1α,25-Dihydroxyvitamin D3 to skew intratumoral levels of immune inhibitory CD34+ progenitor cells into dendritic cells

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with a 5-year mortality rate of approximately 50 percent. Patients with HNSCC have profound immune defects that are associated with increased recurrence.1 Previous work investigated the contribution of CD34+ progenitor cells, whose levels are increased in HNSCC patients, to this immune dysfunction.2 CD34+ cells are hematopoietic progenitor cells that intensely express the CD34 marker as opposed to the dimmer level of expression by endothelial cells. Their numbers are elevated in patients with HNSCC, and they exhibit nonspecific suppression of T-cell function. In healthy individuals, CD34+ cell levels are less than 1 percent of the peripheral blood mononuclear leukocyte population in contrast to patients with HNSCC in whom they compose approximately 5 percent of the peripheral blood mononuclear leukocyte population.3 HNSCC-derived granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the expansion and mobilization of CD34+ cells into the peripheral blood and tumor tissue from bone marrow. CD34+ cells mediate suppression of T-cell functions by secreting transforming growth factor-beta (TGF-β).4 The CD34+ cells from HNSCC patients are progenitor cells because they can grow in soft agar containing colonystimulation factors into colonies containing monocytes and neutrophils.2 Further studies examined the functional and clinical significance of GM-CSF and CD34+ cells in patients with HNSCC. The depletion of CD34+ cells from the peripheral blood of HNSCC patients increased the proliferative capacity of intratumoral T cells.2 Patients with high levels of GM-CSF, the CD34+-mobilizing cytokine, had a corresponding increase in intratumoral CD34+ cells. Examination of 27 patients with HNSCC showed 19 of them to have readily detectable CD34+ cell levels in the peripheral blood, whereas seven control patients did not have detected CD 34+ cells.3 When GM-CSF and CD34+ levels were analyzed in relation to patients’ T stage and nodal status, GM-CSF and CD34+ levels were increased as T stage increased.5 These levels increased more prominently at all T stages with cervical nodal involvement. Twenty HNSCC patients with high levels of GM-CSF and CD34+ cells and 17 HNSCC patients with low levels of GM-CSF and CD34+ cells were evaluated for whether GM-CSF production and the presence of CD34+ cell within primary HNSCC tissue translated into increased recurrence or metastasis during the 2 years after surgical excision.6 This study found a four-fold increase in the level of GM-CSF and a two-and-a-half-fold increase in CD34+ cell levels in the patients who subsequently developed recurrences or metastatic disease. This suggests that increased levels of immune inhibitory CD34+ cells could be decreasing the intratumoral T-cell defenses, leading to decreased disease-free survival. Bone marrow–derived CD34+ progenitor cells have the capacity to differentiate into immune stimulatory dendritic cells.7 Dendritic cells are professional antigen-presenting cells, and they can activate naive T cells. They present antigens in the context of class II MHC and secrete interleukin (IL)-12, a T-cell activating cytokine. Unfortunately, dendritic cell maturation is defective in cancer patients.8 Although immature dendritic cells can induce T-cell tolerance, mature dendritic cells are potent stimulators of the primary immune system and can stimulate antitumor immunity.9 Because of the potent immune stimulatory effects of mature dendritic cells, studies determined if the immune inhibitory CD34+ cells that are mobilized in HNSCC patients could be differentiated into immune stimulatory dendritic cells. CD34+ cells from the peripheral blood of HNSCC patients that were grown in a media containing GM-CSF, stem cell factor, and tumor necrosis factor differentiated into cells that were phenotypically homologous to dendritic cells.10 The efficiency of the differentiation was approximately 25 percent. The functional competence of these cells was shown by their ability to present the tetanus toxoid antigen to autologous T cells and stimulate their intracellular expression of interferon-gamma (IFN-γ).5 Additional studies showed that the supplementation of the CD34+ cell cultures with 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3) further doubled the number of cells that differentiated into dendritic cells.3 In mice bearing tumors, vitamin D3 reduced the levels of CD34+ cells and stimulated antitumor immunity.10 In a phase 1B clinical trial with patients having advanced HNSCC, 25-hydroxyvitamin D3 was shown to reduce the number of peripheral blood CD34+ cells, increase HLA-DR expression (marker for antigen presenting cells), increase plasma IL-12 (a marker of active antigen-presenting cells), increase plasma IFN-γ, and improve activation of patients’ peripheral blood T cells.11 However, this clinical trial was not designed to analyze the effect of vitamin D3 analogs on the composition of CD34+ cells or antigen-presenting cells within the tumor tissue. The present study extended these prior studies by analyzing how 1,25(OH)2D3 affects intratumoral levels of CD34+ cells and dendritic cells. This study found that 1,25(OH)2D3 significantly dropped intratumoral levels of CD34+ cells and levels of immature dendritic cells. In contrast, levels of mature dendritic cells within the HNSCC tissue significantly increased.