Early HIV cohort:

1. 37 HIV-1 infected subjects of the Core 01/Phaedra cohort recruited from Sydney and Melbourne 30 laboratory workers recruited as controls Chronic HIV cohort: Frozen PBMC samples from 32 matched HIV-1 infected subjects: (PBMC collected approx. 21 months after Early sample) (HIV negative or control cohort): Frozen PBMC from 30 HIV- untested laboratory donors Early HIV cohort: Frozen PBMC samples from all 37 HIV-1 infected subjects; (77% of subjects within 6 months of seroconversion) Supplementary FIG 1 Legend. Sample selection for HIV-1 infected and HIV uninfected subjects. Subjects enrolled in the Core 01/Phaedra cohort were antiretroviral therapy naïve subjects who were recruited between the years of 2002-2007. PBMC samples were stored frozen in liquid nitrogen. PBMC from subjects who served as HIV negative controls were stored frozen in liquid nitrogen for at least one week prior to thawing and phenotype staining. Samples with high proportions of dead cells in flow cytometry analysis were excluded from further study. Each sample was subjected to phenotypic and functional studies. * On average, subjects had 2.7 x 105 doublet excluded, live cells within the lymphocyte gate. Subjects with fewer than 65,000 live cells within the lymphocyte gate were excluded from further analysis. Each PBMC sample was split into two aliquots Each PBMC sample was split into two aliquots Each PBMC sample was split into two aliquots 31 subjects with sufficient viable cells * studied for T cell and NKT cell phenotype 30 subjects (26 matched) with sufficient viable cells * studied for T cell and NKT cell phenotype 27 subjects with sufficient viable cells studied for T cell and NKT cell phenotype 28 subjects with sufficient NKT cells studied for NKT cell subset phenotype 21 subjects (20 matched) with sufficient NKT cells studied for NKT cell subset phenotype 27 subjects with sufficient NKT cells studied for NKT cell subset phenotype 27 subjects with sufficient viable cells recovered after in vitro culture 25 subjects with sufficient viable cells recovered after in vitro culture 28 subjects with sufficient viable cells recovered after in vitro culture 20 subjects with sufficient gated NKT events after a-GalCer activation were studied for intracellular cytokines 15 subjects with sufficient gated NKT events after a-GalCer activation were studied for intracellular cytokines 25 subjects with sufficient gated NKT events after a-GalCer activation were studied for intracellular cytokines

2. a c b Supplementary FIG 2 Legend. Titration of CD1d:Ig dimer (Dimer X) loaded with KRN7000 (a-galactosylceramide) using PBMC of healthy controls. PBMC from 4 healthy controls were activated with the above serial dilutions of Dimer X loaded with a-GalCer in a 6-hour activation assay, with the addition of brefeldin A for the duration of activation. (a) Frequency of NKT cells within the T cell population over different ligand concentrations. (b and c) Frequency of total NKT cells expressing IFNg or CD107a, respectively. Frequencies of each donor are background subtracted from their respective control PBMC stimulated with unloaded Dimer X. CD1d:Ig loaded with a-GalCer (ng/ml)