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Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 2007 Dec 1;57(7):987–996. doi: 10.1007/s00262-007-0429-6

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

A Siva 1, H Xin 1, F Qin 1, D Oltean 1, K S Bowdish 1, A Kretz-Rommel 1,✉
PMCID: PMC11030565  PMID: 18060403

Abstract

Background and Objective

Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro.

Methods and Results

CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses.

Conclusion

These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.

Keywords: CD200, Immune evasion, Therapeutic monoclonal antibodies

Introduction

Effective immune responses against tumors are often hampered by negative factors in the tumor environment [1]. We previously demonstrated that the immuno-suppressive molecule, CD200, is upregulated on B-cell chronic lymphocytic leukemia [2], and showed in in vivo models that CD200 expression on tumor cells blocked a productive anti-tumor response, which was restored by administration of an antagonistic (i.e., blocking) anti-CD200 antibody [3]. Furthermore, CD200 appears to be a prognostic factor in multiple myeloma [4] and in acute myeloid leukemia [5].

CD200 is a type 1a membrane protein, related to the B7 family of co-stimulatory receptors, with two extracellular immunoglobulin superfamily (IgSF) domains, a single transmembrane region and a short cytoplasmic tail with no known signaling motifs [6]. It is normally expressed on thymocytes, T and B lymphocytes, some dendritic cells, neurons, kidney glomeruli, syncitiotrophoblast, and endothelial cells [7].

CD200 binds to its receptor, CD200R, which is expressed on cells of the monocyte/macrophage lineage, on T lymphocytes, and on monocyte-derived dendritic cells [7, 8]. The interaction of CD200 with its receptor delivers an inhibitory signal to cells of the macrophage lineage [9]. Studies using CD200 knockout mice have demonstrated expanded macrophage and granulocyte populations in lymphoid organs and increased susceptibility to autoimmune disease [9]. Rodent studies using blocking antibodies or recombinant Fc fusion proteins containing the CD200 or CD200R extracellular domains have shown that CD200 is a potent immunosuppressant [9, 10] implicated in the maintenance of transplant and fetal tolerance [10–12].

We demonstrated that hematologic tumors expressing increased levels of CD200 downregulate Th1 cytokine responses [2]. Th1 cytokines are required for efficient cytotoxic T-cell function [13–15]. As progression of many cancer types has been correlated with a shift from Th1 to Th2 cytokines such as IL-4 [16–20], strategies to reverse this shift are believed to be therapeutically beneficial. CD200 has also been implicated in the induction of regulatory T cells [21–23], which are thought to hamper tumor-specific effector T cell immunity. Thus upregulation of CD200 may be a mechanism used by CD200-expressing tumors to evade eradication by the immune system, and targeting this negative immune regulator in cancer could be therapeutically beneficial.

Since we demonstrated a role of CD200 expression on B-CLL cells and an immunosuppressive effect of CD200 on tumor cells in mouse models, we now addressed whether in addition to hematologic cancers, CD200 protein is also upregulated on solid cancers. As evaluated by flow cytometry of cancer cell lines as well as RTQ-PCR and IHC studies of primary patient samples, we found strong expression of CD200 in malignant cells of melanoma, ovarian cancer, and renal cell carcinoma. Furthermore, we evaluated in vitro whether upregulation of this protein on solid cancers is of biological relevance and provide an important basis for further evaluation of the therapeutic potential of CD200 blockade.

Methods

Antibodies

chB7-G1 is a chimeric antagonistic anti-human CD200 antibody as defined by its ability to block CD200/CD200R interactions. It has a murine variable region and a human constant region. Whereas chB7-G1 has an IgG1 constant region, chB7-G2G4 is an effectorless anti-CD200 antibody that has the same variable region, but has a hybrid IgG2G4 constant region that does not bind to FcR and cannot mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) [24]. The anti-CD200 antibodies were produced in CHO cells and are endotoxin free as demonstrated using the Limulus Amoebocyte Lysate (LAL) test (Cell Sciences, Canton, MA). The Fab fragment of chB7-G1 (Fab C2aB7) was used for IHC staining.

Flow cytometric analysis of CD200 expression on melanoma and ovarian cancer cell lines

Ovarian cancer cell lines PA-1, Caov-3, TOV-21G, TOV-112D, ES-2, SW 626, OVCAR-3, melanoma cancer cell lines SK-MEL 28, SK-MEL 24, SK-MEL 1, prostate cancer cell lines DU145, PC-3, LNCaP, lung cancer cell lines H2228, H1651, astrocytoma cell lines SW1088, SW1783, glioblastoma cell lines U373, T98G, U118MG, the breast cancer cell lines MDA-MB-231, MDA-MB-435, MCF-7, neuroblastoma cell lines IMR-32, SK-N-SH, renal cell carcinoma cell lines SN12C, and ACHN cells were purchased from the American Type Culture Collection (ATCC, Mannassas, VA). Five hundred thousand cells each in FACS buffer (PBS containing 1% BSA and 0.1% NaN3) were stained with Alexa Fluor 488 Zenon labeled (Invitrogen, San Diego, CA) anti-CD200 antibody chB7-G1 according to the manufacturer’s instructions. Cells were incubated at room temperature for 20 min, washed twice in FACS buffer, and then analyzed on a FACSCalibur flow cytometer using BD CellQuest Pro software (Becton Dickinson, San Jose, CA). RAJI cells were used as negative control lacking CD200 expression and primary B-CLL cells as CD200 expressing positive control. Primary B-CLL cells were obtained from Alan Saven (Scripps Cancer Center, La Jolla, CA) after patient informed consent and IRB approval.

Mixed lymphocyte reaction and cytokine production assay

Peripheral blood was obtained from healthy volunteers after informed consent and IRB approval. CD14 + monocytes were isolated from PBMCs using the monocyte isolation kit II from Miltenyi (Auburn, CA). Monocytes were matured to dendritic cells by culturing the cells in the presence of 800 U/mL human recombinant GM-CSF, 500 U/mL human recombinant IL-4 (Stemcell Technologies, Canada) and 100 μg/mL human recombinant IFN-γ (R&D Systems, Minneapolis, MN) for 5 days. Allogeneic T cells were enriched by incubating the cells for 1 h in tissue culture flasks and taking the non-adherent cell fraction. Allogeneic mixed lymphocyte reactions (MLRs) were set up with 2.5 × 105 irradiated (2,000 RAD) monocyte-derived dendritic cells and 1 × 106 T cells in the presence or absence of 2.4 × 105 irradiated cancer cells and 25 μg/mL anti-CD200 antibody chB7-G2/G4 or negative control antibody. Supernatants were collected after 48 h to analyze for the presence of cytokines.

Cytokine analysis

Cytokines such as IL-2 and IFN-γ found in the tissue culture supernatant were quantified using ELISA. Matched capture and detection antibody pairs for each cytokine were obtained from R&D Systems (Minneapolis, MN), and a standard curve for each cytokine was produced using recombinant human cytokine. Anti-cytokine capture antibody was coated on the plate in PBS at the optimum concentration. After overnight incubation, the plates were washed and blocked for 1 h with PBS containing 1% BSA. After three washes with PBS containing 0.05% Tween, supernatants were added at the indicated dilutions in PBS containing 1% BSA. Captured cytokines were detected with the appropriate biotinylated anti-cytokine antibody followed by the addition of alkaline phosphatase conjugated streptavidin and SigmaS substrate. Color development was assessed with an ELISA plate reader (Molecular Devices).

Proliferation assay

For proliferation assays, either MLRs were set up as above, or 100,000 hPBMC were added to 96-well plates and stimulated with 0, 0.0001, or 0.1 μg/mL of anti-CD3 antibody (eBioscience, San Diego, CA) in the presence or absence of anti-CD200 mAb. After 48 h of culture, the cells were pulsed with 1 μCi [3H] thymidine (Amersham, Piscataway, NJ) per well. Twenty-four hours later, the cells were harvested with Filtermate Harvester (UNIFILTER-96, PerkinElmer, Waltham, MA) and thymidine incorporation was measured using a microplate scintillation counter (PerkinElmer).

RNA samples

Total RNA for the normal tissue panel was purchased from BD Biosciences (Palo Alto, CA) and BioChain Institute Inc. (Hayward, CA). Total RNA from melanoma (stage IV) and ovarian cancer patient (one stage IA, one stage IIIA, one stage III, three stage IIIC) samples was purchased from the Cytomyx Corporation (Lexington, MA). All RNA samples were DNaseI-treated, and cDNA was prepared using the High-Capacity cDNA archive kit (Applied Biosystems, Foster City, CA). RNA was extracted from ovarian cancer samples that were greater than 80% tumor, melanoma jejunum metastasis samples greater than 80% tumor, melanoma metastasis to small intestine and lymph node samples greater than 90% tumor, and melanoma lung metastasis samples greater than 75% tumor.

Real-time quantitative PCR

Relative gene expression levels were determined by real-time quantitative PCR (RT-qPCR), using 18S ribosomal RNA (rRNA) as a control for normalization. Assays-on-Demand TaqMan primer/probe sets (Applied Biosystems, Foster City, CA) for CD200 (Hs00245978_m1) and 18S rRNA were used with TaqMan Universal PCR master mix (Applied Biosystems) for amplification of the cDNA according to the manufacturer’s instructions. Amplification and analysis was performed using the ABI 7500 sequence detection system (Applied Biosystems). Expression of CD200 in all samples was determined relative to the CD200 expression level of normal peripheral blood mononuclear cells (PBMC), which was given the expression value of 1.0.

Immunohistochemistry (IHC)

Frozen tissues of four melanoma and three normal skin samples, three ovarian cancer (serous subtype) and three normal ovarian samples, three clear cell renal cell carcinoma and three normal renal samples, three low grade prostate carcinoma (Gleason score < 5) and three normal prostate samples were provided by LifeSpan Biosciences (Seattle, WA). IHC was conducted by LifeSpan Biosciences and reviewed by certified pathologists. Briefly, tissue CD200 expression was evaluated using the murine Fab C2aB7 recognizing human CD200. In antibody titration experiments using 2.5, 5, 10 and 20 μg/mL on frozen tissues, 10 μg/mL was determined as the concentration that results in minimal background and maximum detection of signal. Bound C2aB7 was detected using DAKO Envision peroxidase labeled polymer (Fort Collins, Colorado) with DAB as the chromogen. Tissues were also stained with positive control antibodies (CD31 and vimentin) to ensure that the tissue antigens were preserved and accessible for IHC analysis. Only tissues that stained positive for CD31 and vimentin were selected for the remainder of the study. The negative control consisted of performing the entire IHC procedure on adjacent sections in the absence of primary antibody. An isotype control, using mouse IgG1 at 20 μg/mL as primary antibody, was also included, and did not show specific staining. All sections were visualized with 40× magnification.

Results

CD200 cell surface expression on cancer cell lines

Elevated CD200 protein expression has been found on hematologic cancers such as B-CLL [2], multiple myeloma [4], and acute myeloid leukemia [5]. The present study addressed whether CD200 is also found in solid cancers such as prostate cancer, ovarian cancer, renal cell carcinoma, neuroblastoma, astrocytoma, glioblastoma, and lung cancer. We first evaluated CD200 protein expression on a panel of cancer cell lines using flow cytometry. Two out of eight ovarian cancer cell lines (OVCAR-3 and PA-1) showed about a 5–6-fold increase in CD200 expression over the negative (unstained) control, another three (SK-OV-3, ES-2, and Caov-3) showed about 2-fold increase in CD200 expression, while three cell lines (TOV-21G, TOV-112D, and SW626) were not elevated compared to the negative control (Fig. 1). Out of three melanoma cell lines tested, one cell line (SK-MEL-28) showed high CD200 expression (7–8-fold over the negative control), one showed moderate expression (SK-MEL-24), and two cell lines were at or below the negative control (SK-MEL1 and SK-MEL2). Both neuroblastoma cell lines expressed CD200 (IMR-32, SK-N-SH, 6–7-fold over the negative control). Out of the renal cell carcinoma cell lines tested, SN12C showed moderate CD200 expression (4-fold over the negative control), Caki-1 was 2-fold above the negative control, while ACHN cells did not express detectable levels of CD200. None of the three prostate cancer cell lines (DU145, PC-3, LNCAP), lung cancer cell lines (H2228, H1651), astrocytoma cell lines (SW1088, SW1783), glioblastoma cell lines (U373, T98G, U118MG, U87MG), or breast cancer cell lines tested (MDA-MB-231, MDA-MB-435, MCF-7) expressed CD200 protein at a level above the negative control (data not shown).

Fig. 1.

Fig. 1

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CD200 cell surface expression on a panel of cancer cell lines. Five hundred thousand cancer cells as indicated, or B-CLL primary cells were stained with Alexa Fluor 488-conjugated anti-CD200 mAb, analyzed by flow cytometry, and compared to the negative isotype control

CD200 expression on primary patient samples

To evaluate whether upregulated CD200 expression found on cancer cell lines translates to findings in patient samples, CD200 expression on four malignant melanoma, three ovarian carcinoma (serous subtype), and three ccRCC cancer patient samples was determined by IHC. Normal counterpart tissues for ovary, skin, and kidney were used as controls. IHC revealed strong CD200 staining of solid tumors including melanoma (4/4), ovarian cancer, serous subtype (3/3), and clear cell renal cell carcinoma (2/3) (representative staining shown in Fig. 2). Compared to normal skin, which only showed staining of sweat ducts and hair follicles, CD200 staining on melanoma samples was much stronger. In contrast, moderate to strong anti-CD200 staining of normal ovaries and renal tubular epithelium and glomeruli was observed.

Fig. 2.

Fig. 2

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IHC staining of ovarian cancer, melanoma, and ccRCC patient samples. CD200 expression in a normal ovary (follicle ++, stroma ++/+++), b ovarian cancer, serous subtype (malignant cells: occasional ++/+++, others ++++), c ovarian cancer serous subtype (malignant cells ++++, others ++++), d normal skin (epidermis −), e melanoma (malignant cells +++/++++), f melanoma (malignant cells +++), g normal kidney (glomeruli +++/++++), h ccRCC (malignant cells ++), i ccRCC (malignant cells +++). IHC evaluation key: Negative (−): no stain; Blush (+): a generalized low-level signal, cytoplasm/membrane is translucent, slightly grey; Faint (++): cytoplasm/membrane is stained grey; Moderate (+++): cytoplasm/membrane is black, with some structures of cytoplasm visible; Strong (++++): cytoplasm/membrane is intensely black, and no cytoplasmic structures are visible

The melanoma samples clearly indicated upregulation of CD200. Since surgical excisions of melanomas that have not spread beyond the site at which they developed are highly curable, while melanoma that has spread to distant sites are rarely curable with standard therapy, we evaluated CD200 expression on metastatic samples. Due to the lack of availability of metastatic tissue for IHC staining, CD200 mRNA levels in metastatic melanoma samples were determined by RT-QPCR and compared relative to the normal tissue counterpart of the site of metastases. PBMCs were used as a positive control since CD200 is constitutively expressed on normal B cells [2]. CD200 expression was evaluated on melanoma metastatic samples from jejunum, small intestine, lymph node (Fig. 3a), and lung (Fig. 3b). For several of these samples, normal and tumor tissue came from the same patient (matched normal/tumor patient samples) as indicated by number. Additional samples without matched normal samples were also run for comparison. Jejunum metastatic samples showed about 4–7-fold higher CD200 expression than normal jejunum (Fig. 3a), while the small intestine and lymph node metastatic samples did not consistently show elevated CD200 expression levels compared to the normal organ. For the normal lung/lung tumor-matched samples, two of six samples show 2- to 3.4-fold upregulation of CD200 expression in the tumor compared to its normal counterpart (Fig. 3b); however, there appears to be a wide variance of CD200 expression in the normal lung samples (1.5- to 11.67-fold higher than the level of normal PBMC).

Fig. 3.

Fig. 3

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RT-qPCR of a normal tissue panel, and ovarian cancer and melanoma patient samples. a, b CD200 expression was evaluated in several melanoma metastases samples: jejunum, small intestine, lymph node, and lung, and compared to their normal counterparts. c CD200 expression was examined in ovarian adenocarcinoma with the following subtypes: serous, serous metastatic, papillary serous, endometrioid, mucinous, and clear cell. d CD200 expression in normal tissue by RT-qPCR. All samples were normalized to 18S, and then fold expression relative to normal PBMC was determined. Where indicated by sample number, normal and metastatic samples were from the same patient

We also evaluated CD200 expression levels by RT-QPCR in ovarian adenocarcinoma with the following subtypes: serous, serous metastatic, papillary serous, endometrioid, mucinous, and clear cell, relative to PBMC and compared to normal ovary. Serous/serous metastatic/papillary serous subtypes appeared to have the highest expression of CD200 at approximately 10- to 20-fold higher than normal PBMCs (Fig. 3c). However, of the two normal ovary samples examined, one showed about 5-fold higher CD200 message levels than PBMCs, but the other was 20-fold higher than normal PBMCs, about the same as the tumor samples. CD200 expression was at or below normal ovary expression levels in endometrioid, mucinous, and clear cell samples (1- to 5-fold higher than normal PBMCs).

For comparison, CD200 mRNA levels were evaluated across a panel of normal tissues (Fig. 3d). The highest levels were found in brain and placenta, about 100-fold and 90-fold higher levels compared to normal PBMC, respectively. These data are consistent with what has previously been reported for CD200 tissue expression [7], indicating that mRNA levels correlate with CD200 protein expression. As expected based on B cells constituting about 5% of total PBMC, whole PBMC as a population shows much lower levels (about 15-fold lower levels) of CD200 compared to purified B-cells.

Effect of CD200-expressing tumor cell lines and blocking antibody in MLR

We next evaluated whether CD200 expression on cancer cell lines derived from solid tumors is of biological significance. We previously demonstrated that addition of CD200-expressing primary B-CLL cells shifted cytokine profiles from a Th1 profile to a Th2 profile in allogeneic mixed lymphocyte reactions of dendritic cells and T cells [2]. As Th1 cytokines are required to mount a sufficient cytotoxic T cell response, these studies implicated a role for CD200 in immune evasion. We now examined whether CD200 expressing ovarian cancer and melanoma cell lines would alter Th1 cytokine expression such as IFN-γ and IL-2 in allogeneic mixed lymphocyte reactions of dendritic cells and T cells. We also evaluated production of Th2 cytokines such as IL-4 and IL-10, but levels were generally below the limit of detection. As shown in Fig. 4, all CD200-expressing ovarian cancer and melanoma cell lines downregulated IFN-γ (a) and IL-2 (b) production when added to allogeneic mixed lymphocyte reactions, although the level of downregulation varied. IFN-γ downregulation correlated with the level of CD200 overexpression, and importantly, cell lines lacking CD200 were incapable of shifting cytokine profiles. OVCAR-3 and PA-1 cell lines displaying the highest CD200 expression completely blocked IFN-γ production in this system. SK-OV-3, ES-2, and Caov-3 cells that only showed a 2-fold increase in CD200 expression, still resulted in about 50–60% inhibition of IFN-γ production, while the cell lines lacking CD200, TOV-21G, and SW626, did not affect cytokine production. Similarly, the CD200 expressing melanoma cell lines, SK-MEL-28 and SK-MEL-24, completely inhibited IFN-γ production, while the CD200 negative cell line SK-MEL-1 had no effect. The positive control, primary B-CLL cells, blocked IFN-γ production as expected, and the negative control, RAJI, did not have an effect. Downregulation of IL-2 production was also observed in the presence of CD200 expressing ovarian cancer and melanoma cell lines. IL-2 cytokine production was even strongly downregulated in the presence of cell lines with just 2-fold upregulation of CD200 such as Caov-3, while CD200 negative cell lines did not have an effect. As seen before with B-CLL cells, it appears that there is no dose–response of CD200 expression on cytokine production, but a threshold effect. In all cases, downregulation of Th1 cytokines by CD200-expressing cancer cell lines was prevented by the presence of the antagonistic anti-CD200 antibody, chB7-G2G4. Importantly, the anti-CD200 antibody did not augment Th1 cytokine production in MLR samples without tumor cells above levels observed in MLRs in the absence of antibody.

Fig. 4.

Fig. 4

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CD200 expressing ovarian and melanoma cell lines inhibit Th1 cytokine production in MLRs. Supernatants of allogeneic mixed lymphocyte reactions of T cells and dendritic cells, with or without addition of cancer cell lines were analyzed for the presence of human IFN-gamma and IL-2 levels at 48 h by sandwich ELISA. RAJI cells were used as negative control lacking CD200, whereas B-CLL primary cells were used as positive control. a IFN-gamma production in MLRs with or without addition of cancer cell lines. Ct control, no addition of cancer cell lines. Data represent mean of duplicates. One representative experiment out of two is shown. b IL-2 production in MLRs with or without addition of cancer cell lines. Ct control, no addition of cancer cell lines

Since hyper immune activation is a potential safety concern of immunomodulatory agents, we further verified that not only did the anti-CD200 antibody not augment Th1 cytokine production above normal activation patterns, but also measured proliferative responses in MLRs in the presence or absence of anti-CD200 antibody. As shown in Table 1, no difference in proliferation was observed whether anti-CD200 antibody was present or not. Since in MLRs, the maximum proliferative response might have been reached, we further explored the effect of anti-CD200 on suboptimal stimulation with anti-CD3. Again, no increase in stimulation in the presence of antibody was seen (Table 2), confirming that anti-CD200 treatment does not augment stimulatory immune responses.

Table 1.

Proliferation in mixed lymphocyte reactions in the presence or absence of anti-CD200 mAb chB7-G2G4

MLR Proliferation (cpm)
No antibody 20 μg/mL anti-CD200 mAb
Donor 1 + 2 43,567 ± 1,123 41,501 ± 1,024
Donor 2 + 3 29,876 ± 1,076 30,490 ± 1,129
Donor 3 + 1 54,673 ± 1,239 52,879 ± 1,077
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The data are shown as means of triplicate cultures ± SD. hPBMC from three different donors were used as indicated

Table 2.

Proliferation in response to anti-CD3 treatment in the presence or absence of anti-CD200 mAb chB7-G2G4

Treatment Proliferation (cpm)
0 μg/mL anti-CD3 0.0001 μg/mL anti-CD3 0.01 μg/mL anti-CD3
Medium 563 ± 186 37,676 ± 1,359 57,718 ± 4,789
20 μg/mL anti-CD200 mAb 354 ± 241 36,696 ± 1,815 52,826 ± 3,360
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The data are shown as means of triplicate cultures ± SD. Results from a single donor are shown. The experiment was repeated with three different donors with similar results

Discussion

We identified melanoma as a solid tumor type that upregulates CD200 expression compared to normal tissue. CD200 expression was also found in ovarian and renal carcinoma, but CD200 appears to be expressed at comparable levels on normal ovaries and glomeruli as well. CD200 is an immunosuppressive molecule that is expressed on the cell surface of thymocytes, B cells, T cells, neurons, kidney glomeruli, tonsil follicles, syncytiotrophoblasts and endothelial cells [7]. Although this distribution pattern might not warrant the selection of a therapeutic antibody that can mediate cell killing either directly or through ADCC mechanisms, blockade of CD200 on tumors overexpressing this molecule might be therapeutically beneficial by blocking immune suppression and, therefore, enabling an effective anti-tumor response. We previously demonstrated that CD200 on hematologic tumor cells prevented tumor cell killing by immune cells in a mouse model [2], and treatment with an antagonistic anti-CD200 mAb lacking effector function resulted in effective tumor cell killing. Anti-CD200 therapy might be beneficial in patients where the immune system does mount an immune response against cancer cells, but that response is not sufficient to eradicate tumor cells due to dampening factors in the tumor environment such as CD200. Cytotoxic T cells recognizing tumor cells have been found in both ovarian cancer [25–27] and melanoma patients [28]. Both tumor types, and in particular melanoma, have been fairly extensively explored in immunomodulatory therapy. Treatment of melanoma patients with cancer vaccines or cytotoxic T lymphocytes has shown some success [29], but in the majority of patients these therapies appear to be hampered by negative regulatory factors in the tumor microenvironment [30].

Negative immune regulators such as B7-H4 [31–34] have been identified on ovarian cancer samples, and B7-H1 [35; 36] has been found on both ovarian cancer and melanoma samples. B7-H1 and B7-H4 have also been detected on suppressor dendritic cells in the tumor microenvironment. Both molecules downregulate the effector phase of T cell immunity [37]. Blockade of B7-H1 [38] or B7-H4 [32] on tumor infiltrating macrophages and dendritic cells substantially enhanced the ability of autologous T cells to inhibit human ovarian carcinoma growth in vivo in NOD/SCID mice. Furthermore, Blank et al. [36] showed that blockade of B7-H1 augmented human tumor-specific T cell responses in vitro. These studies suggest that inhibition of immune suppression can be very powerful, and that a strong anti-tumor response can be elicited by blocking just one immunosuppressive molecule. Our in vitro data indicate that it is sufficient to block CD200 on CD200-expressing cancer cell lines to prevent the downregulation of a Th1 response, which is required to elicit a cytotoxic T cell response. In malignant melanoma, Th1/Th2 imbalance coincides with disease progression [19]. It will be interesting to explore whether a combination of anti-B7-H4 or anti-B7-H1 and anti-CD200 will result in an even more potent response in vivo in these solid tumor settings.

CD200 exerts its effects through CD200R found on the macrophage lineage. CD200 has no known signaling motif, and anti-CD200 antibody binding does not signal internally for direct tumor cell killing. Antibody binding to CD200 enables the immune system to kill tumor cells. Interaction of CD200 with its receptor results in shifting cytokine profiles from Th1 to Th2 as well as inducing regulatory T cells [21–23]. Ovarian tumor cells produce macrophage colony stimulating factor, a potent chemoattractant for cells of the macrophage lineage [39, 40]. Cells of the macrophage lineage provide a growth benefit to tumor cells by producing factors that enhance angiogenesis [41, 42] On the other hand, infiltrating dendritic cells capable of raising an anti-tumor response pose a threat to tumor cells. Therefore, upregulation of CD200 and other immunosuppressive molecules might be one of the mechanisms by which the tumor cells prevent an anti-tumor response, yet can take advantage of growth factors produced by dendritic cells.

In contrast to CD200 expression in melanoma, ovarian and renal cancer, CD200 was not present in prostate cancer and was not detectable on the surface of multiple lung cancer, glioblastoma, or breast cancer cell lines. Furthermore, the variability of CD200 expression in melanoma and ovarian cancer cell lines could be either a reflection of natural biological variation in primary tumor expression as is often found for tumor antigens (e.g., Her-2 in breast cancer), or it could be a result of downregulation of CD200 in vitro. CD200 expression in cancer may not be a de novo pathway, but may happen in cells that normally have a basal level of CD200 expression, which can increase in the cancer setting. CD200 is not only found on certain tumor cells, but also on some normal tissues including nerve fibers, brain, kidney glomeruli, and microvascular endothelial cells. Expression on normal tissue might raise concerns of inducing autoimmune disease. However, CD200 KO mice appear normal, although they are more susceptible to the induction of autoimmune disease on exacerbation [9]. Since CD200 is expressed on normal T and B cells, it is conceivable that blocking CD200 might result in increased immune responses in healthy individuals, but our cytokine data suggests that while anti-CD200 can block CD200-mediated immune suppression, it does not augment stimulatory immune responses in the absence of increased CD200 expression on tumor cells. Furthermore, we treated chimpanzees with up to 20 mg/kg of an effectorless antagonistic anti-CD200 mAb (ALXN6000) without autoimmune or other toxicity (data not shown). In order to see an autoimmune effect as a result of anti-CD200 treatment, autoreactive T cells against the tissue have to exist in the first place, which is generally prevented by multiple tolerance mechanisms. In other therapeutic approaches where the goal has been blocking negative regulators of T cell immunity, autoimmunity has been observed. For example, clinical trials using monoclonal antibodies against CTLA-4 demonstrated some efficacy, which correlated with autoimmune phenomena that were manageable by transient treatment with corticosteroids [43]. Interestingly, the combination of blocking negative regulators of T cell immunity combined with vaccines may help to direct the immune response toward the tumor, enhancing clinical efficacy and perhaps reducing treatment-related adverse events [42].

Use of an antagonistic effectorless anti-CD200 mAb to block immune suppression, either alone or in combination could potentially be a treatment for melanoma metastases since CD200 was not only highly expressed on primary melanoma samples, but also a significant number of melanoma metastases tested by RT-QPCR showed higher CD200 expression than their organ of finding. Despite CD200 expression on normal ovaries, anti-CD200 treatment might also be feasible in ovarian cancer, since the primary organ is often removed. Whether CD200 blockade as monotherapy or as combination therapy is indeed an effective therapy can only be evaluated in humans.

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