Flexmap3d

Galectin‐9 and CXCL10 were measured in 50 μl of serum, plasma, or eluate by multiplex assay (xMAP; Luminex). CXCL10 was measured in undiluted material. Galectin‐9 was measured in 10× diluted plasma or serum, except in the serum/plasma samples paired with dried blood spots (in which case galectin‐9 was measured undiluted from the eluate and serum/plasma). The multiplex immunoassay was performed as described previously 49. Heterophilic immunoglobulins were preabsorbed from all samples with HeteroBlock (Omega Biologicals). Acquisition was performed with a Bio‐Rad FlexMAP3D in combination with xPONENT software version 4.2 (Luminex). Data analysis was performed with Bioplex Manager version 6.1.1 (Bio‐Rad).
Between measurement of the internal and external validation cohorts in 2015, the recombinant protein for galectin‐9 was replaced, which affected the standard curve. Therefore, absolute values between these cohorts may not be comparable. Since 2015, the interassay variability has been negligible 50. All biomarker analyses were performed at the UMC Utrecht, thereby minimizing intercenter variation. Treating physicians were blinded with regard to biomarker levels, and technicians performing the multiplex assay were blinded with regard to clinical data.

Cell-bound markers were evaluated at baseline and week 24 using flow cytometry. Briefly, cryopreserved PBMCs were thawed in RPMI 20% FCS, washed using PBS, and incubated with mouse monoclonal antibodies against CD4 (fluorophore BV510; clone RPA-t4; Biolegend, San Diego, CA, USA), CD8 (fluorophore PercP-CY5.5; clone SK-1; BD Pharmingen, Franklin Lakes, NJ, USA), CD38 (fluorophore APC; clone HIT-2; eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), and HLA-DR (fluorophore BV711; clone G46-6; Biolegend, San Diego, CA, USA). Fluorescence minus one (FMO) controls were used to define positive gates for the expression of different proteins. The analysis was performed using BD FACS Diva 9.0.1. For soluble markers, a multiplex immunoassay was used, as previously described [27 (link),28 (link)]. In short, aspecific heterophilic immunoglobulins (IL-6, IL-1b, IP-10, MCP-1 MIP-1a, MIP-1b, sICAM-1, sCD14, sCD163, MIG) were pre-absorbed with HeteroBlock (Omega Chemicals, Hebron, IN, USA). Measurements were performed using a Bio-Rad FlexMAP3D in combination with xPONENT software version 4.1 (Luminex, Austin, TA, USA). Data analysis was performed using Bioplex Manager 6.1.1 (BIO-RAD).

A customized multiplexed microsphere assay to define the characteristics of both Fv and Fc domains of purified serum antibody samples was conducted as previously described method [63 (link)]. Briefly, recombinant protein antigens (Additional file 1: Table S2) were covalently coupled to fluorescently-coded magnetic microspheres, which were incubated with dilute antibody to permit antigen binding, followed by washing and characterization of Fc domains using fluorescent detection reagents that included Fc receptor (FcR) tetramers, lectins, and secondary reagents (Additional file 1: Table S2) to identify total antibody isotypes and IgG subclasses. Data was acquired on a Bio-plex array reader (FlexMap 3D, Bio-Plex Manager 5.0, Bio-Rad). The net median fluorescence intensity (MFI) was reported.

In the cross-sectional HIV-1C study, immune activation-related soluble proteins were measured in EDTA-plasma using multiplex technology (xMAP; Luminex). The multiplex immunoassay was performed as previously described^{60 (link),61 (link)}. In short, aspecific heterophilic immunoglobulins were pre-absorbed with HeteroBlock (Omega Chemicals, Hebron, Indiana). The soluble immunological parameters were CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL12 (SDF-1α), TNFR2, sCD163, CXCL10 (IP-10) and IL-7 (see Table 2). Measurements were performed with a Bio-Rad FlexMAP3D in combination with xPONENT software version 4.1 (Luminex, Austin, Texas). Data analysis was performed with Bioplex Manager 6.1.1 (BIO-RAD).