Original Manuscript

Leukemia (2005) 19, 2264–2272. doi:10.1038/sj.leu.2403975; published online 6 October 2005

Chronic Lymphocytic Leukemia, Normal T and B cells (CLL)

CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation

B T Pittner1, T D Shanafelt2, N E Kay2 and D F Jelinek1

  1. 1Department of Immunology and Division of Hematology, Mayo Clinic College of Medicine, Mayo Graduate School, Rochester, MN, USA
  2. 2Department of Internal Medicine, Mayo Clinic College of Medicine, Mayo Graduate School, Rochester, MN, USA

Correspondence: Professor DF Jelinek, Department of Immunology, Guggenheim 4, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905, USA. Fax: +1 507 266 0981; E-mail: jelinek.diane@mayo.edu

Received 5 January 2005; Accepted 26 August 2005; Published online 6 October 2005.

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Abstract

CD38, a surface protein whose expression increases upon normal B-cell activation, is a marker of disease aggression in B-cell chronic lymphocytic leukemia (B-CLL). Higher percentages of CD38-expressing CLL B cells may be found in lymphoid compartments compared to peripheral blood. Therefore, it is possible that although CLL B cells are resting, CD38 may be a marker of recent cell activation prior to entry into the periphery. To address this hypothesis, we examined the association of CD38 expression with other activation antigens identified in gene expression profiling experiments and include CD18, CD49d, CD20, and subunit 5 of the anaphase-promoting complex/cyclosome. We found that all these markers were more highly expressed in leukemic B cells from CD38-positive CLL patients. Lastly, because interferon is known to modulate CD38 expression, we used IFN-alpha to test the ability of CLL B cells to increase CD38 expression in vitro. Interestingly, IFN stimulation only modulated CD38 expression in CLL B cells that already expressed CD38. Taken together, these data suggest that CD38 is a marker of a more recently activated CLL B cell. This in turn may explain the biological and clinical differences between CD38-positive type B-CLL and CD38-negative type B-CLL.

Keywords:

B-CLL, CD38, interferon, cell activation, cell cycle

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Introduction

In B-cell chronic lymphocytic leukemia (B-CLL), CD38 membrane expression levels serve as a prognostic tool. Several reports suggest that patients with 7–30% CD38-positive leukemic cells exhibit a more aggressive disease course and have an inferior response to treatment.1, 2, 3, 4, 5, 6, 7 However, one study suggests that even lower percentages of CD38-expressing leukemic B cells correlate with poor outcome8 and other studies suggest that CD38 expression levels will change during the disease course.9, 10, 11 Recently, one study showed that CD38-expressing CLL B cells respond to antibody-induced ligation of CD38 with increased cellular signaling.12 To date, it remains unclear whether CD38 signaling plays a direct physiologic role in B-CLL or serves merely as a surrogate marker of disease aggressiveness.

CD38 is a type II transmembrane protein that has several functions in diverse cell types. First, it acts as an ecto-enzyme catalyzing the conversion of NAD+ to cADPR (reviewed in Deaglio et al13) which in turn induces Ca2+ flux into the cell. Secondly, upon antibody ligation, CD38 induces phosphorylation of CD19 and prevents apoptosis of germinal center B cells.14 Lastly, CD38 also exhibits adhesion molecule properties.15

As we believe that the rate of disease progression might be correlated to the proportion of leukemic B cells bearing a more activated phenotype, we have also looked at other activation markers on CLL B cells. A variety of cell surface molecules have been shown to play a role in normal B-cell activation and to increase in expression upon cell activation, for example, CD38, CD20, and integrins.16, 17, 18 Therefore, given that CD38 currently serves as a prognostic marker of B-CLL disease aggressiveness, we sought to determine if these or additional activation markers correlate to CD38 expression. Exploratory experiments comparing the CLL B-cell gene expression profiles of a small cohort of uniformly CD38-positive vs uniformly CD38-negative CLL patients identified several activation marker candidates. These candidates included CD20, CD18, CD49d, and subunit 5 of the cell cycle-associated cyclosome protein, anaphase-promoting complex/cyclosome (APC/C). Although B-CLL is characterized by a massive accumulation of resting leukemic B cells in the peripheral blood, examination of these activation proteins, including CD38, could shed light on the developmental stage and some of the biological capabilities of these leukemic cells, including their response to environmental factors. Indeed, a recent report suggests that signaling through CD38 leads to extensive surface receptor signaling networks that may function to promote a pool of proliferating CLL B cells.19

Since IFN-alpha modulates CD38 expression levels on normal B cells20 and monocytes21 and may increase CD38 expression levels on leukemic cells,22 in this study we chose to examine its ability to modulate CD38 on various CLL B-cell populations that have different levels of membrane CD38 at the onset of culture. Herein, we report that leukemic clones from CD38-positive B-CLL patients display a phenotype congruent with a more recently activated B cell and it is exclusively these CD38-expressing leukemic cells that have a heightened ability to modulate CD38 expression in response to IFN-alpha.

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Materials and methods

Patient and control specimens

Heparinized peripheral blood was obtained from B-CLL patients under the approval of the Mayo Clinic Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density-gradient centrifugation, and PBMC isolated from all B-CLL patients studied were composed of >90% CD5+ CD19+ B cells. All patients were assessed by flow-cytometry using PE-conjugated anti-CD38, APC-conjugated anti-CD19, FITC-conjugated CD5, and PE-, APC-, and FITC-conjugated isotype controls (BD-Pharmingen, San Diego, CA, USA). Normal PBMC were isolated from discarded buffy coats from healthy blood donors. Freshly isolated samples were used in all experiments.

Gene expression profiling of CD38-negative and -positive patients

We performed gene expression profiling (GEP; Affymetrix U133A platform) on primary leukemic B cells isolated from individual CD38pos and CD38neg patients and optimized identification of differentially expressed genes associated with CD38 expression by restricting the gene profiling to patient cells that were unimodally negative and positive for CD38 expression. In both groups of patients, CD19-positive cells were purified using magnetic bead cell sorting on a Miltenyi AutoMacs prior to isolation of total RNA. To determine genes that appear to be differentially expressed in this set of experiments, we used a recently developed model-based algorithm (PAM analysis) that uses the normalized probe-level data as well as information about the underlying experimental design.

Flow-cytometric analysis of surface marker expression

Freshly isolated PBMC from B-CLL patients were stained with APC-conjugated CD19, FITC-conjugated CD5, and either a PE-conjugated isotype control or PE-conjugated CD38, CD20, CD18, or CD49d (BD Pharmingen). CD38 positivity was defined as any patient sample where the quotient of the mean fluorescence intensity (MFI) of the CD38 staining divided by the MFI of the isotype control was greater than 1. For patients displaying CD38 bimodal leukemic populations, the total MFI of CD38 staining was used (ie, the fluorescence intensity was averaged for both peaks). For CD49d staining, 47 CD38neg and 43 CD38pos patient samples were examined. For CD18 staining, 33 CD38neg and 22 CD38pos patient samples were examined. For CD20, 29 CD38neg and 22 CD38pos patient samples were examined. The total MFI of each surface marker or isotype control was determined by analyzing viable CD19-positive/CD5-positive cells. The MFI ratio was calculated as follows: MFI ratio equals the quotient of the total MFI of antisurface marker staining divided by the MFI of the isotype-matched control.

CD38 induction and expression level analysis

Magnetic bead-purified CD19-positive cells or total PBMC were cultured for 48 h in 10% FCS/RPMI with or without 1000 U/ml interferon-alpha 2b (IFN-alpha) or IFN-italic gamma (Schering Corp., Kenilworth, NJ, USA. In all experiments, the results with IFN-italic gamma were congruent with the results with IFN-alpha; therefore, only the IFN-alpha results are included in this report. Fold induction of CD38 expression on viable CD19/CD5-positive cells is defined by a quotient expressed as follows: fold induction=MFIIFN-alpha ratio/MFInil ratio, where the numerator is the MFI of anti-CD38 staining divided by the MFI of the isotype-matched control when cultured with IFN-alpha (MFIIFN-alpha ratio) and the denominator is the MFI of anti-CD38 staining divided by the MFI of the isotype-matched control when cultured in media alone (MFInil ratio).

Flow-cytometric sorting of CD38-negative and CD38-positive cells from bimodal populations

To examine the effects of IFN-alpha on CD38neg vs CD38pos cells isolated from the same patient, CD19-positive magnetic bead-purified B-CLL cells were sorted into CD38neg and CD38pos populations using a gating strategy shown in Figure 5. Post-sort analysis of the CD38neg and CD38pos leukemic cells revealed >99% purity (data not shown).

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CD38-positive vs CD38-negative cells sorted from a bimodal patient respond differently to IFN-alpha. The CD38 staining profile (a) of one of two representative patients shows two discernible populations of leukemic B cells in freshly isolated CD19-purified CLL B cells as well as the gate settings for the CD38-positive vs -negative sort. CD38neg-gated cells (b and d) or CD38pos-gated cells (c and e) were then cultured in the absence (b and c) or presence (d and e) of IFN-alpha for 48 h and then analyzed by flow-cytometry for CD38 expression (gray-shaded histogram). An isotype control staining profile (open-line histogram) is overlaid for each 48-h culture condition.

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Western blotting

Magnetic bead-purified CD19-positive CLL B cells were freshly isolated from B-CLL patients that were either uniformly negative for CD38 expression (n=5) or uniformly positive (n=5). Viable tonsillar mononuclear cells were obtained from discarded tonsillar tissue, and the B-CLL-derived MEC-1 cell line23 was obtained from the DSMZ, Germany. Cells were directly lysed into 2 times SDS sample buffer containing 2-mercaptoethanol, and lysates were boiled for 5 min and then stored at -20°C until further use. After polyacrylamide gel electrophoresis and transfer, the membranes were blotted with anti-APC/C subunit 5 (BioLegend, San Diego, CA, USA), then stripped, and subsequently blotted with anti-beta-actin (Novus Biologicals, Littleton, CO, USA) for a loading control.

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Results

Patient characteristics and CD38 expression patterns in B-CLL

The present study included 47 CD38neg and 43 CD38pos B-CLL patients who were untreated or had received the last dose of chemotherapy more than 1 year previously (Table 1). There were no statistically significant differences in sex distribution, age, Rai stage, time from diagnosis, or treatment status in the two groups defined by CD38 expression. With respect to CD38pos B-CLL patients, the expression patterns of CD38 are known to vary tremendously between B-CLL patients.8, 12 Indeed, the data shown in Figure 1 are illustrative of CD38 expression variability on gated CD19+CD5+ lymphocytes within and between B-CLL patients, for example, uniformly high (Figure 1a), intermediate (Figure 1b), or low (Figure 1c) CD38 expression; uniformly negative CD38 expression (Figure 1d); and bimodal CD38 expression where patient leukemic B cells are both CD38neg and CD38pos (Figure 1e and f).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The patterns of CD38 expression in CLL B cells. (a–f) CD38 staining profiles (gray-shaded histogram) and isotype controls (open-line histogram) represent CLL B-cell populations that are CD38 high, CD38 moderate, CD38 low, CD38-negative, bimodal CD38 low, and bimodal CD38 high, respectively. The percentages of CD38 staining with fluorescence greater than the isotype control are indicated for each example.

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CD38 expression correlates with expression of other activation markers

Recent reports indicate that even low levels of CD38 correspond to disease aggressiveness in B-CLL.8 Interestingly, the patient examples in Figure 1c and e also fit this definition, but they clearly display different profiles; one is CD38 low with a unimodal histogram peak and the other displays bimodal CD38 low expression, with only a small fraction of CLL-B cells expressing CD38 (Figures 1c and e, respectively). To learn more about the possible role of CD38 in this disease, we performed exploratory gene expression profiling on primary leukemic B cells that were unimodally negative for CD38 and those that expressed unimodally high levels of CD38. We initially studied two CD38pos and two CD38neg patients and several genes that are also known as activation markers emerged from this approach. These markers include the integrins, CD49d and CD18, as well as CD20 and the cell cycle-related protein, anaphase promoting complex/cyclosome subunit 5 (APC/C 5). An additional nine patients (six CD38pos and three CD38neg) were profiled and the combined data are graphically shown in Figure 2. In the first set of patients, the difference in gene expression of CD20 was statistically significant; however, upon study of additional patients by this methodology, CD20 no longer displayed statistical significance (Figure 2c). Owing to the important role of CD20 in B-cell biology, we continued to explore this molecule, as well as the integrins and APC/C 5, at the protein level to validate their gene expression profiles.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Gene expression values from CD38neg and CD38pos CLL B cells. The gene expression values, determined with a model-based algorithm that uses normalized probe-set data, are depicted from representative probe-sets for CD49d, CD18, CD20, and APC/C 5 (a, b, c, and d, respectively).

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It has been well established that integrins are major contributors to lymphocyte activation and survival and expression levels increase following cell activation.24 We next determined if CD49d and CD18 were more highly expressed on the leukemic cells of CD38pos B-CLL cases, and indeed this was observed (Figure 3a and b). Of interest, the CD38pos subset of patients included those with expression of CD49d or CD18 at or above the median value of the population, and those with low to negative expression. Moreover, all the patients expressing low levels of CD49d and CD18 also displayed bimodal CD38 expression. The relative expression levels of CD49d or CD18 in this latter subgroup did not correlate to the percentage of the CD38pos leukemic cell fraction (data not shown). Furthermore, there is no evidence of CD49d or CD18 bimodal patterns regardless of CD38 expression patterns (data not shown). Thus, even though two populations may exist within a patient as defined by CD38 expression, both populations display uniform CD49d or CD18 expression.

Figure 3.
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CD38 expression in CLL correlates to other surface activation markers. CLL patients' B cells were triple-stained for CD49d, CD18, or CD20 (a, b, and c, respectively). The mean fluorescence intensity (MFI) ratio is a quotient defined as the total MFI of the surface marker stain divided by the total MFI of the isotype control. The left-most, center, and right-most columns of each panel (open circles, open triangles, and filled triangles, respectively) are from CD38neg B-CLL patients, CD38pos B-CLL patients, or normal PBMC, respectively. The horizontal line in each column represents the median value of the points within that column.

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Normal B-cell expression of CD20 increases early in response to complete activation.16 However, one group showed that CD20 expression may decrease in response to only partial cell activation.25 Additionally, CD20 is lost during terminal differentiation into plasma cells.26 We next determined if CD38 expression correlated to CD20 expression in order to gain insight into the developmental/activation stage of CLL B cells. Figure 3c shows that there is a statistically significant higher expression of CD20 in CD38pos B-CLL. Again, CD38pos patients with CD20 expression levels below the median express bimodal CD38.

Interferon is only able to modulate CD38 expression on CLL B cells from CD38-positive patients

Since interferons are known to stimulate CD38 expression, we next examined the ability of IFN-alpha and IFN-italic gamma to alter CD38 expression in both CD38pos and CD38neg CLL B-cell populations. B-CLL cells from Patient 1 significantly increased CD38 expression in response to IFN-alpha (Figure 4a) as measured by percent CD38pos cells and MFI (4.4-fold induction). However, CLL B cells from patients that were uniformly CD38neg at isolation did not acquire CD38 expression in response to IFN-alpha (Figure 4a, Patient 2). Moreover, in a total of 21 CD38neg and 19 CD38pos CLL patients examined pre- and post-culture with IFN-alpha, the inability of IFN-alpha to upregulate CD38 expression in CD38neg CLL patients remained constant (Figure 4b). B cells from healthy donors (n=4) with low to moderate levels of CD38 responded to IFN-alpha with a significant increase (P<0.001) in CD38 expression compared to CD38neg CLL B cells (Figure 4b). Treatment of B-CLL cells with IFN-italic gamma had similar effects for CD38 induction (data not shown). Additionally, CLL culture with either IFN had no effect on the expression of CD20, CD49d, or CD18 regardless of CD38 status (data not shown). For the CD38neg CLL B cells, it is unlikely that these effects are due to complete IFN unresponsiveness or lack of receptors because IFN-alpha maintained CD38neg cell viability and increased expression of HLA class I molecules (data not shown).

Figure 4.
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IFN-alpha increases CD38 expression only on CD38pos CLL B cells. (a) Freshly isolated CLL B cells were cultured for 2 days in 10% FCS/RPMI in the presence (+IFN-alpha) or absence (nil) of 1000 U/ml IFN-alpha and viable leukemic B cells were analyzed for expression of CD38 by flow-cytometry. Three representative experiments are shown by the upper panels (Patient 1), middle panels (Patient 2), or lower panels (Patient 3) displaying the CD38 staining profile (gray-shaded histogram) in each of the following conditions: freshly isolated CLL B cells (left panels), 48-h media alone (center panels), or 48-h with IFN (right panels). An isotype control staining profile (open-line histogram) is overlaid for each condition. The percent of CD38-stained cells that is captured by the marker is shown in each panel. (b) Cumulative data representing patients whose leukemic cells were either CD38neg (open circles) or CD38pos (open triangles) at onset as well as peripheral blood B cells from normal individuals (black triangles) and cultured for 2 days as in (a) above. Fold induction corresponds to the change in MFI ratios and is calculated as stated in the Materials and methods.

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Interestingly, even minimal CD38 expression on CLL B cells was associated with a CD38 induction in response to IFN-alpha. Thus, the CLL B cells from Patient 3 (Figure 4) had very low-level CD38 expression at onset of culture and IFN-alpha stimulation increased the CD38pos fraction to 44%. We have observed similar levels of CD38 induction in six other B-CLL patients with low numbers of CD38pos B cells, ranging from 5.8 to 16%. Despite the increase in CD38 expression, not all CLL B cells become CD38pos, suggesting that a subpopulation of leukemic cells are resistant to CD38 upregulation.

For Patient 3 it was also conceivable that the cells that increased CD38 expression in response to IFN-alpha could have evolved from CD38neg cells prior to stimulation. To address this, we sorted CD38pos and CD38neg CLL-B cells from a CLL patient, whose leukemic cells displayed a bimodal pattern of CD38, and then immediately cultured these separate populations with or without IFN-alpha (Figure 5). Of note, the B-cell subpopulation that expressed CD38 at the onset of culture was the primary population that acquired increased CD38 expression in response to IFN-alpha (3.7-fold induction; Figure 5c and e). By contrast, the leukemic cells that appeared to lack CD38 expression at isolation were largely nonresponsive to IFN-alpha with greater than 90% of the cells remaining CD38neg. Of interest, although a small increase was noted in the CD38neg sorted population, this response could be due to incidental cosorting of cells that expressed extremely low levels of CD38 at the onset of culture. Alternatively, it could reflect the response of those cells that recently lost CD38 expression and are poised to reacquire it in response to IFN-alpha.

B-CLL CD38 expression correlates to the expression of APC/C 5

Since CD38 expression is higher in CLL cells that are more responsive to external stimuli and because they express other activation markers, it is possible that these cells have more recently exited from or are better primed to re-enter the cell cycle. In support of this concept, the exploratory gene expression analysis identified the expression level of APC/C 5 as higher in CD38pos B-CLL (Figure 2d). APC/C is a multisubunit complex that is expressed throughout the cell cycle and is responsible for degrading cell cycle-related proteins in a timed-critical manner.27 To confirm the gene expression data, we assessed protein expression levels of APC/C 5 by immunoblotting (Figure 6). These data show that CD19-purified, CD38neg CLL B cells express very little to no APC/C 5 (three representative patients shown, lanes 3–5). Conversely, some CD38pos CLL B cells express detectable levels of this subunit (two representative patients shown, lanes 6 and 7). Of the CLL B-cell patient samples tested to date, we found that none of the five CD38neg CLL B-cell samples and three of five CD38pos CLL B-cell samples express detectable levels of APC/C 5. As expected, the MEC-1 CLL cell line displayed high levels (Figure 6, lane 2), while tonsillar mononuclear cells displayed moderate levels of APC/C 5 expression (Figure 6, lane 1).

Figure 6.
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APC/C 5 expression is greater in CD38pos CLL B cells. Whole cell lysates from tonsillar mononuclear cells (T), the CLL-derived MEC-1 cell line (M), three CD38neg CLL B-cells (-), and two CD38pos CLL B-cells (+) were analyzed by immunoblotting for APC/C 5 expression. The blots were then reprobed for beta-actin as a loading control (lower panel). This experiment is representative of a total of five of five CD38neg B-CLL samples and three of five CD38pos B-CLL samples tested.

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Discussion

CD38 is expressed at higher levels in activated germinal center (GC) founder B cells, GC B cells, and early memory B cells within the secondary lymphoid compartment.28 The same study showed that GC founders and early memory B cells exist in peripheral blood; a finding that could reflect the status of the leukemic B cells in B-CLL and other hematological diseases.28, 29 Peripheral blood CD38-positive B cells in B-CLL could arise from a site of B-cell activation with a continued production and release of these GC-like cells into the periphery.

To further understand the underlying biology and developmental stage of CLL B cells as they correlate to CD38 expression, we initiated an exploratory analysis of genes expressed by uniformly CD38pos or uniformly CD38neg CLL B cells from a growing cohort of patients and our results identified a variety of molecules that may be expressed at higher levels in CD38pos B-CLL. This study focused on a small number of these candidate genes and we validate that they are positively associated with CD38 expression levels. These genes include the integrins CD49d and CD18, as well as CD20 and APC/C 5.

CD49d, CD18, CD20, and APC/C 5 expression correlate to CD38 expression

CD49d expression is decreased on resident B cells of the Peyer's patch and other lymphoid compartments, but is upregulated upon activation.18 The fact that CD49d, CD18, and perhaps other adhesion markers are low on CD38neg CLL B cells (Figure 3) suggests that these cells could be more likely found as residents of the peripheral blood lymphoid compartment. As integrins and CD38 have been shown to play a role in migration, the lack of their expression on this subset of B-CLL cells may prevent these cells from recycling back into lymphoid tissues and permanently commits them to a long-lived terminal residency in the peripheral blood. Thus, it can be speculated that CD38pos CLL B cells may have a higher turnover rate. Interestingly, although we found bimodal expression of CD38, we did not find concomitant bimodal expression of other surface activation markers. Additionally, with the integrins, CD18 and CD49d, the bimodal CD38pos CLL B cells displayed lower than median-level expression of these integrins (Figure 3). These findings suggest that the regulation of these other activation markers is different from CD38 regulation in CLL B cells. Moreover, the relative abundance of the CD38pos population had no positive association with the MFI ratio of the integrin staining (data not shown). Of interest, Durig et al30 performed gene expression profiling (approximately 5600 genes) on 25 CD38pos (defined as >20% CD38pos cells) and 45 CD38neg (<20% CD38pos cells) B-CLL patients and concluded that although the vast majority of genes on these chips were expressed at similar levels in the two subsets of patients, 14 genes appeared to be differentially expressed, including CD49d. Our results with CD49d are therefore consistent with this report and we provide validation at the protein level that this integrin is more highly expressed in CD38pos CLL B cells. The results presented in this report underscore the difficulties of determining CD38 expression levels based on a fixed cutoff and highlight the need to carry out gene expression profiling on a larger cohort of uniformly CD38pos vs CD38neg type B-CLL (in progress) to reveal additional genetic differences that may exist between these two subsets of disease that may underlie differential disease progression.

Similarly, when analyzing the association of CD20 expression and CD38 expression we found that there is a significantly higher expression of CD20 in CD38-positive B-CLL. For those CLL B cells with CD20 expression levels below the median of the CD38pos cohort, a bimodal expression of CD38 was detected. In normal B-cell development, CD20 expression increases early in response to activation16 and decreases in response to only partial activation.25 These data suggest that the leukemic cells from CD38pos B-CLL patients may have arrested and undergone tumorigenic transformation at an earlier developmental stage than those from CD38neg patients.

Lastly, the expression of cell cycle-related protein, APC/C subunit 5, correlated with CD38 expression. This protein is part of a multisubunit complex that functions at critical times throughout the cell cycle.27 For instance, when the APC/C is bound to the activator, Cdc20, it promotes the metaphase-to-anaphase transition. Similarly, when the complex is bound to Cdh1, it promotes the G1-to-S phase transition. In eucaryotes, APC/C 5 acts as a regulatory component of the complex, supported by the finding that APC/C 5 mutations cause a pre-metaphase stall in Drosophila development.31 Here, we found that CD38neg CLL B cells expressed very low to undetectable levels of APC/C 5 through immunoblotting, whereas three of five CD38pos CLL B-cell samples displayed detectable levels. Given that APC/C 5 is expressed throughout the cell cycle, these data suggest that CD38pos cells have more recently exited from the cell cycle and/or are better primed to re-enter the cell cycle. Further exploration into the regulated expression of these and other cell cycle-related proteins in B-CLL would be instructive regarding CLL disease progression.

The IFN responsive state of CLL B cells correlates to CD38 expression

CD38pos B-CLL cells are thought to express a more activated phenotype relative to CD38neg cells.32 In addition, CD38pos CLL B cells appear to be more responsive to stimulation through BCR crosslinking than CD38neg B-CLL.33, 34 One possibility for this differential response is that CD38pos cells may also express a higher level of other activation and/or signaling molecules and are poised to respond to environmental stimuli, as recently shown.19 Of interest, our results show that only CD38pos cells, even those with very low-level CD38 expression, respond to IFN-alpha with increased expression of CD38 (Figures 4 and 5). Taken together, these studies lead one to speculate that CD38pos cells are better prepared to respond to cues from their environment, whereas CD38neg cells appear to be in a more dormant, unresponsive state. Further, the role of CD38 as a cofactor in cell activation, due to its ability to act as an adhesion molecule as well as enhance calcium mobilization,13, 35 supports the notion that the CD38pos cells are more sensitive to environmental stimuli, either negatively33 or positively.36 While a small increase was noted in the CD38neg sorted population, from 4% to 10% positivity (Figure 5b and d, respectively), as noted above, this response could be due to incidental co-sorting of cells that expressed extremely low levels of CD38 at the onset of culture. Alternatively, the data in Figure 4 (Patient 3) and Figure 5 suggest that CD38neg CLL B cells from CD38-bimodal patients could reflect cells that have very recently lost CD38 expression but are still poised to reacquire CD38 upon exposure to IFN. Moreover, this may suggest that not all CD38neg cells from bimodal profiles (Figure 1e and f) may be as unresponsive to stimuli as the leukemic cells from homogeneously CD38neg CLL patients (Figure 1d). Similar findings were also observed in some CD38neg patients whose leukemic cells were cultured with IL-2 by Deaglio et al.12 Again, these data underscore the complexity of this disease and suggest that bimodal CD38 leukemic cells may display different biological properties.

Lastly, the failure of IFN stimulation to increase CD38 expression in CD38neg CLL cells from patients who are unimodally CD38neg suggests that the CD38 interferon response locus35 may be inaccessible perhaps due to methylation,37, 38 or that a component of the signaling pathway from the IFN-alpha receptor to the transcription of CD38 gene is missing or dysfunctional. Of interest, recent reports state that CD38 expression is higher in bone marrow and lymphoid tissues, the presumed sites of CLL proliferation and cell activation, compared to peripheral blood.8, 39 These findings suggest that upon CLL B-cell emigration from a lymphoid compartment into blood, the expression of CD38 begins to decrease. In summary, we believe that it is possible that all B-CLL tumorigenic founders are CD38pos and the CD38 B-cell percentage in peripheral blood is directly proportional to the turnover rate with low turnover favoring the presence of CD38neg CLL-B cells, since CD38 expression is associated with a higher activation state. The ability of CLL B cells to proliferate in vivo was recently examined in a study using deuterated water.40 In this report, Messmer and co-workers provide provocative evidence that CLL B-cell turnover rates can differ among patients. This study did not find a correlation to CD38 expression, but additional studies using larger sample sizes are underway. Finally, it must be noted that current published studies and the data presented herein do not rule out that CD38pos B-CLL cases could arise from transformation of CD38neg precursors. These hypotheses require further investigation.

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Acknowledgements

We thank Bonnie Arendt, Nancy Bone, Cheryl Jankiewicz, and Renee Tschumper for their technical assistance, Dr Karla Ballman and Bruce Morlan for their assistance in analyzing the exploratory gene expression study data, and Dr Clive Zent for his insightful comments. This study was supported by the National Institutes of Health Training Grant T32-HL67742 (BTP), R01 CA91942 (NEK), and generous philanthropic support provided by Mr Edson Spencer.