CD200/OX2, a cell surface molecule with immuno‐regulatory function, is consistently expressed on hairy cell leukaemia neoplastic cells (2009)

CD200 (formerly called OX2) is a transmembrane glycoprotein with immunosuppressive functions. It is expressed on normal B-lymphocytes, T-lymphocytes, dendritic cells and several solid tissues (Kawasaki & Farrar, 2008). CD200 receptor expression is limited to myeloid leucocytes and a subset of T-lymphocytes (Kawasaki & Farrar, 2008). In mouse systems, the binding of CD200 to its receptor (i) decreases the production of T-helper cell type 1 (Th1)-like cytokines, such as interleukin (IL)-2 and interferon γ, (ii) increases the release of Th2-like cytokines, such as IL-10 and IL-4 (Gorczynski, 2001) and (iii) promotes the in vitro differentiation of T cells toward CD4+CD25+Foxp3+ Treg lymphocytes (Gorczynski et al, 2008). CD200 is constantly overexpressed on chronic lymphocytic leukaemia (CLL) cells (McWhirter et al, 2006). The addition of CLL cells to mixed lymphocyte reactions causes an immunological shift from a Th1-like response to a Th2-like response, confirming that CD200 plays an important role in controlling T-cytotoxic immune response (McWhirter et al, 2006). Starting from these data, we extended the investigation of CD200 expression to another B-chronic lymphoproliferative disorder i.e. hairy cell leukaemia (HCL). Hairy cell leukaemia is a distinct disease entity in the World Health Organization (WHO) classification, displaying unique clinico-pathological and biological features (Tiacci et al, 2006). As hairy cells display a specific immunophenotype, multicolour flow cytometry is currently the best tool for HCL diagnosis. A total of 10 specimens (six peripheral blood samples and four bone marrow aspirates), collected from 10 patients with newly diagnosed HCL, were studied. As normal controls we analysed 10 peripheral blood specimens and two bone marrow aspirates from 12 healthy donors. An aliquot (50 μl) of each sample was incubated at 4°C for 30 min in the presence of appropriate amounts of monoclonal antibodies. The mixtures were then diluted 1:20 in ammonium chloride lysing solution, incubated at room temperature for 10 min and finally washed prior to flow cytometric analysis with the FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). The following antigens were analysed: CD200, SmIg-kappa, SmIg-lambda, CD45, CD19, CD5, CD23, CD20, CD22, CD103, CD11c, CD25, CD43, CD10, CD3, CD56 and CD81. Hairy cells were gated as CD45brightCD19bright‘monocytoid cells’ (i.e. cells with light scatter features typical of monocytes). In addition, in the majority of cases, we also were able to perform a full immunological gate on CD45brightCD19brightCD103brightCD11cbright cells. With regard to the normal controls included in our study, B-lymphocytes were simply gated as CD45brightCD19bright cells. In all specimens cell doublets and debris were excluded from our analysis by forward-scatter versus side-scatter dot-plot examination. To set the cut-off point to distinguish between CD200 negative and positive cells, we used the ‘Fluorescence Minus One’ technique as described by Perfetto et al (2004). A single case was arbitrarily judged CD200 positive when the percentage of positive cells (PPC) was higher than 30%. All HCL samples were CD200 positive with PPC and median fluorescence intensity (MFI) median values of 99 (25th–75th percentile 92–99) and 3016 (25th–75th percentile 1382–5430), respectively. Although CD200 was positive in 12 out of 12 normal controls, the PPC and MFI median values were of 71 (25th–75th percentile 64–83) and 582 (25th–75th percentile 406–725), respectively. Differences in PPCs and MFIs between HCLs and normal controls were statistically significant (Mann–Whitney U, two-tailed testing, P < 0·0001). Data regarding MFI analysis are shown in Fig 1. Whereas normal controls showed weak CD200 fluorescence intensity with a bimodal distribution, HCL samples showed bright CD200 expression in a homogeneous pattern (Fig 2). CD200 expression on B-lymphocytes in normal controls (CTR) as compared to hairy cell leukaemia (HCL) patients. MFI = median fluorescence intensity. Cytometric plots showing CD200 expression on hairy cells (CD103brightCD19bright) in one case of HCL (quadrants A and B) and on B-lymphocytes (CD45brightCD19bright) in one normal control (quadrants C and D). Note that CD200 expression in HCL is bright and homogeneous, while in normal controls it is faint and bimodal. This is the first documented direct evidence of CD200 overexpression in HCL. As described above, CD200 promotes Th2-like cytokines synthesis. IL-4 and IL-10 are reported to reduce anti-tumour cytotoxic T cell response (McWhirter et al, 2006) and, in addition, IL-4 promotes DNA synthesis in hairy cells (Barut et al, 1990). Moreover, expression of CD200 seems to augment differentiation of T cells toward CD4+CD25+Foxp3+ Treg lymphocytes and increasing evidence indicates that Treg cells inhibit anti-tumour immunity (Wang & Wang, 2007). Moreover, CD200 is already known to be an unfavourable prognostic factor in acute myeloid leukaemia and multiple myeloma and is associated with cancer progression in several solid neoplasms (Kawasaki & Farrar, 2008). Taken together, these data suggest that CD200 overexpression could play a fundamental role in hairy cell survival and proliferation as well as in the inhibition of anti-tumour immunological response. During the last 25 years, four lines of therapy have been identified as effective in patients with HCL: interferon α, cladribine (2-chlorodeoxyadenosine), pentostatine (deoxycoformycin) and rituximab (Golomb, 2008). These drugs provided relevant results in terms of overall responses, progression-free survival and overall survival. However, HCL is still considered incurable and, in a minority of cases, persistence of minimal residual disease with cytopenia as well as disease recurrence still render the therapy of HCL a challenge (Golomb, 2008). A humanized monoclonal antibody directed against CD200 (Anti-CD200 MAb; Alexion Pharmaceuticals, Cheshire, CT, USA) with blocking function is already available. Interesting results have been obtained in in vivo assays testing Anti-CD200 MAb with CD200 positive Burkitt lymphoma cell lines (Kretz-Rommel et al, 2007). In this study, we found CD200 positivity on all hairy cells from all HCL patients studied, with a large amount of this antigen on neoplastic cells. CD200 expression pattern, combined with its molecular connexions toward suppression of T-cytotoxic response and hairy cell growth makes Anti-CD200 MAb an intriguing option for new therapeutic strategies in HCL.