B7-H1 is expressed by malignant T cells. (A) PBMCs from patients with Sezary syndrome were analyzed by flow cytometry. Malignant T cells were identified by the expression of both CD4 and the clonotypic TCR Vβ chain, as shown in the representative dot plot. Samples were stained with an isotype control (closed histogram) or anti–B7-H1 (open histogram) and B7-H1 expression on malignant CD4⁺Vβ⁺ cells determined. For cases in which TCR Vβ use was unknown, malignant CD4⁺ T cells were identified by the aberrant down-regulation of CD7. A representative example (n = 11) is shown. (B) Immunohistochemical staining for B7-H1 was performed in paraffin-embedded biopsy specimens obtained from CTCL (n = 11) and PTCL (n = 144) patients and tumor-associated B7-H1 expression analyzed. Tumor cells were scored as B7-H1⁺ when B7-H1 and T cell–specific antibodies (eg, CD3, CD4, and CD8) both stained an identical population of cells, as determined by an expert hematopathologist. Two representative examples are shown (original magnification ×200).

B7-H1 is expressed by CD14⁺HLA-DR^lo monocytes in CTCL. (A-B) PBMCs from both normal donors (n = 23) and CTCL patients (n = 11) were stained with anti-CD14, anti–HLA-DR, and either an isotype control (closed histogram) or anti–B7-H1 (open histogram). The representative dot plot shown demonstrates a unique population of CD14⁺HLA-DR^lo cells present in CTCL patients. The frequency (percentage of total PBMCs) of HLA-DR^lo monocytes for normal donors and CTCL patients is shown in panel B. B7-H1 expression by the gated populations shown in panel A was determined. (C-D) B7-H1 expression (compared with an isotype control for each sample) by CD14⁺HLA-DR⁺ and CD14⁺HLA-DR^lo cells was analyzed (± 95% confidence interval is shown).

DC-associated B7-H1 promotes the induction of FoxP3⁺ regulatory T cells. (A-B) Purified CD4⁺ T cells were depleted of CD25^hi natural Tregs and cultured alone or with normal donor monocyte-derived iDCs in triplicate for 6 days. TGF-β (25 ng/mL) was included in the cultures shown in panel A or as indicated. Either an isotype control or blocking anti–B7-H1 (4 μg/mL) antibody was included. The frequency of CD25^hiFoxP3⁺ cells was determined by flow cytometry. Representative dot plots are shown in panel A. Data shown are representative of at least 3 similarly performed experiments. (C-D) Immunohistochemical staining for both B7-H1 and FoxP3 was performed on paraffin-embedded PTCL biopsy specimens (n = 48; in triplicate), as described in “Patient samples, immunohistochemistry, and immunofluorescence assay.” B7-H1 and FoxP3 staining from multiple areas of the same biopsy specimen are shown in panel C. (Inset) Original magnification ×200. (D) FoxP3⁺ cells in each high-power field (± 95% confidence interval is shown) were counted and DC staining for B7-H1 determined. For the purpose of our analysis, core biopsy specimens were considered B7-H1⁺ if any portion of the specimen contained B7-H1⁺ DCs.

Tumor-associated B7-H1 inhibits T-cell immunity. (A) Purified allogeneic CD4⁺ or CD8⁺ T cells (4 × 10⁶) were stimulated with irradiated B7-H1⁺ Karpas 299 cells (5 × 10⁵) in 24-well plates. T-cell lines were harvested, washed, counted, and restimulated with newly irradiated tumor cells every 6 to 8 days, at least 3 times. The T-cell lines thus generated were harvested; viable cells counted by exclusion of Trypan Blue and either cultured alone (4 × 10⁵) or restimulated with irradiated Karpas 299 (5 × 10⁴) in triplicate in a 96-well plate and thymidine incorporation determined after 72 hours. On restimulation, either an isotype control or anti–B7-H1 (4 μg/mL) was included, as indicated. Any background thymidine incorporation by irradiated Karpas 299 cells was subtracted from the values shown. (B) CD4⁺ and CD8⁺ T-cell lines were generated, as described in panel A. An isotype control or anti–B7-H1 (4 μg/mL) was included during each stimulation cycle, as indicated. Cells were subsequently harvested and restimulated with irradiated Karpas 299 cells. (C) A CD8⁺ T-cell line (10⁶/well), generated as described in panel A, was left unstimulated or restimulated with irradiated Karpas 299 cells (2.5 × 10⁵/well) for 6 hours in a 48-well plate in the presence of anti-CD107a, as described in “Cell lines, proliferation, and cytotoxicity assays.” An isotype control or anti–B7-H1 (4 μg/mL) was included, as shown. CD107a⁺CD8⁺ cells, identified by flow cytometry, are included in the gates shown (n = 3, P = .048). (D) CFSE-labeled Karpas 299 cells were cocultured with freshly purified CD8⁺ T cells or activated effector CD8⁺ T cells (4 × 10⁵/well) at various E/T ratios (E/T ratio of 200:1 is shown). CFSE⁺ tumor cells were identified by flow cytometry after 4 days of culture. All data shown are representative of at least 3 independently performed experiments.

B7-H1 is expressed by tumor-associated DCs and inhibits T-cell proliferation. (A) As described in Figure 1, immunohistochemical staining for B7-H1 was performed in patient biopsy specimens. B7-H1 expression by stromal monocyte-derived cells, identified by CD11c (shown), S-100, or CD68 staining, was determined. (B) Single-cell suspensions were generated from spleen (shown) or lymph node biopsy specimens obtained from PTCL patients (n = 5). Cells were stained with an isotype control (closed histogram) or anti–B7-H1 (open histogram) and B7-H1 expression on CD11c⁺CD14⁻ DCs determined. A representative example is shown. (C) Tissue specimens obtained from PTCL patients (n = 7) were double stained with antibodies to human CD11c (red) and B7-H1 (green) and viewed by fluorescent microscopy, as described in “Patient samples, immunohistochemistry, and immunofluorescence assay.” A representative example is shown (original magnification ×100). (D-E) Immature or LPS-matured DCs (4 × 10⁵/well) generated from normal-donor monocytes were cocultured with allogeneic, CFSE-labeled T cells (4 × 10⁶/well) for 6 days in the presence of an isotype control or anti–B7-H1 (4 μg/mL), as shown. T-cell activation, demonstrated by increasing forward scatter (D), and proliferation, indicated by CFSE dilution (E), were analyzed. The data shown are representative of at least 3 similarly performed experiments.