Chronic Lymphocytic Leukemia Coverage from Every Angle

Using a Whole-Blood Loop Assay to Characterize Immune Response to Rituximab

By: Lauren Harrison, MS
Posted: Monday, February 1, 2021

A group of researchers in Sweden built a disease-specific cytokine-release syndrome and immune activation profiling ex vivo system using blood from patients with chronic lymphocytic leukemia (CLL). The team utilized a human whole-blood “loop” system to study patient-specific immune responses to rituximab. Sara M. Mangsbo, PhD, of Uppsala University, and colleagues published their work in International Immunopharmacology.

The team recruited six patients with CLL and five age-matched healthy volunteers to donate blood before receiving therapy. Blood from each patient was incubated for 4 hours with rituximab and was monitored for the development of cytokine release, complement activation, and B-cell depletion using a whole-blood loop assay. Flow cytometry; cell counting; and cytokine, complement, and proteomic analyses were performed as well.

Incubation with rituximab induced release of cytokines from natural killer cells in blood from patients with CLL, but not in healthy patients. This cytokine release reflected potential cytokine-release syndrome, with increased release of interferon-γ, tumor necrosis factor-α, interleukin-8, and interleukin-6 levels. Additionally, rituximab was found to induce complement activation in blood samples from patients with CLL. The levels of C3 increased 4.5-fold to 5.8-fold in patients with CLL after rituximab, and C5 levels increased 1.3-fold to 2-fold, which was not seen in healthy controls. Patients with CLL had higher white blood cell counts than healthy controls before rituximab incubation. CLL samples saw a 22.1% drop in white blood cell counts after rituximab compared with 3.0% in healthy samples.

Researchers investigated the killing mechanism of rituximab in patients with CLL with their loop system using blocking agents of complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Blockade of both cytotoxic profiles resulted in skewing of the immune-killing mechanism. There was also downregulation of CD16 (an activating protein) on natural killer cells after rituximab infusion. Cell killing–associated proteins were noted to be found in increased levels in the plasma of blood treated with rituximab.

Disclosure: For a full list of authors’ disclosures, visit

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