These percentages did not switch significantly at the time of C-IRIS event for those who developed C-IRIS (Fig. S3B). CD4+ T-cell counts fold-increase on ART in non-C-IRIS but not C-IRIS patients. Higher frequencies of activated monocytes (CD14+CD86+ or CD14+HLA-DR+; p0.038) were also observed in C-IRIS compared to non-C-IRIS patients and those who failed to clear cryptococci from cerebrospinal fluid pre-ART had higher levels of activated monocytes (CD14+HLA-DR+, p=0.017) compared to those who cleared. In multivariate regression, CD14+HLA-DR+ monocytes were independently associated with C-IRIS (HR=1.055 [1.013-1.098]; p=0.009). Conclusion In contrast to non-C-IRIS patients, C-IRIS patients displayed a lack of association between proportions of IL-7R+ T-cells and several markers of T-cell homeostasis. They also exhibited higher monocyte activation linked to CSF cryptococcal culture positivity pre-ART. These data suggest a role for IL-7/IL-7R signaling pathway dysregulation in the pathogenesis of C-IRIS, possibly linked to monocyte activation and residual pathogen burden pre-ART. strain and then performing an alkaline extraction, as explained 23. ACA include a broad array of antigens, including mannoproteins. The pellet was suspended in 300 l of 20 mM Tris-buffered saline (TBS) made up of a protease inhibitor cocktail of serine, cysteine and metallo-proteases inhibitors (Roche Diagnostic, Boston, USA). Protein concentration Jatrorrhizine Hydrochloride of the Jatrorrhizine Hydrochloride preparations was decided using the bicinchoninic acid. Cells were stimulated using a final concentration of 10 g protein/mL. Circulation cytometry Cryopreserved PBMCs were thawed in RPMI 1640 (Sigma-Aldrich, Johannesburg, South Africa) supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 100 g/mL streptomycin sulfate, FANCE and 1.7 mM sodium glutamate. After 2 hours of resting at 37C in a 5% CO2 incubator, half a million viable cells were aliquoted in a volume of 200 L per well in a 96-well plate. Cells were washed in phosphate-buffered saline (PBS) and then incubated with fixable near infra-red (NIR) staining dye on APC-Cy7 (BioLegend Inc., California, USA) for lifeless cell exclusion for 30 minutes. Cells were then stained with the following antibodies, all from BD Biosciences (California, USA) unless normally indicated: anti-CXCR3 (clone FUN-1) -BV421, anti-CD27 (clone M-T271) (BioLegend) -BV510, anti-CD45RA (clone 5H9) -quantum dot (Qdot)605 (Invitrogen, California, USA), anti-CD4 (clone SK3) -BV711, anti-CD127 (clone M-A251) -BV786, anti-CD8 (clone MAb11) -Alexa F488, anti-PD-1 (clone RPA-T8) -PerCP-Cy5.5, anti-CD25 (clone SK7) -PE, anti-CD3- (clone 5344.111) -PE-CF594, anti-CCR6 (clone G46-6) PE-Cy7 and anti-CCR7 (clone 150503) -Alexa F 700. Cells were then washed and fixed (Perm/fix Medium A, Invitrogen). Separately, one million viable cells/well were stimulated with staphylococcus enterotoxin B (SEB) and lipopolysaccharide (LPS) (both from Sigma-Aldrich) as positive control Jatrorrhizine Hydrochloride at a concentration of 1 1 g/mL each for 4.5 hours (to avoid downregulation of the CD14 molecule by LPS), CMP/ACA at a concentration of 10 g/mL each for 18 hours in 5% CO2 at 37C. Unstimulated unfavorable control (NC) and fluorescence minus one (FMO) control wells were also added. Co-stimulatory antibodies, CD28 and CD49d (1 g/ml each; BD Biosciences) were added to each well. Brefeldin A (BioLegend) was also added to each well after 1 hour of incubation. Cells were surface stained with: anti-CD86 (clone FUN-1) -Amazing violet (BV)421, anti-CD38 (clone HIT2) -BV510, anti-CD14 (clone M5E2) -BV605, anti-CD134 (clone Take action35) -BV650, anti-CD4 (clone SK3) -BV711, anti-CD8 (clone M-A251) -BV786, anti-PD-1-PerCP-Cy5.5 (clone RPA-T8), anti-CD25 (clone SK7) -phycoerythrin (PE), anti-CD16 (clone 3G8) -PE-Cy5, anti-HLA-DR (clone G46-6) PE-Cy7 and anti-CD3 (clone SK7) Jatrorrhizine Hydrochloride -Alexa F 700. Subsequently, PBMCs were washed, fixed (Perm/fix medium A, Invitrogen), permeabilized (Perm/fix Medium B, Invitrogen) and intracellularly stained with anti-TNF- (clone MAb11) -Alexa F488, anti-IL-2 (clone 5344.111) -PE-CF594 and anti-IFN-(Fig. S1). There was no correlation between proportions of T-cells expressing IL-7R and plasma IL-7 Jatrorrhizine Hydrochloride levels pre-cART in both non-C-IRIS and C-IRIS patients (Fig. S2) as would have been expected from previous studies 27,28. We next explored whether there was an association between percentage of IL-7R+ CD4+ T-cells or plasma IL-7 levels pre-cART and pre-cART CD4+ T-cell count or fold-increase in CD4+ T-cell.