SUPPLEMENTAL METHODS
Virus Binding and Infection Conditions
MYXV virion binding to cells was carried out by incubating resting or activated human T
cells with vMyx-Venus/M093L (created by fusing the GFP variant “Venus” protein in
frame to the N-terminus of the M093 protein constituent of the virus particle19). MYXV
infections were performed by incubating human resting or activated T cells with vMyxGFP (a MYXV construct that expresses virus-encoded eGFP at early and late times after
infection,17) or vMyx-GFP/TrFP (a recombinant vMyx that expresses both green
fluorescent protein (GFP) at early and late times plus tomato red fluorescent protein
(TrFP) expressed only at late times, as previously described.18) For this latter virus, the
earliest stages of virus replication can be monitored by GFP expression, and the
progression to the late times of viral infection can be monitored by the dual expression of
both GFP and TrFP. For binding protocols, T cells were adsorbed with Venus-tagged
virus at a multiplicity of infection (MOI) of 10 for one hour on ice to allow virus binding but
not entry. Unbound virus was washed twice with ice-cold 1x-phosphate-buffered saline
(1x-PBS) + 5% FBS. Venus-tagged virus binding was evaluated using flow cytometry.
For virus infection, T cells were exposed to GFP- or GFP/TrFP-tagged virus (live virus)
or either heat-inactivated vMyx-GFP, or UV-inactivated vMyx-GFP at MOI=10 for 1 hour,
at room temperature, to allow virus adsorption. Inactivated vMyx-GFP was prepared by
exposing live vMyx-GFP to UV light for 2 hours (UV-inactivated) or incubating the virus
at 55°C for 2 hours (heat- inactivated).11
For both binding and infection, mock-treated cells were incubated in complete media
containing no virus under the same incubation conditions. Virus infection was evaluated
after 72 or 96 hours of culturing at 37oC and 5% CO2, using fluorescence microscopy
1
and flow cytometry. Unless otherwise indicated, the viruses used were purified using a
36% sucrose cushion. Virus infections were performed in complete 1x-RPMI 1640
culture media (Corning Cellgro, Manassas, VA) supplemented 15% fetal bovine serum
FBS (Atlanta Biologicals, Flowery Branch, GA), 2mM L-glutamine, and 100 Units/mL of
penicillin-streptomycin (Corning Cellgro, Manassas, VA). For flow cytometry at least
100,000 events were acquired in a FACS LSRII flow cytometer (BD Biosciences) and
data were analyzed using FSC Express version 4 (De Novo software).
Cell Lines, Antibodies, and Flow Cytometric Analyses
The human multiple myeloma cell line, U266 was obtained from American Type Culture
Collection (ATCC ® TIB-196TM) and was maintained in complete 1x-RPMI 1640 culture
media. The antibody panel for the in vitro analyses of surface proteins includes Brilliant
Violet 605TM anti-CD3 (clone OKT3), PE/Cy7 anti-CD4 (clone RPS-T4), or PE anti-CD4
(clone SK3), PerCP/Cy5.5 anti-CD8a, (clone RPA-T8), Alexa Fluor®647 anti-FoxP3
(Clone 206D) PE anti Helios (clone 22F6), PercP/Cy5.5 anti CD25 (clone M-A251), PE
anti GATA3 (clone 16E10A23), Pacific BlueTM anti-Tbet (Biolegend, San Diego, CA),
APC anti-CD25, (clone 2A3), PE anti-CD69 (clone FN50), APC anti-RORT (clone
AFKJS-9) (BD Biosciences), PE anti-CD138 (clone B-B4) (MACS Miltenyi Biotec Inc,
Auburn, CA). T cell viability was evaluated with Live/Dead® fixable near-IR dead cell
stain conjugated with the fluorochrome APC/Cy7, (Molecular Probes, Eugene, OR).
Analysis was performed on a LSRII flow cytometer (BD Biosciences).
ELISA, Multiplex Assays and Intracellular IFN-ɣ
Soluble IL-2 and its alpha chain receptor IL-2Rα (sCD25) levels were evaluated by
ELISA. Cytokines IFN-ɣ, IL-4, and IL-10 were quantified from cell culture supernatants in
2
a multiplex format using Luminex100 xMAP System (Austin, TX) with a milliplex® MAP kit
(Human cytokine/Magnetic bead panel) (Millipore). Intracellular IFN-ɣ was evaluated
using flow cytometry (see Supplemental Methods for details).
Staining of naturally occurring regulatory T cells (nTregs)
MNCs from PBMCs of four healthy donors were used to isolate T cells. Cells were
labeled with Cell Trace Violet (CTV) dye, and either mock-treated, or incubated with
vMyx-GFP as described elsewhere in this report. Cells were then incubated with or
without anti-CD3/CD28 beads. After 96 hours of culturing at 37oC and 5% CO2, T cells
were stained for CD4 and CD25, and then fixed and permeabilized for intracellular
FoxP3 and Helios staining as per manufacturer’s protocol (eBiolegend). Proliferation of
CD4+CD25+FoxP3+ and CD4+CD25+Helios+ was determined using flow cytometry. The
gating strategy used to assess proliferation was as follows: (1) Lymphocytes were
identified with a forward-scatter (FSC) vs. side-scatter (SSC) gate. (2) Live T cells were
identified using a CD4 vs. FSC gate. (3) CD25 was identified using a CD4 vs. CD25
gate. (4) FoxP3 or Helios was identified using CD4+CD25+ vs. FoxP3+ gate, or
CD4+CD25+ vs. Helios+ gate. (5) CTV fluorescence histograms were generated gating
on CD4+CD25+FoxP3+ or CD4+CD25+Helios+.
Intracellular staining of Tbet, GATA3 and RORt
MNCs from PBMCs of four healthy donors were used to isolate T cells. Mock- or vMyxGFP-treated T cells with or without anti-CD3/CD28 stimulation were incubated for 96
hours at 37oC and 5% CO2. Cells were first stained for CD3, CD4 and CD8, and then
fixed and permeabilized for intracellular staining of Tbet (Th1), GATA3 (Th2) or RORt
3
(Th17) using nuclear factor fixation and permeabilization buffer set as per
manufacturer’s protocol (eBiolegend).
One-Way Mixed Lymphocyte Reaction (MLR) Assays
De-identified peripheral blood or cord blood was collected from four healthy volunteer
donors (commercially obtained from Life South Community Blood Centers, Gainesville,
FL), and MNCs were separated using Ficoll as described elsewhere in this report. Cells
were resuspended to 1x106 cells/mL and labeled either responder or stimulator.
Stimulators were irradiated using 30 Gy from a Cs157 source to create replication
incompetent stimulator cells. MNCs from responder cells were either mock-treated, or
treated with vMyx-GFP (MOI=10), then 1x105 of responder cells were plated in triplicate
into empty wells, or wells containing 5x105 of irradiated stimulator cells (cell ratio of
responder:stimulator was 1:5). Likewise, 5x105 of irradiated cells were also plated in
triplicate into empty wells. Infection, and proliferation of T cells were assessed using flow
cytometry at 72 hours or 6 days after the MLR assays started. At the indicated time
points, the secretion of soluble cytokines including IL-2, IL-2R, IFN-, IL-4 and IL-10
was evaluated using ELISA or multiplex assays as described elsewhere in this paper.
Primary Human T Lymphocyte Isolation
Anonymous peripheral blood was collected from healthy donors (commercially obtained
from Life South Community Blood Centers, Gainesville, FL) and mononuclear cells
(MNCs) were separated using Ficoll. T lymphocytes were isolated from MNCs using the
EasySepTM negative selection HLA T cell enrichment kit (StemCell Technologies)
according to the manufacturer’s recommendations.
4
T Cell Proliferation Analysis
Proliferation of T lymphocytes was monitored using the cell-tracking dye Cell Trace
Violet (CTV) dye (ex. = 405 nm; em. = 450 nm). CTV diffuses into cells and is cleaved
by intracellular esterases to yield highly reactive, cell-associated fluorescent compounds.
These compounds covalently bind to intracellular amines, which result in stable and well
retained fluorescent label. Each cell division results in sequential halving of
fluorescence, and the proliferation can be monitored with flow cytometry. The
proliferation method used in this study, allowed determination of precursor cell frequency
(this is the proportion of cells in the starting population that are able to proliferate in
response to an stimulus), or the fraction of cells in an experiment that underwent
division. The percentages of proliferation, the proliferation index, and the division index
were determined using flow cytometric analysis (FCS-Express software). Isolated human
CD3+ T cells were labeled with CTV dye as per manufacturer’s recommendations
(CellTraceTM violet cell proliferation kit, Invitrogen). Briefly, about 1x107 cells were
suspended in 1xPBS and CTV stock solution was added to a final concentration of 5 M.
Cells were incubated at 37oC and protected from light for 20 minutes. Unbound dye was
quenched by diluting with 5 volumes of complete culture medium, and incubating for 5
minutes, followed by one washed with tat medium. Next, T cells were either mocktreated, or infected with vMyx-GFP (MOI=10), and plated at 1x105 cells/well in 96-well
round-bottomed plate in complete 1x-RPMI 1640 culture media. Then, mock-treated and
MYXV-treated cells were either stimulated by incubation with Dynabeads human Tactivator -CD3/-CD28 coated microbeads (Gibco® / Life Technologies, Grand Island,
NY) at a cell to bead ratio of 1:1, or unstimulated (without adding the microbeads). Cells
were cultured in a humidified chamber at 37oC and 5% CO2, during 72 or 96 hours.
Proliferation of T cells was evaluated using flow cytometry.
5
ELISA and Cytokine Multiplex Assays
Soluble IL-2 and its alpha chain receptor IL-2Rα (sCD25) levels were evaluated by
ELISA using BD OpTEIATM human IL-2 and human IL-2Rα ELISA kits (BD Biosciences),
respectively and according to manufacturer’s recommendations. Serial dilutions of the
samples were performed in assay diluent containing PBS with 10% FBS, pH 7.0 (BD
Pharmingen). Cytokines IFN-ɣ, IL-4 and IL-10 were quantified from cell culture
supernatants in a multiplex format using Luminex100 xMAP System (Austin, TX) with a
milliplex® MAP kit (Human cytokine/Magnetic bead panel) (Millipore). When required,
samples were diluted 1:5 in complete RPMI 1640 culture media prior to analysis.
Analysis of Intracellular IFN-ɣ
Isolated T lymphocytes were stimulated with -CD3/-CD28 microbeads for 66 hours
followed by further 6 hours incubation in the presence of 2 M BD Golgi stop (BD
Biosciences), which contains the protein transport inhibitor monensin at 37oC and 5%
CO2 in complete RPMI 1640. Unstimulated controls were incubated in complete RPMI
1640 in similar fashion as the stimulated samples and 2 M of BD Golgi stop was added
during the last 6 hours of incubation. After staining surface antigens with antibodies
against human CD3 and CD4, the lymphocytes were fixed using BD Cytofix (BD
Biosciences). Fixed cells were permeabilized with1x-Perm/Wash buffer (BD bioscience)
according to manufacturer’s instructions. Intracellular staining of IFN-ɣ was performed
using the PE/Cy7 anti-human IFN-ɣ (cloneB27) (Biolegend). Data analysis was
performed by selecting lymphocytes in in a forward scatter A (FSC-A) / side scatter A
(SSC-A) dot plot. The percentage of CD3+ and CD4+expressing IFN- are reported. In
this latter case CD4+ was gated on CD3+.
6
Co-culture T Lymphocytes with U266 cells
MNCs from PBMCs of six different healthy donors were used to isolate T cells. On day
one, T cells were isolated and exposed to vMyx-GFP/TrFP (MOI=1) for 1 hour at room
temperature, unbound virus was washed as previously described in this manuscript and
right after this, 10 g/mL of AraC (+AraC) or vehicle only (-AraC) were added to the T
cells with or without -CD3/-CD28 stimulation. After 48 hours of culturing T cells +/AraC, U266 cells were added to the T cell culture at a cell ratio of 1:1 and incubated at
37oC 5% CO2 for additional 48 hours. Infection was evaluated using florescence
microscopy and flow cytometry. MM cell dead was evaluated using flow cytometry. The
gating strategy used to assess MM cell dead was as follows: (1) Lymphocytes and U266
cells identified with a forward-scatter (FSC) vs. side-scatter (SSC) gate. (2) U266 cells
were identified using CD138+ vs. FSC gate. (3) Infected MM cells were identified using
CD138+ vs GFP+ gate. (4) Likewise, non-infected MM cells were identified using CD138+
vs GFP- gate. (5) MM cell dead was identified either using CD138+GFP+ vs. Nearinfrared (IR) fluorescent gate, or CD138+GFP- vs. Near-infrared (IR) fluorescent gate.
MM cell death was analyzed using flow cytometry and Invitrogen Live/Dead Fixable near
IR stain (Life Technologies catalogue: L10119).
Statistical Analyses
Statistical analyses were performed using Prism software (GraphPad Software, Inc., La
Jolla, CA). Statistical differences between different experimental groups were
determined by the Student’s t-test. The reported values represent the mean ± standard
error of the mean (SEM). P values < 0.05 were deemed statistically significant.
7
SUPPLEMENTAL FIGURES AND TABLES
Figure S1
Figure S1. Suppression of proliferation by MYXV is donor dependent and not the
result of experimental variation. (A)-(D), To determine if suppression of proliferation
was donor dependent and no the result of experimental variation, T cells from four
healthy donors, were analyzed for proliferation using flow cytometry. Each sample was
split in three, and the proliferation of activated and infected T cells were evaluated using
flow cytometry as described elsewhere in this paper. Data indicate that MYXV either (A)
and (B), completely suppressed the proliferation of T cells as compared with mocktreated cells, or (C) and (D) MYXV partially suppressed the proliferation of T cells as
compared with mock-treated cells.
Figure S2
Figure S2. MYXV affects reduces differentation into Th1, Th2 and Th17
phenotypes. Lymphocytes (N=4 subjects) were incubated with MYXV or mock
treatment, and then exposed +/- to anti-CD3/CD28 beads: unstimulated (Unst.) cells vs.
stimulate (Stim. cells). 96 hours after treatment, cells were analyzed for Th differentiation
by staining for intracellular transcription factors and then analyzed by flow cytometry.
Upon stimulation of T cells there was reduced percentage of Th1 cells (Tbet+) in MYXV
vs. mock in the (i.e., 40.21 ± 6.50% vs. 63.57 ± 6.59%, NS), Th2 cells (GATA3+) in
MYXV vs. mock (i.e., 57.04 ± 0.16% vs. 62.66 ± 8.54%, NS), and Th17 (RORt+) (73.52
± 4.16% vs. 88.70 ± 5.97% P = 0.05). On the other hand, in the unstimulated T cells,
the percentages of expression of each transcription factor were below 6.0% for both
MYXV-treated, and mock-treated T cells. Data shown correspond to the mean ± SEM of
4 independent experiments, each one performed in duplicate.
TABLES
Unstimulated T cells
% of MYXV Binding
Donor # 1
62.93
Donor # 2
56.53
Donor # 3
13.00
Donor # 4
19.01
Donor # 5
12.00
Table 1. MYXV binds to unstimulated T cells. The levels of binding of
vMyx-Venus/M093L to unstimulated T cells is donor dependent.
Lineages
CD3+
CD4+ gated
on CD3+
CD8+ gated
on CD3+
CD25+ gated
on CD4+
CD25+ gated
on CD8+
CD69+ gated
on CD4+
CD69+ gated
on CD8+
Unstimulated cells
(mean  SEM)
4.57  0.59
Stimulated cells
(mean  SEM)
93.00  2.97
5.04  0.60
92.71  1.95
4.00  0.36
93.63  2.56
not expressed
93.93  2.75
not expressed
93.89  2.53
not expressed
91.91  3.92
not expressed
92.27  3.16
Table 2. MYXV infects stimulated T cells. Levels of infection of different populations of T
cells including CD3, CD4, and CD8 were quantified using flow cytometrty. While vMyx-GFP
pooly infects the unstimulated T cells, the virus productively and significantly infects
stimulated T cells (P < 0.05, when compared unstimulated with stimulated T cells, according
to the Student’s t-test). Likewise, we evaluated the levels of infection of lymphocyte
activation proteins like CD25 and CD69, and found that MYXV also infected these
populations. As expected, these proteins were not expressed in the unstimulated samples.
The mean  of the standard error of the mean (SEM) of at least six independent experiments
is reported.
Full
responders
Unstimulated cells
ls
% of
gated
PI
DI
cells
Stimulated cells
% of
gated
PI
cells
DI
Mock_72 h
1.5  0.6
1.0  0.0
-
47.6  8.5
2.0  0.01
3.0  0.3
MYXV_72 h
1.3  0.5
1.0  0.0
-
13.4  3.4
1.4  0.1
2.0  0.02
Mock_96 h
1.3  0.4
1.0  0.0
-
67.0  0.3
2.5  0.2
3.6  0.2
MYXV_96 h
1.5  0.4
1.0  0.0
-
13.3  0.3
1.5  0.2
2.0  0.0
Partial
responders
% of
gated
cells
PI
DI
% of
gated
cells
PI
DI
Mock_72 h
1.0  0.2
1.0  0.0
-
61.6  1.9
2.8  0.2
3.5  0.2
MYXV_72 h
1.1  0.4
1.0  0.0
-
43.4  3.6
2.8  0.5
3.2  0.3
Mock_96 h
1.1  0.6
1.0  0.0
-
84.0  3.8
5.2  0.9
6.2  0.9
MYXV_96 h
1.0  0.6
1.0  0.0
-
68.8  5.4
3.4  0.7
4.5  0.6
Table 3. Effect of MYXV in the proliferation of T cells. Unstimulated or stimulated T
cells were mock-treated or vMyx-GFP-treated. Cells were stained with cell trace violet
dye to tract cell proliferation. After culturing for 72 hours or 96 hours flow cytometry was
used to determine the proliferation profiles. The proliferation patterns were donor
dependent. Indeed, for certain donors (N=4) the proliferation of T cells was inhibited
upon infection with myxoma virus. These donors were named as full responders. On
the other hand, for other donors (N=4), the proliferation of T cells was slightly affected
by MYXV infection. These donors were designed as partial responders. The table
summarizes the mean  SEM.
PI refers to the proliferation index
DI refers to the division index.
Gated on
CD4+
Mock
MFI
(mean  SEM)
vMyx-GFP
MFI
(mean  SEM)
72 h
17383 1847
15460  2243
96 h
Gated on
CD8+
23214  5148
Mock
MFI
(mean  SEM)
25579  7772
vMyx-GFP
MFI
(mean  SEM)
72 h
15814  3832
14923  1689
96 h
15488  3398
16022  5597
Table 4. The expression of surface CD25 was not affected in partial
responders. The mean fluorescent intensity (MFI) was generated from the
histograms shown in Figure 5H. Data indicate that MYXV did not affect the
surface expression of the activation protein CD25. Data is reported as mean
 SEM of at least 4 different donors.
Mock
Unstimulated
cells
vMyx-GFP
Unstimulated
cells
Mock
Stimulated
cells
vMyx-GFP
Stimulated
cells
Donor # 1
Partial
responder
0.13
0.01
5.83
1.16
Donor # 2
Partial
responder
1.35
0.05
7.10
5.80
Donor # 3
Full
responder
0.14
0.30
23.08
7.09
Mock
Unstimulated
cells
vMyx-GFP
Unstimulated
cells
Mock
Stimulated
cells
vMyx-GFP
Stimulated
cells
Donor # 1
Partial
responder
0.62
0.01
5.92
1.12
Donor # 2
Partial
responder
1.13
0.05
5.01
3.70
Donor # 3
Full
responder
0.11
0.17
23.34
6.10
CD3+
CD4+
Table 5. MYXV downregulates the levels of intracellular IFN-. The effect of MYXV
on the expression of intracellular IFN- was determined by flow cytometry. Data from
three independent experiments corresponding to three different healthy donors are
shown. The percentages of IFN- produced by CD3+ and CD4+ cells are reported. Data
reveals that MYXV downregulates the intracellular expression of this cytokine.