Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab

In nonhuman primates, CD49d-blockade with humanized antifunctional antibodies results in rapid (peak < 1 day) and prolonged (> 10 days) increase in circulating hematopoietic stem/progenitor cells (HSPCs).^1,2  Elevated circulating HSPCs were likewise observed in anti-CD49d antibody-treated mice.²  Furthermore, mice conditionally ablated for CD49d sustain more than 8-fold elevated levels of circulating HSPCs without evidence of progressive accumulation or of bone marrow (BM) depletion for their life span.³  Even though the homing efficiency of anti-CD49d-mobilized HSPC was reduced, if sufficient numbers of cells were used, these cells provided short- and long-term engraftment. Studies in humans treated with anti-CD49d antibody, particularly after protracted blockade of CD49d, have not been reported, nor have any other studies of prolonged administration of mobilizing agents been performed. A clinical-grade humanized mouse-antihuman function-blocking CD49d antibody (natalizumab, Tysabri;Biogen/Idec, Cambridge, MA) is available under a special restricted distribution program for treatment of relapsing-remitting multiple sclerosis (MS) patients who failed to respond to or did not tolerate first-line therapeutics.⁴  As trials of natalizumab in healthy volunteers are not justifiable, because of prolonged immune-modulating effects of the antibody, including the possibility of rare, but potentially fatal progressive multifocal leukencephalopathy,⁵  we made observations in a cohort of MS patients receiving/scheduled to receive disease-modifying monotherapy with natalizumab.

Adults with MS receiving/scheduled to receive disease-modifying therapy with natalizumab (300 mg intravenously once every month) at the University of Washington Departments of Neurology/Rehabilitation Medicine were eligible for participation. Exclusion criteria were other disease-modifying therapy, steroids, or lithium. After written informed consent was obtained in accordance with the Declaration of Helsinki, immediately preceding the next scheduled natalizumab infusion blood was drawn from “untreated” patients (before the first infusion) and “chronic” patients (≥ 5 prior doses). In some patients, a second blood draw was done after infusion, generally on the subsequent day. A cohort of healthy controls was also recruited. Blood draws were anonymous; except for classification as “untreated/first-dose” or “chronic” recipient, no subject information was collected. The study was approved by the University of Washington Internal Review Board.

Colony-forming unit-culture (CFU-C) assays were performed as described.⁶  Side-scatter low/CD34bright (CD34⁺) cells were quantified by flow cytometry, as described.⁷  The presence of competitive repopulating units (CRU) was tested in xenotransplants 9 to 10 weeks posttransplantation, as described. Transwell migration of CFU-C toward SDF-1 (100 ng/mL) was enumerated as described.⁶  Cell-cycle status on flow-sorted CD34⁺ cells was analyzed by Acridine Orange staining.⁸ 

Before the first natalizumab dose, circulating CD34⁺ cells and CFU-C in “untreated” MS patients (Figure 1A,D) were within the range reported for healthy subjects⁹  and our healthy controls studied concurrently (ie, MS per se is not associated with elevated circulating HSPCs). Subjects on “chronic” natalizumab treatment had 5- to 7-fold elevated circulating CD34⁺ cells and CFU-C (Figure 1A,D) 1 month after infusion. In a subgroup of “chronic” subjects, circulating HSPCs were analyzed before and 1 day after the monthly dose of natalizumab. Renewed infusion did not result in significant further augmentation of circulating CD34⁺ cells or CFU-C (analyzed 1 day after the infusion, Figure 1C,F), indicating continuous functional satiation of CD49d on BM-HSPC with standard natalizumab dosing.

Comparison of natalizumab-recipients before and 1 day after the first infusion (“1st dose”) revealed 5- to 6-fold increased CD34⁺ cells and CFU-C after the first infusion (Figure 1B,E). Mean postinfusion values after the first dose were no different from those in “chronic” natalizumab-recipients before or after repeated natalizumab infusions, documenting achievement of maximal levels of circulating HSPCs within 24 hours of a single natalizumab infusion. Because circulating CD34⁺ cells and CFU-C in untreated MS patients were normal, the increased frequency of circulating HSPCs in natalizumab-treated MS patients is apparently the result of drug effects.

CD34⁺ counts in natalizumab-treated MS patients were thus approximately one-sixth of those in MS patients mobilized with granulocyte colony-stimulating factor (G-CSF).¹⁰  However, the relative frequency of clonogenic cells appeared to be higher among natalizumab-mobilized than among G-CSF-mobilized CD34⁺ cells (1 in 3 CD34⁺ cells for natalizumab vs 1 in 10 for G-CSF mobilized CD34⁺ cells),¹¹  likely because the G-CSF induced proliferation/differentiation dilutes clonogenic cells with more mature CD34⁺ subsets. Thus, the difference in HSPC mobilization potency may be less pronounced than CD34⁺ numbers suggest. CD34⁺ counts in natalizumab patients were one-third of those achieved with the CXCR4-antagonist AMD3100 in healthy volunteers.⁹ 

The sustained HSPC levels in natalizumab recipients contrast with mobilization kinetics after G-CSF, in which after the peak on day 5, circulating HSPCs numbers are regressive despite continued administration.^12,13  It indicates that, without CD49d-mediated BM retention, a new equilibrium is established between HSPCs in BM and those in circulation. In natalizumab recipients, a total of approximately 50 × 10E6 CD34⁺ cells (8 CD34⁺ cells/μL × 6 L of blood) are in circulation at any given time. Assuming that the transit time of normal circulating HSPCs is short¹⁴  and not very different from normal in natalizumab-treated patients (supported by unpublished data for CD49d^−/− mice), several million CD34⁺ cells are trafficking through the blood of the average natalizumab recipient every day. A similar pattern of sustained elevation of circulating HSPCs was observed in CD49d-deficient mice.³ 

Most CD34⁺ cells in blood from natalizumab patients are quiescent (Figure 1H), a phenotype associated with favorable homing and/or engraftment.^15,16  Similar data were reported for unmobilized¹⁷  or G-CSF-mobilized CD34⁺ cells¹⁸  (ie, may represent a general feature of circulating HSPCs). Peripheral blood CFU-C from natalizumab-treated patients did not migrate in in vitro transwell assays, whether spontaneously or SDF-1–directed, in contrast to normal BM HSPC (Figure 1G). We attribute the lack of migration to continuous saturation by natalizumab, as several previous observations show absence of migration with anti-CD49d antibody-treated cells.

To establish whether peripheral blood of natalizumab-treated patients contained any CRU, xenotransplants of mononuclear cells from 7 to 8 mL of blood (containing 50 000-60 000 CD34⁺ cells) were performed. Human engraftment of more than 1% was observed in 3 of 3 recipients (not shown), although no quantitative conclusions about CRU can be drawn from these studies. The engraftment data are of interest in view of the cells' inability to migrate in vitro. Similar to our data, cells mobilized with the chemokine Groβ did not migrate in vitro but engrafted well,¹⁹  reinforcing the view that in vitro behavior of HSPCs cannot predict their in vivo performance.

The role, or clinical consequence, of elevated circulating HSPCs for natalizumab-treated MS patients is unclear. Potential effects of HSPC on remodeling/repair of nonhematopoietic organs have been reported.^20,21  It is thus tempting to hypothesize that circulating HSPCs may contribute to the anecdotal cases of neurologic improvement under therapy, which may not be explained by the main mechanism of natalizumab in MS (ie, attenuated lymphocyte recruitment).^22,23  Although in natalizumab-treated MS patients significant numbers of HSPCs continuously circulate outside the protective BM environment, their likelihood to acquire deleterious mutations is probably small, given their G₀ cycling status, but this possibility needs to be considered. Thus far, an increased propensity of natalizumab patients for hematologic malignancies has not been reported; however, further long-term observations are needed.

The usefulness of natalizumab as a priming agent for HSPC peripheralization is likely restricted only to patients receiving natalizumab for their underlying illness, given the risk of immunization against natalizumab²⁴  and prolonged immune-modulating effects. However, CD49d blockade with short-acting small-molecule CD49d inhibitors, if efficacious, could be theoretically considered as treatment strategy for patient groups intolerant or unresponsive to G-CSF,^10,25  especially in combination with other mobilizing agents, such as the CXCR4 antagonist AMD3100.²⁶ 

The authors thank the nursing staff of the 8SE infusion station at University of Washington Medical Center for their support.

This work was supported by a Core Center of Excellence in Hematology grant from the FHCRC (National Institutes of Health [NIH] DK56465 to H.B.) and by NIH grant HL58734 (T.P.).

National Institutes of Health

Contribution: H.B. conceived of the studies, wrote the IRB protocol, obtained informed consent, performed experiments, collected and analyzed data, and wrote the paper; A.W. wrote the IRB protocol and recruited subjects; K.-H.C. performed experiments; S.L. recruited subjects; T.P. analyzed data and wrote the paper.

Conflict-of-interest disclosure: S.L. serves on the Speaker's Bureau for Biogen/Idec, manufacturers of natalizumab. The other authors declare no competing financial interests.

Correspondence: Halvard Bonig, Johann-Wolfgang-Goethe University, Institute for Transfusion Medicine and Immune Hematology, Sandhofstrasse 1, 60528 Frankfurt; e-mail: hbonig@u.washington.edu or hbonig@blutspende.de.