Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts

Ninety healthy HLA-haploidentical parents of children with hematological disorders were enrolled in the present study. Our treatment algorithm is detailed in Figure 1. Donors received MZ on a compassionate basis, outside the approved label of the drug, after providing written informed consent. The study was reviewed and approved by the Institutional Ethics Committee (protocol #938-LB). We opted for an ‘immediate salvage’ strategy administering 0.24 mg/kg MZ to donors failing to achieve the predefined cutoff of ≥40 CD34⁺ HSCs/μL of blood after 4 daily split-doses of 12 μg/kg G-CSF (Figure 1A) and/or donors with suboptimal predicted apheresis yields on the expected day of HSC collection (≤12x10⁶ CD34⁺ HSCs/kg of recipient’s body weight; Figure 1B) [27]. MZ was administered at 12:00 PM (day +4) and leukapheresis was performed on day +5, following the morning dose of G-CSF (i.e., 9 hours after MZ injection). Peripheral blood was collected prior to G-CSF (day 0) and at peak CD34⁺-cell counts (days +4 and +5). Normal BM samples were collected from 10 consented donors performing BM donation under general anesthesia for matched-sibling HSCT.

FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-17A, PerCP-Cy5.5-conjugated anti-CD4 and APC-conjugated anti-IL-4 monoclonal antibodies (mAbs; Human Th1/Th2/Th17 Phenotyping Kit), Lineage Cocktail-1 (a mixture of FITC-conjugated mAbs directed against CD3, CD14, CD16, CD19, CD20 and CD56), BD™ Stem Cell Enumeration Kit™, mAbs directed against human CD14, CD16, CD45RO, CD62L, CD19, IgD, CD27 and CD184 (12G5 clone), Cytofix/Cytoperm™ solution and Golgi Plug Protein Transport Inhibitor™ were purchased from BD Biosciences (Mountain View, CA). 6-sulfo-LacNAc⁺ (Slan)-DCs were identified using a PE-conjugated anti-M-DC8 mAb (DD-1 clone; Miltenyi Biotec, Bologna, Italy). Fluorochrome-conjugated mAbs directed against BDCA-1 (CD1c; AD5-8E7 clone; type 1 myeloid DCs or MDC1), BDCA-2 (CD303; AC144 clone; plasmacytoid DCs), BDCA-3 (CD141 or thrombomodulin; AD5-14H12 clone; type 2 myeloid DCs or MDC2) and BDCA-4/neuropilin-1 (CD304; AD5-17-F6 clone; plasmacytoid DCs) were from Miltenyi Biotec. Phorbol myristate acetate (PMA) and ionomycin were purchased from Sigma-Aldrich (Milan, Italy). The mAb panel and mAb combinations used for graft characterization are detailed in Table 1.

CD34⁺ HSCs in donor PB, leukapheresis products and manipulated grafts were counted using the ISHAGE protocol [28].

Leukapheresis collections containing <60.0x10⁹ nucleated cells were washed by centrifugation at 300 g for 15 minutes with PBS-EDTA-0.5% human serum albumin (Clini-MACS® washing buffer; Miltenyi Biotec) and were treated with γ-globulins to minimize the non-specific antibody binding to Fc receptors, before the addition of the biotin-conjugated, anti-TCR-αβ antibody. Cells were then incubated with magnetic beads conjugated to anti-biotin and to anti-CD19 antibodies, were re-suspended at <300.0x10⁶/mL and were applied to the fully automated Clini-MACS® device [29].

The following B-cell subsets were monitored in HSC donors: naïve B cells (CD19⁺CD27^-IgD⁺), switched memory B cells (CD19⁺CD27⁺IgD^-), non-switched memory B cells (CD19⁺CD27⁺IgD⁺) and double-negative memory B cells (CD19⁺CD27^-IgD^-). [30] Based on CD45RO and CD62L expression, T cells were allotted to either of the following subpopulations: naïve T cells (T_N; CD45RO^-CD62L⁺), effector memory T cells (T_EM; CD45RO⁺CD62L^-), central memory T cells (T_CM; CD45RO⁺CD62L⁺) and terminally differentiated effector T cells (T_EFF; CD45RO^-CD62L^-) [31].

Three NK-cell subsets were identified and counted. Based on their reciprocal expression of CD16 and CD56, CD3^- cells within the lymphoid gate were subdivided into fully mature NK cells (CD56⁺CD16⁺), tissue-resident NK cells (CD56⁺CD16^-) and immature NK cells (CD56^-CD16⁺) [32].

Three monocyte subsets were analyzed based on CD14 and CD16 expression: classical (CD14⁺⁺CD16^-), intermediate (CD14⁺⁺CD16⁺) and non-classical monocytes (CD14⁺CD16⁺⁺) [9]. Intermediate and non-classical monocytes were collectively referred to as CD16⁺ monocytes. To monitor DC mobilization, cells were stained with the ‘Lineage Cocktail-1’ and with mAbs directed against CD1c, CD303, CD141, or CD304 [33]. After gating on Lineage^- events, DCs were enumerated and their frequency was expressed as a percentage of total leukocytes [9],[33]. 6-sulfo-LacNAc⁺ (Slan)-DCs were counted with the anti-M-DC8 antibody recognizing an O-linked carbohydrate modification of P-selectin glycoprotein ligand-1 [34].

Cytokine production at the single-cell level was assessed with mAbs directed against IFN-γ, IL-17 and IL-4. CD4⁺ cells were activated for 4-6 hours with 50 ng/mL PMA and 1 μg/mL ionomycin, in the presence of inhibitors of protein transport. Following fixation and permeabilization, cells were labeled with cytokine-specific mAbs for 30 minutes at 4°C and then analyzed by flow cytometry.

After staining for surface antigens with mAbs at 4°C for 30 minutes, cells were incubated with 0.9% ammonium chloride for 5 minutes to lyse residual red blood cells. Cells were then extensively washed with PBS - 1% BSA and were run on a FACS Canto II® flow cytometer (BD Biosciences) with standard equipment. A minimum of 50,000 events was collected and acquired in list mode using the FACS Diva® software package (BD Biosciences).

The approximation of data distribution to normality was tested preliminarily using statistics for kurtosis and symmetry. Data were presented as median and range, and comparisons were performed with the Mann-Whitney U test for paired or unpaired data, or with the Kruskal-Wallis test with Bonferroni’s correction for multiple comparisons, as appropriate. P values ≤ 0.05 denoted statistical significance.

We initially enumerated circulating CD34⁺ cells in healthy donors receiving G-CSF for 4 consecutive days. Overall, 61 out of 90 donors (68%) failed to achieve the predefined threshold of 40 CD34⁺ HSCs/μL of blood and were operationally defined as ‘poor mobilizers’ (PM; Figure 1C and Figure 2). However, 31 out of these 61 donors (51%) were re-assigned to the ‘good mobilizer’ (GM) group, since the prediction algorithm favored an apheresis yield >12x10⁶ CD34⁺ HSCs/kg of recipient’s body weight (Figure 1C). By contrast, 29 out of the 90 donors (32%) had post-mobilization CD34-cell counts greater than the cutoff value of 40 CD34⁺ HSCs/μL on day +4, and were classified as ‘good mobilizers’ (GM). Collectively, 60 donors fell into the GM category, whereas the remaining 30 PMs (33% of the whole donor cohort) received single-dose MZ.

In GMs and PMs, the frequency (Figure 2A) and number (Figure 2B) of circulating CD34⁺ HSCs were 0.10% (range 0.02-0.25) and 0.05% (0.01-0.09; p < 0.0001), and 36.5 cells/μL (5-110) and 16.0 cells/μL (2-36; p < 0.0001), respectively, on day +4 of G-CSF administration. Figure 2B also illustrates that CD34⁺-cell counts further increased on day +5 in GMs given G-CSF alone, when compared with those recorded on day +4. PMs consistently achieved >40 CD34⁺ cells/μL of blood after single-dose MZ, and their CD34⁺ counts on day +5 were even higher when compared with those measured in GMs given G-CSF alone [135.0 cells/μL (40-277) vs. 99.0 cells/μL (29-232); p = 0.0089]. Interestingly, PMs given single-dose MZ had higher day-5 monocyte and lymphocyte counts compared with GMs receiving G-CSF alone (Figure 2C-D). The role of MZ in mobilizing monocytes and lymphocytes was further reinforced by the observation that PMs and GMs had similar monocyte and lymphocyte counts on day +4, before receiving single-dose MZ (Figure 2C-D).

As expected, larger numbers of CD34⁺ HSCs were collected, with one single apheresis session, after mobilization with G-CSF + MZ compared with G-CSF alone (Table 2). However, children whose HSC donors were given MZ had a significantly greater body weight compared with children transplanted with G-CSF-mobilized HSC products [45.0 kg (18-74) vs. 20.5 kg (4-66); p < 0.0001].

It is presently unknown whether single-dose MZ affects T-cell and B-cell phenotype in G-CSF-treated healthy subjects [17],[18]. We thus extensively characterized PB samples from donors receiving either G-CSF alone or G-CSF and MZ. No statistically significant differences in the relative proportion of TCR-αβ and TCR-αβ-expressing CD3⁺ T cells were detected, when comparing baseline [94.0% (88.8-98.8) and 5.4% (1.0-9.1), respectively] and post-mobilization samples [95.6% (86.5-99.3) and 4.0% (0.6-12.7), respectively], irrespective of the mobilization protocol (Additional file 1). Moreover, the frequency of T_N, T_CM, T_EM and terminally differentiated effectors was comparable in baseline and post-mobilization samples. Specifically, T_N cells accounted for 29.7% (2.4-62.2) and 30.9% (1.0-58.7), and for 32.2% (2.4-56.4) and 26.6% (2.0-53.4) of the CD4⁺ and CD8⁺ T cells before and after treatment with G-CSF (Additional file 1), indicating that G-CSF administration had no apparent effect on the re-circulation of individual T-cell subsets. We also quantitated B-cell subpopulations in mobilized donors. Again, no appreciable differences were detected in the frequency of naïve B cells, double-negative B cells, switched memory B cells and non-switched memory B cells (Additional file 2). Furthermore, the addition of MZ to the G-CSF-based mobilization regimen exerted no measurable effect on the relative frequency of T-cell and B-cell subsets (data not shown). Previous studies showed that G-CSF skews T-cell function towards a regulatory profile, both in mice and in humans [15],[17]. We thus measured the frequency of CD4⁺ T cells expressing intracellular IFN-γ, IL-17 and IL-4 at baseline and after mobilization with G-CSF. Figure 3A-B illustrates that the frequency of IFN-γ-expressing CD4⁺ T cells was significantly lower in mobilized PB samples compared with baseline. By contrast, IL-4- and IL-17-producing T cells were unchanged. Intriguingly, no differences were found in the frequency of IFN-γ-expressing CD8⁺ T cells (Figure 3C). When data were dichotomized based on the mobilization regimen used, no statistically significant differences were detected in the frequency of CD4⁺ and CD8⁺ T cells expressing intracellular IFN-γ (Figure 3D).

As shown in Figure 4A-B, the overall frequency of PB NK cells was significantly lower after HSC mobilization compared with baseline. This reduction was mainly accounted for by the subgroup of PMs who were given MZ (Figure 4B). Notably, both fully mature CD56⁺CD16⁺ NK cells and tissue-resident CD56⁺CD16^- NK cells were significantly lower after G-CSF administration, whereas immature CD56^-CD16⁺ NK cells were not affected by the HSC mobilization regimen (Figure 4C). Mirroring the above data on NK cells, the frequency of NK-like CD56⁺ T cells was also reduced after G-CSF mobilization, especially in PMs given single-dose MZ (Figure 4D). Collectively, these experiments suggest that NK cells and NK-like T cells may be less represented in healthy donors given G-CSF-based mobilization and that this phenomenon is accentuated by the co-administration of MZ.

We then aimed at enumerating monocyte and DC subsets in mobilized donors. Classical, intermediate and non-conventional monocytes were labeled and quantitated as shown in Figure 4E. Although the frequency of conventional CD14⁺CD16^- monocytes was not significantly different in baseline compared with post-mobilization samples, the proportion of CD16⁺ monocytes increased after G-CSF administration, both in the GM and in the PM group (Figure 4F-G). Slan-DCs (Figure 5A) were not appreciably mobilized into PB by treatment with G-CSF, either alone or combined with MZ (data not shown). Because G-CSF induces CXCR4 cleavage and disrupts the CXCR4/SDF-1α interaction during HSC mobilization, [35] we reasoned that a differential regulation of CXCR4 expression on the surface of Slan-DCs could account for low-level Slan-DC mobilization after combined treatment with G-CSF and MZ. As depicted in Figure 5B, CXCR4 expression significantly decreased on monocytes from donors given G-CSF, after labeling with CD184-12G5 antibodies. Comparable reductions were measured on other leukocyte subsets, such as lymphocytes and neutrophils (data not shown). By contrast, CXCR4 was not apparently down-regulated on circulating Slan-DCs, irrespective of the mobilization regimen (Figure 5C).

Finally, no changes were recorded in the frequency of MDC1, MDC2 or plasmacytoid CD303⁺ or CD304⁺ DCs when comparing baseline and post-mobilization samples, irrespective of the mobilization regimen (Additional file 3) and in keeping with previous reports showing no changes in the frequency of BDCA-2⁺ DCs in donors given G-CSF [36]. Collectively, these experiments suggest that single-dose MZ exerts no major effects on circulating B-cell and T-cell subsets and on monocyte/DC subpopulations, although it may reduce the frequency of blood NK cells and NK-like T cells, when compared with G-CSF alone.

In 70 randomly selected HSC donors (43 GMs given G-CSF only and 27 PMs receiving G-CSF plus MZ) undergoing a standard large-volume (15-20 L) leukapheresis, the graft was extensively characterized in terms of immune effector cells with particular relevance to the setting of haploidentical HSCT, such as NK cells, CD3⁺CD56⁺ NK-like T cells, monocytes and DCs. [37]-[39] For the purpose of comparison, we also enumerated immune effectors in 10 BM samples from healthy HLA-identical sibling donors. TCR-αβ/CD19-depleted grafts obtained after either mobilization regimen were highly enriched with NK cells compared with normal BM samples (Figure 5D). However, the frequency and absolute numbers of NK cells were comparable in grafts collected after the administration of either G-CSF alone or G-CSF and MZ (Figure 5D-E). Although grafts from donors assigned to the GM group had a higher frequency of NK-like CD56⁺ T cells compared with those from the PM group (Figure 5F), the overall number of CD56⁺ NK-like T cells harvested was similar (Figure 5G).

Both conventional and CD16⁺ monocytes were contained at higher frequency in TCR-αβ/CD19-depleted grafts, compared with normal BM samples (Figure 6A-B). As shown in Figure 6C-D, both monocyte populations were more abundant in G-CSF + MZ-mobilized grafts compared with grafts collected after G-CSF alone. 6-sulfo-LacNAc⁺ (Slan) DCs constitute 0.5-2% of all PBMCs, are CD14^lowCD16⁺ and are a highly phagocytic monocyte subset inducing potent pro-inflammatory Th1 and Th17 responses [40]. As illustrated by Figure 6E- F, the frequency of Slan-DCs was significantly lower in grafts mobilized with G-CSF + MZ (0.85% of total leukocytes, 0.06-1.3) compared with G-CSF alone (1.4%, 0.5-4.15; p = 0.0045), although this did not translate into the collection of lower numbers of Slan-DCs.

Finally, Figure 7 shows that both MDC2 and plasmacytoid DCs were significantly more represented in grafts collected after mobilization with G-CSF and MZ. Thus, the number of MDC1, MDC2 and plasmacytoid DCs collected from donors given G-CSF and MZ was significantly greater. Also, the frequency of DC subsets was higher in TCR-αβ/CD19-depleted grafts compared with normal BM samples (Figure 7). Table 3 summarizes the absolute numbers of cells infused in children receiving the TCR-αβ/CD19-depleted haploidentical grafts collected after G-CSF alone or G-CSF + MZ. Whereas the number of monocytes and DCs infused in the two patient groups was similar, children given G-CSF-mobilized grafts received significantly greater numbers of NK cells and NK-like T cells, as well as pro-inflammatory Slan-DCs, compared with children given HSCT from donors mobilized with G-CSF + MZ (Table 3). These differences can be accounted for by the significantly higher number of nucleated cells collected from GMs treated with G-CSF alone (Table 3). Also, the fact that children receiving haploidentical HSCT from GMs had a lower body weight may have translated into the infusion of higher numbers of NK cells, NKT cells and Slan-DCs.

From a clinical standpoint, neither the HSC mobilization regimen (G-CSF alone vs. G-CSF and MZ) nor the number of monocytes and DCs infused correlated with either the occurrence of acute GVHD or the reactivation of viral infections (data not shown). By contrast, patients who developed chronic GVHD had received lower numbers of conventional CD14⁺CD16^- monocytes (3.76×10⁶/kg, 1.7-10.1) compared with children without chronic GVHD (11.45×10⁶/kg, 0.6-144.2; p = 0.016].