Relationship between B7-H4, Regulatory T Cells, and Patient Outcome in Human Ovarian Carcinoma Ilona Kryczek, 1 Shuang Wei, 1 Gefeng Zhu, 2 Leann Myers, 3 Peter Mottram, 3 Pui Cheng, 3 Lieping Chen, 2 George Coukos, 4 and Weiping Zou 1 1 Department of Surgery, University of Michigan, Ann Arbor, Michigan; 2 Departments of Dermatology and Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 3 Tulane University Health Science Center, New Orleans, Louisiana; and 4 Division of Gynecologic Oncology, University of Pennsylvania, Philadelphia, Pennsylvania Abstract B7-H4 is a recently identified B7 family member. We previously showed that ovarian tumor and associated macro- phages expressed B7-H4; tumor B7-H4 + macrophages and CD4 + CD25 + FOXP3 + regulatory T cells (Treg cells) suppressed tumor-associated antigen–specific T-cell immunity. To deter- mine the pathologic relationship between B7-H4, macro- phages, and Treg cells in the tumor environment, in addition to Treg cell numbers, we quantified B7-H4 expression in the tumor and tumor-associated macrophages in 103 patients with ovarian carcinoma. We observed that the intensity of B7-H4 expression in macrophages was significant- ly correlated with Treg cell numbers in the tumor. Further, both Treg cells and macrophage B7-H4, but not tumor B7-H4, were negatively associated with patient outcome. Tumor Treg cells enabled macrophages to spontaneously produce inter- leukin (IL)-10 and IL-6. Tumor macrophages stimulated B7-H4 expression in an autocrine manner through IL-10 and IL-6. Our previous work showed that tumor-associated macro- phages spontaneously produced chemokine CCL22 to mediate Treg cell trafficking into tumor, and Treg cells induced B7-H4 on antigen-presenting cells (APC) including macrophages. Altogether, our data support the concept that there is a mechanistic interaction between Treg cells and macrophage, and that Treg cells may convey the suppressive activity to APCs through B7-H4 induction in human ovarian cancer. [Cancer Res 2007;67(18):8900–5] Introduction B7-H4 (B7x, B7S1) was identified in 2003 (1–3). B7-H4 mRNA expression was found to be widely distributed in the peripheral tissues, including kidney, liver, lung, spleen, thymus, and placenta. However,B7-H4proteinexpressiononthecellsseemstobelimited (1, 4). Interestingly, several groups have shown that human ovarian cancersexpresshighlevelsofB7-H4protein(4–9).Additionally,high levelsofB7-H4werefoundinnon–small-celllungcancer(10),ductal and lobular breast cancer (9, 11), and renal cell carcinoma (12). Antigen-presenting cells (APC) are critical for initiating and maintaining tumor-associated antigen (TAA)–specific T-cell immu- nity. Tumor-associated macrophages markedly outnumber other APCs, such as dendritic cells, and represent an abundant popu- lation of APCs in solid tumors (13–16). We recently reported that human ovarian cancer–associated B7-H4 + macrophages inhibited TAA-specific T-cell immunity, in part through B7-H4 (5). We have now further quantified B7-H4 expression in tumor and tumor- associated macrophages, and analyzed the relationship between B7-H4 expression, Treg cells, and patient outcome. Materials and Methods Human subjects. We studied 103 previously untreated patients with epithelial ovarian carcinomas, International Federation of Gynecology and Obstetrics stage I to IV (Table 1). Patients gave written, informed consent. The study was approved by local institutional review boards. Clinical information is available for 70 patients. Human cells and ovarian tumor tissues. Cells were obtained from ascites, fresh ovarian tumor tissues, and peripheral blood as previously described (17–19). CD3 + T cells and CD14 + cells were purified with paramagnetic beads (StemCell Technology) and sorted with FACSaria using DiVa software (Becton Dickinson Immunocytometry Systems). Lin À EpCam + CD45 À CD14 À epithelial ovarian tumor cells were sorted on a FACSaria using DiVa software (Becton Dickinson). Cell populations were >98% pure as confirmed by flow cytometry (LSR II, Becton Dickinson). Ovarian tumor tissues and associated cells were collected for immuno- fluorescence analysis and cytokine detection by reverse transcription-PCR (RT-PCR). Immunofluorescent staining. Immunofluorescence analysis was done as previously described (19). Tissues were stained with mouse anti-human B7-H4 (hH4.1, IgG1, 4 Ag/mL; ref. 1), mouse anti-human Ham56 (Ham56, IgM,1/20;DAKO),followedbyAlexaFluor488–conjugatedgoatanti-mouse IgG1 and Alexa Fluor 568–conjugated goat anti-mouse IgM (all 2 Ag/mL, Molecular Probes). The same antibody concentrations were used for all the tissue staining throughout this work. Quantification of B7-H4 expression. B7-H4 expression was detected with the Leica DMIRE2 confocal microscope. Laser intensity was calibrated foreachobservationsessionusingacontroltestslidewithCaliBRITEbeads (Alexa 488, Alexa 568). The intensity of the laser was adjusted to a normalized value of the mean fluorescent intensity (10 F 0.1) using the same beam settings, Alexa 488 (10%), Alexa 568 (10%), PMT 1 and PMT 2 value (700), offset value PMT 1 and PMT 2 (0), Z thickness (10 Am), numberofslices(10),andaverageofslices(2).Beamsettingswereadjusted using the slide with the most fluorescent intensity to Alexa 488 29%, PMT1 733, Offset-1; and Alexa 568 23%, PMT2 (701), Offset-1. The scanner was engaged and the peak fluorescence intensity of B7-H4 staining with Alexa 488waslocatedbyadjustingthe Z position to reveal the most intense pixel concentration. A series of eight slices containing fields with the most intense pixel concentration were selected with a total Z thickness of 3 Am. Two scans for each of the eight slices were captured and the average projection of all 16 slices was used to quantify mean fluorescent intensity. Five adjacent consecutive fields were captured for each of the specimens. The Leica Confocal Simulator software contains functions to quantify specific values located in designated regions of interest (ROI). Background fluorescence was normalized adjusting the threshold of the average projection of each series. An ROI with an area of 2,400 F 100 Am 2 was Note: I. Kryczek and S. Wei contributed equally to this work. Requests for reprints: Weiping Zou, University of Michigan School of Medicine, C560B MSRB II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0669. Phone: 734-763-6402; Fax: 734-763-0143; E-mail: wzou@umich.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-1866 Cancer Res 2007; 67: (18). September 15, 2007 8900 www.aacrjournals.org Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/67/18/8900/2575172/8900.pdf by guest on 19 June 2022