Characterization of Splenic CD34⁺ Cells From Patients with Primary Myelofibrosis

Abstract 2810

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extramedullary hematopoiesis. The mechanism by which HSC/HPC preferentially migrate, reside, proliferate and differentiate in the spleen remains poorly understood. We phenotypically and functionally characterized PMF splenic CD34+ cells (PMFSC) (n=8). Greater numbers of CD34+ (5.9±1.9%), CD34+CD38- (3.2±1.6%), CD34+CD90+ (2.0±1.5%), CD34+Lin- (4.4±1.8%) cells and assayable HPCs (4513±240/10⁶ mononuclear cells (MNC)) were detected in PMF spleens as compared with PMF peripheral blood (PB) (CD34⁺: 1.4±0.6%; CD34+CD38-: 0.8±0.6%; CD34+CD90+: 0.1±0%; CD34+Lin-: 0.8±0.4%; HPC: 2496±677/10⁶ MNCs). In addition, the expression of CXCR4 by CD34⁺ cells present in the spleen (20.8±3.7%) was shown to be greater than that previously observed in PMF PB CD34+ cells (PMFPBC) (7.4±2.2%, P<0.05). We next examined PMF spleens for the presence of SCID repopulating cells by analyzing human cells in the marrow and spleen of NOD/SCID γc^null mice transplanted with PMFSC using flow cytometry. Five months following transplantation, 12.1% of the marrow cells were hCD45+ in mice receiving 2×10⁵ PMFSC as compared to 3.4% in mice receiving same number of PMFPBC from the same patient. The percentage of hCD45+ cells in the marrow significantly increased to 40.6% in mice receiving 2×10⁶ PMFSC, while 2.7% hCD45+ cells were detected in the marrow of mice receiving similar number of PMFPBC. Surprisingly, 0.6% of the cells were hCD45+ in the spleens of mice receiving 2×10⁴ PMFSC, while 33.9% of the cells were hCD45+ in the spleens of mice receiving 2×10⁵ PMFSC. By contrast, as few as 0.1% hCD45+ cells were detected in the spleens of mice transplanted with equal numbers of PMFPBC. Moreover, the spleens of mice transplanted with PMFSC contained greater number of hCD34+ cells, while no hCD34+ cells were observed in the spleens of mice transplanted with PMFPBC. The human cells in the marrow of mice receiving PMFSCs were further found to be capable of differentiating into not only myeloid cells (CD33+, 8.7%; glycophorin A+, 1.1% and CD34+, 1.3%) but also B (2.3%) and T cells (2.9%). While, the human cells in the marrow of mice receiving similar numbers of PMFPBC were composed of myeloid cells (CD33+, 0.5%; glycophorin A+, 0.1% and CD34+, 0.1%) but very few CD19+ (0.1%) and no CD3+ cells. Moreover, both myeloid cells (CD33+, 14.5%; glycophorin A+, 3.9% and CD34+, 6.8%) and CD19+ (6.7%) and CD3+ cells (23.1%) were detected in the spleens of mice receiving PMFSC. The engraftment of human cells in the spleen and marrow of mice receiving PMFSC and the distinct differentiative potential of PMFSC in mice were further confirmed by immunohistochemical analysis. Greater numbers of hCD45+ cells were observed in the marrow and spleen of mice receiving PMFSC and the degree of human cell engraftment was consistent with the number of PMFSC transplanted. Moreover, the white pulps of the spleens of mice receiving PMFSC were composed primarily of human lymphocytes. Both human granulocytic cells and human lymphocytes were observed in red pulps of the spleens of mice transplanted with PMFSC. In addition, following the infusion of 5×10⁵ PMFSC and PMFPBC, reduced numbers of PMFSC (90.3±16.8/10⁶ BMCs) were detected in the marrows of these mice as compared with G-CSF mobilized PB (mPB) CD34+ cells (196±19/10⁶ BMCs; P<0.05), and the number of PMFSC that homed to the marrow of the mice was lower than PMFPBC (101.7±8.8/10⁶ BMCs; P=0.09). By contrast, similar numbers of PMFSC and PMFPBC and mPB CD34⁺ cells were detected in the spleens of these mice. Recently the use of JAK2 inhibitors has been shown to dramatically reduce the size of the spleen in patients with PMF. PMFSC and PMFPBC were treated with a JAK2 inhibitor, AZD1480. Similar reductions in the number of total cells, CD34+ cells and assayable HPCs were observed as compared with cells exposed to cytokines alone. These findings suggest that multipotent HSCs are present in the spleen but not the PB of PMF patients and that PMFSC have a distinct differentiative program and migratory behavior that distinguishes them from PMF and normal PB CD34+ cells. In addition treatment with JAK2 inhibitors does not appear to lead to a reduction of splenomegly by preferentially inhibiting splenic HPCs.