Rituximab therapy modulates IFN‐γ and IL‐4 gene expression in a patient with acquired haemophilia A

Several published observations have demonstrated that acquired haemophilia A can be successfully treated with rituximab (Franchini, 2007). Interestingly, emerging reports demonstrated that T cell-mediated diseases, such as ulcerative colitis and psoriasis, are sometimes exacerbated by the use of rituximab (Dass et al, 2007; Goetz et al, 2007). Currently, the impact of anti-B-cell therapy on the immune system is not clearly understood. T-helper (Th) cells are divided into Th1, Th2 and Th17 subtypes producing c-interferon (IFN-c), interleukin (IL)-4 and IL-17, respectively. Alterations affecting the balance between Th cell types need to be monitored as these may cause the emergence of pathologies (Lucey et al, 1996). Here, we investigate the impact of rituximab on the gene-expression of key T-helper cytokines: IFNG, IL4 and IL17A. This is the case of an 18-year-old woman, previously healthy G1P2 (delivered twins 18 months earlier) who presented with excessive bleeding after appendectomy. On work-up, she was found to have a prolonged activated partial thromboplastin time (aPTT), normal prothrombin time (PT), and factor VIII (FVIII) of 3Æ5%; a FVIII inhibitor was measured at 7 Bethesda units (BU). Treatment with high doses (60 units/kg per h of FVIII concentrate via continuous infusion provided good haemostasis with average FVIII levels of 83% during a complicated 2 month hospital course; however, the inhibitor persisted during this period. A decision was made to attempt inhibitor eradication with rituximab. The study was approved by the Tulane University Institutional Review Board and written informed consent for participation was obtained from the patient. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) gradient separation from heparinized blood. Cells were surface-stained with the following fluorochrome-conjugated anti-human antibodies: CD3-peridinin chlorophyll protein Cyanin 5Æ5 (PerCp-Cy5Æ5) (clone SK7; BD Biosciences, San Jose, CA, USA), CD4-AmCyan (clone SK3; BD Biosciences), CD14-phycoerythrin-texas red (ECD) (clone RMO52; Beckman coulter, Miami, FL, USA), CD19-allophycocyanin (APC) (clone HIB19; BD Biosciences). Data acquisitions were performed using a BD LSRII Cytometer (BD Biosciences) and results were analysed with DiVa Software (BD Biosciences). Expression of IFNG, IL4 and IL17A mRNA was monitored using reverse transcription polymerase chain reaction (RTPCR). Total RNA was isolated from whole blood using the PaxGene blood RNA isolation kit (BD-Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. RNA quality was assessed using Experion Automated Electrophoresis Station (Bio-Rad, Hercules, CA, USA). The reverse transcription reaction was performed on 1 lg total RNA using iScript cDNA synthesis Kit (Bio-Rad). RT-PCR was performed on a Bio-Rad iCycler with iQ Supermix (BioRad) according to manufacturer’s instructions. The PCR programme cycle was the following: 50 C 2 min, 95 C 3 min, 40 cycles of 95 C 15 s 60 C 1 min. The following TaqMan probes from Applied Biosystems (Foster City, CA, USA) were used: Hs99999903_m1 (ACTB); Hs00174143_m1 (IFNG); Hs00929862_m1 (IL4) and Hs00936345_m1 (IL17A). Cycle thresholds were normalized internally to ACTB. The proportion of transcript present in the samples was calculated using the 2 DD CT calculation method. The final results represent the relative amount of amplicon in samples (fold increase) with the level of transcripts collected at the onset of the treatment set to 1. The FVIII inhibitor became undetectable 4 days after the first dose of rituximab (375 mg/m) and FVIII levels initially increased as the FVIII inhibitor decreased (Fig 1). The FVIII level then gradually decreased again, while the FVIII inhibitor rose slightly. The patient therefore received subsequent doses of rituximab on days 26, 36 and 48. With these infusions, the FVIII level increased gradually to 112% on day 110 and remained in the normal range (116%) on day 235 (Fig 1). As expected, the initial dose of rituximab profoundly depleted CD19 B-cells in the blood (data not shown).