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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.
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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
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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+.
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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
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% HAD 24 Not determined
Note: Superscript asterisk indicates significance difference (p<0.001, T test) between
the cohorts for the indicated demographic.
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Table e-2. Neuropsychological test battery and normative data.
Neuropsychological Domain and Tests Normative Data Sources
Abstraction/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
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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
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