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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