SUPPLEMENTARY MATERIAL
CD300a identifies a CD4+ memory T cell subset with a higher susceptibility to HIV-1 infection
Joana Vitallé, Laura Tarancón-Díez, María Reyes Jiménez-Leon, Iñigo Terrén, Ane Orrantia, Cristina Roca-Oporto, Luis López-Cortés, Ezequiel Ruiz-Mateos, Olatz Zenarruzabeitia& Francisco Borrego.
SUPPLEMENTARY METHODS Subjects and samples
In this work, freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors (n=8) and naïve for combined antiretroviral therapy (cART) HIV-1 infected patients (n=6) were studied. Whole blood samples from HIV-1 infected patients were collected from patients attending to the Infectious Diseases Unit at Virgen del Rocío University Hospital in Seville (Spain) and buffy coats from healthy donors were obtained from the Regional Center of Blood Transfusion of Seville. The study was approved by the Basque Ethics Committee for Clinical Research (PI2014017 and PI2013108) and the Virgen del Rocío University Hospital Ethics Committee for Research (15/2009). All subjects provided written and signed informed consent in accordance with the Declaration of Helsinki. Clinical data of HIV-1 infected subjects are described in Supplementary Table 1.
Laboratory measurements
Plasma HIV-1 RNA levels were measured using quantitative PCR (Cobas Ampliprep/Cobas TaqMan HIV-1 test; Roche Molecular Systems, Basel, Switzerland) with a detection limit of 20 HIV-RNA copies/ml. Absolute numbers of CD4+ T cells were determined with an Epics XL-MCL flow cytometer (Beckman-Coulter).
Cell isolation and cell culture
PBMCs from all subjects were isolated by a density gradient. Cell preparation tubes (CPT) (BD, Vacutainer®) were used to obtain cells from untreated HIV-1 infected patients and Ficoll-Paque (Sigma-Aldrich) was utilized for healthy donors. Then, PBMCs were counted and CD4+CD45RA- (CD4+RA-) T lymphocytes were enriched with the Memory CD4+ T cell Isolation Kit (Miltenyi Biotec) following the manufacturer’s protocol. Enriched CD4+RA- T cells from both healthy donors and infected patients were cultured at a concentration of 106 cells/ml in culture flasks of 25 cm2 in R10 medium (RPMI 1640 containing 10% of Fetal Bovine Serum, 1% GlutaMax and 1% of penicillin/streptomycin). Afterwards, cells were activated with 5 µg/ml of
phytoheamagglutinin (PHA) (Sigma-Aldrich) at 37 ̊C and 5% CO2 for 24 hours (Supplementary Figure 2). In three healthy donors, the sorting of CD300a+ and CD300a- subsets from CD4+RA- T lymphocytes was carried out before the activation with PHA, utilizing the FACSAria Flow Cytometer using FACS Diva software (BD Biosciences).
In this set of experiments, cells were cultured in 24 well plates in the same conditions. As a negative control an uninfected condition was included for each experiment. As CD4+RA- T cells from patients were already infected in vivo with HIV-1, in order to achieve a suitable number of infected cells for our analysis, after the activation with PHA cells were washed with R10, rested overnight and expanded with 10 ng/ml of interleukin (IL)-2 (R&D Systems) for 13 days. Regarding CD4+RA- T cells from healthy donors, after the activation with PHA, cells were washed and infected overnight with HIV-1 (BaL) (NIH AIDS Reagent Program [https://www.aidsreagent.org/index.cfm]) at a MOI of 200:1. Lastly, cells were incubated with 10 ng/ml of IL-2 in the presence of the virus for 7 days (Supplementary Figure 2). In all cases, the R10 medium with IL-2 was renewed each 3 days.
Flow cytometry
Flow cytometry-based procedures were performed in order to study the cell populations of interest at different time points. Specifically, regarding CD4+RA- T cells from HIV-1 infected patients the percentage of CD300a+ cells within HIV-1 infected cells was analyzed at day 7 and day 13 of incubation (Supplementary Figure 2). The percentage of HIV-1 infected cells at day 3 and day 7 and the expression of CCR5, HLA-DR, CD38 and PD1 receptors at day -2, day 3 and day 7 were analyzed within CD300a+ and CD300a- CD4+RA- T cells from healthy donors (Supplementary Figure 2). For that, cells were first stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Thermofisher) to detect dead cells following the manufacturer’s protocol, washed with PBS with 2%
Bovine Serum Albumin (BSA) and then stained for 30 min at 4 ̊C in the dark with fluorochrome-conjugated monoclonal antibodies (mAbs) to determine the expression of surface receptors. The mouse anti-human mAbs utilized for the extracellular staining were PerCP-Cy5.5 anti-PD1 (clone EH12.1), APC anti-CD38 (clone HIT2), BV421 anti- CCR5 (clone 2D7) and BV510 anti-HLA-DR (clone G46-6) from BD Biosciences; PE- Cy7 anti-CD4 (clone RPA-T4) from Thermofisher and PE anti-CD300a (Clone E59.126)
from Beckman Coulter. After the staining of surface receptors, cells were washed with PBS with 2% BSA and were fixed and permeabilized using Cytofix/Cytoperm Plus Kit (BD Biosciences) following manufacturer’s instructions. Then, they were incubated for 30 min at 4 ̊C in the dark with a FITC-conjugated anti-p24 mAb (clone KC57, Beckman Coulter) for the detection of HIV infected cells. Lastly, stained cells were washed again, resuspended in 250 µl of PBS and acquired with the FACS Canto II flow cytometer using FACS Diva software (BD Bioscience).
Statistical analysis and data representation
Data obtained from the flow cytometer were analyzed utilizing FlowJo software (version 10.0.7) (Treestar, Ashland, OR) and graphical representation and statistical analysis were carried out with GraphPad Prism software (version 6.01). Due to the sample size, the non- parametric Wilcoxon matched-pairs test was used for all the analysis. Correlations were assessed using the Spearman’s rho correlation coefficient.
SUPPLEMENTARY MATERIAL
Supplementary Table 1. Clinical data HIV-1 infected patients
Patient Sex
Age
(years) CD4+ T cells/mm3
Viral load (RNA copies/ml)
VIH-1 Male 32 612 4,350
VIH-2 Male 57 524 2,390
VIH-3 Male 32 495 33,600
VIH-4 Female 53 588 9,310
VIH-5 Male 49 252 19,100
VIH-6 Male 37 393 26,800
Supplementary Figure 1. Gating strategy used for the identification of CD300a- and CD300a+ subsets. First, previously purified CD4+RA- T cells were electronically gated according to forward and side scatter parameters and single cells were selected based on their area and height. Then, dead cells were discarded gating the negative cells for the viability marker. Finally, CD300a- and CD300a+ subsets were selected.
Supplementary Figure 2. Schematic representation of cell culture protocol for memory CD4+RA- T cell activation, HIV-1 infection and expansion. (A) PBMCs from naïve for cART HIV-1 infected patients were freshly isolated by density gradient and CD4+RA- T cells were purified by negative selection (day -2). Then, cells were stimulated with PHA for 24 hours, rested in R10 overnight and then they were cultured during 13 days with IL-2 in order to expand HIV-1 infected cells. The medium with IL-2 was renewed each 3 days and samples were analyzed by flow cytometry at day 7 and day 13. (B) PBMCs from healthy donors were freshly isolated by density gradient and CD4+RA- T cells were purified by negative selection (day -2). Afterwards, cells were stimulated with PHA for 24 hours, and then they were infected with HIV-1 (BaL) overnight and cultured during 7 days with IL-2. An uninfected condition was included for each experiment as a negative control. The medium with IL-2 was renewed each 3 days and samples were analyzed by flow cytometry at day -2, day 3 and day 7.
Supplementary Figure 3. Percentage of CD300a+ and HIV-1 infected memory CD4+RA- T cells at different time points. Before-after graphs representing the percentage of CD300a+ (A) and p24+ (B) CD4+RA- T cells from healthy donors before activation and infection (day -2) and at day 3 and day7. Each dot represents a subject.
**p<0.01.
Supplementary Figure 4. Correlation between the percentages of HIV-1 infected memory CD4+RA- T cells with CCR5 and activation markers expressing cells.
Correlations between the percentage of p24+ cells at day 3 after infection with the percentage of CCR5+, CD38+, HLA-DR+ and PD1+ cells at day 3 within CD300a- and CD300a+ CD4+RA- T cells from healthy donors.