CD200 is a transmembrane molecule with an important immunoregulatory role that is overexpressed on most chronic lymphocytic leukemia (CLL) cells. In this study, we characterized a previously unknown soluble form of this molecule in human plasma termed sCD200. Levels of sCD200 were elevated in the plasma of patients with CLL as compared with healthy controls, and there was a significant correlation with CLL disease stage. Infusion of sCD200hi CLL plasma into severely immunocompromised NOD.SCIDγcnull (NSG) mice enhanced the engraftment of CLL splenocytes as compared with mice receiving sCD200lo normal plasma. CLL cells were detected in both the spleen and peritoneal cavity of animals for up to 75 days. Engraftment of CLL cells did not occur after infusion of CLL plasma depleted of sCD200 and was abolished in mice treated with anti-CD200 or OKT3 monoclonal antibody (mAb), suggesting a role for both sCD200 and T cells in CLL engraftment. Notably, anti-CD200 mAb was as effective as rituximab in eliminating engrafted CLL cells when administered 21 days after engraftment. Taken together, our findings point to sCD200 as a novel prognostic marker and therapeutic target for CLL. Furthermore, the humanized mouse model described here may prove valuable to preclinically assess new treatment regimens for CLL. Cancer Res; 72(19); 4931–43. ©2012 AACR.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by the accumulation of malignant CD5+CD19+ B cells in peripheral blood, bone marrow, and secondary lymphoid organs. Some patients have a benign clinical course, whereas others die of this disease within a short time from diagnosis. Improved understanding of the biology of CLL may help identify other variables predicting which patients may have a poor disease outcome.

We have previously shown that CD200, an immunoregulatory molecule overexpressed on a number of solid and systemic tumors, as well as cancer stem cells, played a functional role in suppressing cytotoxic killing of CD200+ lymphoma and CLL cells (1–6). Increased expression of CD200R, which is required for signaling mediated following CD200 engagement, was detected on a subpopulation of CD4+ T cells in the spleen of patients with CLL relative to controls (6). Thus, CD200+ CLL cells and CD200R+ CD4+ T cells seem to colocalize in the tumor microenvironment.

The CLL microenvironment is crucial for CLL survival and proliferation (7). Nonmalignant constituents of the microenvironment, mostly T cells, mesenchymal stromal cells, and CD14+ nurse-like cells provide antigenic signals, cytokines, and other CLL survival factors, such as B-cell activating factor (BAFF), to support CLL survival and proliferation (8–11). T cells in the CLL microenvironment are also known to express CD40L, which, through stimulation of CD40 on CLL cells, induces CLL proliferation and antiapoptotic pathways (12). A number of studies have shown that CLL cells can directly modulate T-cell function by expression of cell-surface molecules and/or production of soluble factors (13, 14). On the basis of these data, we hypothesized that CD200 may be one of the important factors modulating the CLL microenvironment (2, 3, 5).

A number of membrane-bound molecules with immunomodulatory functions are known to exist also in soluble forms where they are believed to play a functional role in a number of disease states, including cancer. In CLL, soluble forms of CD23, CD44, and CD14 have been reported to augment the survival of CLL cells in vitro (15–17). We found that CD200 could be shed from CLL cells after stimulation by phorbol 12-myristate 13-acetate and TLR7 agonists, inferring that a soluble form of CD200 (sCD200) might be present in human plasma including that of patients with CLL (6). The studies described later were designed to investigate if soluble CD200 levels were increased in patients with CLL relative to healthy controls and whether those levels were related to disease stage. We also investigated whether sCD200 present in patients with CLL plasma might contribute to growth/survival of CLL cells in NOD.SCIDγcnull (NSG) mice.

Mice

NSG mice were bred and maintained under sterile conditions at the Toronto Medical Discovery Tower, MaRs Centre (Toronto, ON, Canada). All mice were used at 8 to 13 weeks of age.

Human splenocytes and CLL cells

The spleen from consenting patients undergoing splenectomy at Sunnybrook Health Sciences Centre (Toronto, ON, Canada) was harvested at surgery and single cell suspensions prepared in AIMV medium (Invitrogen). Cells were washed (×2), counted, and stored frozen at −80°C in freezing medium [AIMV + 40% FBS+ 10% dimethyl sulfoxide (DMSO)] at a concentration of 1 × 108 cells/mL either in 1.5 mL aliquots in cryovials or in 30 mL aliquots in 50 mL Falcon tubes. At least 1011 cells in total were harvested from each spleen. An aliquot (107) of fresh splenocytes was retained for cell-surface phenotype analysis in fluorescence-activated cell sorting (FACS). For in vivo studies in NSG mice, aliquots of splenocytes were rapidly thawed at 37°C, washed in PBS, and cell aggregates separated by centrifugation on Ficoll 400 density media (Sigma). Cells recovered after centrifugation were resuspended in PBS at appropriate concentrations for injection.

Some experiments in which cells from the peripheral blood of patients with CLL were used for reconstitution, CD19+CD5+ CLL cells were purified from fresh blood as described previously (18). All protocols were approved by Institutional Review Board.

Human plasma

Plasma samples from 82 patients diagnosed with CLL at Sunnybrook Health Sciences Centre from 1996 to 2008 (Table 1) were stored at −20°C and used for retrospective analysis of sCD200 levels in CD200 ELISA (see later).

Table 1.

Clinical characteristics of patients in plasma sCD200 analyses

Pt IDAgeSexRai stageaWBCb% CD38+β2McCytogeneticsdTreatments
61 IV 225 17 13q-, 17p- Fx2, CP, FC 
68 25 NA NA None 
72 IV 175 5.7 13q-/17p- S/CHOP/CVP 
84 III 200 46 NA 13q- P/C, S/R 
62 IV 121 2.2 13q- CP, CVP, S, FC 
66 21 1.2 NA None 
71 IV 25 NA T12, 11q- CVPx2, splen, FC 
74 IV 220 NA NA 17p- FR, splen, S 
77 IV 10 40 NA T12 C/P, splen, FC 
10 71 IV 54 NA 13q- CVP 
11 72 32 NA 13q-, 17p- None 
12 55 IV 23 81 2.3 13q-, 17p- CHOP, P 
13 57 IV 22 1.4 13q-, 17p- CP, CHOP, FCx3, S 
14 71 II 186 72 NA 11q-, 13q- None 
15 51 IV 22 NA 17p- Splen 
16 69 IV 189 NA Normal CP 
17 55 III 55 10 NA 13q- CP, FCR 
18 88 IV 35 71 NA 11q-, 13q- CVP, FC 
19 57 IV 50 21 8.1 17p- FCR, DHAP, S, R 
20 58 III 55 NA NA CP 
21 48 IV 62 NA Monosomy 11, 17p- CP, FC, CHOP 
22 54 IV 4.3 Normal CP, FC, FCR, S 
23 53 III 10 2.1 T12 CVP, FC 
24 73 IV 100 4.6 13q- Cx4, Fx3 
25 56 IV 78 NA 13q- Cx3, Fx2, CHOP 
26 48 III 17 72 NA T12 CP, FCR 
27 82 III 125 NA T12 None 
28 62 III 110 NA 13q- Splen, CP 
29 67 II 25 2.6 13q- None 
30 66 IV 122 54 NA 13q- Splen, CP 
31 69 120 Normal None 
32 51 18 NA Normal None 
34 58 IV 70 19 5.8 Normal FC, S, R 
35 64 II 20 50 2.8 11q-, 13q- FCR 
36 59 IV 10 2.7 NA CP 
37 58 II 25 25 NA Normal None 
38 55 IV 135 18 2.9 T12 CPR 
39 74 III 185 25 3.7 13q- 
40 52 42 NA 13q- None 
41 59 III 35 40 1.7 Normal CP, splen 
42 89 25 NA NA NA 
44 61 III 25 51 2.6 Normal CP 
45 60 IV 100 6.1 NA Fx2, CP, Revl, CHOPR 
46 55 III 121 3.1 T12 None 
47 61 33 1.4 13q- None 
48 70 IV 25 NA NA NA None 
49 60 IV 61 30 NA Normal None 
51 80 15 14 2.8 NA None 
52 91 18 28 2.8 T12 
53 61 III 22 91 NA 13q-, T12 None 
54 64 II 22 NA NA T12 None 
55 63 23 1.5 13q- None 
56 62 10 1.6 NA None 
57 77 IV 120 NA 8.5 13q- CP 
58 54 IV 60 13q- None 
59 68 III 85 53 2.4 T12, 17p- None 
60 88 IV 62 1.7 13q-, 17p- None 
61 81 III 89 11.3 Normal 
62 61 IV 165 3.4 13q- CP 
63 62 IV 12 71 4.3 13q-, 11q- CP/FC 
64 69 IV 81 NA 3.6 Normal CP, CVP, FC 
65 61 IV 140 18 NA 13q-, 11q- Splen 
66 60 III 5.7 13q-, T12 CP, FC, FCR 
67 57 III 10 10 NA 13q- CP 
68 62 20 11 1.1 13q- None 
69 47 II 65 NA 13q- Splen 
70 78 III 33 13 2.2 13q- None 
71 57 II 21 1.9 T12, 13q- None 
72 48 13 1.5 NA None 
73 53 IV 65 NA 13q- None 
74 77 III 250 2.7 13q- None 
75 63 II 22 71 1.7 NA None 
76 58 II 17 1.1 NA None 
77 38 II 37 63 NA Normal None 
78 72 IV 80 13 3.8 Normal CP, FCR 
79 51 IV 140 3.1 13q- Cx2 
80 51 II 88 13q- None 
81 64 III 160 12 3.3 11q- CPx2 
82 74 II 45 3.5 11q- None 
Pt IDAgeSexRai stageaWBCb% CD38+β2McCytogeneticsdTreatments
61 IV 225 17 13q-, 17p- Fx2, CP, FC 
68 25 NA NA None 
72 IV 175 5.7 13q-/17p- S/CHOP/CVP 
84 III 200 46 NA 13q- P/C, S/R 
62 IV 121 2.2 13q- CP, CVP, S, FC 
66 21 1.2 NA None 
71 IV 25 NA T12, 11q- CVPx2, splen, FC 
74 IV 220 NA NA 17p- FR, splen, S 
77 IV 10 40 NA T12 C/P, splen, FC 
10 71 IV 54 NA 13q- CVP 
11 72 32 NA 13q-, 17p- None 
12 55 IV 23 81 2.3 13q-, 17p- CHOP, P 
13 57 IV 22 1.4 13q-, 17p- CP, CHOP, FCx3, S 
14 71 II 186 72 NA 11q-, 13q- None 
15 51 IV 22 NA 17p- Splen 
16 69 IV 189 NA Normal CP 
17 55 III 55 10 NA 13q- CP, FCR 
18 88 IV 35 71 NA 11q-, 13q- CVP, FC 
19 57 IV 50 21 8.1 17p- FCR, DHAP, S, R 
20 58 III 55 NA NA CP 
21 48 IV 62 NA Monosomy 11, 17p- CP, FC, CHOP 
22 54 IV 4.3 Normal CP, FC, FCR, S 
23 53 III 10 2.1 T12 CVP, FC 
24 73 IV 100 4.6 13q- Cx4, Fx3 
25 56 IV 78 NA 13q- Cx3, Fx2, CHOP 
26 48 III 17 72 NA T12 CP, FCR 
27 82 III 125 NA T12 None 
28 62 III 110 NA 13q- Splen, CP 
29 67 II 25 2.6 13q- None 
30 66 IV 122 54 NA 13q- Splen, CP 
31 69 120 Normal None 
32 51 18 NA Normal None 
34 58 IV 70 19 5.8 Normal FC, S, R 
35 64 II 20 50 2.8 11q-, 13q- FCR 
36 59 IV 10 2.7 NA CP 
37 58 II 25 25 NA Normal None 
38 55 IV 135 18 2.9 T12 CPR 
39 74 III 185 25 3.7 13q- 
40 52 42 NA 13q- None 
41 59 III 35 40 1.7 Normal CP, splen 
42 89 25 NA NA NA 
44 61 III 25 51 2.6 Normal CP 
45 60 IV 100 6.1 NA Fx2, CP, Revl, CHOPR 
46 55 III 121 3.1 T12 None 
47 61 33 1.4 13q- None 
48 70 IV 25 NA NA NA None 
49 60 IV 61 30 NA Normal None 
51 80 15 14 2.8 NA None 
52 91 18 28 2.8 T12 
53 61 III 22 91 NA 13q-, T12 None 
54 64 II 22 NA NA T12 None 
55 63 23 1.5 13q- None 
56 62 10 1.6 NA None 
57 77 IV 120 NA 8.5 13q- CP 
58 54 IV 60 13q- None 
59 68 III 85 53 2.4 T12, 17p- None 
60 88 IV 62 1.7 13q-, 17p- None 
61 81 III 89 11.3 Normal 
62 61 IV 165 3.4 13q- CP 
63 62 IV 12 71 4.3 13q-, 11q- CP/FC 
64 69 IV 81 NA 3.6 Normal CP, CVP, FC 
65 61 IV 140 18 NA 13q-, 11q- Splen 
66 60 III 5.7 13q-, T12 CP, FC, FCR 
67 57 III 10 10 NA 13q- CP 
68 62 20 11 1.1 13q- None 
69 47 II 65 NA 13q- Splen 
70 78 III 33 13 2.2 13q- None 
71 57 II 21 1.9 T12, 13q- None 
72 48 13 1.5 NA None 
73 53 IV 65 NA 13q- None 
74 77 III 250 2.7 13q- None 
75 63 II 22 71 1.7 NA None 
76 58 II 17 1.1 NA None 
77 38 II 37 63 NA Normal None 
78 72 IV 80 13 3.8 Normal CP, FCR 
79 51 IV 140 3.1 13q- Cx2 
80 51 II 88 13q- None 
81 64 III 160 12 3.3 11q- CPx2 
82 74 II 45 3.5 11q- None 

Abbreviations: NA, not available; CVP, cyclophosphamide/vincristine/prednisone; CHOP, cyclophosphamide/vincristine/doxorubicin/prednisone; DHAP, dexamethasone/cytarabine/cisplatin; FC, fludarabine/cyclophosphamide; C, chlorambucil; P, prednisone; F, fludarabine; R, rituximab; Revl, Revlimid (lenalidomide); S, solumedrol; Splen, splenectomy.

aRai stage 0, lymphocytosis; I, with adenopathy; II, with hepatosplenomegaly; III, with anemia; IV, with thrombocytopenia.

bWBC, white blood cell count (106 cells/mL) in peripheral blood.

cβ2M, plasma β2-microglobulin level, mg/L.

dT12, trisomy 12.

For in vivo studies in NSG mice, plasma obtained at routine clinical follow-up from a group of patients at late disease stage (Rai stage III–IV), and/or with high white cell count, were pooled into batches (>8 donors per batch). The control plasma used was pooled from a group of 10 healthy volunteers. The sCD200 levels in all plasma samples were assessed by CD200 ELISA. The sCD200 levels in pooled normal plasma were in the range 0.5 ± 0.2 ng/mL, whereas in various pooled CLL plasma batches levels were consistently approximate to 10-fold higher (5.0 ± 1.3 ng/mL). Where absorbed plasma was used, the pooled CLL plasma was absorbed overnight at 4°C with anti-CD200 (1B9)-conjugated CNBr-activated Sepharose beads (Cedarlane), a method previously shown to be effective in absorbing sCD200 from plasma (6).

Antibodies

The rat anti-hCD200 monoclonal antibodies 1B9 and 3G7 were described previously (19). 1B9 was previously shown to be effective in blocking CD200 function in vitro (6). For in vivo use, Fab fragments of 1B9 were prepared using a Fab preparation kit (Thermo Fisher Scientific Inc.).

The polyclonal rabbit anti-hCD200 serum, absorbed to deplete all anti-Fc reactivity (rabbit anti-CD200Fc), was described elsewhere (6). A rabbit polyclonal antibody specific for the extracellular region (v+c) of CD200 (rabbit anti-CD200v+c) was generated by immunization of rabbits with protein expressed from Chinese hamster ovary cells transfected with an expression vector encoding only this extracellular domain of CD200. The immunoreactivity and specificity of both sera was characterized by Western blot analysis. Both antisera were used as detection antibodies in CD200 ELISA described later.

Mouse anti-CD200R1-FITC antibody was purchased from R&D systems, whereas all other monoclonal antibodies used for cell-surface phenotype characterization (CD45, CD19, CD5, CD20, CD40, CD23, CD38, CD49d, CD4, CD8, CD14, and CD56) were purchased from BioLegends. Rituxan (Roche Canada) was obtained from the hospital pharmacy. OKT3, used for depletion of T cells in in vivo studies, was purchased from Ortho-McNeil Pharmaceuticals.

CD200 sandwich ELISA

High binding 96-well EIA/RIA plates (Corning Life Sciences) were coated with the capture anti-CD200 mAb1B9 at 1.25 μg/mL overnight at 4°C in Tris–HCI, pH 8.1. Plates were then blocked for 1 hour at room temperature with the blocking buffer, 5% FBS in PBS, washed, and different concentrations of either pure CD200Fc (standard curve) or plasma samples (diluted 1:4 in blocking buffer) were added. Plates were incubated for 2 hours at room temperature, followed by 2 hours of incubation with the detection antibody (rabbit anti-CD200Fc or rabbit anti-CD200v+c) at a 1:2,000 dilution. Goat antirabbit immunoglobulin G–horseradish peroxidase antibody at a 1:12,500 dilution was added and plates incubated at room temperature for 30 minutes. At least 5 washes with wash buffer (PBS + 0.01% Tween20) were conducted between each step. After the final wash, 3,3′,5,5′-tetramethylbenzidine substrate was added. All reactions were stopped by addition of 50 μL of 0.2 mol/L sulfuric acid per well after 5 minute of incubation at room temperature in the dark. All plates were read at 450 nm in a Multiskan Ascent 96/384 plate reader (MTX Lab Systems).

Engraftment of human CLL splenocytes in NSG mice

On the day of experimentation, mice received 2.4 Gy of γ-irradiation, followed by 1 × 108 human splenocytes intraperitoneally (i.p.), and 0.8 mL of pooled CLL plasma or control plasma, also given i.p. Subsequent infusions of CLL plasma or control plasma were conducted biweekly throughout the course of the experiment. Mice were sacrificed at various time points to assess for CLL engraftment. Spleen and bone marrow were harvested from individual animals and cells in the peritoneal cavity were recovered by flushing the peritoneum with 8 mL PBS. Single cell suspensions were prepared from all 3 compartments, and cells enumerated in a hemocytometer.

For immunohistochemistry (IHC) an aliquot of fresh spleen tissue was fixed in 10% formalin, and slides were prepared and processed by the Pathology laboratory at Sunnybrook Health Sciences Centre. Engraftment of CLL cells was analyzed by multicolor FACS staining using single cell suspensions and the various monoclonal antibodies (mAbs) discussed earlier.

CD200 blockade, CD200Fc, and T-cell depletion in vivo studies

For CD200 blockade experiments, mice were randomly assigned into 3 groups after infusion of human splenocytes. Two of the 3 groups received sCD200hi CLL plasma, whereas a 3rd group received sCD200 absorbed CLL plasma. Of the 2 groups that received CLL plasma one group also received 50 μg (i.v.) of Fab anti-CD200 mAb 1B9 the day after spleen cell injection, and on 2 subsequent occasions at 72-hour intervals. The experiments in which CD200F was used, purified CD200Fc was prepared in normal plasma at a concentration of 50 μg/mL, and was infused i.p. at the same dosing schedule as that of CLL plasma.

For T-cell depletion experiments, mice received human splenocytes and sCD200hi CLL plasma, or sCD200 absorbed CLL plasma. The following day animals were randomly assigned to receive 20 μg OKT3 (anti-CD3) antibody i.v., with 2 additional infusions at 72-hour intervals.

In studies comparing the therapeutic efficiency of 1B9 and rituxan, mice were engrafted with human splenocytes along with biweekly infusion of sCD200hi plasma. Twenty-eight days following CLL infusion, mice were randomly assigned to receive saline, 1B9 (50 μg/mouse) or rituxan (50 mg/kg per mouse), all delivered i.v. in 300 μL. All treatments were repeated at 72-hour intervals for a total of 4 treatments. Animals were sacrificed 8 days after the last treatment.

For each in vivo experiment, mice of both sexes were used, with at least 3 mice per group. All experiments were repeated individually with splenocytes from at least 2 different patients.

FACS analyses

CD200 cell-surface staining was conducted using a rat anti-CD200 mAb (3G7) as previously described (6). Multicolor FACS analyses were conducted to characterize engrafted human cells. The optimal concentration of antibody for staining was determined individually for each antibody. Single color controls were included in each experiment for compensation purposes, and all samples were analyzed in a Coulter FC500 flow cytometer.

Statistics

Spearman rank correlation test and a Mann–Whitney U test were used to determine the correlation between sCD200 levels and various clinical markers in CLL. All clinical analyses were done using SPSS Statistics software. For in vivo studies, the absolute count of each engrafted cell population (CLL or T cells) was calculated from [total cell count × frequency], with frequency based on FACS staining profiles. Unpaired t tests were used to determine significance between sample means. Analysis of in vivo studies was conducted using GraphPad Prism 5.0 software.

Identification of sCD200 in plasma from CLL patients

CD200 is normally considered a membrane molecule (20). We have previously shown that CD200 was shed following activation of CLL cells in vitro, suggesting it might also exist in a soluble form in the plasma (6, 19). To explore evidence for a plasma form of CD200 (sCD200), we established a sandwich ELISA using 1B9 as capture antibody and a rabbit anti-hCD200 detection polyclonal antibody (rabbit anti-hCD200Fc) as described in the Materials and Methods section. The sensitivity of this ELISA using pure CD200Fc protein as standard was found to be 0.05 ng/mL. CD200Fc was used to generate standard curves in the range 0.05 to 10 ng/mL for quantitation of sCD200 in samples in all ELISA studies. The reproducibility and validity of sCD200 measurements was confirmed in a separate ELISA using a different antiserum (rabbit anti-hCD200v+c) as detection antibody.

The sCD200 levels in plasma from 25 healthy controls ranged from 0.4 ± 0.2 ng/mL. These levels were independent of age (20–64 years) or gender (data not shown). Elevated sCD200 levels were observed in plasma samples from patients with CLL across all clinical stages (Fig. 1A).

Figure 1.

Identification of sCD200 and clinical analysis of plasma sCD200 in CLL; sCD200 levels in plasma samples were measured by ELISA. All P values were obtained from Mann-Whitney U test unless specified otherwise. A, plasma from patients with CLL at various clinical stages of disease (n = 82) showed significantly higher levels of sCD200 than plasma from age-matched healthy controls (n = 27). *, P < 0.001. B, patients with CLL with Rai stage III (P = 0.015) and IV (P = 0.002) diseases showed higher plasma sCD200 levels than patients with early disease (stage 0-I). No significant difference in plasma sCD200 level was found between patients at stage 0-I and stage II. C, patients requiring more than 2 courses of treatments had higher plasma sCD200 levels than patients with more indolent disease requiring no (P < 0.0001) or 1 (P = 0.0027) treatment. D, plasma sCD200 levels strongly correlated with plasma β2-microglobulin levels (P < 0.0001, Spearman rank correlation test).

Figure 1.

Identification of sCD200 and clinical analysis of plasma sCD200 in CLL; sCD200 levels in plasma samples were measured by ELISA. All P values were obtained from Mann-Whitney U test unless specified otherwise. A, plasma from patients with CLL at various clinical stages of disease (n = 82) showed significantly higher levels of sCD200 than plasma from age-matched healthy controls (n = 27). *, P < 0.001. B, patients with CLL with Rai stage III (P = 0.015) and IV (P = 0.002) diseases showed higher plasma sCD200 levels than patients with early disease (stage 0-I). No significant difference in plasma sCD200 level was found between patients at stage 0-I and stage II. C, patients requiring more than 2 courses of treatments had higher plasma sCD200 levels than patients with more indolent disease requiring no (P < 0.0001) or 1 (P = 0.0027) treatment. D, plasma sCD200 levels strongly correlated with plasma β2-microglobulin levels (P < 0.0001, Spearman rank correlation test).

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Serum sCD200 levels are associated with disease stage in CLL

Although CD200 expression was detected in CLL cells from all individuals in a cohort of 25 patients at various stages of disease, the cell-surface expression level was not correlated with either tumor burden or other clinical parameters of disease (6). To determine whether sCD200 levels correlated with CLL clinical parameters, plasma samples obtained at diagnosis from 82 patients with CLL were tested in the sandwich ELISA for sCD200 levels. The patient age in this cohort ranged from 38 to 91 years (median age 61 years). Correlation analyses were conducted to compare sCD200 levels with other parameters linked to clinical outcome in CLL (see Table 1 for patient details).

Independent of the expression of CD200 on the cell surface, the sCD200 levels were correlated with tumor burden (see Supplementary Table SI), with patients in later stages of disease (Rai stage III and IV) having significantly higher sCD200 levels than patients at early stages (Fig. 1B). In general, clinical treatment of CLL is reserved for patients with late disease and/or rapidly progressive disease (21). Using the number of treatments received by each patient in our cohort, regardless of the nature of that treatment, as a surrogate marker for aggressive disease, we pooled patients into groups who had received 2 or more courses of treatments versus those with no (or only 1 course of) treatment. The former had significantly higher serum sCD200 levels than those with more indolent disease as defined by no treatment (P < 0.0001, Fig. 1C) or only 1 treatment (P = 0.0027, Fig. 1D).

Of the conventional prognostic markers for CLL, the sCD200 levels correlated most strongly with serum β2-microglobulin levels (P < 0.0001, Fig. 1D). CD38 expression levels did not correlate with sCD200 levels (22). The presence of intermediate- to high-risk cytogenetic abnormalities (trisomy 12 or deletions of regions of chromosomes 11 or 17) also did not correlate with sCD200 levels. However, patients with CLL cells that exhibited either a normal karyotype or 13q deletions, usually considered to be indicative of a more benign clinical course, were treated more often if they also had high levels of sCD200 (P < 0.0001, see Supplementary Table S1).

Cultured CLL cells seemed to release sCD200 at variable levels in a time-dependent manner (Supplementary Fig. S1); in addition, the level of sCD200 detectable in CLL supernatant correlated significantly with sCD200 level in the corresponding patient plasma (Spearman r = 0.8858; P = 0.033; n = 5; Supplementary Fig. S1). Importantly, sCD200 release by CLL cells was inhibited by the global protease inhibitor GM6001 (Supplementary Fig. S1). These observations support the hypothesis that CD200 is shed from CLL cells via mechanisms of ectodomain shedding with the involvement of proteases (23).

Development of a xenograft model for CLL

Given that sCD200 levels were higher in patients with late stage and/or aggressive disease, we hypothesized that sCD200 in CLL plasma might play a role in vivo in fostering CLL growth. In a series of preliminary studies, we infused 1 × 108 purified circulating peripheral blood lymphocytes (PBL)–derived CLL cells from 5 individual patients with high white cell counts into each of 4 per group NSG mice, with subsequent biweekly infusion of pooled sCD200hi CLL plasma, or sCD200lo normal plasma pooled from healthy volunteers. Although, animals receiving CLL plasma had greater engraftment of CLL cells than animals receiving normal plasma, the number of human cells recovered was generally low (data not shown), consistent with other reports in the literature (24, 25). We considered that a possible explanation for this poor engraftment might be the absence of supporting cells that would be present in proliferation centers but not in the blood.

In an attempt to provide a proposed “microenvironment factor,” we next attempted reconstitution of mice with splenocytes harvested from patients with CLL (Table 2; ref. 26). The cellular composition of each spleen sample was analyzed by FACS. In all cases, CD19+CD5+ CLL cells were the predominant cells in the spleen, although the frequency of CLL cells varied widely in a range from 25% to 95% (Fig. 2A: 4 representative spleens). Expression of CD20 and CD40 on CLL cells also varied, whereas CD200 was expressed on CLL cells from all spleens tested (Fig. 2A, bottom; CD40 staining not shown). CD4+ T cells were the next most common population detected, with frequencies ranging from 3% to 20% (Fig. 2B). CD8+ T cells and CD56+ natural killer cells were also detectable (Fig. 2B, data not shown). Low levels of CD14+ cells, previously reported to produce BAFF to support CLL survival in vitro, were found in all spleens studied (data not shown; ref. 27).

Figure 2.

Characterization of CLL splenocytes harvested from splenectomized patients (Table 2). A total of 2 × 106 fresh CLL splenocytes were characterized for relative frequency of CLL (CD20, CD19, CD5, and CD200) and T cells (CD4 and CD8) in multicolor FACS analyses. Results from 4 representative spleens are shown (Sp 1, 4, 5, and 6). A, frequency of CD19+CD5+ CLL cells from the 4 spleens ranged from 23% to 90% (top). CD19+CD5+ CLL cells were gated and analyzed for CD20 and CD200 expression, the results of which are shown in the bottom. CLL cells from all 4 patients stained brightly for CD200, whereas CD20 expression varied among the patients (bottom). B, distribution of CD4+ and CD8+ T cells in the same patient spleens as A. Frequency of CD4+ T cells ranged from 3% to 17%, whereas frequency of CD8+ T cells ranged from less than 0.5% to more than 10%.

Figure 2.

Characterization of CLL splenocytes harvested from splenectomized patients (Table 2). A total of 2 × 106 fresh CLL splenocytes were characterized for relative frequency of CLL (CD20, CD19, CD5, and CD200) and T cells (CD4 and CD8) in multicolor FACS analyses. Results from 4 representative spleens are shown (Sp 1, 4, 5, and 6). A, frequency of CD19+CD5+ CLL cells from the 4 spleens ranged from 23% to 90% (top). CD19+CD5+ CLL cells were gated and analyzed for CD20 and CD200 expression, the results of which are shown in the bottom. CLL cells from all 4 patients stained brightly for CD200, whereas CD20 expression varied among the patients (bottom). B, distribution of CD4+ and CD8+ T cells in the same patient spleens as A. Frequency of CD4+ T cells ranged from 3% to 17%, whereas frequency of CD8+ T cells ranged from less than 0.5% to more than 10%.

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Table 2.

Clinical characteristics of patients whose spleens were harvested and used in in vivo studies

Spleen #Pt IDSexAgeTime (y)Rai stageaWBCb% CD38β2McCytogeneticsTreatmentsdAIHAe
102 72 III 88 40 13q CP, steroids Yes 
113 42 IV 76 13q None No 
114 60 10 IV 300 13q None No 
125 72 IV 25 45 17p, 13q FC, HDP No 
153 71 15 IV 120 44 13q CVP, HDP Yes 
154 64 22 III 75 11q Revl, CP yes 
Spleen #Pt IDSexAgeTime (y)Rai stageaWBCb% CD38β2McCytogeneticsTreatmentsdAIHAe
102 72 III 88 40 13q CP, steroids Yes 
113 42 IV 76 13q None No 
114 60 10 IV 300 13q None No 
125 72 IV 25 45 17p, 13q FC, HDP No 
153 71 15 IV 120 44 13q CVP, HDP Yes 
154 64 22 III 75 11q Revl, CP yes 

aRai stage 0, lymphocytosis; I, with adenopathy; II, with hepatosplenomegaly; III, with anemia; IV, with thrombocytopenia.

bWBC, white blood cell count (106 cells/mL) in peripheral blood.

cβ2M, plasma β2-microglobulin level, mg/L.

dTreatments received before splenectomy: C, chlorambucil; P, prednisone; CVP, cyclophosphamide/vincristine/prednisone; FC, fludarabine/cyclophosphamide; HDP, high dose prednisone; Revl, Revlimid (lenalidomide).

eAIHA, documented autoimmune hemolytic anemia.

Recapitulating the observation made using CLL cells engrafted from PBL of patients, superior engraftment of CD19+CD5+ CLL cells at 28 and 54 days after infusion of CLL splenocytes was seen in animals receiving CD200hi CLL serum compared with controls receiving CD200lo normal human serum (Fig. 3A, day 28; Fig. 3B, day 54). This difference was particularly pronounced in the peritoneal cavity compartment (P = 0.003, Fig. 3B). As noted, splenocytes produced greater engraftment of CLL cells in both the spleen and peritoneal cavity of NSG recipients compared with engraftment seen using PBL (data not shown). Substantial patient-to-patient variability was observed in engraftment of both CLL and T cells (Fig. 3B and C, engraftment of spleens 1 and 5 shown). Accordingly, in all subsequent studies described detailed analysis was restricted to use of frozen aliquots of splenocytes from only 3 patients (spleens 1, 5, and 6).

Figure 3.

Engraftment of human CLL cells and T cells in NSG mice. Irradiated NSG mice at 8 to 13 weeks of age were infused with 1 × 108 thawed CLL splenocytes i.p., followed up biweekly with infusion of sCD200lo normal plasma, pooled from age-matched healthy volunteers, or sCD200hi CLL plasma. Mice were scarified at designed time points to assess for CLL and T-cell engraftment. P value was calculated from unpaired t test. A, FACS analysis of CLL (top) and T-cell (bottom) engraftment at day 28 in the peritoneal cavity of animals given either sCD200hi plasma or sCD200lo normal plasma. Spleen 1 was used in this experiment; result from 1 representative animal per group is shown. B, absolute count (×105 cells) of CD19+CD5+ CLL cells in the peritoneal cavity of animals that received either sCD200hi plasma or sCD200lo control plasma, in addition to spleens 1 or 5, at day 54 (data from 2 independent experiments were pooled). CLL cell counts were obtained by multiplying total cell count with %CD19+CD5+ cells as found in FACS. Mice that received sCD200hi plasma showed elevated engraftment of CLL cells in comparison with mice that received sCD200lo control plasma at this time point (**, P = 0.003). C, comparison of CLL and T-cell engraftment in mouse peritoneal cavity at day 54 by CLL spleens 1 (top) and 5 (bottom). D, immunohistochemical analysis of mouse spleens at day 54. Top, H&E staining; middle, CD20 staining; bottom, CD3 staining. E, CD19, CD5 (left column), and CD200 staining (right column) on human CD45+ cells engrafted in mouse peritoneal cavity at day 70 (bottom) as compared with CD45+ cells in original spleen (top). The data shown are representative from 1 individual mouse, reconstituted with Sp6, in a study that has been repeated 3 times. Results from 1 representative animal are shown in D and E.

Figure 3.

Engraftment of human CLL cells and T cells in NSG mice. Irradiated NSG mice at 8 to 13 weeks of age were infused with 1 × 108 thawed CLL splenocytes i.p., followed up biweekly with infusion of sCD200lo normal plasma, pooled from age-matched healthy volunteers, or sCD200hi CLL plasma. Mice were scarified at designed time points to assess for CLL and T-cell engraftment. P value was calculated from unpaired t test. A, FACS analysis of CLL (top) and T-cell (bottom) engraftment at day 28 in the peritoneal cavity of animals given either sCD200hi plasma or sCD200lo normal plasma. Spleen 1 was used in this experiment; result from 1 representative animal per group is shown. B, absolute count (×105 cells) of CD19+CD5+ CLL cells in the peritoneal cavity of animals that received either sCD200hi plasma or sCD200lo control plasma, in addition to spleens 1 or 5, at day 54 (data from 2 independent experiments were pooled). CLL cell counts were obtained by multiplying total cell count with %CD19+CD5+ cells as found in FACS. Mice that received sCD200hi plasma showed elevated engraftment of CLL cells in comparison with mice that received sCD200lo control plasma at this time point (**, P = 0.003). C, comparison of CLL and T-cell engraftment in mouse peritoneal cavity at day 54 by CLL spleens 1 (top) and 5 (bottom). D, immunohistochemical analysis of mouse spleens at day 54. Top, H&E staining; middle, CD20 staining; bottom, CD3 staining. E, CD19, CD5 (left column), and CD200 staining (right column) on human CD45+ cells engrafted in mouse peritoneal cavity at day 70 (bottom) as compared with CD45+ cells in original spleen (top). The data shown are representative from 1 individual mouse, reconstituted with Sp6, in a study that has been repeated 3 times. Results from 1 representative animal are shown in D and E.

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Characterization of CLL in humanized NSG mice

Hematoxylin and eosin (H&E) stain staining of spleen tissue taken from mice at day 54 showed aggregates of small lymphocytes resembling proliferation centers by H&E staining that were absent in control animals (Fig. 3D, top). CD20+ CLL cells were also more abundant in the spleen of experimental animals by IHC (Fig. 3D, middle). All CLL cells found in the peritoneal cavity and spleen of both experimental and control groups expressed CD40, CD38, and CD200 at similar levels to the starting population (data not shown).

Moreover, splenic CLL cells colocalized with T cells by IHC, in a pattern reminiscent of that observed in proliferation centers and in the spleen of patients with CLL themselves (Figs. 2B and 3D; ref. 28). Note that T cells were found in the spleens of mice injected with normal plasma, although CLL cells did not engraft in this case (Fig. 3C and D, bottom). Engraftment of cells other than CLL and T cells was minimal regardless of the source of plasma.

In a study monitoring longer-term engraftment, in which biweekly infusion of CLL plasma was continued to 1 week before experimental endpoint, we observed persistence of CLL cells in both peritoneal cavity and spleen at 70 days post spleen cell infusion (Fig. 3E, engraftment of spleen 6 shown). Engrafted CLL cells express the CLL markers CD19, CD5, and CD200 (Fig. 3E), as well as CD40, CD20, and CD38 at levels similar to that on CLL cells from the original spleen (Supplementary Fig. S2). In contrast, increased expression of CD49d was detected in a subpopulation of engrafted CLL cells (Supplementary Fig. S2). Engrafted human CD45+ cells in the bone marrow were mostly CD4+ and CD8+ T cells at this time point (Supplementary Fig. S3).

Anti-CD200 and OKT3 mAbs abrogate engraftment of CLL in NSG mice

To assess whether sCD200 in CLL plasma was an important factor contributing to engraftment of CLL cells in vivo, animals receiving CLL splenocytes and sCD200hi serum also received Fab anti-CD200 mAb 1B9. An independent group of mice received CLL splenocytes, and CLL plasma that had been depleted of CD200 by passage though an anti-CD200 CNBr column. sCD200 absorption from the serum was confirmed independently by ELISA (>97% depletion). Both anti-CD200 mAb and depletion of sCD200 from plasma attenuated the engraftment of CLL cells in vivo (Fig. 4A, spleen engraftment; Supplementary Fig. S4, peritoneal cavity engraftment).

Figure 4.

Effects of sCD200 and/or T-cell depletion on CLL engraftment in NSG mice. A, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and then given either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. A separate group of animals was given 1B9, an anti-CD200 mAb, in addition to sCD200hi CLL plasma for in vivo sCD200 depletion. Both methods of sCD200 depletion effectively reduced CLL engraftment in peritoneal cavity. B, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and one of sCD200lo normal plasma alone or normal plasma plus 50 μg/mL CD200Fc, or sCD200hi CLL plasma on a biweekly basis. CLL engraftment in mouse spleen is shown in absolute cell count; data from 1 of 2 independent experiments using spleen 6 is shown (**, P = 0.04; *, P = 0.05). C, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. Both groups of animals were subdivided to receive either OKT3 i.v. for T-cell in vivo depletion or control saline. OKT3 was given a total of 4 times in 2 weeks. T-cell depletion seemed to abrogate CLL engraftment regardless of the presence of sCD200 in supplemented plasma. Data from 1 representative experiment using sp1 with engraftment of CLL cells in mouse peritoneal cavity is shown (mouse spleens showed similar engraftment pattern). CLL engraftment was assessed by CD19, CD5, and CD200 staining. Mice were sacrificed at day 21 for A and C, and at day 54 for B.

Figure 4.

Effects of sCD200 and/or T-cell depletion on CLL engraftment in NSG mice. A, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and then given either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. A separate group of animals was given 1B9, an anti-CD200 mAb, in addition to sCD200hi CLL plasma for in vivo sCD200 depletion. Both methods of sCD200 depletion effectively reduced CLL engraftment in peritoneal cavity. B, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and one of sCD200lo normal plasma alone or normal plasma plus 50 μg/mL CD200Fc, or sCD200hi CLL plasma on a biweekly basis. CLL engraftment in mouse spleen is shown in absolute cell count; data from 1 of 2 independent experiments using spleen 6 is shown (**, P = 0.04; *, P = 0.05). C, irradiated NSG mice were infused with 1 × 108 CLL splenocytes and either sCD200hi CLL plasma or sCD200 absorbed CLL plasma. Both groups of animals were subdivided to receive either OKT3 i.v. for T-cell in vivo depletion or control saline. OKT3 was given a total of 4 times in 2 weeks. T-cell depletion seemed to abrogate CLL engraftment regardless of the presence of sCD200 in supplemented plasma. Data from 1 representative experiment using sp1 with engraftment of CLL cells in mouse peritoneal cavity is shown (mouse spleens showed similar engraftment pattern). CLL engraftment was assessed by CD19, CD5, and CD200 staining. Mice were sacrificed at day 21 for A and C, and at day 54 for B.

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To confirm further that the presence of CD200 in human plasma was sufficient to enhance CLL engraftment, a recombinant form of CD200, CD200Fc, was used. The addition of CD200Fc to sCD200lo normal plasma, at a concentration within the range, which had previously been shown to be effective in promoting survival of skin allografts in mice (50 μg/mL, i.p.), enhanced engraftment CD19+CD5+ CLL cells in the spleens NSG mice (P = 0.04, Fig. 4B, CLL engraftment in mouse spleen from 1 representative experiment using spleen 6 shown; ref. 29). The effect of CD200Fc at this dosage seemed more pronounced than that of CLL plasma, although infusion of sCD200hi CLL plasma consistently resulted in enhanced engraftment of CLL cells when compared with sCD200lo normal plasma (P = 0.05, Fig. 4B). The effect of CD200Fc for engraftment in peritoneal cavity was less pronounced, although followed a similar pattern to that shown for spleen (Supplementary Fig. S5).

CD200 is known to deliver downstream signals through a receptor CD200R, and we had previously reported that CD200R was detected mostly on splenic CD4+ T cells but not on CLL cells (6, 20). The absence of CD200R on CLL cells has been independently confirmed by reverse transcriptase PCR (RT-PCR; data not shown). We investigated the role of T cells in CLL engraftment by treating mice in the early period post spleen reconstitution with OKT3 antibody in vivo, analyzing CLL and T cells 4 weeks later. Interestingly, in vivo depletion of T cells abrogated engraftment of CLL cells, despite continuous infusion of sCD200hi CLL serum (Fig. 4C, spleen engraftment; Supplementary Fig. S6, peritoneal cavity engraftment).

Comparison of anti-CD200 and rituximab in eliminating engrafted CLL cells

In a final study, we asked whether CD200 blockade could eliminate engrafted CLL cells, thus highlighting CD200 as a potential therapeutic target. Specifically, we compared the efficacy of rituxan, a clinically approved monoclonal antibody targeting CD20 on CLL cells, with anti-CD200 mAb therapy as treatment in mice with established spleen-cell derived CLL engraftment (30).

At 28 days following CLL splenocyte injection and biweekly sCD200hi CLL serum infusion, independent groups of mice received rituxan (50 μg/mouse) or Fab antimouse CD200 mAb (i.v.). A total of 4 injections were given, at 72-hour intervals over 2 weeks. Animals were maintained for 10 days after the last dose of treatment, again with ongoing injection of CLL serum on a biweekly basis, and were sacrificed at day 45. The FACS analyses on cell suspensions harvested from the spleen and peritoneal cavity of these animals showed that both rituxan (P = 0.0026) and anti-CD200 mAb (P = 0.0057) were effective in reducing CLL engraftment in both tissue compartments (Fig. 5A and B). Essentially all CLL cells engrafting in the peritoneal cavity were depleted, with significant attenuation of engraftment in the spleen (>70%). Although, the overall frequency of CD3+ T cells in either compartment did not seem to be significantly affected (Fig. 5C), the CD4:CD8 ratio in mice treated with either rituxan or anti-CD200 mAb differed from that in untreated mice (Supplementary Fig. S7).

Figure 5.

Therapeutic efficacy of rituxan and 1B9 on CLL engraftment in NSG mice. Irradiated NSG mice were engrafted with 1 × 108 CLL splenocytes and infused biweekly with sCD200hi CLL plasma. At day 21, mice were divided into 3 groups and given one of rituxan, 1B9, or saline i.v. 4 times in 2 weeks. Engraftment of CLL and T cells was assessed at day 45. P values were calculated from unpaired t test. Both rituxan and 1B9 were effective in eliminating CLL engraftment in both peritoneal cavity (*, P = 0.0057; **, P = 0.0026; A) and spleen (B) without having significant effect on CD3+ T cells (C). Results were pooled from 2 independent experiments engrafted with splenocytes from Sp1 and 5.

Figure 5.

Therapeutic efficacy of rituxan and 1B9 on CLL engraftment in NSG mice. Irradiated NSG mice were engrafted with 1 × 108 CLL splenocytes and infused biweekly with sCD200hi CLL plasma. At day 21, mice were divided into 3 groups and given one of rituxan, 1B9, or saline i.v. 4 times in 2 weeks. Engraftment of CLL and T cells was assessed at day 45. P values were calculated from unpaired t test. Both rituxan and 1B9 were effective in eliminating CLL engraftment in both peritoneal cavity (*, P = 0.0057; **, P = 0.0026; A) and spleen (B) without having significant effect on CD3+ T cells (C). Results were pooled from 2 independent experiments engrafted with splenocytes from Sp1 and 5.

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The studies described in this chapter implicate a novel soluble form of CD200 (sCD200) in the progression of CLL. sCD200 is present at high levels in CLL plasma compared with normal plasma and promotes the growth of CLL cells in immunodeficient mice, suggesting it may be a novel therapeutic target for this malignancy.

CD200 expression has been linked with outcome in many malignancies (3, 31, 32). Biochemical analysis has suggested that sCD200 is likely shed from the cell surface by proteolytic cleavage, with a disintegrin and metalloproteinase family of proteases being prime candidates in this mechanism (Supplementary Fig. S1; K. Wong, unpublished data). We found that plasma sCD200 levels, but not surface expression of CD200 on CLL cells, correlated strongly with tumor burden, disease stage, and an aggressive disease course as reflected in the requirement for multiple treatments. Plasma sCD200 was also correlated with plasma β2-microglobulin levels, known to be an independent predictor of progression-free survival and overall survival in CLL (33, 34). The value of plasma sCD200 as an independent prognostic marker against current clinical prognostic markers remains to be determined.

The hypothesis that sCD200 is important in CLL disease is supported by evidence for its contribution to successful engraftment of spleen (and PBL)-derived CLL cells in vivo in NSG mice. PBL-derived CLL cells generally fail to engraft in immunocompromised hosts, although some engraftment was recorded following combinations of i.p. and i.v. infusion of PBL-derived CLL cells or following injection of an Epstein-Barr virus (EBV+) transformed CLL cell line (35, 36). Quantitative comparison of engraftment using spleen or PBL from the same patient has not been conducted to assess engraftment potential of CLL cells from these 2 tissue sources. Nevertheless, it is clear that sCD200hi CLL plasma augments engraftment of CD19+CD5+ CLL cells in both peritoneal cavity and spleen, in contrast to the inferior engraftment seen using sCD200lo plasma pooled from healthy volunteers with 10-fold lower sCD200 levels (∼0.5 ng/mL). Those CLL cells that engraft in both sets of animals express CD20, CD40, CD23, CD38, and CD200 at levels similar to the CLL cells from the starting spleen population, suggesting no selection for survival of unique subpopulations occurred in vivo under these conditions. Interestingly, an increase in the expression of CD49d, α4 subunit of the lymphocyte integrin VLA-4, which has been shown to play a role in CLL homing to bone marrow in vivo, was observed in a subpopulation of CLL cells in both spleen and peritoneal cavity at 70 days postreconstitution (37). Because few CLL cells engraft in the bone marrow, the significance of this CD49d expression remains unclear.

Engraftment of CLL cells in a xeno-microenvironment often reflects persistence of donor cells, rather than proliferation of CLL cells in the host, and engrafted CLL cells gradually decline with time (24, 25, 36). The use of Epstein-Barr virus–transformed CLL cell lines circumvents this issue, although these lines often exhibit very different biologic properties from those of primary cells, particularly with the absence of CD5 expression (35, 38). In contrast, we did not observe a significant decline in the absolute number of engrafted CLL cells from day 25 to day 70 (data not shown). It remains to be determined whether CLL engraftment in our model reflects both survival and in vivo proliferation of CLL cells.

Because the engrafted CLL cells continue to express CD200 at high levels, it is possible that sCD200 may be released in ongoing fashion from grafted CLL cells in vivo—this hypothesis is supported by recent findings from assaying sCD200 levels in mice at >6 months postreconstitution (unpublished data). We have not investigated whether continual infusion of sCD200hi CLL plasma is needed to sustain CLL engraftment, or whether the engrafted CLL cells produce sufficient amount of sCD200 in vivo to maintain themselves in the absence of exogenous sCD200.

Normal plasma and CLL plasma differ significantly in their content of multiple molecules, including many proteins and fatty acids. A number of such molecules, including the B-cell growth factors BAFF and a proliferation-inducing ligand, and soluble CD14, have direct effects on CLL cells (15, 39, 40). Although, we do not rule out contributions by other factors besides sCD200 in the support of CLL growth, 2 lines of evidence from this study supports the hypothesis that sCD200 is a key contributor. First, preabsorption of sCD200 from CLL plasma minimized CLL engraftment. Second, addition of exogenous CD200Fc, a soluble, recombinant form of CD200, to sCD200lo normal plasma, which by itself had no effect on CLL engraftment, resulted in enhanced CLL in vivo survival.

Our data also support a role for T-cell involvement in this mechanism of action, as T-cell depletion (OKT3 in vivo) abrogated CLL engraftment, despite continuous infusion of sCD200hi CLL plasma. This may help explain why, in preliminary experiments, infusion of purified CLL cells alone, even with continuous sCD200hi CLL serum supplement, produced only minimal engraftment of CLL cells. The role of nonmalignant T cells in CLL has been investigated by a number of groups (41–43). T cells harvested from patients with CLL differ from normal T cells by their high production of IL4 and reduced expression of costimulatory molecules (42). The observation that CLL cells fail to engraft in the absence of T cells despite sCD200 infusion suggests T cells, a subpopulation of which have previously been shown to express CD200R, may represent an important target of sCD200 (6). Whether such sCD200-targeted CD200R+ T cells could affect CLL growth directly, or indirectly through the action of other T or non-T populations, remains unknown.

Studies comparing the efficacy of anti-CD200 mAb (Fab) or rituxan as therapy for NSG mice reconstituted with CLL splenocytes showed both were effective in treating preexisting disease. Although rituxan induces lysis of CLL cells by complement-dependent and antibody-dependent cell-mediated cytotoxicity, anti-CD200 (Fab) likely regulates host resistance to growth (44). In addition, both rituxan and anti-CD200 mAb treatment seemed to affect the CD4:CD8 ratio in engrafted T cells with a particularly marked increased frequency of CD8+ cells observed in anti-CD200 mAb treated mice. The functional and therapeutic significance of these observations remains to be determined.

In summary, our data suggest that sCD200, present in patient plasma, is associated with disease progression in patients with CLL, and its presence can be used to establish a novel xenograft model for CLL, which may be useful for preclinical testing.

No potential conflicts of interest were disclosed.

Conception and design: K.K. Wong, D.E. Spaner, R.M. Gorczynski

Development of methodology: K.K. Wong, A. Chesney, D.E. Spaner, R.M. Gorczynski

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.K. Wong, F. Brenneman, A. Chesney, D.E. Spaner, R.M. Gorczynski

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.K. Wong, A. Chesney, D.E. Spaner, R.M. Gorczynski

Writing, review, and/or revision of the manuscript: K.K. Wong, A. Chesney, D.E. Spaner, R.M. Gorczynski

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Chesney, R.M. Gorczynski

Study supervision: D.E. Spaner, R.M. Gorczynski

This research was supported by grants from CIHR and CCSRI (to R.M. Gorczynski), CIHR-TPRM Graduate student Fellowship (to K.K. Wong), CIHR (#190633), and the Leukemia and Lymphoma Society of Canada (LLSC; to D.E. Spaner).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data