Bone Marrow Transplantation (2015), 1–6 © 2015 Macmillan Publishers Limited All rights reserved 0268-3369/15 www.nature.com/bmt

REVIEW

Mobilized peripheral blood grafts include more than hematopoietic stem cells: the immunological perspective F Saraceni1,2, N Shem-Tov2, A Olivieri1 and A Nagler2 Although stem cell mobilization has been performed for more than 20 years, little is known about the effects of mobilizing agents on apheresis composition and the impact of graft cell subsets on patients’ outcome. With the increasing use of plerixafor and the inclusion of poor mobilizers in autologous transplant procedures, new parameters other than CD34+ stem cell dose are emerging; plerixafor seems to mobilize more primitive CD34+/CD38− stem cells compared with G-CSF, but their correlation with stable hematopoietic engraftment is still obscure. Immune recovery is as crucial as hematopoietic reconstitution, and higher T and natural killer cells infused within the graft have been correlated with better outcome in autologous transplant; recent studies showed increased mobilization of immune effectors with plerixafor compared with G-CSF, but further data are needed to clarify the clinical impact of these findings. In the allogeneic setting, much evidence suggests that mobilized T-cell alloreactivity is tempered by G-CSF, probably with the mediation of dendritic cells, even though no clear correlation with GVL and GVHD has been found. Plerixafor is not approved in healthy donors yet; early data suggest it might mobilize a GVHD protective balance of immune effectors, but further studies are needed to define its role in allogeneic transplant. Bone Marrow Transplantation advance online publication, 9 February 2015; doi:10.1038/bmt.2014.330

INTRODUCTION Hematopoietic stem cell transplant is a potentially curative treatment for many hematological diseases; in the past two decades, G-CSF-mobilized PBSC have largely replaced BM as the graft source, for both autologous and allogeneic stem cell transplant.1 Taking into account the fact that stem cell mobilization has been widely used for more than 20 years, there is relatively scarce literature investigating graft cell subsets other than those associated with hematopoietic engraftment. So far, the main focus has been in the CD34+ stem cell dose, and this is still the only generally accepted indicator of graft quality; however, knowledge in apheresis composition is increasing as many authors recently tried to shed some light on graft CD34+ subpopulations, immune cell subsets and their influence on engraftment, immune recovery, anti-tumor activity and patients’ outcome. Furthermore, there is growing evidence that different mobilizing agents have a particular effect on graft composition, an issue that is becoming of great importance with the increasing use of plerixafor in clinical practice. Similarly, in the allogeneic transplant setting, many fascinating data are emerging about the effect of mobilizing agents on collected dendritic cells, natural killer (NK) and T-cell subpopulations and their impact on tolerance, GVL and GVHD. This review will focus on the more recent insights into mobilized graft composition in terms of stem and immune cell subsets, and on how different mobilizing strategies shape graft characteristics and accordingly may influence patients’ outcome.

CD34+ STEM CELL DOSE FOR AUTOLOGOUS TRANSPLANT IN THE PLERIXAFOR ERA Up to date, CD34+ stem cell content in mobilized peripheral blood product remains the most important parameter of graft quality, as it is the only recognized predictor of stable hematopoietic engraftment after stem cell transplantation.2–4 The optimal CD34+ cell dose is widely accepted to be 5 × 106/kg, even though in many centers a threshold of 2 × 106/kg CD34+ cells is used; however, stem cell dose has not been established yet and remains a matter of debate. Further, the increasing inclusion of plerixafor in mobilizing regimens is moving forward the issue of CD34+ count, as plerixafor-mobilized grafts seem to show different characteristics compared with those obtained with G-CSF; moreover, with the inclusion of poor mobilizers in auto-SCT procedures, there is a need to identify new parameters that can predict successful engraftment even with a lower CD34+ dose. In a recent meta analysis of two phase III trials, which compared the combination of plerixafor and G-CSF with G-CSF alone, CD34+ stem cell dose was associated with better long-term platelet recovery after auto-SCT, but did not correlate with the time of neutrophil or platelet engraftment, long-term neutrophil or hemoglobin recovery or patients’ survival.5 These results are somewhat inconsistent with previously published data, which showed a strong positive association between CD34+ apheresis content and hematopoietic recovery.3 This finding may be due to a particular plerixafor effect on mobilized progenitor subpopulations. Among the CD34+ stem cells, the CD34+/CD133+/CD38− most primitive subsets are known to have high self-renewal and repopulation capacity,6,7 and are thought to be responsible of

1 Department of Hematology and Bone Marrow Transplantation, Ospedali Riuniti, Ancona, Italy and 2Department of Hematology and Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel. Correspondence: Dr F Saraceni, Department of Hematology and Bone Marrow Transplantation, Ospedali Riuniti, Via Conca 71, Ancona 60126, Italy. E-mail: [email protected] Received 17 November 2014; revised 10 December 2014; accepted 12 December 2014

Mobilized PBSC: the immunological perspective F Saraceni et al

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rapid engraftment after auto-SCT.8 Several reports suggest that plerixafor may increase mobilization of this CD34+/CD38− stem cell subpopulation when compared with G-CSF alone or G-CSF combined with chemotherapy (chemomobilization)9 (Table 1). Fruehauf et al.10 found on a small series of patients with multiple myeloma and non-Hodgkin lymphoma (NHL) an eightfold increase in primitive CD34+/CD38− cell mobilization after administration of G-CSF plus plerixafor compared with G-CSF alone; complete hematologic recovery was observed in 91% of patients within 35 days, with no difference between the groups. These data were confirmed in three recent retrospective studies by Varmavuo et al.11,12 and Roug et al.; 13 they all showed higher proportions of CD38− among the CD34+ cells in patients who received plerixafor, compared with G-CSF only or chemomobilization, with similar engraftment kinetics. The clinical relevance of these observations is at present unclear. Further studies on larger series are needed to clarify whether higher proportions of CD38− cells among precursors mobilized by plerixafor provide faster engraftment and whether this difference is due to plerixafor peculiar effect on BM niche or is just a consequence of mobilization variables leading to plerixafor use.

Table 1.

AUTOLOGOUS GRAFT IMMUNE CELL SUBSETS AND OUTCOME CD34+ hematopoietic stem cells are just one out of many members of the graft crowded and various family; graft composition is still largely obscure and little is known about immune cell subsets contained in the apheresis product and their impact on immune recovery, anti-tumor activity and long-term outcome after auto-SCT. Lately, a more detailed characterization of graft immune cell subpopulations has been the subject of intense investigation; several studies14–17 correlated higher number of T lymphocytes and NK cells infused within the graft with a clinical benefit in terms of speed of immune recovery and outcome after auto-SCT (Table 2). Absolute lymphocyte count on day 15 (ALC-15) has been reported to be an independent prognostic factor for OS in patients undergoing auto-SCT,18,19 and has been shown to be strictly related to the number of lymphocytes infused within the graft (A-ALC), both in multiple myeloma and NHL patients. In a prospective study on 50 NHL patients mobilized with G-CSF, Porrata et al.20 found a correlation between ALC-15 4500/mL and superior OS and PFS (3-year OS 80 vs 37%, P o 0001; 3-year PFS 63 vs 13%, P o 0.0001). Multivariate analysis identified, among the different ALC-15 lymphocyte subsets, CD16+/56+/CD3− NK cells as

Studies comparing G-CSF and plerixafor mobilization of the most primitive CD34+/38− stem cell subset

Author

Study

10

Fruehauf et al. Varmavuo et al.11 Varmavuo et al.12 Roug et al.13 Taubert et al.9

Prospective Prospective Retrospective Prospective Prospective

Disease(s)

No. of patients

MM, NHL NHL MM MM, NHL, HD MM

15 34 21 22 8

Proportion of CD34+/38 − mobilized (% of all CD34+ stem cells) G-CSF

G-CSF+plerixafor

0.5 1.6a 0.6a

4 2.9a 3.5 Higher mobilizationb 2,8-Fold increase compared with G-CSF only

P value

0.004 0.09 0.02 0.03 n.a.

Abbreviations: G-CSF = granulocyte-colony stimulating factor; HD = Hodgkin disease; MM = multiple myeloma; n.a. = not available; NHL = non-Hodgkin lymphoma. aWith the addition of chemotherapy. bValues not reported.

Table 2. Graft cell subset

Impact of mobilizing regimens on autologous graft immune effector cells and patients’ outcome Authors

Mobilizing regimen used and relative efficacy (if compared) CT+ G-CSF

T CD3+

B NK Treg DC

Holtan et al.,32 Varmavuo et al.33 and Gaugler et al.27 Varmavuo et al.11,29 Varmavuo et al.12 Porrata et al.20 Varmavuo et al.11,12,29 Gaugler et al.27 Gaugler et al.27 and Gazitt et al.28

G-CSF +MZ

CT+GCSF +MZ

●

Porrata et al.22 Porrata et al.21

GCSF

●a ● ● ● ●

●● ●● ●●b

● ● ●

● ●●

Outcome implications (if evaluated)

●●b

A-ALC40.5 × 109/kg predicts better OS and PFS A-ALC independent prognostic factor for OS and DFS22 A-ALC40.5 × 109/kg predicts better OS and PFS A-ALC independent prognostic factor for OS and PFS21 No relapses in G-CSF+MZ group, 15/19 in the G-CSF group32 Not significative33 NK-15480/μL predicts better OS and DFS NK-15 independent prognostic factor for OS20

Abbreviations: A-ALC = absolute lymphocyte count in the graft; ALC-15 = absolute lymphocyte count at day 15 post autologous transplant; B = B lymphocytes; CT = chemotherapy; DFS = disease free survival; MZ = plerixafor; NK = natural killer cells; NK-15 = NK cell count at day 15 post autologous transplant; T CD3+ = T lymphocytes; Treg = regulatory T cells; ●● = higher mobilization compared with ●. aPatients received either G-CSF or GM-CSF, 8/267 received only GM-CSF. b Four of nine patients did not receive chemotherapy but only G-CSF+plerixafor.

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3 the only independent predictor of survival; furthermore, high NK-15 count was associated with a lower risk of relapse and superior survival, regardless of disease status at transplant. In previous studies,21,22 the same group reported a strong correlation between infused A-ALC and both T and NK cell counts at day +15; interestingly, no association was found between CD34+ stem cells and ALC-15. These findings indicate that a higher content of T and NK cells might be desirable in auto-SCT to achieve a quicker immune recovery; this may be of importance to prevent infection complications and enhance anti-disease activity, especially in patients receiving immune-modulating agents after auto-SCT.23 Further, different mobilizing agents might shape graft characteristics and have a strong impact on immune cell subsets collected and subsequently infused to the patients.24 CY, often used as part of chemo-mobilizing regimens, is well known to be heavily lymphotoxic and to dramatically reduce the number of mobilized T cells,25 NK and B cells;12 however, its powerful apoptotic effect might spare regulatory T cells within the T-cell population, as showed by recent evidences in the haploidentical setting.26 Recent data on plerixafor showed increased mobilization of T and NK cells, as well as DCs independently of CD34+ cell yield.27,28 In two retrospective studies on NHL patients,11,29 preemptively given plerixafor added to chemo-mobilization resulted in a significant increase in CD3+CD4+, CD3+CD8+ T cells and NK cells when compared with chemo-mobilized grafts, while CD34+ cell mobilization was comparable between the groups. This effective immune cell mobilization is consistent with the finding that CXCL12/CXCR4 chemokine signaling controls T and NK-cell trafficking,30 implying that plerixafor is able to overcome CXCR4-mediated retention of these cells in the BM niche. Furthermore, plerixafor seems to be capable of mobilizing not just more, but fully functional NK cells, as recently showed by Sheng et al.31 in a mouse model. Given the previously mentioned data showing better survival after auto-SCT for patients receiving more immune cells within the apheresis product,20 plerixafor might appear superior to G-CSF in this setting; however, only few controversial data are available about the clinical impact of plerixafor mobilization on patients’ outcome. Holtan et al.32 showed better PFS in a small series of patients mobilized with G-CSF and plerixafor when compared with G-CSF alone; this result was not confirmed in a recent retrospective study33 conducted on 89 NHL patients, in which PFS at 1 year after auto-SCT was comparable (79% and 86% in the plerixafor group and the control group, respectively). It is worth noting that both studies by the Finnish group11,29 were conducted on patients who mobilized poorly with G-CSF, with or without chemotherapy; the difference in lymphocyte subsets content may therefore be an intrinsic characteristic of the Table 3.

graft obtained by poor mobilizers and not a consequence of plerixafor action itself. Randomized comparisons between groups of patients with comparable features are required to confirm these findings and to assess whether different agents have particular impacts on mobilized immune effectors and subsequently influence patients’ outcome. ALLOGENEIC GRAFT IMMUNE CELL SUBSETS AND TOLERANCE G-CSF-mobilized PBSC has largely replaced BM as stem cell source for patients undergoing allo-SCT, based on a great body of data showing faster hematopoietic recovery, decreased relapse and increased disease-free survival compared with collected BM; however, except in patients with advanced disease, these advantages did not lead to a benefit in OS due to a higher incidence of GVHD.34–37 This has been related to the much higher amount of T cells carried in PBSC compared with BM, up to 10-fold more, despite similar content of CD34+ stem cells. Nonetheless, this quantitative discrepancy does not provide a satisfactory explanation of the whole picture; indeed, immune effectors contained in mobilized PBSC and different cytokine profiles within the T-cell population might have a major role. Up to date, the only validated parameters to evaluate allogeneic PBSC graft quality are CD34+ stem cell and CD3+ T-cell doses, and little is known about the effects of mobilizing agents on allograft immune cell subpopulations, their alloreactivity and impact on GVL and GVHD (Table 3). G-CSF is currently the only approved agent to mobilize healthy donors’ stem cells for allo-SCT; it is known to promote diverse immunomodulatory effects on a broad range of cell types, with a profound influence on T-cell differentiation and cytokine production.38 A large body of evidence suggest that G-CSF exposure of donors tempers collected T cells alloreactivity by skewing them toward a Th2 cytokine profile (IL-4, IL-5, and IL-10) rather than type 1 (IFN-γ, TNF-α, and IL-2).39–41 The actual mechanism by which G-CSF alters T-cell function remains unclear; some data suggest that its action on T cells might be mediated by mobilized myeloid and plasmacytoid DCs (pDCs).42–44 Lonial et al.45 recently reported slower T-cell recovery in patients receiving higher numbers of G-CSF-mobilized pDCs within the graft; further, high content of DCs in BM graft, but not in PBSC, have been associated with increased incidence of disease relapse.46 These data have raised the concern that DC-mediated G-CSF action on mobilized T cells could impair their anti-leukemic activity. However, mobilized lymphocytes maintain their GVL effect via different pathways, 47 and Abbi et al.48 recently showed that G-CSF-mobilized donor lymphocyte infusions possess similar therapeutic activity compared with conventional unmobilized donor lymphocyte infusions.

Reported impact of G-CSF and plerixafor on allogeneic graft immune effector cells

Cell subset Role

G-CSF influence

Plerixafor influence

T CD3+

Increased mobilization compared with G-CSF62,64 without affecting function52 Increased mobilization compared with G-CSF.62 No impact64 Increased mobilization compared with G-CSF.62 No impact64 Increased mobilization of pDC and regulatory mDC compared with G-CSF.62,63 Increased mobilization compared with G-CSF.62 Reduced mobilization63

T reg

Immune recovery, early post transplant GVL, acute GVHD Immune recovery, GVHD protective

TEM

Immune recovery, GVL

Skewed to Th2 cytokine production39–41 Promoted expansion, modulated transcription51 Not reported

DC

T- and NK-cell priming, temper T-cell alloreactivity, may impair GVL efficacy Immune recovery, GVL

Induced IL-4 and IL-10 production42,44 Impaired cytotoxicity55

NK

Abbreviations: mDC = monocyte-derived dendritic cells; pDC = plasmacytoid-derived dendritic cells; NK = NK cells; T CD3+ = T lymphocytes; TEM = T effector memory; Th2 = T helper 2 lymphocytes; Treg = regulatory T cells.

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4 On the other hand, mobilized DCs might induce the desirable development of a GVHD protective T-cell repertoire; nonetheless, their adoptive transfer during transplantation, even though significantly affected T-cell alloreactivity, failed to confer protection from GVHD in animal models.49 Further, Waller et al.50 recently found an association between the number of pDCs within BM graft and OS, but failed to show any correlation between pDCs in PBSC and outcome or GVHD. G-CSF mobilization seems to modulate also CD4+/CD25+ /FoxP3+ regulatory T cell activity and to promote their expansion in donor and recipient.51,52 Regulatory T cells temper T CD8+ alloreactivity and are involved in induction and maintenance of tolerance without impairment of GVL effect.53 Among immune effectors contained in mobilized allograft, NK cells have a key role; they have been recognized to carry strong GVL power without inducing GVHD in T-deplete haploidentical stem cell transplant, and their infusion within the graft provides both anti-leukemic and anti-viral activity in the early posttransplant period.54 There is evidence of a detrimental effect of G-CSF on their function, which seems to be transient and not to affect late-phase GVL activity.55 Given the variety of immune cells contained in the apheresis product, many strategies have been developed in order to rationally select graft effectors and provide designed adoptive immunotherapies, especially in the haploidentical stem cell transplant setting.56–58 In this scenario, Schumm et al.59 recently introduced an innovative approach of graft manipulation, which consist in depleting the leukapheresis product of only TCR αβ+ T cells and CD19+ B cells, thus retaining large numbers of crucial immune effectors such as TCR γδ+ T cells, NK cells and DCs; this method has been safely tested in a cohort of children by Locatelli and colleagues.60 Although G-CSF mobilization is known to alter phenotype and cytokine polarization of transplanted immune cells, very few data are available on the impact of plerixafor on allogeneic graft cell subsets, as it is not approved for administration in healthy donors yet. Two recent studies in mice compared the effect of G-CSF and plerixafor on T-cell alloreactivity, showing that alterations in the T-cell phenotype and cytokine gene expression profile characteristic of G-CSF mobilization do not occur with plerixafor.51,61 Interestingly, in the study by Lungvist et al.,61 mice which received plerixafor-mobilized PBSC had a significantly higher incidence of skin GVHD compared to the ones tranplanted with G-CSF mobilized graft. In apparent contrast with these results, a study on a rhesus macaque model by Kean et al.62 reported a higher content of regulatory subpopulations such as regulatory T cells, pDCs and CD8+ effector memory T cells in plerixafor-mobilized graft, when compared with apheresis obtained with G-CSF. In healthy donors, Rutella et al.63 recently found greater numbers of myeloid DCs and pDCs in T-cell-depleted grafts mobilized with plerixafor and G-CSF compared with G-CSF alone; most mobilized DCs showed a regulatory profile, with a very low proportion of pro-inflammatory 6-sulfo-LacNAc+ (Slan) DCs. This results are consistent with Gaugler’s data,27 which evidenced a potential regulatory capacity of pDCs mobilized by plerixafor. These findings might represent an immune background for the unexpected results of a phase II study by Devine et al.,64 in which 25 patients received PBSC from HLA-identical sibling donors mobilized with a single dose of plerixafor as the only agent; patients were compared with a historical group who received standard G-CSF mobilized product. The authors observed a higher content of T CD3+ and T CD4+ cells in plerixafor-mobilized grafts, with no apparent polarization toward a particular lymphocyte subset. Immune reconstitution was prompt (T CD3+CD4+4200/μL at day +28), and despite lower numbers of CD34+ cells being collected with plerixafor, engraftment kinetics were comparable to Bone Marrow Transplantation (2015), 1 – 6

the historical group. Surprisingly, recipients of plerixafor-mobilized grafts showed rates of GVHD that were not significantly different from historical controls, despite more T cells were mobilized and infused to the patients. The fascinating hypothesis that plerixafor-mobilized PBSC may possess a GVHD protective balance of immune effectors needs to be tested in a rigorous comparison between groups of patients receiving either G-CSF- or plerixafor-mobilized graft, in order to clarify hematopoietic and immune reconstitution kinetics, relative risks of GVHD and relapse in patients transplanted with each type of mobilized stem cell product. CONCLUSION Graft cell composition is still largely obscure and little is known about its impact on hematopoietic engraftment, immune recovery and long-term outcome. CD34+ stem cell dose is still the only validated parameter to evaluate graft quality in the auto-SCT setting; however, increasing knowledge in apheresis product’s composition and use of new mobilizing agents may lead to development of new predictors of hematopoietic engraftment. Inclusion of poor mobilizers in transplant procedures represents a new challenge in the assessment of graft quality and might change the perspective on CD34+ stem cell dose evaluation. The recent finding that plerixafor mobilizes more CD34+/CD38− precursors needs to be challenged in larger series and related with engraftment. If confirmed, less CD34+ cells might be needed in patients mobilized with plerixafor, and evaluation of CD34+ /CD38− cell dose might become a helpful parameter, together with the CD34+ total dose, to assess graft quality in patients who mobilize poorly. Further, changing the perspective on auto-SCT from being just a means to overcome high-dose chemotherapy to be viewed as effective immunotherapy will provide new insights on the influence of different mobilizing agents on graft variegate cell subsets. There is evidence of better survival for patients receiving more T and NK cells within the apheresis product; different mobilization strategies result in various cell subset compositions, and plerixafor seems to mobilize more T and NK cells compared with G-CSF. Whether this provides a graft with enhanced immune properties, which can have an impact on patients’ outcome, needs to be determined prospectively in future studies. In the allogeneic transplant setting, G-CSF significantly shapes graft characteristics, specifically tempering T-cell alloreactivity by modulation of cytokine production. These regulatory effects are highly desirable to promote tolerance mechanisms, with the downside of possible decreased anti-disease activity; however, despite many studies addressing this crucial issue, the actual pathways of its action and consequences on GVL–GVHD balance are largely unknown. Plerixafor is not approved for stem cell mobilization in healthy donors yet; recent observations on small series suggest it can be safely administered as a single agent and may have the potential to mobilize a GVHD protective cell repertoire, although data are contradictory and no conclusion can be drawn yet. However, the capacity of plerixafor to induce an enhanced mobilization of T and NK cells is undoubtedly fascinating, both in autologous and allogeneic settings; as an example, this property could be exploited to obtain higher yield of regulatory T cells and NK cells before ex vivo expansion for adoptive immune cell therapies. Furthermore, the coming of new mobilizing agents in the near future may lead us to rethink the way we obtain mobilized peripheral blood cells; with the availability of multiple molecules, transplant physicians may be able to choose for each patient the agent that can provide a graft cell profile which best fits patient characteristics, such as disease status or factors predicting poor stem cell yield. The design of the graft is just at the beginning. © 2015 Macmillan Publishers Limited

Mobilized PBSC: the immunological perspective F Saraceni et al

CONFLICT OF INTEREST The authors declare no conflict of interest.

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