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Expression of Adhesion Molecules on CD34⁺ Cells in Peripheral Blood of Non-Hodgkin's Lymphoma Patients Mobilized with Different Growth Factors

University of Texas Health Science Center and Wilford Hall Medical Center, San Antonio, Texas, USA

Key Words. Mobilization • CD34⁺ • VLA-4 • L-selectin • NHL

Yair Gazitt, Ph.D., Department of Medicine/Hematology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA. Telephone: 210-567-4848; Fax: 210-567-1956: email:gazitt{at}uthscsa.edu

	ABSTRACT

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Adhesion molecules on CD34⁺ cells were implicated in the process of peripheral blood stem cell (PBSC) mobilization and homing. We studied the mobilization of CD34⁺Thy1⁺ cells, CD34⁺ very late-acting antigen (VLA)4⁺ cells, and CD34⁺L-selectin⁺ cells in non-Hodgkin's lymphoma patients mobilized with cyclophosphamide plus G-CSF, GM-CSF, or GM-CSF followed by G-CSF. The mean percentage of CD34⁺ cells in the bone marrow (BM) expressing Thy1 was 23.6% ± 11% and 17.8% ± 8% in the PB before mobilization, and was markedly decreased to 4.5% ± 3.3% in the apheresis collections. Similarly, the mean percentage of CD34⁺ cells expressing L-selectin was 35.8% ± 4.3% in the BM, 21.6% ± 4.1% in the PB before mobilization and was markedly decreased to 9.1% ± 2.5% in the apheresis collections. Patients in the three arms of the study had a similar pattern of CD34⁺Thy1⁺ and CD34⁺L-selectin⁺ cell mobilization. Also, a similar pattern of coexpression of CD34⁺Thy1⁺ and CD34⁺L-selectin⁺ cells was observed when the patients were regrouped as "good mobilizers" (2 x 10⁶ CD34⁺CD45^dim cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10⁶ CD34⁺CD45^dim cells/kg, in two collections).

The mean percentage of CD34⁺ cells expressing VLA-4 in the BM and PB was relatively high (73.4% ± 12% and 65.4% ± 6.6%, respectively) and dropped considerably in the PBSC collections to 43.5% ± 7.1% with a similar pattern observed for patients in arms A, B, and C. However, when the patients were regrouped as "good mobilizers" and "poor mobilizers," a higher percentage of CD34⁺ cells expressing VLA-4 was observed in the PBSC of the pooled "good mobilizers" (50.5% ± 9% versus 36.3% ± 6.4%; p = 0.01). We conclude that release of CD34⁺ cells to the PB involves a general downregulation of Thy1, L-selectin and VLA-4 on CD34⁺ cells, irrespective of the growth factor used for mobilization. However, good mobilizers had a relatively higher percentage of CD34⁺ cells expressing the VLA-4 antigen.

	INTRODUCTION

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Autologous peripheral blood stem cells (PBSCs) provide a rapid and sustained hematopoietic recovery after the administration of high-dose chemotherapy or chemoradiotherapy in patients with hematological malignancies. PBSCs have become the preferred source of stem cells for autologous transplantation because of the shorter time to engraftment and the lack of a need for surgical procedure necessary for bone marrow (BM) harvesting [1-5].

Mobilization of CD34⁺ in PBSCs can be achieved by the administration of G-CSF or GM-CSF alone [3, 6-10], or in combination with chemotherapy [11-13], in which case a higher yield of CD34⁺ cells can be obtained resulting in a decrease in the number of collections required for prompt engraftment. Recent studies advocated the use of a concurrent or sequential combination of G-CSF and GM-CSF, which in some reports appears to be superior over one growth factor alone, in normal allogeneic donors [8, 14-16] and cancer patients [17]. Others found no difference in the potency of G-CSF or GM-CSF in mobilizing CD34⁺ cells [18]. Our own experience in a randomized study of non-Hodgkin's lymphoma (NHL) patients suggested similar efficacy for mobilization of CD34⁺ cells for patients mobilized with G-CSF, GM-CSF, or sequential GM-CSF followed by G-CSF, in NHL patients primed with cyclophosphamide (Cy) [19].

CD34⁺ hematopoietic stem cells subsets, characterized immunophenotypically as CD34⁺Thy1⁺ and CD34⁺CD38^– cells [20-22], have been shown to be capable of self-renewal in vitro and in vivo in severe combined immunodeficent mice and are thought to play an important role in short-term and long-term engraftment [23-25]. These cells are relatively abundant in fetal tissues and cord blood [26-28] and were recently detected in the PBSC collections of patients mobilized with G-CSF [29], or GM-CSF [11, 30]. More recently, we have shown that low doses of CD34⁺Thy1⁺ cells purified from PBSC collections of myeloma patients were capable of engrafting these patients following high-dose chemotherapy and stem cell support [31].

The CD34 antigen has been shown to have adhesion properties, although the exact ligand was not yet characterized [25]. Similarly, the Thy1 antigen (CDw90) is thought to have adhesion properties [21, 29]. Other subsets of CD34⁺ cells express well-defined adhesion molecules such as L-selectin and very late-acting antigen (VLA)4 which are thought to play an important role in the release of CD34⁺ cells to the PB during mobilization and in homing to the BM in the process of engraftment. The extent of expression on CD34⁺ cells of adhesion molecules, such as VLA-4 (CD49d) and L-selectin (CD62L), has been shown to correlate with mobilization of PBSC [32-37] and in vivo treatment of mice with antibodies to VLA-4 [38] or VCAM-1 [39] were capable of inducing mobilization of CD34⁺ stem cells to the PB.

In this study we compared the mobilization of CD34⁺Thy1⁺cells, CD34⁺VLA-4⁺ cells, and CD34⁺L-selectin⁺ cells in NHL patients primed with Cy and G-CSF, GM-CSF, or sequential GM-CSF followed by G-CSF, relative to the steady-state levels of these adhesion molecules on BM and PB CD34⁺ cells. We observed a decrease in the percentage of CD34⁺ cells expressing Thy1, VLA-4, and L-selectin in all three arms of the study with a similar pattern of these CD34⁺ subsets observed in all patients, regardless of the mobilization regimen used.

	METHODS

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Study Design
We designed a randomized study of stem cell mobilization to compare the efficacy of PBSC mobilization with Cy (3 g/m²) plus G-CSF (10 µg/kg, arm A), Cy plus GM-CSF (250 µg/m², arm B), or Cy plus GM-CSF (250 µg/m²) for seven days, followed by eight days of G-CSF (10 µg/kg, arm C), in 60 NHL patients (20 patients in each arm), undergoing high-dose chemotherapy and PBSC rescue. Patients were randomized for the mobilization regimen. Patients were excluded from the study if they were over 70 years of age, had therapy for over two years, or received therapy within the last four weeks. Patients with abnormal heart, kidney, or liver function and pregnant women were also excluded. Patients who collected 2 x 10⁶ CD34⁺CD45^dim cells/kg in one to four days were defined as "good mobilizers" [10, 19]. These patients, by and large, collected 1 x 10⁶ CD34⁺CD45^dim cells/kg in two days of apheresis. In contrast, patients who collected 0.4 x 10⁶ CD34⁺CD45^dim cells/kg in the first two days of apheresis were considered "poor-mobilizers" and subsequently were dropped off protocol after two collections and were remobilized later by other mobilization protocols at the discretion of the referring physician [10, 19]. These definitions of "good mobilizers" and "poor mobilizers" were adopted following our previous observation that patients who collected 0.4 x 10⁶ CD34⁺CD45^dim cells/kg in the first two days of apheresis had >95% probability of failure to collect the desired amount of 2 x 10⁶ CD34⁺CD45^dim cells/kg in 4 days of apheresis [10, 19].

Patient Population
Thirty-five patients enrolled in the study at the Audie L. Murphy Memorial VA Hospital, University Hospital, and Wilford Hall Medical Center at San Antonio. Thirteen patients were enrolled in arm A, 10 in arm B, and 12 in arm C. Median age in arm A was 46 years and in arm B and C was 52 and 55 years, respectively. Greater than 75% of patients in each arm were males (mostly VA patients). Fifty-four percent, 80% and 83% of patients in arms A, B, and C, respectively, were in relapse at the time of transplant and 42%, 40%, and 50% of patients in arms A, B, and C, respectively, had 3 regimens of therapy prior to mobilization. The distribution of patients with follicular lymphoma was similar in the three arms of the study (41%, 30%, and 40%, in arms A, B, and C, respectively) (Table 1). All patients were consented as required by our Institutional Review Board.

Dual Color Staining for CD34⁺Thy1⁺, CD34⁺CD49d⁺, and CD62L⁺ Cells
Staining was done as outlined before for CD34⁺/CD45^dim cells [10] except that anti-CD34 antibody (HPCA-2-PE, Becton Dickinson; San Jose, CA; http://www.bd.com) was matched with anti-CD62L-fluorescein isothiocyanate (FITC) (Becton Dickinson), or anti-CD90w (Thy1-FITC, Immunotech; Marseilles, France), anti-CD49d-FITC (Immunotech). One hundred thousand cells were collected to ensure >100 cells in the double-positive quadrant. An example for dual staining for CD34/CD49d, CD34/CD62L, and CD34/CD90w of an apheresis collection from a good mobilizer patient is shown in Figure 1.

View larger version (35K): [in this window] [in a new window]   Figure 1. Dual color staining for CD34/CD49d, CD34/CD62L, and CD34/CD90w cells. Figure 1 is an example of dual staining of an apheresis collection from a "good mobilizer" patient. Left upper panel is the light scatter gating on the lymphoblastoid population; right upper panel is the isotype controls. Middle left panel is staining for CD34+-only cells (0.83%), CD34/CD49d+ cells (0.90%), middle right panel is staining for CD34+-only cells (1.31%), CD34/CD62L+ cells (0.40%). Lower middle panel is staining for CD34+-only cells (1.75%) and CD34/CD90w+ cells (0.15%). In this example (52% of total CD34+ cells), 23% and 7.9% were dual positive for CD34/CD49d, CD34/CD62L, and CD34/CDw90, respectively. Hundred thousand cells were analyzed. For further details, see Methods section.

	RESULTS

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To assess the recent expression by CD34⁺ cells of CD49d (VLA-4), CD62L (L-selectin), or CDw90 (Thy1), we used dual color immunofluorescence, and results were analyzed by flow cytometry. An example is shown in Figure 1. The left upper panel is the light scatter gating on the lymphoblastoid population; in the right upper panel are the isotype controls. Middle left panel is staining for CD34⁺-only cells (0.83%), CD34/CD49d⁺ cells (0.90%); middle right panel is staining for CD34⁺-only cells (1.31%), CD34/CD62L⁺ cells (0.40%). The lower middle panel is staining for CD34⁺ only cells (1.75%) and CD34/CD90w⁺ cells (0.15%). In this example (52% of total CD34⁺ cells) 23% and 7.9% were dual-positive for CD34/CD49d, CD34/CD62L, and CD34/CDw90, respectively. Note that the total CD34⁺ cells is very similar in the three different combinations (1.73%, 1.71%, and 1.90%). Dual-stained cells appear in the upper right quadrant of each square.

Mobilization of CD34⁺Thy1⁺ Hematopoietic Stem Cells
Coexpression of the Thy1 antigen on CD34⁺ cells was studied in patients mobilized with A) Cy plus G-CSF, B) GM-CSF, and C) GM-CSF followed by G-CSF in 35 NHL patients. The results are depicted in Figure 2. The mean percentage of CD34⁺ cells expressing Thy1 in the steady-state BM was 19.9% ± 4.5%, 18.8% ± 4.9%, and 22.6% ± 13.4% in patients in arms A, B, and C, respectively. The mean percentage of CD34⁺ cells expressing Thy1 in steady-state PB was similar to that observed in BM (18.2% ± 5%, 17% ± 6.2%, and 17.3% ± 6.7%), in patients in arms A, B, and C, respectively. The mean percentage of CD34⁺ cells expressing Thy1 in four apheresis collections dropped to 12.3% ± 1.9%, 10.6% ± 3.0%, and 10.4% ± 1.2%, in patients in arms A, B, and C, respectively (BM versus PBSC; p = 0.006). No major differences were observed between the four days of collection or the three arms of the study (Fig. 2). The mean percentage of CD34⁺ cells expressing Thy1 in the PBSC collections obtained from four normal donors (primed with 10 µg/kg of G-CSF x 4 days) was low compared to unprimed BM obtained from three normal donors (6.6% ± 3.5% versus 13.1% ± 4.2%; BM versus PB p = 0.01) (Fig. 2, gray bars). No differences were observed in the mobilization of CD34⁺Thy1⁺ cells between the "good mobilizers" (2 x 10⁶ CD34⁺CD45^dim cells/kg, in four collections) and "poor mobilizers" (<0.4 x 10⁶ CD34⁺CD45^dim cells/kg, in two collections).

View larger version (33K): [in this window] [in a new window]   Figure 2. Mobilization of CD34+Thy1+ stem cells in NHL patients. The results presented are from the patients in arms A, B, and C as described in Figure 1. Staining for CD34+Thy1+ cells was determined as described in the Methods section. The gray bars represent the CD34+Thy1+ cells in the bone marrow (N-BM) of unprimed normal donors, or in the PB on G-CSF-primed normal donors (N-PB) as described in the text. d1 to d4 are apheresis collections in four consecutive days. Error bars represent + SD.

View larger version (32K): [in this window] [in a new window]   Figure 3. Expression of CD62L antigen on mobilized CD34+ cells. The results presented are from the patients in arms A, B, and C as described in Figure 1. Staining for CD34+CD62L+ cells was performed as described in the Methods section. d1 to d4 are apheresis collections in four consecutive days. The gray bars represent the CD34+CD62L+ cells in the bone marrow (N-BM) of unprimed normal donors, or in the PB of G-CSF primed normal donors (N-PB) as described in the text. Error bars represent ± SD.

View larger version (37K): [in this window] [in a new window]   Figure 4. Expression of CD49d antigen on mobilized CD34+ cells: comparison between the three arms. Staining for CD34+CD49d+ cells was performed as described in the Methods section. d1 to d4 are apheresis collections in four consecutive days. Error bars represent ± SD.

View larger version (20K): [in this window] [in a new window]   Figure 5. Correlation between CD34+ cell mobilization and percent expression of VLA-4 on CD34+ cells. Data for percentage of CD34+CD49d+ cells were taken from Figure 4. Data for mobilization of CD34+CD45dim cells/kg in patients in arms A, B, and C were published elsewhere [19]. A weak correlation was observed between CD34+CD49d+ cells and CD34+CD45dim cells/kg (r = 0.39; p = 0.04).

View larger version (26K): [in this window] [in a new window]   Figure 6. Coexpression of CD49d antigen on mobilized CD34+ cells: "good mobilizers" versus "poor mobilizers." The results presented are from the pool of patients in arms A, B, and C as described in Figure 4, but grouped as "good mobilizers" (2 x 106 CD34+CD45dim cells/kg, in four collections), versus "poor mobilizers" (<0.4 x 106 CD34+CD45dim cells/kg, in two collections). Other details are as in Figure 4.

	DISCUSSION

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Mobilization of CD34⁺ Thy1⁺ Hematopoietic Stem Cells
We studied the mobilization of CD34⁺Thy1⁺ cells in NHL patients primed with Cy and G-CSF, GM-CSF, or sequential GM-CSF followed by G-CSF, relative to the steady-state levels in the BM and PB, before mobilization. Our results indicate a general decrease in PBSC collections of the percentage of CD34⁺Thy1⁺ cells relative to the percentage observed in steady-state BM or PB. These results are in agreement with previous observations by Tsuchia et al. [45], Humeau et al. [22], and Thong et al. [46] and disagree with the results published by Haas et al. [29], who reported an increase in the percentage of CD34⁺ cells expressing Thy1 PBSC collections from patients with different types of cancer. Our observation was consistent in all three arms of the study, regardless of the growth factor used, and with similar distribution observed in the "good mobilizers" and "poor mobilizers" groups of patients. The fact that patients receiving PBSC engraft faster than patients receiving BM, which contain relatively higher percentage of CD34⁺Thy1⁺ cells, suggests that these cells are less important for short engraftment compared to the more mature CD34⁺ progenitor cells which are more abundant in PBSC collections. This conclusion is supported by our previous findings that relatively high doses of purified CD34⁺Thy1⁺ cells/kg are required for prompt engraftment compared to unmanipulated PBSC collections [31]. Further support is also evident in recent findings that patients transplanted with cord blood experience delayed engraftment compared to patients receiving PBSC products, despite the fact that cord blood products have higher percentages of CD34⁺Thy1⁺ cells, but lower amounts of total CD34⁺ cells/kg [27, 47-48].

Mobilization of CD34⁺CD62L⁺ Cells and CD34⁺CD49d⁺ Cells
We observed a marked decrease in the expression of L-selectin on CD34⁺ cells in the PBSC apheresis collections relative to the steady-state PB and BM. This decrease was observed in apheresis collections of all patients, regardless of the growth factor used for mobilization, and is consistent with results obtained by other groups [32-38]. The fact that "good mobilizers" and "poor mobilizers," as well as PBSCs from normal donors, had a similar pattern of coexpression of this molecule on CD34⁺ cells suggests a role for this molecule in the process of release of stem cells to the PB.

The expression of VLA-4 on CD34⁺ cells in the apheresis collections was slightly decreased compared to CD34⁺ cells in the BM; however, only a weak correlation was observed between mobilization of CD34⁺ cells and the extent of expression of VLA-4 on CD34⁺ cells. Nevertheless, the decrease in the percentage of CD34⁺ cells coexpressing VLA-4 was consistent in the PBSC collections of all patients, regardless of the growth factor used for mobilization. Others reported a similar decrease for VLA-4 in mobilized PB [32-38]. However, in our study the "good mobilizer" group of patients had a higher percentage of cells coexpressing VLA-4 on CD34⁺ cells compared to the "poor mobilizer" group of patients. In this respect, Zeller et al. [49] found a relatively high percentage of CD34⁺CD49d⁺ and CD34⁺CD49e⁺ cells in NHL and Hodgkin's disease (HD) patients compared to breast cancer and testicular cancer patients. However, we and others have shown that NHL and HD patients are poorer mobilizers of CD34⁺ cells compared to breast or testicular cancer patients, or patients with other solid tumors [2, 10, 42, 44]. The differences between the Zeller et al. results and our results could be disease-related and not directly associated with expression of adhesion molecules. Nevertheless, in our study we compared the "good mobilizers" and the "poor mobilizers" within the same group of patients with NHL. Thus, our findings, using our criteria for "good mobilizers" and "poor mobilizers," if confirmed using larger numbers of "poor mobilizers," are more clinically relevant for NHL patients. Furthermore, based on our results, we propose that the release of CD34⁺ cells from the marrow is contingent upon an extensive decrease in L-selectin and moderate decrease in VLA-4 expression on CD34⁺ cells and therefore, VLA-4 might have a role in facilitation of extravasation and release of CD34⁺ cells from the marrow into the blood stream.

L-selectin as well as VLA-4 were implicated in homing and engraftment of PBSCs, in particular high expression of L-selectin and VLA-4 was implicated in successful homing and rapid engraftment [33, 36, 38, 50-52]; however, upon release to the PB CD34⁺ cells express reduced levels of VLA-4 and L-selectin. The apparent paradox can be resolved by recent findings indicating that a great variety of hematopoietic growth factors upregulates the expression of VLA-4 and L-selectin in vitro [32].

In addition to downregulation of adhesion molecules in PBSC collections, we have recently shown that downregulation of CXCR4, the chemokine receptor to SDF-1, is also a prerequisite for mobilization [53]. In this respect, it is interesting to note that the chemokine SDF-1 can activate VLA-4 and VLA-5 on CD34⁺ cells to bind their corresponding ligands [52], in addition to downregulation of the expression of its own receptor [51, 54]. On the other hand, upregulation of CXCR4 occurs in vitro by growth factors such as stem cell factor, interleukin 3 (IL-3), erythropoietin, thrombopoietin, and flt3-L [55]. In addition, IL-1, tumor necrosis factor, and interferon , ß, and [56] were recently reported. Thus, it is reasonable to assume that regulation of the CXCR4 receptor and adhesion molecules may occur also in vivo by endogenous growth factors or by growth factors such as G-CSF and GM-CSF, administered routinely to transplant patients to facilitate engraftment.

Finally, the fact that a similar decrease in the expression of adhesion molecules was also observed in PBSC collections from normal donors primed for mobilization with G-CSF suggests a general role for these molecules in normal hematopoiesis in the BM microenvironment and in the release of hematopoietic cells to the circulation.

	ACKNOWLEDGMENTS

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This study was partially funded by a grant from Immunex Corporation. The authors would like to thank Mr. C. Thomas for running the flow cytometer.

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