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CCR2 on CD14+CD16+ Monocytes Is A Biomarker of HIV Associated Neurocognitive Disorders
Dionna W. Williams, PhD, Desiree Byrd, PhD, Leah H. Rubin, PhD, Kathryn Anastos, MD, Susan Morgello, MD, Joan W. Berman, PhD
APPENDIX e-1
Supplementary Material:
Cell Isolation:
Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) and density gradient centrifugation within four hours of blood draw.
Monocyte Identification by Flow Cytometry:
PBMC were analyzed by flow cytometry using fluorochrome coupled monoclonal antibodies specific for human CD14 (clone M5E2), CD16 (clone 3G8), CD3 (clone HIT3a), CD19 (clone HIB19), CD56 (clone B159), CD66b (clone G10F5), HLA-DR (clone G46-6) or corresponding isotype matched negative control antibodies (all from BD Biosciences, San Jose, CA). Antibodies were titrated to determine optimal concentrations for staining. PBMC (1-3 x 105) were stained with the appropriate antibodies and fixed with 2% paraformaldehyde prior to acquisition with a BD Canto II flow cytometer. FlowJo software (TreeStar v 10.0.6, Ashland, OR) was used for analyses. Forward and side scatter were used to determine monocyte gating. Monocytes were defined as CD14 and HLA-DR positive and CD3, CD19, CD56, and CD66b negative. Monocytes were then discriminated according to CD14 and CD16 expression. The monocyte subsets were identified as CD14+CD16-, CD14+CD16+, and CD14lowCD16+.
CCR2 Analysis by Flow Cytometry:
PBMC (1-3 x 105) were washed once with FACS buffer (PBS (Gibco, Grand Island, NY) supplemented with 1% BSA (Thermo Scientific, Waltham, MA)). Additional blocking was not necessary. To determine CCR2 expression, the cells were stained using a fluorochrome coupled monoclonal antibody specific for human CCR2 (0.125 μg/μL/test, clone 48607, R&D Systems, Minneapolis, MN) for 5 minutes, covered, on ice. The corresponding isotype matched antibody was used as a negative control. Following addition of the CCR2 antibody, CD14 and CD16 fluorochrome coupled monoclonal antibodies were added to the PBMC and the cells stained, covered, on ice for an additional 30 minutes. After staining the cells were washed with FACS buffer, fixed with 2% paraformaldehyde, and stored covered at 4°C. The samples were acquired within two weeks of staining. CCR2 surface expression was analyzed on each monocyte subset using FlowJo software (TreeStar v 10.0.6, Ashland, OR) using the monocyte gating strategies indicated above.
The order in which the antibodies are added is critical for CCR2 staining. Optimal results are obtained when adding the CCR2 antibody prior to any others as it may be “outcompeted” by the additional antibodies resulting in diminished expression 6. Additionally, it is important to add the proportionate amount of antibody when deviating from the listed cell number per test. The intensity of the CCR2 signal may become quenched when staining more than the recommended number of cells.
Transmigration Assay Across the BBB Model
An in vitro model of the human BBB was used to assay monocyte transmigration. This model consists of coculturing human brain microvascular endothelial cells (Applied Cell Biology Research Institute, Kirkland, WA) and human cortical astrocytes (obtained with an ongoing protocol established at the Albert Einstein College of Medicine) on opposing sides of a tissue culture insert with 3 μm pores as previously described 7-9.
PBMC (4 x 105) from HIV seropositive individuals were added to the top of each tissue coculture insert to assay transmigration. The cells migrated across the BBB model in response to media alone or CCL2 (200 ng/mL, Invitrogen, Grand Island, NY) located at the bottom of the coculture chamber. Each transmigration condition was performed with 4 replicate cocultures. After 24 hours, the cells that transmigrated were collected from the bottom of the chamber, immunostained for CD14 PE, CD16 PE-Cy7, and CCR2 APC, fixed with 2% paraformaldehyde, and quantified by flow cytometry. Dose response and kinetic analyses were performed to determine the optimal concentration of chemokine and the duration of the transmigration assay.
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Figure e-1: Gating Strategy for Identification of Monocyte Subsets.
PBMC from a representative HIV seropositive individual was analyzed by flow cytometry. (A) Forward and side scatter characteristics were used to determine identify monocytes from the other PBMC populations. (B) Isotype matched negative control antibodies were used to determine appropriate gating of the monocyte subpopulations. Mouse IgG2a (blue) and IgG1 (red) irrelevant control antibodies were used for the determination of CD14 and CD16 positivity, respectively. Using the indicating gating strategy, the three monocyte subsets (green) were identified as CD14+CD16-, CD14+CD16+, and CD14lowCD16+.
Table e-1. Demographic, immunovirologic, and neurocognitive characteristics of HIV infected participants.
MHBB(n=27)
Mean ± SD (%) / WIHS
(n=33)
Mean ± SD (%)
Patient Demographics
Age, years / 55 ± 7 / 49 ± 7*
% Female / 50 / 100*
% Black/Hispanic / 82 / 94
Immunovirologic Information
CD4+ T-Cell Count, cells/μL / 479 ± 329 / 547 ± 309
Plasma HIV RNA, log copies/mL / 2.30 ± 1.45 / 2.22 ± 1.29
% Undetectable Viral Load / 79 / 73
% on cART / 100 / 78
% HCV/HBV Seropositive / 32 / 24
Cognitive Information
% No Cognitive Impairment / 29 / 27
% HAND/Cognitive Impairment / 71 / 73
% ANI / 12 / Not determined
% MCMD / 65 / Not determined
% HAD / 24 / Not determined
Note: Superscript asterisk indicates significance difference (p<0.001, T test) between the cohorts for the indicated demographic.
Table e-2. Neuropsychological test battery and normative data.
Neuropsychological Domain and Tests / Normative Data SourcesAbstraction/Executive Functioning
Wisconsin Card Sorting Task-64 Item Version
Trail Making Test (Part B) / 10 A,B
11 A,B,C
Attention/Working Memory
WAIS-III Letter Number Sequencing
PASAT Total Correct / 12 A
13 A,B,C,D
Learning
Hopkins Verbal Learning Test-Revised (Total Recall)
Brief Visuospatial Memory Test-Revised (Total Recall) / 14 A
15 A
Delayed Recall
Hopkins Verbal Learning Test (Delayed Recall Trial)
Brief Visuospatial Memory Test-Revised (Delayed Recall Trial) / 14 A
15 A
Motor
Grooved Pegboard Time (Dominant Hand)
Grooved Pegboard Time (Non-Dominant Hand) / 11 A,B,C
Speed of Information Processing
WAIS-III Digit Symbol
WAIS-III Symbol Search
Trail Making Test (Part A) / 12 A
11 A,B,C
Verbal Functioning
Controlled Oral Word Association Test (F-A-S) / 16 A,B,D
Note: Wechsler Adult Intelligence Scale (WAIS); Paced Auditory Serial Arithmetic Test (PASAT). Normative data corrects for the following demographic characteristics indicated by superscript letter: A. Age; B. Education; C. Gender; D. Ethnicity