Differences in the phenotype, cytokine gene expression profiles, and in vivo alloreactivity of T cells mobilized with plerixafor compared with G-CSF

Abstract

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Figures

Figure 1. Mobilization of blood mononuclear cells after a single dose of plerixafor in healthy subjects

Blood samples were collected prior to the start of mobilization and 6 hours after a single injection of 240 µg/kg of plerixafor immediately before apheresis. Each symbol represents an individual subjects. **p

Figure 2. Th1 and Th2 gene expression profiles in CD3+ T cells assessed by real-time PCR

(A) Heat map showing real-time PCR expression levels of 16 genes in CD3+ T cells that changed significantly from baseline following G-CSF mobilization in 5 healthy subjects. All samples were normalized to the center of the mean of the pre G-CSF samples with red denoting up-regulated expression and green denoting down-regulated expression (10% false discovery rate). (B) Gene expression levels of plerixafor did not change from baseline in the 84 measured Th1-Th2-Th3 genes, including the 16 genes that were altered with G-CSF treatment which are displayed.

Figure 3. The impact of mobilization on alloreactivity

The alloreactivity of PBMCs before and after mobilization with plerixafor (A and B) or G-CSF (C and D) against 3rd party PBMC stimulators was measured using a 3H Thymidine uptake (A and C), or CFSE dilution assay gated on viable CD3+ T cells (B and D).

Figure 4. The impact of mobilization on CD4+ FoxP3 expression

FoxP3 expression following mobilization compared to baseline in CD4+ cells mobilized with plerixafor or G-CSF showed no fold-change in expression from baseline in healthy subjects mobilized with either plerixafor or G-CSF alone. Line represent the median change from baseline in 13 samples obtained after plerixafor mobilization and 7 samples obtained after G-CSF mobilization.

Figure 5. Animal transplant model

Donor B10.d2 mice were injected subcutaneously with either 10ug G-CSF daily for 5 days, one injection of 100ug plerixafor, or saline control (HBSS). (A) Number of splenocytes and complete blood counts were obtained from G-CSF and plerixafor mobilized donors. (B) The alloreactivity of donor B10.d2 splenocytes in response to ConA blasts derived from C57BL/6 donors was assessed by MLR and showed no significant differences between the three groups. (C) Incidence of skin GVHD in recipients receiving 15×106 mobilized total unfractionated splenocytes (p<0.05 G-CSF compared to HBSS controls).