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Page 8 of 32 Noor et al. Neuroimmunol Neuroinflammation 2019;6:10 I http://dx.doi.org/10.20517/2347-8659.2019.18

calibrators (provided by the kit) or samples (100 μg protein from each experimental sample per well) were
loaded onto a “multi-spot” plate in duplicates. Each plate-well is pre-coated with antigen-specific “capture”
antibodies on independent, spatially well-defined “spots” that are in turn connected to a working electrode
surface. Following incubation with protein lysates, immobilized proteins were recognized by SULFO-
TM
TAG -conjugated antigen-specific “detection” antibodies. Samples were read using a Quickplex SQ120
Imager (MesoScale Discovery).

Preparation of Naïve CD4 T cell suspension
To further investigate whether LFA-1 contributes to T cell differentiation and their functional responses,
CD4 T cells were cultured with or without BIRT377 (500 ng/mL). A total of 20 mice (wildtype, FFID: IMSR_
JAX:000664; 10 females and 10 males, 8-10 week-old) were compared in this study. In each experiment,
5 male and 5 female mice were used, with two repeat experiments (total of 10 male and 10 female mice).
No handling occurred with these mice. Mice were sacrificed with CO asphyxiation, followed by cervical
2
dislocation. Under sterile conditions, spleens and lymph nodes (cervical, inguinal and brachial) were
collected in tubes containing ice-cold PBS with 2% fetal bovine serum (FBS; Gibco, Thermofisher Scientific,
MA, USA). Spleens and lymph nodes were disrupted using a micro-plunger to press the tissues through a
8
70 µm nylon mesh. Cells were centrifuged at 300 × g for 10 min at 4 °C and resuspended at 1 × 10 nucleated
cells/mL in PBS with 2% FBS and 1mM EDTA (ethylene diaminetetraacetic acid). Naïve CD4 T cells
TM
+
low
high
(CD4 CD44 CD62L ) were isolated using EasySep Naïve CD4 T Cell Isolation Kit, per manufacturer’s
instructions (Stemcell Technologies, BC, Canada). In this technique, non-naïve T cells were labeled with
biotinylated antibodies and magnetic particles, allowing for the collection of desired naïve T cells using an
EasySep magnet (Stemcell Technologies, BC, Canada). Live cells were counted on a hemocytometer.
TM
CD4 T cell culture with BIRT377
Our prior data demonstrated that BIRT377 (500 ng/mL) induces a switch of stimulated macrophage
(RAW264.7) from a proinflammatory bias to an anti-inflammatory state . To examine effects of blocking
[42]
LFA-1 actions on T cells in vitro, isolated male or female derived CD4 T cells were resuspended with
complete RPMI media and treated with BIRT377 (500 ng/mL). A 24-well tissue culture plate was pre-
[51]
coated with anti-CD3 antibody (10 µg/mL in sterile PBS; R&D systems), and stored overnight at 4 °C. The
tissue culture plate was pre-warmed and washed 3 × with sterile PBS before use. One million CD4 T cells
were plated per well, with 2-3 well-replicates per experimental condition. Cells were cultured with either
Th17 or Treg differentiation conditions, as described previously [64,65] , with minor modifications. Briefly,
in the presence of TCR (T cell receptor) stimulation by anti-CD3 antibody, a cocktail of Th17 polarizing
cytokines was applied as follows: anti-mouse CD28 (5 µg/mL), TGF-β1 (2.25 ng/mL), IL-1β (20 ng/mL),
IL-6 (30 ng/mL), IL-23 (30 ng/mL), anti- IFNγ (10 µg/mL). For Treg differentiation, a cocktail containing
CD28 (2 µg/mL), IL-2 (20 ng/mL), and TGF-β1 (5 ng/mL) was added to the cells. BIRT377 treatment
(500 ng/mL) was applied simultaneously with the Th17 or Treg cytokine T cell stimulation mixtures
throughout the culture timecourse (4 days). Anti-CD3, anti-CD28, and anti-IFNγ were purchased from
eBioScience (Thermofisher Scientific, MA, USA), and TGF-β1, IL-1β, IL-6, IL-23 and IL-2 were purchased
form PeproTech (NJ, USA). Pooled CD4 T cells from naïve mice (5 male or 5 female mice maintaining sex-
separate tubes) were used for two independent experiments, followed by flow cytometry procedures similar
to that detailed in prior reports [66-69] .

Intracellular staining and flow cytometry
On Day 4 of T cell differentiation culture, cells were collected, washed with 1 × PBS (300 × g, 5 min, 4 °C),
kept on ice to prevent cytokine secretion, and stained for intracellular levels of pro- or anti-inflammatory
cytokine or transcription factor (protein) production. Proinflammatory markers included retinoid-related
orphan receptor-ɣt (RORɣt), the Th17-associated transcription factor, and the cytokines, IL-17A and TNF.
Anti-inflammatory markers included the cytokines IL-10 and TGF-β1. Cells were first stained for viability,