Patients with anti-IFN-γ autoantibodies have impaired IFN-γ signaling leading to severe disseminated infections with intracellular pathogens especially nontuberculous mycobacteria. High-titer neutralizing anti-IFN-γ autoantibodies affect the same pathway and cause a syndrome of disseminated nontuberculous mycobacterial and other opportunistic infections.2-5 These patients can have severe and progressive disease despite prolonged antimicrobial therapy which can result in drug toxicity and therapeutic failure. Rituximab is a chimeric mAb directed against human CD20 on mature B cells and plasmablasts that causes rapid and sustained depletion of circulating and tissue-based B cells.6 Diseases for which rituximab is approved by the Food and Drug Administration include B-cell lymphoma dosed Rupatadine Fumarate at 375 mg/m2 for 4 or 8 doses 6 7 or rheumatoid arthritis dosed at 1000 mg for 2 doses on day 1 and day 15.8 Rituximab has also been used off-label for the treatment of diseases caused by pathogenic autoantibodies including the anti-desmoglein 3 autoantibodies of pemphigus vulgaris 9 the anti-acetylcholine or anti-muscle-specific kinase autoantibodies of myasthenia gravis 10 11 the anti-GM-CSF autoantibodies of pulmonary alveolar proteinosis 12 and the anti-erythropoietin autoantibodies of pure red cell aplasia.13 We used rituximab in 4 patients with high-titer anti-IFN-γ autoantibodies who had progressive refractory nontuberculous mycobacterial disease despite aggressive anti-infective treatment. Our initial approach was to treat according to a lymphoma regimen given our aim of depleting B cells. Additional doses of rituximab were given for signs and symptoms consistent with infection persistence or relapse. Methods Subjects The 4 patients were seen at the National Institutes of Health and consented to evaluation and treatment of disseminated nontuberculous mycobacterial infection under protocol 01-I-0202. Control plasma and PBMCs were obtained though the National Institutes of Health Blood Bank under appropriate protocols. Clinical monitoring Patients had routine laboratories including HIV testing complete blood count with differential serum electrolytes renal and hepatic function chemistries inflammatory markers quantitative immunoglobulin levels and lymphocyte markers that included total T cells (CD3+; BD PharMingen) CD4+ T cells (Immunotech) CD8+ T cells (Immunotech) CD20+ B cells (BD PharMingen); and CD16+ or CD56+ natural killer cells (BD PharMingen). Disease activity was determined by evidence of active infection on computed tomographic scan bone scan culture or smear as indicated. Treatment and clinical data were collected by review Rupatadine Fumarate of electronic chart records. Salient clinical features of disease activity were compiled retrospectively into a scoring system. Plasma collection Plasma from each subject was collected and stored at ?80°C before during (2-9 months after start) and after treatment (1-4 years Rupatadine Fumarate after start) with rituximab. All samples were tested at first thaw. For each patient the same 3 plasma collection dates for before during and after rituximab treatment were used across experiments. For patient 2 plasma and clinical data had been collected and was studied at a time of relapse followed by retreatment. Determination of IFN-γ autoantibody titers Relative titers of IFN-γ autoantibody were determined by performing serial 10-fold dilutions of plasma and measuring Rupatadine Fumarate IFN-γ autoantibody levels by a particle-based technique as previously described.14 Briefly magnetic beads (Bio-Rad) conjugated to recombinant human IFN-γ (R&D Systems) were incubated 1 hour with subject or control plasma washed and CCR5 incubated with mouse anti-human IgG conjugated to PE (eBioscience). The beads were run on the Bio-plex (Bio-Rad) instrument and the fluorescence intensity was plotted at each dilution to generate titration curves for each patient (GraphPad Prism Version 5.0c). For all patients a dilution factor of 1 1:5000 yielded a fluorescence intensity within the dynamic range of the assay and was chosen as a standard dilution factor to test Ab levels. First-thaw aliquots of plasma for each patient from each date were tested ≥ 3 times at 1:5000 dilution with error bars.