones was assessed by stimulating 25,000 T cells with 1 μg/mL plate-bound anti-CD3 (OKT-3; Ortho Biotech) and 1 μg/mL plate-bound anti-CD28 (clone L293; BD Biosciences) in the presence or absence of 5 μg/mL plate-bound recombinant human B7-H1/Fc chimera (R&D Systems). The effect of B7-H1 on the regulatory capacity of the CD4+Foxp3+ T-cell clone was tested in a classic suppression assay (7) in which the clone C148.31 was stimulated with 1 μg/mL plate-bound anti-CD3 in the presence or absence of 5 μg/mL plate-bound recombinant human B7-H1/Fc chimera for 24 h. Subsequently, the stimulated C148.31 regulatory T cells were washed to prevent spillover of B7-H1 and put into a coculture with CD4+CD25- responder cells in the presence of 1 μg/mL soluble anti-CD3 and APC (7). After 48 h, the supernatants of triplicate wells were harvested and pooled for the analysis of IFN-γ production by the activated responder cells using enzyme-linked immunosorbent assay (ELISA).

Correlations between B7-H1 or B7-DC expression with clinicopathologic parameters or the (high or low) number of tumor-infiltrating cells was done by the χ² or, where appropriate, the Fisher's exact test. Patient groups were based on the median (50th percentile) of the numbers of infiltrating immune cells per square millimeter because none of the data for the TIL subtypes followed a normal distribution pattern. Analyses of differences in the numbers of subpopulations of PD-1+ TIL in B7-H1–positive or–negative tumors were done by the nonparametric Mann-Whitney test. All reported Ps are two sided. A P < 0.05 was considered significant. Cumulative 5-y survival rate was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Statistical analyses were done with the SPSS software package 16.

To assess the expression and impact of B7-H1 and B7-DC in cervical cancer, we studied a group of 115 well-characterized patients whose clinicopathologic characteristics are shown in Table 1. The mean age of the patients was 48.5 years, with a range between 24 and 87 years at the time of surgery. Fifty-one patients received postoperative radiotherapy because of either tumor-positive lymph nodes or a combination of two of the following parameters: depth of infiltration ≥ 15 mm, tumor size ≥ 40 mm, and presence of vasoinvasion. At the end of the 5-year follow-up period, 23 patients had died of disease, 85 were alive, 5 patients had a recurrence, and 2 died of causes unrelated to the primary disease but showed no evidence of disease.

The expression of B7-H1 and B7-DC in the tumors of these patients was determined by immunohistochemistry. Specific examples of B7-H1 and B7-DC staining are shown in Fig. 1. Examination of the entire group of 115 patients revealed the expression of B7-H1 in 22 (19%) tumors, whereas B7-DC was expressed in 34 (29%) cases. The expression of B7-H1 and B7-DC did not correlate (P = 0.604). Notably, B7-DC expression was associated with cervical adenocarcinomas and a more advanced stage of cervical cancer (Table 1).

The number and subtype of intraepithelial TIL per square millimeter of tumor within this group of 115 patients has already been quantified (8), and this enabled us to analyze the impact of B7-H1 and B7-DC expression on the number and type of intraepithelial TIL in these tumors. The expression of B7-H1 was associated with higher intraepithelial infiltration by Foxp3+ T cells (P = 0.022) but not with CD8+ T cells (Table 2). In contrast, there was no significant association between TIL and B7-DC expressed by tumor cells (Table 2).

Negative regulation of the tumor-specific T-cell response by B7-H1 expressing tumor cells requires the intraepithelial infiltrating T cells to express PD-1. Therefore, PD-1 expression by intraepithelial TIL was tested in a group of patients with a B7-H1–positive tumor, as well as in a group of patients with a B7-H1–negative tumor, which were matched with respect to the number of tumor-infiltrating CD8+ T cells, CD4+ T cells, and regulatory T cells (Table 3). In addition, there was no difference in the CD8/Foxp3 ratio (P = 0.959) between these two groups, allowing their comparison with respect to PD-1 expression. The number of single-, double- and triple-positive cells for CD8, Foxp3, and PD-1 was analyzed by triple fluorescent immunohistochemistry. This revealed that, in both groups of patients, more than half of the infiltrating CD8+ T cells and half of the Foxp3+ T cells expressed PD-1 (Table 3). Although, on the whole, the patient group with B7-H1–positive tumors displayed somewhat more intraepithelial PD-1+ T cells, this was not significantly different.

Retrospective analyses of patients with different types of malignancies showed a link between B7-H1 expression on tumors and poor prognosis (13–17). A similar analysis of the overall survival of patients with cervical cancer did not show such a direct relationship (P = 0.690; Fig. 2A). Notably, we recently reported that the ratio between intraepithelial CD8+ T cells and regulatory (Foxp3+) T cells is an independent prognostic factor for survival in this group of patients with cervical cancer, in which patients with a low CD8/regulatory T cell ratio had the worst survival (ref. 8; Fig. 2B). As a consequence, a potential detrimental effect of B7-H1 expression on survival is more likely to be observed within patients with well-infiltrated tumors. No effect of B7-H1 was seen in the subgroups divided either on the basis of CD8+ T-cell infiltration (P = 0.584) or on the number of regulatory T cells (P = 0.100; data not shown). In contrast, subdivision based on the CD8+/regulatory T-cell ratio revealed that the overall survival of patients with a B7-H1–positive tumor and a low CD8+/regulatory T-cell ratio was significantly better than in patients with a B7-H1–negative tumor and a low CD8+/regulatory T-cell ratio (P = 0.033; Fig. 2C). These data indicate that B7-H1 expression does not have a direct detrimental effect on the overall survival of patients with cervical cancer but on the contrary may improve survival of a small subgroup of cervical cancer patients with tumors relatively heavily infiltrated by regulatory T cells.

The association of B7-H1 expression by tumor cells and the enhanced survival of a small subgroup of patients with a low CD8+/regulatory tumor-infiltrating T-cell ratio (Fig. 2C) suggest that the function of regulatory T cells is affected by B7-H1. To test this hypothesis, we made use of a HPV16-specific CD4+Foxp3+ regulatory T-cell clone, which we had isolated from a HPV16+ patient with cervical cancer (7) and which expressed PD-1 at its surface (Fig. 3A), similar to what we observed in situ in cervical cancer (Fig. 3B). To determine if B7-H1 ligation has an effect on PD-1 expressing T cells, this CD4+Foxp3+PD-1+ regulatory T-cell clone was stimulated with anti-CD3 in the presence or absence of recombinant B7-H1 protein to test its proliferative capacity. Whereas the proliferation of a PD-1–negative helper T-cell clone was not affected, the PD-1–positive regulatory T-cell clone proliferated less well when B7-H1 was present in the culture (Fig. 3C). To assess whether B7-H1 ligation also inhibited the suppressive capacity of PD-1+ regulatory T cells, the HPV16-specific CD4+Foxp3+PD-1–positive regulatory T cells were stimulated with anti-CD3 in the presence or absence of recombinant B7-H1 protein and then cocultured with CD4+CD25- responder cells (Fig. 3D). Responder cells alone produced high amounts of IFN-γ, but this capacity was suppressed when activated regulatory T cells were added to the culture in a dose-dependent fashion. The presence of recombinant B7-H1 during the activation of the regulatory T cells had a clear negative impact on their suppressive capacity because the IFN-γ production by the responder cells was partly restored (Fig. 3D). These data show that, in principle, PD-1+ regulatory T cells can be incapacitated with respect to their suppressive function when engaged by B7-H1.

Following the initial reports that tumor-associated B7-H1 could serve as an immune escape mechanism in a mouse tumor model through downregulation of tumor-specific T-cell responses (19, 20), several retrospective studies in human cancer cohorts showed that B7-H1 expression was associated with clinicopathologic markers for poor prognosis (13, 17) or with lower overall survival (14–16). Our study indicates that most (80%) cervical cancers are B7-H1 negative and that the survival of B7-H1–positive cases is not negatively affected (Figs. 1 and 2). This suggests that tumor-expressed B7-H1 does not play a significant role in T-cell antitumor immune response in cervical cancer. Rather, our data show that more than half of the tumor-infiltrating CD8+T cells are positive for PD-1, which has previously been shown to be indicative for chronic antigen stimulation and T-cell exhaustion (9).

Although studies reported a negative impact of B7-H1 on the overall survival of patients with either renal or esophageal cancer (15, 16), we did not observe such a direct link between B7-H1 and survival in cervical cancer. A direct effect of B7-H1 on survival likely requires that this survival is associated with CD8+ T-cell infiltration and that these CD8+ T cells express PD-1. Indeed, in esophageal cancer, the infiltration with CD8+ T cells is an independent prognostic factor (21). It will be of interest to determine the proportion of PD-1 expressing CD8+ T cells in renal and esophageal cancer for a better assessment of their role in the observed negative association between B7-H1 and survival in these cancers. The fact that, in cervical cancer, the ratio between CD8+ T cells and coinfiltrating regulatory T cells functions as an independent prognostic factor (8) suggests that the interplay between the two cell types and, as such, the regulation of both cell types is important for survival in cervical cancer. In this view, we made an interesting observation that, among cervical cancer patients whose tumors were infiltrated with relatively higher number of regulatory T cells (low CD8:Foxp3 ratio), tumor-expressed B7-H1 may have conferred a survival benefit (Fig. 2C). Although the underlying mechanism remains to be elucidated, our data provide evidence that it may involve the functional impairment of regulatory T cells because a substantial portion of these cells express the receptor PD-1 in situ (Table 3, Fig. 3) and engagement of this receptor through B7-H1 decreases the capacity of an HPV16-specific CD4+Foxp3+ regulatory T-cell clone to suppress the function of effector cells in vitro (Fig. 3D). The absence or presence of B7-H1 in the group of patients with a high CD8+/regulatory T-cell ratio did not affect the overall survival (Fig. 2B), suggesting either that B7-H1 does not play a role in patients with a high number of tumor-infiltrating CD8+ T cells and a low number of regulatory T cells or that the stimulatory interaction between B7-H1 and B7.1 on CD8+PD-1- T cells (22) balances the negative interaction between B7-H1 and PD-1+ CD8 T cells within the local tumor environment. It seems, therefore, that B7-H1–mediated impairment of PD-1+ T cells particularly affects the PD-1–expressing regulatory T cells, thereby releasing the brake on the tumor-specific CD8+ T cells. Future experiments using primary tumor-infiltrating T-cell cultures may shed more light on this. Recent studies support the notion that PD-1 expression on regulatory T cells is associated with improved survival (22) and B7-H1–mediated inhibition of proliferation and function of regulatory T cells (23).

From a clinical standpoint, this study represents an unselected series of patients. Given the interest to target PD-1 or B7-H1 for the immunotherapy of cancer, our study suggests that treatment with PD-1– or B7-H1–blocking antibodies is a viable option. Although we have studied PD-1 expression in patients with stage IB or stage II disease, PD-1– or B7-H1–targeted immunotherapy is likely to be applied in a more advanced stage of cervical cancer. Although we speculate that PD-1 expression will be even more pronounced in advanced stage of cervical cancer, it will be of interest to determine PD-1 expression on T cells at this stage too. The PD-1–B7-H1 pathway also plays an important role in dendritic cell–T-cell interactions (9). One can easily envisage that B7-H1+ dendritic cell, cross-presenting tumor antigen (e.g., E6 and E7 of HPV), may impair the function of responding PD-1+ tumor–specific T cells.

No potential conflicts of interest were disclosed.