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Stem Cells, Vol. 19, No. 5, 443-452, September 2001
© 2001 AlphaMed Press

Engraftment of Human T-Cell Acute Lymphoblastic Leukemia in Immunodeficient NOD/SCID Mice Which Have Been Preconditioned by Injection of Human Cord Blood

Deno P. Dialynasa, Li-en Shaoa, Glenn F. Billmanb, John Yua,c

a Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California;
b Department of Pathology, Children's Hospital, San Diego, California;
c Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

Key Words. Leukemia engraftment • Immunodeficient mouse • Chemokine receptor CXCR4 • IL-2R {gamma} chain

John Yu, M.D., Ph.D., Department of Molecular and Experimental Medicine, MEM 265, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 858-784-7924; Fax: 858-784-7977; e-mail: JohnYu{at}scripps.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Childhood T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers. Study of leukemia biology, as well as preclinical testing of potential therapeutic regimens directed at T-ALL, has been impeded by the lack of an efficient in vivo model of primary leukemia. We have reported elsewhere some observations that human cord blood conditioned medium enhances leukemia colony formation in vitro and preconditioning of sublethally irradiated nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice with cord blood mononuclear cells (MNCs) facilitates the subsequent engraftment of primary T-ALL cells in these mice. Here we characterize in greater detail this in vivo xenograft model of human leukemia in NOD/SCID mice. Consistent with the thesis that cord blood facilitates engraftment, the engraftment of human leukemia can be shown to increase with increasing number of cord blood MNCs injected. In addition, we documented the expression of chemokine receptor CXCR4 by primary T-ALL from patients and found that the presence of these receptors did not result in the transmigration of T-ALL cells induced by stromal cell-derived factor-1{alpha}. Finally, we show that in this xenograft system T-ALL cells recovered from engrafted bone marrow are characterized by upregulated expression of interleukin 2 receptor {gamma} chain, suggesting that cord blood preconditioning may function in part to increase T-ALL responsiveness to growth factor(s).


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
T-cell acute lymphoblastic leukemia (T-ALL) comprises approximately 20% of ALL [1], with ALL being the most common type of cancer in children. A better understanding of the biology of T-ALL and regulatory signals from the microenvironment would facilitate the development of selective therapy that exploits specific biological properties of the leukemia, thereby improving the outlook for this disease. The ability to engraft T-ALL cells directly from patient samples into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice would be uniquely valuable for expansion of primary leukemia cells for subsequent ex vivo or in vivo studies of these cells, as well as for developing individualized therapeutic strategies. Even though a number of leukemia cell lines of T-cell origin have been established from patients [2-5], difficulty in maintaining primary cultures of leukemia cells from patients has impeded study of the biology of the disease.

Leukemic progenitor cells have been implicated in the maintenance and expansion of leukemic blast populations [6, 7]. These clonogenic blast cells comprise only 0.05% to 1.5% of the bulk marrow or peripheral blood blasts from ALL patients [6, 8], identified on the basis of their ability to proliferate and form colonies in semisolid media in response to specific growth factors [8, 9]. It is generally assumed that the colony-forming blasts represent the in vitro counterparts of the in vivo ALL blast progenitors [6]. Despite these in vitro studies, leukemia-initiating cells were not demonstrated in vivo until recently [10-13]. The availability of a robust in vivo mouse model for T-ALL would expedite characterization of the corresponding leukemia-initiating cell, as well as delineation of cellular hierarchy within the leukemia.

We have reported elsewhere some observations that human cord blood conditioned medium enhances leukemia colony formation in vitro [14] and preconditioning of sublethally irradiated NOD/SCID mice with human cord blood mononuclear cells (MNCs) facilitates the subsequent engraftment in these mice of primary T-ALL cells [15]. Here we characterize in greater detail this novel in vivo model of human leukemia engraftment. We show that the level of engraftment depends on both the number of cord blood MNCs and T-ALL cells injected. In addition, we demonstrate that despite the high level of chemokine receptor CXCR4 expression by primary T-ALL cells obtained from patients, chemokine stromal cell-derived factor-1{alpha} (SDF-1{alpha}) does not induce the transmigration of these T-ALL primary cells in vitro. Finally, we show that in this system human T-ALL cells recovered from engrafted mouse bone marrow are characterized by upregulated expression of interleukin 2 receptor {gamma} chain (IL-2R{gamma}), suggesting that cord blood preconditioning may function in part to increase T-ALL responsiveness to growth factor(s).


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
NOD/SCID Mice
The NOD/SCID mice [16] were bred and maintained in a specific pathogen-free environment at The Scripps Research Institute vivarium in sterile Micro-Isolator cages and ventilated mouse racks (Lab Products; Seaford, DE) without antibiotics. Five- to six-week-old mice of either sex (but matched within a given experiment) were used in the present study.

Primary Leukemia Cells
Heparinized peripheral blood or bone marrow samples were obtained from patients with childhood T-ALL who enrolled in protocol #9400 Pediatric Oncology Group. Primary leukemia samples from a total of nine patients with T-ALL were used in the present studies. The MNC fraction from peripheral blood/bone marrow was isolated by Ficoll-Paque density gradient separation (Pharmacia; Piscataway, NJ; http://www.pnu.com). The content of lymphoblasts, as determined by Wright stain, was generally >90%. In some cases, MNCs of leukemia samples were cryopreserved and stored in liquid nitrogen before use in the studies. Viability on thawing was generally greater than 80% as determined by Trypan blue dye exclusion.

Human Cord Blood
Fetal cord blood samples were obtained from umbilical cord blood scheduled for discard, according to procedures approved by our Institutional Review Board. After Ficoll-Paque density gradient centrifugation, the MNCs were collected and washed with RPMI 1640 medium containing 2% fetal calf serum (FCS). Cord blood was used for injection as described below.

Mouse Injections
The protocol for human cord blood preconditioning of NOD/SCID mice consists of irradiation and injection of human cord blood as outlined in Figure 1Go. Briefly, prior to leukemia implantation, the mice received 350 rads total body irradiation from a 137Cs {gamma}-irradiator. Immediately thereafter, 10-25 x 106 fetal cord blood MNCs from a normal healthy newborn were injected in 0.25 ml sterile phosphate-buffered saline (PBS) via tail vein. For a given experiment, cord blood from a single donor was used for all mice. Six to nine days later, 1-5 x 106 viable primary leukemia cells from a patient were suspended in 0.25 ml PBS and injected via tail vein. Primary leukemia cells from a total of nine patients with T-ALL were used. For a given experiment, same lots of cord blood MNCs were used for preconditioning, and leukemia cells from a single donor were used for all mice. Experimental mice were typically set up as two to four replicates in a group of 12 mice in a given experiment. In addition, for a given primary patient specimen, experiments were repeated usually two to four times. Mice were sacrificed when they became moribund with disseminated leukemia or electively between 5 and 7 weeks after the leukemia cell injection. Necropsies were performed, and the burden of leukemia cells in mouse tissues was determined by flow cytometry and histocytochemistry as described below.



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  		Figure 1. Protocol for cord blood preconditioning of NOD/SCID mice and analysis of the engraftment of primary human leukemia.

 
Flow Cytometry
Multi-parameter analysis of single-cell suspensions from mouse bone marrow and spleen was carried out using a FACScan flow cytometer (Becton Dickinson; Franklin Lakes, NJ; http://www.bd.com). Two-color immunofluorescence was used to identify human leukemia cells. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mouse anti-human monoclonal antibodies (mAbs) were obtained from PharMingen (San Diego, CA; http://www.pharmingen.com), with the exception of PE-conjugated anti-TCR Vß2 (clone MPB2D5, Coulter; Miami, FL; http://www.coulter.com). The mAbs used in the work presented here include those directed against human CD5 (clone UCHT2), CD7 (M-T701), CD19 (HIB19), and CD45 (HI30). In addition, conjugated mAbs for IL-2R{alpha} (mAb M-A251), IL-2Rß (mAb 3D7), IL-2R{gamma} (mAb AG184) and CXCR4 (12G5) were used in flow cytometric analysis for some other experiments. During analysis, red blood cells and debris were gated out on the basis of forward angle and 90° side scatter. At least 15,000 events were collected for each sample. Isotype-matched control mAbs (FITC- or PE-conjugated IgG1 [clone MOPC021]) were used to determine the appropriate cursor settings for analysis. Using CellQuest 3.2.1 software (Becton Dickinson), data were analyzed and displayed by two-dimensional plots and by one-dimensional histograms.

Transmigration of T-ALL Leukemia Cells Toward an SDF-1 Gradient
Human CD34+ cells were purified from mobilized peripheral blood with CD34 mAb to a purity of 82%, as determined from fluorescence-activated cell sorter analysis (generous gift from Dr. P. Low at University of California at San Diego [UCSD]). Transmigration of primary T-ALL, HL60, or purified CD34+ cells was carried out as described elsewhere [17, 18]. Briefly, 5 x 104 cells were added to the 30 mm Millicell-PCF culture plate inserts (3 µm pore; Millipore; Bedford, MA; http://www.millipore.com), which were subsequently placed into 6-well plates (Millipore) containing 0 or 100 ng/ml of SDF-1{alpha} (R&D Systems; Minneapolis, MN; http://www.rndsystems.com) in 10% FCS, RPMI medium. The cells in culture plate inserts were allowed to migrate across the 3-µm pore membrane at 37°C and 5% CO2. After 0, 2, 4, 12, and 24 hours, the culture plate inserts were carefully removed. Cell numbers and concentrations were assessed using a hemocytometer, and the viability of the cells was assessed by trypan blue dye exclusion, according to published procedures [17]. The percent migration of T-ALL or other types of cells was calculated relative to the numbers of these populations on control plates. Early studies indicated that transmigration reached a plateau level after 4 hours of incubation [18].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Engraftment of Primary Human T-ALL in Preconditioned NOD/SCID Mice
We have reported elsewhere that human cord blood conditioned medium enhances leukemia colony formation in vitro [14] and preconditioning of sublethally irradiated NOD/SCID mice with cord blood MNCs facilitates the subsequent engraftment in these mice of primary leukemia cells obtained from patients with T-ALL [15]. Out of a total of nine patients' samples, a typical profile of T-ALL engrafted mouse bone marrow and spleen, as assessed by flow cytometry, is presented in Figure 2Go. CD45 expression is indicative of total human hematopoietic cell engraftment. CD19 is indicative of engrafted human cells of the B-cell lineage. As indicated by the corresponding histograms, CD45+CD7+ engrafted T-ALL cells comprise approximately 83% of bone marrow and 68% of spleen cells harvested from engrafted mice (Fig. 2Go). It should be noted that there are very few CD19+ cells (approximately 2% in bone marrow and 4% in spleen) in the T-ALL engrafted mouse, suggesting that expansion of the T-ALL overtakes the expansion of normal CD19+ cells developing from engrafted cord blood MNCs [19-23]. Similar findings of the paucity of CD19+ cells in mouse bone marrow/spleen after injection of leukemia cells were observed in the experiments using samples taken from the other patients. Therefore, the results presented here, along with those obtained using mAbs directed against CD2, CD3, CD5, CD20, CD38, or glycophorin A (not shown), indicate that to the extent studied, the phenotype of the cells recovered from engrafted mouse bone marrow and spleen is identical to that of the injected primary T-ALL cells obtained from the nine patients under study.



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  		Figure 2. Leukemia-engrafted bone marrow and spleen from a cord blood preconditioned mouse injected with primary T-ALL cells. Mice were preconditioned with 10 x 106 cord blood MNCs. Seven days later, the mice were injected with 5 x 106 primary T-ALL cells. Six weeks after the T-ALL injection, the mice were sacrificed and analyzed by flow cytometry for T-ALL engraftment in bone marrow (A) and spleen (B). As described in Materials and Methods, flow cytometric analysis was carried out with the determination of the cells expressing fluorescence intensity using specific mAbs against CD45, CD7, and CD19 respectively. In addition, for each panel, the filled histogram curve corresponds to the indicated experimental mAb and is superimposed over an open histogram corresponding to the isotype control mAb. The fraction of cells staining positive for the experimental mAb was determined by subtraction of the curves, using CellQuest 3.2.1 software. The percentage of human CD45+, CD7+, and CD19+ cells in mouse bone marrow (A) and in mouse spleen from (B) is indicated in the panels.

 
Current studies also characterized the level of T-ALL engraftment in mouse bone marrow for these nine primary T-ALL donors (Fig. 3Go). In these studies, the level of human leukemia engraftment was determined at 6 weeks following injection of primary T-ALL cells into mice, which had been preconditioned with cord blood. When more than 1 x 106 primary T-ALL cells were injected, efficient engraftment in mouse bone marrow was typically observed. In Figure 3Go, samples from six patients were shown to achieve more than 77% engraftment in mice, and the samples from the other three patients had engrafted in mice to a level more than 29%. We then addressed the issue of whether the level of engraftment in mouse bone marrow and spleen at 6 weeks is dependent on the number of cord blood MNCs and the number of primary T-ALL cells injected (Fig. 4Go). In Figure 4Go, two different experiments were set up, with the same primary T-ALL donor but different cord blood donors. From Figure 4AGo it is apparent that the level of T-ALL engraftment in mouse bone marrow and spleen at 6 weeks is dependent on the number of cord blood cells used for preconditioning. Analogously, from Figure 4BGo it is apparent that the level of T-ALL engraftment in bone marrow and spleen at 6 weeks is dependent on the number of primary T-ALL cells injected.



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  		Figure 3. Engraftment of primary leukemia obtained from nine patients with T-ALL in mouse bone marrow. Experiments were set up and analyzed similarly as described in Figure 1Go, using nine different primary T-ALL donors. The level of T-ALL engraftment was determined by flow cytometry, on the basis of CD45, CD7, and CD5 expression in percent of bone marrow cells harvested from engrafted mouse bone marrows. Individual symbols represent data obtained from experiments using the same T-ALL donors.

 


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  		Figure 4. The level of engraftment in mouse bone marrow and spleen is dependent on both the number of cord blood MNCs and the number of T-ALL cells. Experimental mice were set up similarly as described in Figure 1Go. In (A), mice were injected with the indicated number of cord blood MNCs 7 days prior to the injection of 2.5 x 106 primary T-ALL cells; the mice were sacrificed for analysis 6 weeks after injection of the T-ALL cells. In (B), mice were injected with 10 x 106 cord blood MNCs 7 days prior to the injection of the indicated number of primary T-ALL cells; the mice were sacrificed for analysis 6 weeks after injection of the T-ALL cells. The percentage of T-ALL in engrafted bone marrow ({square}-{square}-{square}) and spleen (--) was determined flow cytometrically as CD7+CD5+ cells, the phenotype of the primary T-ALL cells in each case.

 
Expression of Chemokine Receptor CXCR4 by Primary T-ALL
Prior to expansion of the T-ALL in engrafted bone marrow in preconditioned mice, the T-ALL initially must home to the bone marrow. The high level of engraftment that we attain in preconditioned mice affords us a window for delineating at the molecular level not only signals driving expansion per se, but also requisite events upstream of expansion such as trafficking of the injected T-ALL to bone marrow. As chemokines play a dominant role in cellular trafficking in vivo [24], it was of interest to characterize expression of CXCR4 by the primary T-ALL cells. CXCR4 is the receptor for SDF-1 [25, 26], a chemokine released by bone marrow stromal cells [26, 27] and shown to play a role in the homing of hematopoietic stem cells to bone marrow [18, 28].

To this end, six different primary T-ALL samples were analyzed for the expression of CXCR4 by flow cytometry. For each of the samples studied to date, the majority of the primary T-ALL cells clearly expressed CXCR4, although there was variability both in the level of CXCR4 expression and in the percentage of cells expressing CXCR4 (e.g., three samples presented in Fig. 5AGo). As the CXCR4 profile in three of the samples shown in Figures 5A-C corresponds to that of gated CD7+ cells (T-ALL) (Fig. 5Go legend), cells staining negative for CXCR4 in the Figure cannot readily be ascribed to non-T-ALL cells within the patient's MNCs but, instead, most likely correspond to a subset of the primary T-ALL cells.



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  		Figure 5. Expression of chemokine receptor CXCR4 and transmembrane migration by primary T-ALL leukemia cells obtained from patients. (A) MNCs isolated by Ficoll-Paque centrifugation from the peripheral blood of three different primary T-ALL donors (#1-3) were analyzed for the expression of the chemokine receptor, CXCR4, as described in Materials and Methods. For each of the T-ALL donors, T-ALL blasts comprised >90% of the MNCs. In each panel, the filled histogram curve corresponds to the CXCR4 profile of gated CD7+ cells and is superimposed over an open histogram curve corresponding to the isotype control mAb. The fraction of CD7+ (T-ALL) cells staining positive for CXCR4 was determined by subtraction of the curves, using CellQuest 3.2.1 software. (B) A total of 5 x 104 CD34+ cells (82% purity), HL60, or primary T-ALL leukemia cells were added to the 30 mm Millicell-PCF culture plate inserts (3 µm pore) of the transmembrane system [17, 18]. The transmigration assay has been described in detail in Materials and Methods. After 4 hours of incubation, transmigrated cells recovered from the lower chamber were enumerated. Spontaneous migration (addition of control RPMI medium containing 10% FCS to the lower chamber) was compared with SDF-1-induced migration (addition of SDF-1{alpha}-containing medium to the lower chamber). CD34+ hematopoietic cells and CD34– HL60 leukemia cells showed significant migration. SDF-1{alpha}-induced migration of primary T-ALL was minimal. SDF-1{alpha} at 100 ng/ml was used in these experiments. A total of three experiments using three T-ALL donors was performed, and error bars refer to the standard error of these independent experiments.

 
In the following experiments, the chemotactic effect of SDF-1 on the transmigration of T-ALL primary leukemia cells was examined. As shown by others [18], the SDF-1-induced transmembrane migration of human primitive hematopoietic cells occurred in 3 hours of incubation. Following these procedures [17, 18], transmigrated cells were recovered from the lower chamber of the transmigration system after 4 to 24 hours of incubation and enumerated. As shown in Figure 5BGo, purified CD34+ cells from mobilized peripheral blood (generous gift from Dr. P. Low at UCSD Medical Center) efficiently transmigrated the membrane in vitro when SDF-1{alpha}-containing medium was added to the lower chamber of the transmigration system. A significant level of CXCR4 (54.7%) was expressed also in CD34– leukemia cell line, HL60; it was shown that these cells responded to the transmembrane SDF-1{alpha} gradient with significantly enhanced migration (Fig. 5BGo).

In contrast, however, no migration was observed when the CXCR4+ T-ALL leukemia cells were added to the upper chamber of the transmigration system and with SDF-1{alpha}-containing medium in the lower chamber (Fig. 5BGo), despite the high level of CXCR4 expression in these T-ALL cells (averaged at 73.0% in these samples).

Upregulated Expression of IL-2R{gamma} by Engrafted T-ALL Cells in Preconditioned Mice
Although the expression of lineage markers by T-ALL in engrafted bone marrow and spleen resembles that of the patients' T-ALL used for injection, we investigated whether an influence of the bone marrow microenvironment in preconditioned mice could be discerned at the level of an activation marker. We reasoned that this approach might also shed light on the signals driving in vivo expansion of T-ALL in engrafted bone marrow.

In Figure 6Go, an analysis of IL-2R chain expression by engrafted T-ALL is presented. For the two independent experiments corresponding to Figures 6B and CGo, the mice were injected with primary T-ALL cells (Fig. 6AGo), but with different batches of cord blood MNCs. Two-color flow cytometric analysis of IL-2R expression by T-ALL was carried out using mAb specific for the {alpha}, ß, or {gamma} chain of IL-2R in conjunction with anti-CD7 mAb to identify T-ALL cells. It was found that primary T-ALL cells obtained from the patient express IL-2R{alpha} (Fig. 6AGo) at a level indistinguishable from background; similarly, the patient's T-ALL cells recovered from engrafted mouse bone marrow express IL-2R{alpha} (Figs. 6B & CGo) at a level only barely distinguishable from background. Fourteen percent of the patient's primary T-ALL cells express abundant IL-2Rß (Fig. 6AGo, not apparent in the contour plot), with the majority of the cells expressing IL-2Rß at a level indistinguishable from background. The fraction of T-ALL cells recovered from engrafted bone marrow that express abundant IL-2Rß is essentially the same at 17% (Fig. 6BGo) and 10% (Fig. 6CGo, not apparent in the contour plot), with the majority of cells again expressing IL-2Rß at a level barely distinguishable from background.



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  		Figure 6. Expression of IL-2R{alpha}, IL-2Rß, and IL-2R{gamma} by primary T-ALL cells and by T-ALL cells recovered from engrafted bone marrow. The expression of surface IL-2R{alpha}, IL-2Rß, and IL-2R{gamma} by primary T-ALL cells obtained from a patient (A) is compared with that of the same patient's T-ALL cells harvested from engrafted bone marrow (two independent experiments, B & C). Isotype-matched control mAbs (FITC- or PE-conjugated IgG1) were used to determine appropriate cursor settings for analysis. The percentage of total cells mapping to each of the two CD7+ (T-ALL) quadrants is indicated. The same batch of staining reagents was used to generate all of the contours presented here. Similar experiments were performed three times, and two different primary T-ALL donors were used.

 
Most striking is the behavior of IL-2R{gamma}. Primary T-ALL cells obtained from the patient express IL-2R{gamma} at a level barely distinguishable from background (Fig. 6AGo). By comparison, T-ALL cells recovered from engrafted mouse bone marrow (Figs. 6B & CGo) are characterized by a uniform upregulation of surface IL-2R{gamma} expression similar in magnitude to what has been reported for activated normal T cells [29, 30]. The results presented here for IL-2R{gamma} are representative of five mice, encompassing three different experiments and two different primary T-ALL donors.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this study we have further characterized the properties and likely utility of the human cord blood preconditioned NOD/SCID mouse model for the in vivo study of human leukemia. We have shown elsewhere that human cord blood facilitates the subsequent engraftment of primary childhood T-ALL cells obtained from patients [15]. We have used several independent approaches to conclude that the engrafted cells are of human leukemia origin and are not derived from cord blood. In addition to phenotypic markers using more than nine different markers (e.g., present data), the other criteria [15] include: A) progression/dissemination of human leukemia in mice, resulting in fatal leukemia; B) gene usage of TCR Vß in cells harvested from mice; C) the presence of morphologically identifiable human leukemia cells in bone marrow and in the circulating peripheral blood, and D) microarray analysis of cDNA expression (J. Yu et al., unpublished observation). On the basis of these results to date, our working hypothesis is that engrafted human cells of cord blood origin provide important microenvironmental signals that in the NOD/SCID mouse are otherwise limiting with respect to engraftment of human primary T-ALL. The demonstration here that the level of T-ALL engraftment can be shown to increase with an increasing number of injected cord blood MNCs is consistent with this hypothesis. In related work, we have also shown that human cord blood conditioned medium significantly increases both the number and burst size of primary T-ALL colonies in methylcellulose culture [14, 31].

The efficient engraftment and subsequent expansion of the leukemia within cord blood preconditioned mice affords a viable window for addressing at the molecular level all events up to and including the expansion. For example, we should be able to address in vivo whether engagement of chemokine receptor CXCR4 by SDF-1 plays a role in homing of the leukemia-initiating cell to mouse bone marrow, as has been reported to be the case for the normal human hematopoietic stem cell [18, 28]. Clearly, to the extent studied to date, the majority of primary childhood T-ALL cells appear to express CXCR4, although there is heterogeneity in expression. Although mouse SDF-1 is active on human cells, it is conceivable that in the bone marrow of preconditioned mice engrafted human cells of cord blood origin release additional SDF-1 that significantly augments the level of T-ALL engraftment. Recently, CXCR4 has been shown to be variably expressed on primary acute myelogenous leukemia cells [17]. However, it is important to point out that CXCR4 expressed by the primary T-ALL cells was shown in present studies to be ineffective in signaling the SDF-1{alpha}-induced transmigration of leukemia cells in vitro. In this respect, there is precedent for this scenario in the response of chronic myeloid leukemia (CML) cells to the chemokine macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), wherein the proliferation of CML cells cannot be suppressed by MIP-1{alpha} due to a signaling defect downstream of the relevant chemokine receptor [32, 33].

On the other hand, our mouse model should facilitate identification of signals driving in vivo expansion of leukemia clonogenic cells. Experiments in many laboratories [2, 5, 8] indicate that in vitro childhood T-ALL cells are growth-factor dependent (e.g., requiring IL-2 and an additional signal(s)). The receptor for IL-2 (IL-2R) can occur in several configurations that differ in their affinity for IL-2 and in their signaling capacity [34]. The high-affinity IL-2R is composed of an {alpha} chain (IL-2R{alpha}), a ß chain (IL-2Rß), and a {gamma} chain (IL-2R{gamma}). IL-2Rß is found also in the receptor for IL-15 [35]. IL-2R{gamma} is a signaling component common to the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 [36, 37], cytokines that can function as T-cell growth factors. As activation of normal T cells in the course of a mitogenic response leads to upregulated expression of IL-2R, including IL-2R{gamma} [29, 30], we characterized cell surface expression of IL-2R{alpha}, IL-2Rß, and IL-2R{gamma} by primary T-ALL cells obtained from patients and by T-ALL cells in bone marrow harvested from NOD/SCID mice preconditioned with cord blood and implanted with the patients' T-ALL leukemia.

It was found that the T-ALL cells recovered from engrafted bone marrow of preconditioned NOD/SCID mice were characterized by a uniform upregulation of surface IL-2R{gamma} expression relative to the primary T-ALL cells obtained from the same patients. The magnitude of IL-2R{gamma} upregulation observed here is similar to that reported for phytohemagglutinin (PHA)-activated normal T cells in the course of a mitogenic response [29, 30]. As we find that in our mouse model the expression of IL-2R{alpha} by T-ALL cells in engrafted bone marrow is not significantly upregulated, it will be interesting to determine whether the expression of any of the other cytokine receptor specificity-determining chains associated with IL-2R{gamma} (IL-4R{alpha}, IL-7R{alpha}, IL-9R{alpha}, or IL-15R{alpha}) is upregulated in parallel with IL-2R{gamma}. A corollary of our working hypothesis that engrafted cells of human cord blood origin provide otherwise limiting signals for leukemia engraftment and/or expansion is that cord blood preconditioning might also facilitate engraftment/expansion of other leukemias that require at least some of these limiting signals. Consistent with this hypothesis, we have found that human cord blood cells secrete a factor(s) that markedly enhances in vitro both colony number and burst size of the T-ALL clonogenic progenitors from patients [14]. Further characterization of this enhancing activity should enable us to study the growth properties of primary T-ALL cells and possibly identify some factors required for the engraftment of T-ALL cells in NOD/SCID mice.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
We have characterized in greater detail the properties and likely utility of our human cord blood preconditioned NOD/SCID mouse model for human T-ALL leukemia engraftment. As further evidence that cord blood facilitates engraftment, we have demonstrated that the level of primary T-ALL engraftment increases with an increasing number of cord blood MNCs injected. In particular, we have documented that chemokine receptor CXCR4 is expressed by primary T-ALL cells obtained from patients, but may not play a role in the homing of leukemic progenitor cells to mouse bone marrow. Finally, we have provided evidence that upregulated expression of IL-2R{gamma} and the likely consequential upregulated responsiveness to T cell growth factor may be relevant to T-ALL expansion in vivo.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We thank Judith Preston for secretarial assistance in the preparation of this manuscript. We are grateful to investigators from the Pediatric Oncology Group for providing primary T-ALL samples, to PharMingen for generously providing antibodies, and to Scripps GCRC for flow cytometry support. This work was supported by the Leukemia Society of America #6226 and NIH MO1-RR00833. This is publication number 13030-MEM from The Scripps Research Institute.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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Received April 16, 2001; accepted for publication June 11, 2001.




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