Poster Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Determining the neutralising capabilities of anti-cytokine antibodies on STAT phosphorylation by flow cytometry using patient serum and donor cells. (24549)

Samuel Neeling 1 , Christine Bundell 1 2 , Andrew McLean-Tooke 1 3 4
  1. PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, Western Australia, Australia
  2. University of Western Australia, School of Biomedical Sciences, Nedlands, Western Australia, Australia
  3. Clinical Immunology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia
  4. Clinical Immunology, Perth Children's Hospital, Nedlands, Western Australia, Australia

Background: Autoantibodies against interferon gamma (IFNg) and granulocyte macrophage colony stimulating factor (GM-CSF) cytokines are a known cause of adult onset immunodeficiency. These antibodies can be detected in serum by enzyme linked immunosorbent assay (ELISA), however, this method does not determine the neutralising capacity of the antibody. The current method for assessing neutralising anti-cytokine antibodies is by detecting signal transducer and activator of transcription phosphorylation (pSTAT) in monocytes following cytokine stimulation using flow cytometry. This is performed using patient blood cells in the presence or absence of autologous serum to determine if antibodies present in the serum neutralise the cytokine thereby preventing activation and pSTAT expression. Logistics for collection and testing of patient samples limits the utility of the current confirmatory approach to assess the neutralising capability.

Approach: We aimed to optimise a method assessing neutralising effect of anti-cytokine antibodies in patient serum using cryopreserved healthy donor peripheral blood mononuclear cells (cPBMCs). pSTAT1 and pSTAT5 expression in cPBMCs after IFNg or GM-CSF stimulation was evaluated, and the minimum cytokine concentration required was determined. Mean fluorescence intensity of intracellular pSTAT expression was measured in cPBMCs using commercial anti-STAT1 or STAT5 PE conjugated antibody (BD Biosciences). This was performed after stimulation with the relevant cytokine in the presence of anti-cytokine antibody positive or negative control serum.

Results: cPBMCs incubated with negative control serum expressed normal pSTAT after stimulation. In contrast, pSTAT expression was not detected in cPBMCs after stimulation when cells were incubated with anti-cytokine antibody positive serum.

Discussion: This confirms that the neutralising activity of anti-cytokine antibodies can be determined without the use of autologous patient cells. An optimised method using patient serum alone is possible for a routine flow cytometry laboratory.