Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape

Abstract

Although many human cancers such as melanoma express tumor antigens recognized by T cells, host immune responses often fail to control tumor growth for as yet unexplained reasons. Here, we found a strong association between melanocyte expression of B7-H1 (PD-L1), an immune-inhibitory molecule, and the presence of tumor-infiltrating lymphocytes (TILs) in human melanocytic lesions: 98% of B7-H1(+) tumors were associated with TILs compared with only 28% of B7-H1(-) tumors. Indeed, B7-H1(+) melanocytes were almost always localized immediately adjacent to TILs. B7-H1/TIL colocalization was identified not only in melanomas but also in inflamed benign nevi, indicating that B7-H1 expression may represent a host response to tissue inflammation. Interferon-γ, a primary inducer of B7-H1 expression, was detected at the interface of B7-H1(+) tumors and TILs, whereas none was found in B7-H1(-) tumors. Therefore, TILs may actually trigger their own inhibition by secreting cytokines that drive tumor B7-H1 expression. Consistent with this hypothesis, overall survival of patients with B7-H1(+) metastatic melanoma was significantly prolonged compared with that of patients with B7-H1(-) metastatic melanoma. Therefore, induction of the B7-H1/PD-1 pathway may represent an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous antitumor activity and may explain how melanomas escape immune destruction despite endogenous antitumor immune responses. These observations suggest that therapies that block this pathway may benefit patients with B7-H1(+) tumors.

Fig. 1

Patterns of B7-H1 expression observed in melanocytic lesions stained with the anti–B7-H1 mAb 5H1. (A) No B7-H1 expression (brown chromogen) by melanocytes in a benign nevus. Original magnification, ×200 (scale bar, 50 µm). Note the paucity of TILs in this case. (B) B7-H1 expression by both melanocytes and TILs at the advancing edge of an invasive primary melanoma, nodular histologic subtype. Original magnification, ×40 (scale bar, 200 µm). (C) Original magnification of the boxed area shown in (B), ×400 (scale bar, 20 µm). Overall, 10% of total melanoma cells in this specimen expressed B7-H1 (scoring was performed as described in Materials and Methods). The inflammatory host response to tumor was graded as “moderate” in this case and included B7-H1⁺ TILs. (D) Diffuse B7-H1 expression by melanocytes in a subcutaneous melanoma metastasis to the scalp, associated with singular TILs. Original magnification, ×200 (scale bar, 50 µm). Representative photomicrographs of CD3 staining for TILs corresponding to each of these examples are shown in fig. S1. The interface pattern with B7-H1 expression by both melanocytes and TILs at the advancing edge pre-dominated in this study.

Fig. 2

Geographic association of B7-H1 expression and TILs. (A) Primary nodular melanoma with associated “severe” grade of lymphocytic infiltration, highlighted by CD8 immunostaining. The dark line demarcates the area of colocalizing T cells and B7-H1⁺ tumor cells. Overall, 30% of the tumor cells demonstrated cell surface B7-H1 expression. Note that the melanoma cell cytoplasm is heavily pigmented due to abundant melanin (see isotype control). Original magnifications, ×200 for left and middle column panels (scale bars, 50 µm) and ×400 for right column panels (scale bars, 25 µm). (B) Metastatic deposit of melanoma in a lymph node, associated with “mild” grade of lymphocyte infiltration shown by CD3 immunostaining. Original magnifications, ×100 for left column panels (scale bars, 100 µm), ×200 for middle column panels (scale bars, 50 µm), and ×400 for right column panels (scale bars, 25 µm). Arrows highlight the geographic area of concordance of CD3⁺ TILs with B7-H1 expression by melanoma, and also point to the area shown at higher magnification in the right-hand panels. Five percent of tumor cells demonstrated B7-H1 expression in this case.

Fig. 3

Distribution of intensity of B7-H1 expression by melanocytic cells and immune infiltrates in 150 lesions. (A) Percent B7-H1 expression by melanocytes ≥5% was considered positive. (B) AIS, defined as the intensity of intratumoral inflammation including TILs and histiocytes (graded 0 to 3, see text) multiplied by the percent B7-H1⁺ inflammatory cells.

Fig. 4

Correlation of overall patient survival with B7-H1 expression in melanomas. (A) There was no significant difference in survival related to melanoma cell B7-H1 expression in 43 patients with primary invasive melanomas (P = 0.33). (B) However, among 56 patients with metastatic disease, those whose metastatic tumors expressed B7-H1 had significantly improved survival compared to the B7-H1⁻ cohort (P = 0.032). Similar results were seen in analyzing the AIS, representing B7-H1 expression in lymphohistiocytic tumor infiltrates, as well as in analyzing the presence of TILs (mild, moderate, and severe lymphocyte infiltrates with associated histiocytes/macrophages). (C and E) No correlation of AIS or TILs was observed with survival in patients with invasive primary melanomas [P = 0.25 (C) and 0.32 (E)]. (D and F) However, there was a significant correlation in patients with metastatic disease [P = 0.014 (D) and 0.017 (F)]. B7-H1⁺, ≥5% of tumor cells with membranous expression; B7-H1⁻, <5% membranous tumor cell expression.