Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells

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Myxoma Virus Suppresses Proliferation of Activated T Lymphocytes Yet Permits

Oncolytic Virus Transfer to Cancer Cells

Running Title: MYXV Suppresses T Cells Yet Permits Virus Transfer

Nancy Y. Villa,1 Clive H. Wasserfall,2 Amy Meacham,1 Elizabeth Wise,1 Winnie Chan3,

John R. Wingard,1 Grant McFadden,3 Christopher R. Cogle1*

1Division of Hematology & Oncology, Department of Medicine, University of Florida,

Gainesville, FL 32610 2Department of Pathology, Immunology and Laboratory Medicine, University of Florida,

Gainesville, FL 32610 3Department of Molecular Genetics and Microbiology, University of Florida, Gainesville,

FL 32610

Corresponding author: Christopher R. Cogle, M.D. 1600 SW Archer Road Box 100278 Gainesville, FL 32610-0278 E-mail: [email protected] Tel: 352-273-7493 Fax: 352-273-5006 Keywords: graft versus host disease, transplant, oncolytic virus,

T lymphocyte

Blood First Edition Paper, prepublished online April 22, 2015; DOI 10.1182/blood-2014-07-587329

Copyright © 2015 American Society of Hematology

For personal use only.on May 26, 2015. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

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KEY POINTS • MYXV binds human T lymphocytes but does not enter and infect T cells until after

activation. • MYXV-infected T lymphocytes proliferate less and secrete less inflammatory

cytokines; but effectively deliver oncolytic virus to augment GVM.




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ABSTRACT

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain

hematological malignancies, but the risk of graft-versus-host disease (GVHD) is a major

limitation for wider application. Ideally, strategies to improve allo-HCT would involve

suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-

versus-malignancy (GVM). Recently, using a xenograft model we serendipitously

discovered that MYXV prevented GVHD while permitting GVM. In this study, we show

that MYXV binds to resting, primary human T lymphocytes but will only proceed into

active virus infection after the T cells receive activation signals. MYXV-infected T

lymphocytes exhibited impaired proliferation after activation with reduced expression of

interferon-γ, interleukin-2 and soluble IL-2Rα, but unaffected IL-4 and IL-10. MYXV

suppressed T cell proliferation in two patterns (full vs. partial) depending on the donor. In

terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively

deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by

delivery of active oncolytic virus to residual cancer cells. Given this dual capacity of

reducing GVHD plus increasing the anti-tumor effectiveness of GVM, ex vivo virotherapy

with MYXV may be a promising clinical adjunct to allo-HCT regimens.




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INTRODUCTION

Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for patients with

certain hematological malignancies. However, graft-versus-host disease (GVHD)

remains a major challenge after allo-HCT.1-3 An increasing number of experimental

GVHD prophylaxis efforts have exploited T cell depletion strategies.4-7 Unfortunately,

these approaches delay the time to donor engraftment, increase risk for disease relapse,

and increase risk for opportunistic infections.

Recently, we discovered that ex vivo virotherapy with the oncolytic poxvirus, myxoma

virus (MYXV), selectively targets malignant human hematopoietic cells like acute

myeloid leukemia and multiple myeloma, while sparing normal human hematopoietic

stem and progenitor cells.8-10 MYXV is a viral oncolytic agent that is non-pathogenic to

humans and mice but has natural tropism for a variety of human cancers.11-13 In the

course of developing MYXV as an ex vivo purging agent for transplant, we

serendipitously discovered that NSG mice receiving human HCT xenografts treated ex

vivo with MYXV developed no GVHD, lived longer, and yet still exhibited robust human

hematopoietic engraftment in the recipient bone marrow.14 We hypothesized that MYXV

impaired the GVHD capacity of alloreactive donor T lymphocytes. To test this prediction

and dissect mechanisms by which MYXV suppresses GVHD, we focused on human T

lymphocyte responses after MYXV exposure.




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METHODS

Virus Binding and Infection Conditions

MYXV virion binding to cells was carried out by incubating resting human T cells with

vMyx-Venus/M093L at a multiplicity of infection (MOI) of 10 for one hour on ice.15 MYXV

infections were performed by incubating human resting or activated T cells with vMyx-

GFP16 or vMyx-GFP/TrFP17 at MOI=10 for 1 hour at room temperature. For both binding

and infection, mock-treated cells were incubated in complete media containing no virus

under the same incubation conditions. Furthermore, heat- and UV-inactivated vMyx-GFP

were used as controls to assess if virus replication competency is needed for the

inhibition of T cell proliferation (see Supplemental Methods for details).

Proliferation Analysis and One-Way Mixed Lymphocyte Reaction (MLR) Assays

Isolated human CD3+ T cells were first labeled using the CellTraceTM violet (CTV) cell

proliferation kit (Invitrogen), as per manufacturer’s recommendations (see Supplemental

Methods for details). Next, T cells were either mock-treated, or infected with vMyx-GFP

(MOI=10), and plated in 96-well round-bottomed plate. Then, cells were either stimulated

(i.e., by adding α-CD3/α-CD28 coated microbeads) or left unstimulated. 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 (see Supplemental Methods

for details). One-way mixed lymphocyte reaction (MLR) assays were performed using

mononuclear cells (MNCs) derived from PBMCs or cord blood (CB) from healthy donors

(see Supplemental Methods for details).18, 19

Graft-versus-Malignancy Assays

Mock-treated or MYXV-treated T lymphocytes (either unstimulated or anti-CD3/CD28

activated) were cultured for 48 hours at 37oC, 5% CO2. At this point, the human multiple




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myeloma cell line U266, was mixed with the T cells at a ratio of 1:1, and this mixture was

cultured for additional 48 hours at 37oC, 5% CO2. MM cell infection was analyzed by

analyzing GFP+ fluorescence in CD138+ cells using direct microscopy and flow

cytometry (see Supplemental Methods for details).

RESULTS

Myxoma Virus Binds to Human T Lymphocytes but Stimulation of T Lymphocytes

Is Required for Productive Infection

Our first question was whether MYXV can bind or infect resting human T lymphocytes.

Primary human CD3+ T cells, isolated from healthy donor peripheral blood, were

incubated with fluorescently labeled MYXV (vMyx-Venus/M093L15) for one hour. After

one hour adsorption, the T cells were washed of free virus and then analyzed by flow

cytometry for evidence of MXYV binding. The T lymphocytes showed Venus-tagged

MYXV binding (Figure 1A), ranging from 13.00% to 62.93% that varied by donor

(Supplemental Table 1). Since the lower limit of sensitivity of this binding assay with

Venus-tagged MYXV is approximately 500 virus particles per cell, these binding

percentages are likely underestimations of the actual percentage of T lymphocytes with

bound MYXV.

We next questioned whether MYXV actively infects these human T lymphocytes using a

vMyx-GFP that expresses GFP encoded in the viral genome and driven by a synthetic

early/late viral promoter, so that the very earliest stages of virus replication can be

monitored by the expression of GFP. When human T lymphocytes were in an

unstimulated state, MYXV initiated its infection cycle in only a very small fraction of the T

cells by 72 or 96 hours after incubation (Figure 1B, middle panels). In contrast, upon




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stimulation of T cells with α-CD3/α-CD28 beads, the GFP-tagged MYXV infected the

activated T cells at much higher levels (GFP+) (Figure 1B, lower panels). Together,

these data show that MYXV binds resting human T cells, but enters and initiates

infection only after T cell activation. In contrast, in the absence of stimulation, there is a

very early block in MYXV replication prior to early viral gene expression. Importantly,

nearly 100% of the activated T cells became infected with GFP-tagged MYXV (Figure

1C), confirming that some input virus was initially bound to essentially all of the available

T cells in the culture, regardless of whether the donor exhibited high (50-60%) or low

(10-20%) binding levels of input Venus-tagged virus.

These results were confirmed with flow cytometric analysis. In all cases, after 72 hours,

MYXV successfully infected over 90% of stimulated T cells (Figure 1E) as compared to

unstimulated T cells (Figure 1D). Infection of CD4+ T cells and CD8+ T cells by MYXV

were similar (Figure 1F). Interestingly, 2D-plots of infected and stimulated T cells

showed different subpopulations and levels of infection of T cells, as assessed by GFP

intensities (right panels of Figure 1E). Within activated lymphocyte subsets, 94% of

CD25+ cells were infected with MYXV and 92% of CD69+ cells were infected (Figure 1G,

Supplemental Table 2). Thus, we observe no particular bias amongst the various

subclasses of CD3+ T lymphocytes, in terms of their ability to become infected by MYXV

following cell activation with anti-CD3/CD28.

MYXV Replication Generates Low Levels of Progeny Virus in Stimulated Human T

Lymphocytes

To determine whether the viral replication cycle was completed with the concomitant

production of new infectious progeny virus, we performed single step viral growth

analysis (Figure 1H). To do this, unstimulated or stimulated T cells were incubated with

MYXV, washed, sampled at serial time points, pelleted and harvested. The infectious




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virus was released by sequential freeze-thaw, and the titre of live virus in each sample

was determined as previously described.20 Notably, we observed that even though

MYXV effectively initiated infection in stimulated T lymphocytes, only low amounts of

new viral progeny were produced by the activated T cells. As expected, in unstimulated

T lymphocytes essentially no new viral progeny were detected. These data show that

MYXV is unable to replicate in resting T cells, but shows a limited capacity to

productively replicate in activated cells and generate progeny virus.

To confirm these results, we performed fluorescence microscopy analysis (Figure 1I)

following incubation with vMyx-GFP/TrFP, a recombinant MYXV expressing both green

fluorescent protein (GFP) driven by a synthetic early/late viral promoter and tomato red

fluorescent protein (TrFP) driven by poxvirus late viral promoter. Thus, the successful

progression of the virus replication from early to late times can be monitored by the

progression of infected cells from green (GFP+) to green plus red (GFP+ + TrFP+). We

found that MYXV replication efficiently progresses in stimulated T lymphocytes from

early stages (i.e., GFP+) to the late viral stages (i.e., TrFP+). Our conclusion is that all

stages of viral replication occur in stimulated T cells, but the extent of final progeny virus

assembly is somewhat less efficient than in fully permissive mammalian cells (such as

rabbit cells or many classes of human cancer cells).

Inhibition of Activation-Induced T Lymphocyte Proliferation by MYXV Infection

We next analyzed the impact of this infection on lymphocyte proliferation in response to

the T cell activation signals. As expected, unstimulated donor human T lymphocytes,

whether mock-treated or MYXV-treated, showed no proliferation after 72 hours or 96

hours in culture (Figure 2A, 2C). Stimulated T cells that were mock-treated showed the

expected increase in cell proliferation at 72 and 96 hours (Figure 2B, red histograms).

But, when stimulated T cells were MYXV-treated, two different proliferation patterns




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were found depending on the donor: some donors were classified as “full responders”,

because MYXV completely inhibited the activation-induced proliferation of their T

lymphocytes (Figure 2B, green histograms). Interestingly, for these full responders,

MYXV’s inhibitory effect on activation-induced proliferation was unchanged over time.

Some normal donors, however, were classified as “partial responders” because MYXV

decreased but did not fully suppress the proliferation of stimulated T cells (Figure 2D).

These data (Figure 2, Supplemental Figure 1A-D and Table 3) support the concept that

although MYXV mitigates a proliferative response for all donors tested (N=8), the extent

of anti-proliferative effects (i.e., full vs. partial) is donor dependent.

Additionally, we found that live virus is required for the suppression of proliferation of

stimulated T cells, (Figure 2E). In contrast, when cells were treated with the inactivated

viruses, the T cells proliferate similarly to mock-treated stimulated cells (Figure 2E).

Proliferation of naturally occurring Tregs (nTregs) of at least four healthy donors were

also evaluated and revealed that MYXV does not suppress nTregs as shown in the

histograms (data of one representative donor) (Figure 2F, left and right panels) and the

bar grafts (CD4+CD25+FoxP3+: 74.84 ± 2.79%; CD4+CD25+Helios+: 66.13 ± 5.56%,

MYXV-treated cells) as compared to mock controls (CD4+CD25+FoxP3+: 70.14 ± 3.41%;

CD4+CD25+Helios+: 60.69 ± 6.38%), (Figure 2 F-1 and F-2).

In order to investigate whether MYXV affects the differentiation of T lymphocytes, T cells

of four different donors were mock-treated, or MYXV-treated +/- CD3/CD28 stimulation.

Quantification of intracellular transcription factors and proliferation of Tregs of four

different donors, each one in duplicate, was determined using flow cytometry. Compared




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to mock treatment, MYXV treatment reduced differentiation into Th1 (Tbet-expressing)

cells (mock: 63.57 ± 6.59%, vs. MYXV: 40.21% ± 6.50, NS), Th2 (GATA3-expressing)

cells (mock: 62.66 ± 8.54%, vs. MYXV: 57.04% ± 0.16, NS), and Th17 (RORγt-

expressing) cells (mock: 88.70 ± 5.97%, vs. MYXV: 75.52 ± 4.16%, P = 0.05),

(Supplemental Figure 2). Together, the results indicate a preferential attenuation of Th1

polarization and Th17, with relatively low attenuation of Th2, and Treg differentiation.

MYXV Infection of Activated Human T Cells Affects Viability

MYXV infection plus stimulation with anti-CD3/CD28 beads resulted in increased

percentage of non-viable T lymphocytes for all donors tested (Figure 3). Specifically, this

additive effect was observed in T cell samples from either full responders (Figure 3A and

3B) or from partial responders (Figure 3C and 3D), suggesting that such augmented

decline in T cell viability is donor independent (Figure 3E and 3F). Even though a slightly

higher frequency of cell death was observed as a trend for full responders vs. partial

responders infected with MYXV and stimulated with α-CD3/α-CD28 beads, it was not

statistically significant (i.e., at 72 hours after culturing: 30.00 ± 8.73% full responders vs.

19.59 ± 1.60% partial responders, P = 0.32; and at 96 h after culturing: 49.54 ± 8.01%

full responders vs. 37.73 ± 7.14% partial responders, P = 0.12). On the other hand,

unstimulated T cells mock-treated or MYXV-treated, exhibited low and very similar

frequencies of cell death (Figure 3E and 3F).

MYXV Infection of Stimulated Human T Lymphocytes Downregulates a Subset of

Activation-Inducible Cytokines

Excessive levels of IL-2 have been implicated in the pathophysiology of acute GVHD.21

As expected, supernatants of unstimulated T lymphocytes treated with MYXV, or mock-




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treated controls, showed no detectable levels of secreted IL-2 (not shown). However,

supernatants from stimulated T lymphocytes, either mock-treated or infected with MYXV,

now contained measurable levels of IL-2 (Figure 4A and 4B). For donors assessed as

full responders, in terms of activation-induced proliferation, MYXV treatment of

stimulated T lymphocytes significantly reduced IL-2 expression compared to mock

treatment of stimulated T cells by 72 and 96 hours after infection and stimulation. For

partial responders, MYXV also decreased the IL-2 expression after 72 and 96 hours

after infection and stimulation (Figure 4B). Thus, both full and partial responders

exhibited significantly reduced levels of secreted activation-induced IL-2.

In addition, supernatants of unstimulated T lymphocytes treated with MYXV, or mock-

treated, showed no detectable levels of IL-2Rα (not shown). Supernatants of stimulated

but uninfected T lymphocytes showed high levels of activation-induced IL-2Rα. However,

MYXV-treated stimulated T lymphocytes secreted significantly less IL-2Rα than mock-

treated stimulated controls. Notably, this inhibitory pattern was observed in both full

responders (Figure 4C) and in partial responders (Figure 4D) at 72 and 96 hours after

infection and stimulation.

When we analyzed the samples of full responder donors, we found that MYXV

decreased the expression of IL-2Rα receptor (CD25) (Figure 4E) by approximately 50%

on CD4+ lymphocytes and by approximately 40% on CD8+ lymphocytes (Figure 4F and

4G). Interestingly, MYXV did not affect the activation-induced surface levels of CD25 of

partial responders (Figure 4H, Supplemental Table 4).

Besides IL-2, other lymphocyte-derived cytokines involved in GVHD include interleukin-4

(IL-4), inteleukin-10 (IL-10) and interferon-gamma (IFN-�).22-24 Of these cytokines, only




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activation-induced soluble IFN-� was significantly decreased after MYXV infection as

compared to mock-treated and stimulated samples (Figure 4). Notably, we observed a

similar inhibitory pattern for T cells derived from both types of donors at 72 hours (Figure

4J) and at 96 hours (Figure 4K). Intracellular staining of IFN-� confirmed these findings

(Supplemental Table 5).

Infection with MYXV Decreases the Proliferation and Cytokine Production of Allo-

Stimulated T cells

We next used mixed lymphocyte reactions to recapitulate allo-stimulation conditions in

GVHD. Responder cells were labeled with Cell Trace Violet (CTV) dye to track

proliferation. Mock- or MYXV-treated responder cells were mixed with irradiated

unmatched cells (stimulator), and the levels of infection and proliferation were quantified

using flow cytometry after 72 hours or 6 days after culturing MLRs. Upon allo-stimulation

of MNCs from PBMCs, we observed that 30.00 ± 1.30% of CD4+ T cells and 30.00

±1.72% of CD8+ T cells in the MLR were infected with MYXV (Figure 5A). In contrast,

only 3.76 ± 0.79% responder CD4+ T cells alone and 4.41 ± 1.71% responder CD8+ T

cells alone were infected (Figure 5A). As expected, only 1.44 ± 0.38% of stimulator CD4+

T cells alone and 1.37 ± 0.0 of stimulator CD8+ T cells alone were infected (Figure 5A).

When the MNCs from cord blood were allo-stimulated by MLR, 19.19 ± 1.77% of CD4+ T

cells and 16.00 ± 2.10% of CD8+ T cells were infected with MYXV (Figure 5B). As

expected 2.15 ± 0.43% responder CD4+ T cells alone and 3.25 ± 0.62% of responder

CD8+ T cells alone were infected (Figure 5B). Likewise, 2.15 ± 0.43% of CD4+ stimulator

T cells alone and 1.99 ± 0.39% of stimulator CD8+ T cells alone were infected (Figure

5B).




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Furthermore, infection with MYXV and allo-stimulation of cells from PBMCs or CB via

MLR resulted in significant inhibition of proliferation of responder T lymphocytes (Figure

5C and 5D, respectively) as compared to mock-treated cells. As expected, responder or

irradiated stimulator T cells alone without MLR did not proliferate. Next, we performed

ELISA or multiplex assays to determine the levels of secreted cytokines in MLRs. Low

levels of soluble IL-2, IL-2Rα, IL-4, IL-10 and IFN-γ were observed after infection and

allo-stimulation of either PBMCs (Figure 6A, 6B and 6E) or CB cells (Figure 6C, 6D and

6F). These results were consistent with those obtained upon stimulation of T cells with

anti-CD3/CD28 beads (Figure 2A-D and Figure 4). In terms of infection, proliferation

pattern and cytokine production no major differences were found for PBMCs or CB cells,

suggesting that both PBMCs and CB are almost equally susceptible to MYXV.

Activated T Lymphocytes Efficiently Transfer MYXV and Kill Susceptible Cancer

Cells

The purpose of allo-HCT is two-fold: (1) to replace the hematopoietic system of the

recipient using hematopoietic stem/progenitor cells from a closely matched donor, and

(2) to attack and eliminate residual cancer cells in the recipient by means of alloreactive

donor T cells. Any viable adjunct therapy to allo-HCT to prevent GVHD should not come

at the price of either reducing engraftment of the normal stem cells or reducing the

efficiency of GVM. Previously, when we used a xenograft model of human multiple

myeloma (MM) in immunodeficient mice, we found that ex vivo treatment of the donor

human bone marrow with MYXV, while leaving normal stem cell engraftment unaffected,

not only prevented GVHD but also preserved GVM against pre-seeded myeloma.14

However, the mechanisms of GVM after MYXV treatment of the donor transplant were

unknown in our prior study. Therefore, we designed in vitro modeling experiments to

examine the mechanistic basis for the observed GVM preservation (Figure 7A). Briefly,




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human T lymphocytes were isolated by negative selection from healthy donor PBMCs.

The T cells were then mock-treated or treated with vMyx-GFP for one hour at room

temperature to allow for virus binding. Unbound virus was removed by washing and the

cells were cultured in complete media, either with or without α-CD3/α-CD28 stimulation

for 48 hours at 37oC. Next, the T cells were mixed with human multiple myeloma (MM)

cells (U266 cells) that have previously shown susceptibility to MYXV oncolysis.10, 14, 15

This mixture of T cells and MM cells was incubated at 37oC for an additional 48 hours.

As controls, T cells alone or MM cells alone were subjected to the same MYXV

treatment, +/- anti-CD3/CD28 activation and culture conditions. The levels of MYXV

infection in the MM cells (monitored as CD138+) were evaluated at 96 hours after MYXV

treatment of unstimulated or stimulated T cells (corresponding to 48 hours after mixing T

cells with MM cells). MYXV infection levels of either the donor T cells or MM cells were

assessed using fluorescence microscopy and flow cytometry. As expected, MYXV did

not productively infect unstimulated T cells (Figure 7B, left middle panel). Although a

slight increase in the number of MYXV infected T cells was observed when

unstimulated/infected T cells were mixed with MM cells, (Figure 7B, right middle panel),

the percentage of infection in all cells (i.e., T cells and MM cells) was only 1.39% (Figure

7C, top right panel). The percentage of infection of MM cells in this mixture was only

0.78% (Figure 7C, bottom right panel). This low level of MYXV infection was expected

since only a very small fraction of unstimulated T cells were infected with MYXV.

In contrast, when MYXV-treated stimulated T lymphocytes were mixed with MM cells,

there was a significant increase in the percentage of infected MM cells (Figure 7B,

bottom right panel). Under conditions of T cell stimulation, the level of MYXV infection in

MM cells increased 100-fold (from 0.78% to 21.13%) (Figure 7D, bottom right panel). T

lymphocytes from 3 different donors were tested in a similar fashion and showed

reproducibly consistent infection levels of the target MM cells (Figure 7E). Furthermore,




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we show evidence that CD138+ cell death is induced by MYXV infection (i.e.,

CD138+GFP+) under both unstimulated and stimulated conditions. For instance, up to

21.74 ± 3.50% and 27.00 ± 1.15% of CD138+ MM cells die, under unstimulation and

stimulation conditions, respectively (Figure 7E-1). These results confirm the oncolytic

effects of MYXV infection on human MM cells. Interestingly, we also show enhanced and

significant killing of even non-infected MM cell (i.e., CD138+GFP-) after MYXV infection

of T cells and stimulation with anti-CD3/CD28 44.96 ± 6.94%) as compared with mock-

treated and stimulated T cells (i.e., 14.52 ± 2.93%), **P=0070, (Figure 7E-2). On the

other hand, when comparing MM cell killing of un-infected CD138+ MM cells (i.e., GFP-)

under the unstimulated conditions, no significant differences were found between MYXV-

treated resting T cells (i.e., 19.30 ± 4.99%) and mock-treated resting T cells (i.e., 14.58 ±

2.57%), P = 0.3984, NS. (Figure 7E-2). This latter result suggests that MYXV-

infected/stimulated T cells that do not donate virus to MM are nevertheless now better

cytotoxic killers of MM cells than mock-treated/stimulated T cells. We speculate that T

cells exposed to MYXV are now also better armed to kill cancer cells by cytotoxic T

lymphocyte killing. Together, our results indicate that MYXV enhances the beneficial

effects of GVM.

To determine if input and/or progeny virus are transferred from infected/stimulated T

cells to MM cells, cytosine arabinoside (AraC), a known inhibitor of viral DNA replication

and late gene expression of MYXV, was used. When T cells were exposed to vMyx-

GFP/TrFP followed by the addition of AraC and stimulation with anti-CD3/CD28 beads,

the late gene expression of MYXV (TrFP+) was inhibited (Figure 7F), which resulted in

very low levels of viral late gene expression in both MM cells (Figure 7G) and T cells

(Figure 7H). Lower levels of early and late gene expression (GFP+) were observed in

activated T cells treated with AraC, indicating that GFP expression was aborted by the




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AraC, as expected (Figure 7H). Importantly, GFP expression in the MM cells, which are

infected by MYXV donated from the stimulated T cells, was reduced but not eliminated

by the AraC treatment. This means that both progeny virus (inhibited by AraC) and input

virus (unaffected by AraC) can infect the target MM cells. Together, these results

support the notion that both input and progeny virus, derived from stimulated T cells are

handed-off or delivered into the MM cells, which results in their productive infection.

DISCUSSION

A major clinical challenge after allo-HCT is the prevention or control of GVHD. Since

GVHD is driven by resident CD3+ T lymphocytes in the donor allograft, one of the most

effective treatments for GVHD is the prophylactic depletion or inhibition of alloreactive T

cells.24, 25 However, intensifying T cell purging by conventional methods increases the

risk for life-threatening infections due to delayed immune recovery, graft failure, and

disease relapse.26, 27 At a minimum, optimizing outcomes after allo-HCT simultaneously

require control of GVHD, sparing of normal hematopoietic stem/progenitor cell

engraftment, and permission for GVM.

We previously demonstrated efficient human hematopoietic engraftment with no GVHD

after xenotransplant of MYXV-treated primary human hematopoietic stem/progenitor

cells.14 The safety of using MYXV with human hematopoietic stem/progenitor cells has

been correlated to the virus’ inability to bind or infect normal human CD34+

hematopoietic cells.9, 10 Of the hundreds of immunocompromised mice that we have

transplanted with MYXV-treated cells, none have shown pox ulcerations or any

semblance of viral infection whatsoever. Moreover, MYXV has been widely distributed in

the Australian environment as a means to control feral populations of European rabbits,

and no human infections have ever been reported.28




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The data in this report are the first to reveal mechanisms by which ex vivo virotherapy

with MYXV controls GVHD yet does not compromise GVM.14 Herein, we present direct

evidence that MYXV binds unstimulated human CD3+ T lymphocytes but T cell activation

is required to initiate productive virus infection, which can then be delivered to

susceptible cancer cells.

In addition to efficiently infecting stimulated human T lymphocytes from all normal

donors tested, MYXV also impaired T cell functionality by (1) reducing T cell proliferation

and (2) down-regulating T cell signaling pathways of known importance in GVHD.21

Specifically, we found that MYXV infection consistently decreased activation-induced T

lymphocyte secretion of IL-2, IL-2Rα and IFN-γ, but not the secretion of IL-4 or IL-10.

Inhibition of secretion of IFN-γ is consistent with lower levels of Tbet (Th1) expression,

and not variation in the expression of GATA 3 (Th2).

The reason(s) why MYXV completely suppresses the proliferation of stimulated T cells in

some donors (full responders) and only partially inhibits the proliferation of stimulated T

cells in other donors (partial responders) is still unknown, and requires more

investigation. We found that the levels of initial virus binding could not be directly

correlated with the type of donor (i.e., full responder vs partial responder). Since the

limit of detection of binding of Venus-tagged MYXV to cells by FACS is in the order of

several hundred virus particles per cell, we can only note that the majority of T cells from

all donors tested became infected following T cell stimulation, suggesting that sufficient

virus binds all of the cells to at least initiate infection following cell activation.




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Although depletion or inhibition of T cell activation helps to control GVHD, a detrimental

result of this strategy is that the beneficial effects of GVM may also be compromised. A

previous report by our group demonstrated that ex vivo virotherapy with MYXV can

control GVHD in NSG mice xenografted with human PBMCs and yet still retain the

beneficial effects of GVM against pre-seeded human MM in the bone marrow of the

recipients.14 Results in our present study support the notion that both input and progeny

MYXV derived from infected/stimulated T cells can be efficiently transferred to

susceptible target human cancer cells and mediate oncolytic effects against these

cancer cells via the antigenic stimulation of the donor T cells. Our results are somewhat

distinct from those of Cole et al., in which viral vectors hitchhike on non-activated T cells,

and virus delivery to cancer cells does not involve virus replication.29 In contrast, we

report here that activation of T cells is required to efficiently deliver both input and

progeny oncolytic MYXV to target human myeloma cells.

Overall, our results provide new insights into the specific mechanisms used by MYXV to

control GVHD after allo-HCT. We now show that the ex vivo virotherapy regimen has the

potential to arm resident resting T cells residing in the donor allograft with adsorbed

MYXV, which is then triggered into the replication cycle only after the T cells encounter

antigenic stimulation. At this point, if the stimulating host cells are from normal tissues,

the triggered virus replication retards or blocks subsequent T cell proliferation. But if the

stimulating host cells are cancerous, the oncolytic virus is efficiently transferred from T

lymphocytes to target and kill cancer cells. In essence, GVM by the donor transplant

now becomes augmented by virus-versus-malignancy (VVM). Since productive viral

infection of a tumor can also induce an in situ vaccine effect and initiate systemic anti-

tumor immunity, an added benefit of using the ex vivo MYXV approach is that infection

of malignant cells in vivo via T cell mediated delivery might also create an in situ




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vaccination effect against tumor antigens. Therefore, for all these reasons, ex vivo

MYXV pre-treatment of donor allografts may be a promising clinical adjunct to allo-HCT.




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CONFLICT OF INTEREST CRC and GM have filed intellectual property rights to the University of Florida for

prevention of GVHD by MYXV virotherapy.

ACKNOWLEDGEMENTS

We thank The Diabetes Institute at the University of Florida, Department of Pathology,

Immunology and Laboratory Medicine for providing us with reagents, tools, and

instrumentation, and very helpful discussions. We also thank Dr. Shannon Wallet from

the department of Oral Biology at UF for providing the Luminex instrument facility. The

Leukemia & Lymphoma Society supported CRC with a Scholar in Clinical Research

award (2400-13). NYV was supported by a Chagnon Fellowship in Blood & Marrow

Transplant. This study was also supported by Florida Bankhead-Coley Cancer Research

Program grant 1BT02 and NIH/NCI grant R01 CA138541-01 to GM. This work was also

supported by the Gatorade Trust, which was administered by the University of Florida

Department of Medicine.

CONTRIBUTIONS

CRC, GM, and NYV conceived the concept of the study, designed the experiments,

analyzed the results, and wrote the manuscript. NYV, CW, AM, EW, and WC conducted

the experiments and edited the manuscript. JRW edited the manuscript.




21

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26

FIGURE LEGENDS Figure 1. MYXV binds to unstimulated human T lymphocytes but activation of

human T lymphocytes is required for MYXV replication (A), To investigate whether

MYXV binds to unstimulated human T lymphocytes, T cells were isolated using an

EasySep negative selection HLA T cell enrichment kit (up to > 95% purity).

Approximately 1x106 of these negatively-isolated T lymphocytes were incubated with

recombinant vMyx-Venus/M093L at MOI of 10 for 1 hour on ice to allow virus binding but

not entry. After this, unbound virus was washed twice with cold 1X-PBS + 5% FBS. T

cells were then stained with anti-CD3 antibody, and the levels of Venus+ labeling in the

CD3+ population were determined by flow cytometry (bottom panel). A representative

experiment from one donor is shown.

To investigate virus infection, approximately 3-4x106 isolated human T lymphocytes

were incubated with recombinant vMyx-GFP at MOI of 10 for 1 hour at room

temperature to allow virus adsorption. After this, mock treated and infected T cells were

stimulated with α-CD3/α-CD28 beads at a cell:bead ratio of 1:1. This was followed by

incubation at 37oC for 72 hours or 96 hours. The unstimulated (i.e., without adding

beads) mock- and MYXV-treated T lymphocytes were subjected to the same culturing

conditions. (B), 72 and 96 hours after culturing, expression of virus-expressed GFP was

monitored using fluorescence microscopy. (C)-(F), To quantify the levels of infection of

different T populations, 72 hours after vMyx-GFP exposure, cells were stained with

antibodies against CD3, CD4 and CD8, and the levels of GFP+ in each population were

quantified by using flow cytometry. Likewise (G), levels of infection of T lymphocyte

activation proteins CD25 and CD69 were also quantified using flow cytometry. Data

reported are representative of al least six independent experiments. Significance (i.e., P

< 0.05) was determined using the Student’s t-test. To investigate whether MYXV

launches productive virus replication in stimulated human T lymphocytes we performed




27

one-step growth curves. (H), T cells were infected with vMyx-GFP at a MOI of 10.

Infected/unstimulated T cells, and infected/stimulated T cells were harvested, cells were

lysed using repeated freeze-thaw, and the amount of infectious virus in each sample

was quantified using foci formation on BSC40 cells. (I), Stimulated or unstimulated T

cells were infected with recombinant vMyx-GFP/TrFP at MOI of 10. Expression of GFP

(expressed at both early and late times post-infection) and TrFP (expressed only at late

stages of virus infection) was determined 72 hours after infection using fluorescence

microscopy.

Figure 2. MYXV impairs activation-induced proliferation of human effector T

lymphocytes. To determine if MYXV can impair the post-activation functions of T

lymphocytes, the levels of cell proliferation of stimulated T lymphocytes were assessed

using flow cytometry. T cells were pre-loaded with the tracking dye Cell Trace Violet

(CTV) at 37oC for 20 min and then either mock-treated (with or without MYXV infection),

or incubated with +/- anti-CD3/CD28 microbeads (with or without MYXV infection) as

described in Materials and Methods. T lymphocytes were then incubated in a humidified

chamber at 37oC, and 5% CO2 for 72 hours or 96 hours to allow for the proliferation of

stimulated T cells. At the indicated time points, cells were stained for CD3, CD4, CD8,

CD25 and CD69. FCS-Express version 4 was used to analyze the characteristic

subpopulations of dividing lymphocytes, and to determine the percentage of proliferation,

the proliferation index (PI) and the division index (DI) (See Supplemental Methods for a

detailed description). (A), Histograms showing populations of mock-treated T

lymphocytes (black outline) and MYXV-treated T lymphocytes (blue diagonal) in

unstimulated conditions at 72 and 96 hours. The histograms reveal no CTV shift to the

left, indicating low numbers in proliferation, and complete overlap when treated with

MYXV, indicating no effect of MYXV on lymphocyte proliferation of unstimulated T cells.




28

(B) In stimulated conditions, the mock-treated T lymphocytes (red) proliferate as

evidence by leftward CTV stain shifting of the population. However, the MYXV-treated T

cells (green diagonal) remains unchanged, indicating full suppression of T cell

proliferation. This case is representative a Full Responder Donor. (C) Control

treatments, in unstimulated conditions, showing lack of T cell proliferation in a Partial

Responder donor. (D) Under stimulated conditions with a Partial Responder donor, the

mock-treated T lymphocytes (red) proliferate as evidence by leftward shifting of the CTV

stained population. However, the MYXV-treated T cells (green diagonal) exhibit an

intermediate CTV shifted pattern, indicating partial suppression proliferation by MYXV.

(E) To determine if live virus is needed to suppress the proliferation of T cells, T

lymphocytes were incubated with live vMyx-GFP, heat-inactivated vMyx-GFP, or UV-

inactivated vMyx-GFP at an equivalent MOI = 10 and +/- anti-CD3/CD28 beads. After 72

hours, the proliferation of CTV-tagged T cells was evaluated using flow cytometry. Data

indicate that proliferation of T cells is suppressed only in the presence of live MYXV. In

contrast, inactivated MYXV did not affect the activation-induced proliferation of T cells.

(F) To investigate if MYXV can affect Tregs, proliferation of naturally occurring regulatory

T cells (nTregs) was evaluated using flow cytometry. (F), Histograms of one

representative donor, showing the proliferation patterns of CTV-tagged

CD4+CD25+FoxP3+ (left panels) and CD4+CD25+Helios+ (right panels). (F-1), and (F-2)

summarize the percentage of proliferation of CTV-tagged CD4+CD25+FoxP3+ and

CD4+CD25+Helios+, respectively of different donors (N=4).

Figure 3 MYXV infection and stimulation of human T Cells reduces their viability

Cell death of T cells was evaluated 72 hours and 96 hours after mock-, or MYXV-

treatment, and +/- α-CD3/α-CD28 stimulation, using flow cytometry. To assess cell

viability, T cells were labeled with the Live/Dead near-infrared (IR) fluorescent dye, an




29

amine reactive dye that binds covalently to intracellular and extracellular amines,

generating a bright signal that allows the distinction between live/dead cells in a single

channel. In addition, the staining pattern of this dye is preserved following cell fixation.

(A) and (B), Correspond to a representative full responder donor. (C) and (D),

Correspond to a representative partial responder donor. The percentage of cells was

evaluated under unstimulated (top panels) and stimulated conditions (bottom panels).

The data revealed that MYXV infection plus stimulation of T cells increased the

percentage of cell death of the CD3+ population in culture. (E) and (F), Summarizes the

profile of cell death among donors (N=4 for each type of donor).

Figure 4. MYXV downregulates the expression IL-2, IL2Rα and IFN-γ in activated

human T cells. To determine if MYXV infection affects the expression of IL-2 and the IL-

2 alpha chain receptor (IL-2Rα, a.k.a CD25), about 1x106 of mock-treated (i.e., without

adding virus) or MYXV-treated human T cells and stimulated with α-CD3/α-CD28 coated

microbeads were culturing for 72 hours, or 96 hours at 37oC, 5% CO2. Supernatants

were collected and analyzed using human IL-2 or human IL-2Rα ELISA. (A and B),

MYXV decreases the secretion of IL-2 compared to mock-treated and stimulated T cells.

(C and D), Soluble IL-2Rα was significantly downregulated upon infection of activated T

cells with MYXV at the indicated time points. (E), Histograms generated from the flow

cytometric analysis suggest that MYXV also inhibits the expression of the surface IL-2Rα

(CD25) in stimulated and full responder samples (green histograms) as compared to

mock-treated and stimulated samples (red histograms). Black histograms correspond to

mock-treated T cells and unstimulated T cells, while blue histograms correspond to

MYXV-treated and unstimulated T cells. (F) and (G) From the histograms of stimulated

samples shown in (E), the mean fluorescent intensity (MFI) was calculated and is




30

reported as the percentage relative to mock. (F) Shows the MFI of CD25 gated on CD4+

and (G), corresponds to the MFI of CD25 gated on CD8+. Results represent the mean ±

SEM of at least four different donors. (H) MYXV did not affect the levels of expression of

CD25 in the surface of activated lymphocytes of partial responders.

(I and J) MYXV affects the expression of cytokines IFN-γ, IL-4 and IL-10 in activated

human T cells. After culturing activated T cells for 72 hours or 96 hours, with or without

virus infection, 1x106 of cells were pelleted and the supernatants of both (I) full

responders and (J) partial responders used to evaluate the levels of secreted cytokines

such as IL-4, IL-10, and IFN-� utilizing a Luminex platform. MYXV inhibited the secretion

of IFN-� in all donors tested (I and J) at 72 hours, or 96 hours following stimulation as

compared to mock-treated samples; whereas the secretion of the cytokines IL-4 and IL-

10 was not affected by MYXV-treated, vs. mock-treated T cells. Results shown

correspond to the mean ± SEM of at least 3 different full responder donors, and 3

different partial responder donors.

Figure 5. Effect of MYXV on infection, proliferation, IL-2 and IL-2Rα secretion of T

cells allo-stimulated via MLR. To investigate if MYXV affects the functionality of T cells

in the context of allo-stimulation, in vitro myxed lymphocyte reaction (MLR) assays were

carried out. PBMCs and cord blood from healthy donors were used to isolate MNCs.

Stimulator cells were irradiated using 3000 cGy from a Cs157 and 5x105 of cells were

plated in triplicate into 96 well plates. Responder cells were mock-treated or MYXV-

treated and then 1x105 cells were seeded in triplicate into empty wells or in wells

containing irradiated stimulator cells. After culturing for 72 hours or 96 hours, levels of

infection (A and B) and proliferation (C and D) were evaluated using flow cytometry.




31

Figure 6. Effect of MYXV on the expression of IFN-γ, IL-4 and IL-10 by T cells allo-

stimulated via MLR. To determine if MYXV infection affects the expression of IL-2 and

the IL-2 alpha chain receptor (IL-2Rα, a.k.a CD25) in the setting of allo-stimulation via

MLR. PBMCs and cord blood (CB) cells, from healthy donors were used to isolate

MNCs. 1x105 mock-treated (i.e., no virus) responder cells or MYXV-treated responder

cells were mixed with 5x105 irradiated stimulator cells. Co-cultures were incubated for 72

hours or 6 days at 37oC, 5% CO2. At the indicated time points, supernatants were

collected to carried out ELISA and multioplex assays. (Aand B) ELISA assays of IL-2

and IL-2Rα. of PBMCs. (C and D) ELISA assays of IL-2 and IL-2Rα. of CB. (E and F),

Multiplex assays were used to quantify the levels of secretion of IFN-γ, IL-4 and IL10

from PBMCs and CB cells, respectively.

Figure 7. Input MYXV and virus progeny from activated human T cells are both

efficiently transferred to human multiple myeloma cells. To investigate if MYXV

infection of unstimulated vs. activated T cells can secondarily target and infect virus-

susceptible human U266 MM cells, an in vitro virus transfer assay was perfomed and is

described in diagram (A), Experimental schematic depicting human T lymphocytes

incubated with MYXV in the presence or absence of activating anti-CD3/CD28

microbeads, (1) MYXV binding to T cells (allo-reactive T cells), free MYXV washed from

culture, (2) admixture of human multiple myeloma (U266 cells). As a result a dual action

of MYXV is proposed: (3) MYXV mediates infection/suppression of allo-reactive T cells

when they interact with host U266 antigens, and (4) the infection of malignant cells by

passing of virus from activated T cells to myeloma cells (GFP+). (B) Fluorescence

micrographs showing minimal increase in MYXV infection (GFP+) in unstimulated

conditions when T cells were mixed with multiple myeloma cells (middle panels).




32

However, after stimulation there was a significant increase in MYXV infection of all cells

(bottom panels). (C) Flow cytometry was used to quantify infection in cell subsets.

CD138+ myeloma cells showed only minimal MYXV infection in unstimulated conditions

(bottom right plot). (D) In stimulated conditions, there was a significnat increase in the

percentage of CD138+ myeloma cells with MYXV infection (bottom right panel).

Compared to unstimulated conditions, activated T lymphocytes caused more than a 25-

fold increase in myeloma infection with MYXV (from 0.78% to 21.13%). (E) Bar graph

showing percentage of CD138+ myeloma cell population with MYXV infection in the

unstimulated (white) versus stimulated (black) conditions. (E-1), Bar graph showing

percentage of MM dead (CD138+) induced by MYXV infection (GFP+) (i.e., gating on

CD138+GFP+) under unstimulated (white) versus stimulated (black) conditions (i.e., 21%

vs. 27%, respectively). (E-2), Gating on CD138+GFP-, bar graph showing percentage of

MM dead (CD138+). Mock-treated (white), or MYXV-treated (black) T cells and under

stimulation with anti-CD3/CD28 resulted on 19.30% and 44.96% of MM dead,

respectively. On the other hand, mock-treated (white), MYXV-treated (black) T cells -

CD3/CD28, resulted in less than 6% of MM died. T cells from 3 different donors were

tested and showed reproducibly consistent virus-transfer and killing results. (F) To

determine if progeny and/or input virus is tranferred from activated T cells to multiple

myeloma cells, late gene expression and therefore, the generation of progeny MYXV

was blocked by using cytosine arabinoside (AraC). Briefly, after exposure of T cells with

vMyx-GFP/TrFP and then,10 μg/mL of AraC (+AraC), or vehicle only (-AraC) were

added to the T cells with or without α-CD3/α-CD28 stimulation. After 48 hours of

culturing T cells +/- AraC, U266 cells were added to the culture and incubated at 37oC

5% CO2 for additional 48 hours (see supplemtal methods for more details). Infection was

evaluated using florescence microscopy. (G) Flow cytometry was used to quantify the




33

levels of infection of multiple myeloma cells (CD138+) and (H) T cells (CD3+) Data

indicate that after infection of activated T cells, MYXV replicates, but both input virus

(unaffected by AraC) and virus progeny (inhibited by AraC) are both then tranferred to

myeloma cells to initiate infection of these cells. At least 3 independent experiments

were performed.

























doi:10.1182/blood-2014-07-587329Prepublished online April 22, 2015;

Grant McFadden and Christopher R. CogleNancy Y. Villa, Clive H. Wasserfall, Amy Meacham, Elizabeth Wise, Winnie Chan, John R. Wingard, permits oncolytic virus transfer to cancer cellsMyxoma virus suppresses proliferation of activated T lymphocytes yet

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Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells