Intravenous immunoglobulin treatment of spleen cells from patients with immune thrombocytopenia significantly increases the percentage of myeloid-derived suppressor cells.

Abstract

The response of myeloid-derived suppressor cells (MDSCs) to intravenous immunoglobulin (IVIG) treatment was evaluated in the spleens of patients with immune thrombocytopenia (ITP) and compared with spleens removed for trauma. Exposure to IVIG significantly increased the percentage of MDSCs in cultures of splenocytes from patients with ITP. IVIG also significantly increased MDSCs in the spleen cells from trauma patients, confirming the effects of IVIG. The response to IVIG was similar to that seen in patients with ITP treated with dexamethasone. This finding suggests that IVIG may also exert its effects and contribute to its salutary effects in ITP by increasing splenic MDSCs. We had the opportunity to study frozen spleen cell specimens from seven ITP and seven trauma patients. Although the exact therapies of each patient with ITP were unknown, we could at least assume that all patients that underwent splenectomy had received all of the standard treatments. MDSC phenotype was analysed by flow cytometry for the cell surface markers CD11b, CD33 and HLA-DR. Human spleen cells (5 × 104 cells/well) from ITP and trauma control patients were cultured in complete medium for 90 h in the presence or absence of IVIG. Titrations of IVIG were added to respective wells and the plates were incubated in humidified air with 5% CO2 at 37°C in HEPES buffer at a pH of 6·5. The concentration of IVIG ranged from 5 to 70 mg/ml and encompasses the approximate 25 mg/ml level in plasma that is clinically used. Cells cultured in medium alone without IVIG were run in parallel as non-treated controls. At 90 h, ITP and trauma human spleen cells were surface stained with HLA-DR, CD33 and CD11b. A mixture of 50 μl of diluted stains [HLA-DR (allophycocyanin), CD11b (phycoerythrin), CD33 (fluorescein isothiocyanate)] was added to the respective wells. Plates were re-incubated for 30 min at room temperature, centrifuged, and the pellets re-suspended in 200 μl of staining buffer. Cell viability and number were evaluated using Vi-CELL™ cell viability analyser (Beckman Coulter, Brea, CA, USA). At 90 h incubation 60–70% cells were viable. MDSCs in the HLA-DR negative gate were used for analysis (Fig 1) using a BD FACSort analyser (BD Biosciences, Mississauga, ON, Canada). A minimum of 10 000 events was collected, and the percentage of HLA-DR− CD33+ CD11b+ expressing MDSCs was evaluated. Results are expressed as the percentage of the total HLA-DR negative populations. In view of the fact that IVIG is an important agent in the treatment of ITP, we explored the effect of increasing concentrations of IVIG on the expression of MDSCs in spleen cells from patients with ITP and control trauma patients. The isolation sequence is shown in Fig 1 and described in its legend. The baseline MDSCs in the spleen cells from patients with ITP (0·28%) was 40% of that in the spleen cells from trauma patients (0·7%) (P < 0·02). The percentage of MDSCs in ITP spleen cells increased significantly with increasing concentrations of IVIG (Fig 2). Cells from spleens removed for trauma also showed an increase in MDSCs after treatment with IVIG. This finding confirmed the effect of IVIG on increasing the MDSC population in the spleen. The concentration most closely related to a pharmacological dose of 1 g/kg IV was estimated to be 25 mg/ml based on a plasma distribution space of 40 ml/kg. Of interest, compared with spleen cells from the ITP patients, the spleen cells from trauma patients had a more robust IVIG-induced MDSC response in vitro, suggesting that the cells from ITP patients may, in some way, be defective in their responsiveness. This may reflect either an inherent defect to respond to IVIG in vitro or that the previous therapies of the patients with ITP reduced their responsiveness. More studies, however, are required to confirm these possibilities. During the last decade, MDSCs have emerged as key regulatory cells in the context of tumour growth, inflammation, transplantation and autoimmunity (Gabrilovich & Nagaraj, 2009; Cripps & Gorham, 2011; Greten et al, 2011; Fujii et al, 2013), and MDSC number and function are reduced in patients with ITP (Hou et al, 2016). MDSCs may play a decisive role in the immunological pathogenesis of human ITP because a defect in these regulators of adoptive immunity may augment T-lymphocyte-mediated destruction of platelets in concert with reduced T-regulatory cells (Hou et al, 2016; Yazdanbakhsh, 2016) and increased B-regulatory cells (Aslam et al, 2016). These data are consistent with the finding that high-dose dexamethasone increases the number and function of MDSCs in patients with ITP (Hou et al, 2016). Both blood and splenic MDSCs were increased in patients who received high-dose dexamethasone, and these observations were supported in a mouse model (Hou et al, 2016; Semple, 2016). Our data support the concept that reduced numbers of MDSCs may be an important factor in the pathogenesis of adult type chronic ITP. The findings of an increase in MDSCs as a result of exposure of spleen cells to IVIG is novel and suggests that, in addition to altering the function of the macrophage Fc receptor population, IVIG may also exert an effect similar to that recently reported with dexamethasone by increasing MDSCs (Hou et al, 2016). This work was supported by a Health Canada/Canadian Blood Services Research Grant (J.W.S.). R.B. is the recipient of grant award P50 CA130805 and an “Upstate New York Translational Research Grant” administered through UL1 RR024160 from the National Center for Advancing Translational Sciences of the National Institutes of Health for supporting specimen acquisition. M.J.M. is the recipient of a Canadian Blood Services Doctoral Fellowship and a Vanier Doctoral Fellowship. G.B.S. and R.A. conceived and designed the experiments, performed research, analysed data and wrote the manuscript. R.B., S.A.S. and M.M. provided samples for study. JWS is the principal investigator of the research laboratory and provided funds, facilities and edited the final manuscript. The authors declare no competing financial conflicts.