“The following small study describes a new way of screening for neutralizing antibodies, or NABs, to interferon beta. When interferon beta binds to its receptor on cells it causes changes in the cell that can be detected using a new technique called phosphoflow. If you have have NABs that stop interferon-beta binding to its receptor on the cell surface you stop the changes occurring in the cell. Will this replace NAB testing? Unlikely as this technique will need to be validated and we will need to optimise it to give us a level of antibody. The study does however highlight the issue of NABs and it should remind you to ask your neurologist if you are on interferon beta to ask your neurologist to test for NABs. If you are NAB positive you are more likely to have MRI activity, relapses and disease progression compared to subjects who are NAB negative.”
Gavasso et al. Deficient phosphorylation of stat1 in leukocytes identifies neutralizing antibodies in multiple sclerosis patients treated with interferon-Beta. PLoS One. 2014 Feb 19;9(2):e88632. doi: 10.1371/journal.pone.0088632. eCollection 2014.
BACKGROUND: Anti interferon-beta (IFN-β) neutralizing antibodies (NAb) affect efficacy of treatment of MSers, but exactly when the detrimental effects of NAbs offset therapeutic efficacy is debated. Quantification of intracellular pathway-specific phosphorylation by phospho-specific flow cytometry (phosphoflow) is a promising tool for evaluation of these effects in primary immune cells from treated patients at the single-cell level.
METHOD: Samples for phosphoflow and gene expression changes were collected before administration of IFN-β and at four, six, and eight hours thereafter. MSers were NAb negative (n = 3) or were NAb positive with low/medium (n = 1) or high (n = 2) NAb titres. Levels of phosphorylation of six Stat transcription factors (pStat) in seven cell subtypes and expression levels of 71 pathway-specific genes in whole blood were measured. The data was subjected to principal component analysis (PCA), fifty-fifty MANOVA, ANOVA, and partial least square regression (PLSR).
RESULTS: PCA of pStat levels clustered MSers according to NAb class independently of time. PCA of gene expression data clustered MSers according to NAb class but was affected by time and treatment. In the fifty-fifty MANOVA, NAb class was significant for both pStat levels and gene expression data. The ANOVA identified pStat1 protein in several cell subtypes as significantly affected by NAb class. The best fitting model for NAb prediction based on PLSR included pStat1 in monocytes, T cells, or lymphocytes and pStat3 in monocytes (r = 0.97). Gene expression data were slightly less predictive of NAb titres.
CONCLUSION: Based on this proof of concept study, we hypothesize that NAb effects can be monitored by evaluation of a single biomarker, pStat1, in either monocytes or T cells by phosphoflow directly after IFN-β administration. The method will significantly reduce cost relative to labor intensive in vitro methods and offers a patient-specific approach to NAb evaluation.
“The following is a scientific description of the phosphoflow method for NAB detection; for most of your this will be gibberish. Please don’t feel obliged to read it. The diagram shows the times and type of sample collection before and after IFN-β administration and sample processing and analysis. Concentrations of NAbs (against IFN-β) and IFN-β were measured in blood using a standard NAB assay. mRNA used for gene expression measurements was extracted from whole blood. For signalling pathway analysis by phosphoflow, immune cell subtypes were identified and phosphorylation levels of various Stat proteins quantified in single cells. If present in sera, NAbs prevent the initiation of the IFN-β signal at the cell surface. In the absence of NAbs IFN-β binds to its cognate receptor and forms an activated receptor complex with associated kinases that phosphorylate Stat proteins at specific residues. The Jak/Stat pathway is the major signalling pathway activated by IFN-β.”