The CD40-Receptor Is Upregulated on Monocytes from Patients with Low-Risk Myelodysplastic Syndromes and Its Inhibition by a Monoclonal Anti-CD40 Antibody Can Increase Colony Formation.

There is increasing evidence that the immune system can promote cytopenia in myelodysplastic syndromes (MDS) to a variable extent. The interaction between CD40 and its natural ligand CD40L (CD154) plays a critical role in the regulation of humoral and cellular immune responses. The aim of this study was to investigate the role of the CD40/CD40L interaction in the pathogenesis of MDS. For this purpose, we looked at the presence of the CD40 receptor and its ligand in MDS peripheral blood (PB) and bone marrow (BM), and we studied the in vitro effects of inhibition and stimulation of the CD40 receptor on MDS monocytes. 1) Expression of CD40/CD40L. We analysed PB samples of 40 patients with MDS (19 RCMD, 15 RCMD-RS, 3 RAEB-1, 3 5q- syndrome) and 29 age-matched control subjects by flow cytometry. We observed that MDS patients have significantly higher percentages of circulating CD40+/CD14+ cells (8.34 % +/− 1.28 vs. 2.78 % +/− 0.31, p=0.0004) and CD40L+ T helper cells (4.44 % +/− 0.52 vs. 2.16 % +/− 0.22, p=0.001). In addition, we found higher percentages of CD40+/CD14+ cells (9.47 % +/− 1.96 vs. 3.53 % +/− 0.63, p=0.03) and CD40L+ T helper cells (5.13 % +/− 0.86 vs. 2.60 % +/− 0.53, p= 0.008) in BM of 22 patients (1 RA, 11 RCMD, 4 RCMD-RS, 5 RAEB-1 and 1 5q- syndrome) compared to 11 donor samples. 2) Stimulation of CD40. Selected CD14+ cells from PB of 21 patients (1 RA, 12 RCMD, 6 RCMD-RS, 2 RAEB-1) and 25 control subjects were stimulated for 24h with LPS or an agonistic anti-human CD40 antibody (clone 64, PanGenetics NV, The Netherlands). We observed that anti-CD40 induced significantly higher TNF-alpha levels in patients than in controls (436 vs. 64 pg/mL, p=0.008), whereas LPS induced comparable TNF-alpha levels. 3) Blocking of CD40. We cultured 5x10⁵ BM mononuclear cells of 22 patients (1 RA, 12 RCMD, 3 RCMD-RS, 4 RAEB-1 and 2 5q- syndrome) in methylcellulose supplemented with growth-factors in the presence or absence of 5D12 (antagonist chimeric monoclonal anti-human CD40 antibody, PanGenetics NV, The Netherlands). The presence of 5D12 significantly increased colony-formation at day 14 (140 vs. 112, p=0.002). 5D12 did not affect colony formation in CD14-depleted cultures, nor in controls. In conclusion, we have demonstrated elevated levels of CD40 receptor on PB and BM monocytes of patients with MDS compared to age-matched control subjects. In addition, we have demonstrated increased levels of its ligand CD40L on T helper cells in PB and BM. Stimulation of CD40 on monocytes from patients induces significantly higher TNF-alpha levels, a cytokine known to inhibit colony formation. Moreover, we demonstrated that inhibition of this CD40 receptor with 5D12 can increase colony formation and that its effect is specific monocyte-dependent. Our observation together with the proven safety of 5D12 in phase 1 trials for patients with inflammatory bowel disease, might justify a phase I trial of 5D12 to improve cytopenia in patients with MDS.