Analysis of soluble immune checkpoint proteins using quantitative multiplex microbead-based immunoassays

Abstract

Abstract Immune-checkpoint receptors play critical roles in immune regulations and maintenance of immune homeostasis. Recently, a series of soluble immune checkpoint proteins have been found in circulation, and are putative biomarkers in infection, cancer, and autoimmune diseases. We have developed two multiplex immuno-oncology (I/O) panels. Human I/O Checkpoint Protein Panel 1 (17-plex) consists of 17 targets: BTLA, CD27, CD28, CD40, CD80, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2. Human I/O Checkpoint Protein Panel 2 (31-plex) consists of 31 targets: 4-1BBL, CD73, APRIL, Arginase-1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, BAFF, CD25, CD30, CD40L, 4-1BB, CD226, E-Cadherin, FGL1, Gal-1, Gal-3, Granulysin, Granzyme B, IDO1, MICA, MICB, Nectin-2, Nectin-4, OX40, Perforin, CD155, Siglec-7, and Siglec-9. Using these multiplex arrays, we have simultaneously quantitated the expression levels of the above 48 key immune regulators in cancer serum samples and in cell culture supernatants. Soluble checkpoint protein signatures generated from this multiplex approach revealed differential expression of sBTLA, sCD27, sCD40, sCD86, sHVEM, sPD-1, sTIM-3, sTLR-2, sCD40L, Gal-3, Gal-1, FGL1, BAFF in the colorectal or breast cancer serum samples, compared to healthy serum controls. Heatmaps demonstrate the detection of checkpoint proteins in the conditioned media collected from human tumor cell lines and stimulated PBMCs. In summary, our results demonstrate that these two multiplex immunoassays we developed are useful research tools for the simultaneous quantitation of circulating immune checkpoint proteins, as well as for its potential application in biomarker discovery and translational research.