Clinical Research Development and Phase I Unit; CREA ... · Kokubun1, Tarek H. Mouhieddine1,...
Transcript of Clinical Research Development and Phase I Unit; CREA ... · Kokubun1, Tarek H. Mouhieddine1,...
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Blocking IFNRA1 inhibits multiple myeloma-driven Treg expansion and immunosuppression. Yawara Kawano*1, 2, Oksana Zavidij*1, Jihye Park1, Michele Moschetta1, Katsutoshi Kokubun1, Tarek H. Mouhieddine1, Salomon Manier1, Yuji Mishima1, Naoka Murakami3, Mark Bustoros1, Romanos Sklavenitis Pistofidis1, Mairead Reidy1, Yu J. Shen1, Mahshid Rahmat1, Pavlo Lukyanchykov3, Esilida Sula Karreci3, Shokichi Tsukamoto1, Jiantao Shi4, Satoshi Takagi1, Daisy Huynh1, Antonio Sacco1, 5, Yu-Tzu Tai1, Marta Chesi6, P. Leif Bergsagel6, Aldo M. Roccaro1, 5, Jamil Azzi3 and Irene M. Ghobrial1 1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 2Department of Hematology, Kumamoto University Hospital, Kumamoto, Japan 3Transplantation Research Center, Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA 4Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 5Clinical Research Development and Phase I Unit; CREA Laboratory, ASST Spedali Civili di Brescia, Brescia, BS, Italy 6Comprehensive Cancer Center, Mayo Clinic, Scottsdale, AZ, USA. * Equally contributed Corresponding Authors: 1-Irene M. Ghobrial, MD Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School 450 Brookline Ave Boston, MA, 02115 Phone: (617)-632-4198 Fax: (617)-632-4862 Email: [email protected] 2- Jamil Azzi MD Transplantation Research Center, Brigham and Women’s Hospital, Harvard Medical School 221 Longwood Ave, Boston, MA, 02115 Phone: (617)-525-7359 Fax: (617)-732-5254 Email: [email protected]
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ABSTRACT
Despite significant advances in the treatment of multiple myeloma (MM), most patients
succumb to disease progression. One of the major immunosuppressive mechanisms that
is believed to play a role in myeloma progression, is the expansion of regulatory T-cells
(Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and
activation by secreting type-1 interferon (IFN). Blocking IFNAR1 (interferon alpha and
beta receptor 1) on Tregs significantly decreases both, myeloma-associated Treg
immunosuppressive function and myeloma progression. Using syngeneic transplantable
murine myeloma models and bone marrow (BM) aspirates of multiple myeloma patients,
we found that Tregs were expanded and activated in the BM microenvironment at early
stages of myeloma development. Selective depletion of Tregs led to a complete remission
and prolonged survival in mice injected with myeloma cells. Further analysis of the
interaction between myeloma cells and Tregs using gene sequencing and enrichment
analysis uncovered a feedback loop, wherein myeloma-cell-secreted type-1 IFN induced
proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and
IFNAR1 knockout Tregs, we demonstrated a significant decrease in myeloma-associated
Treg proliferation, which was associated with longer survival of myeloma-injected mice.
Our results thus suggest that blocking type-1 IFN signaling represents a potential strategy
to target immunosuppressive Treg function in MM.
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INTRODUCTION
Immunotherapy has shown promising results in clinical trials against both solid(1) and
hematological malignancies(2), such as Hodgkin lymphoma(3). Even though targeting
the immune microenvironment can lead to significant improvements in response and
patient survival, our understanding of its regulatory mechanisms is incomplete. The role
of immunosurveillance in controlling cancer growth thus remains under active
investigation in many cancer types, including Multiple Myeloma (MM).
Multiple myeloma is characterized by clonal expansion of malignant plasma cells in the
bone marrow (BM). It is preceded by two precursor states, Monoclonal Gammopathy of
Undetermined Significance (MGUS) and Smoldering MM (SMM)(4), which represent
stages of tumor progression with no evidence of end-organ damage. Unfortunately, MM
remains an incurable disease despite significant advances in treatment. Therefore,
immunotherapies may offer a promising strategy that could lead to significant responses,
especially in patients with poor-risk cytogenetics who do not benefit from standard of
care options or in patients at early stages of disease, like Smoldering MM. On the other
hand, MM might not be as immunogenic as other cancers, such as melanoma or lung
cancer(5, 6). Therefore, a better understanding of the immune microenvironment within
the bone marrow niche is critical for the development of novel immune therapies for not
just MM, but other cancers that infiltrate the bone marrow.
Recent success of immune checkpoint inhibitors indicates that anti-tumor T-cell responses
are functionally inhibited by poorly understood immunosuppressive mechanisms(7). One
of the major immunosuppressive mechanisms in tumor progression is the expansion of
regulatory immune cells, particularly regulatory T-cells (Tregs). Tregs are a subset of
CD4+ T lymphocytes characterized by the expression of Factor Forkhead Box P3
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(FOXP3)(8). They play an important role in the maintenance of self-tolerance and the
control of immune homeostasis; however, they can be co-opted by cancer cells to aid in
immune suppression and evasion. Tregs can inhibit tumor-specific CD8+ and CD4+ T-cell
effector functions through cell-cell contact and/or the production of anti-inflammatory
cytokines. Additionally, they can induce effector T-cell exhaustion, which is frequently
seen in the tumor microenvironment(9).
In MM, the role of Tregs in regulating tumor progression remains under active
investigation(10, 11). Despite controversy in previous reports regarding the number and
function of Tregs in the peripheral blood of MM patients(10-13), there is growing
evidence supporting a role for Tregs in MM progression(13, 14). Recent data have shown
a link between Tregs and sustenance of BM plasma cells, indicating that Treg activity
directly affects homeostasis of the plasma cell population(15), while in vitro experiments
have demonstrated that myeloma cells are capable of inducing Treg activation,
predominantly in a contact-dependent manner(16, 17). Moreover, CD38-expressing Tregs
have been suggested as suppressive modulators of anti-tumor immune responses in MM,
while CD38-targeting has been shown to inhibit immunosuppression in ex vivo co-
cultures(17). In this context, understanding Tregs homeostasis and specifically, how to
overcome their suppressive function is critical in developing effective immune therapies
for myeloma and other hematological malignancies infiltrating the bone marrow.
In the present study, we demonstrate that myeloma cells are responsible for Tregs
expansion and activation via secretion of type-1 interferon (IFN). Using the Vk*MYC
transplantable mouse model, we show that Tregs are critical for MM disease progression
and that depletion of Tregs leads to a complete remission. To further validate our findings,
we show that blocking IFNAR1 (interferon alpha and beta receptor 1) signaling
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significantly decreases myeloma-associated Treg proliferation and activation, which is
associated with prolonged survival of myeloma-injected mice. Our results, thus, suggest
that blocking type-1 IFN signaling represents a potential immune-therapeutic strategy in
MM.
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RESULTS
Increased number of Tregs at early stages of disease progression in the BM of a
transgenic MM transplantable mouse model and BM aspirates of MM patients.
To examine the T-cell immune microenvironment in MM during different stages of
disease progression, we injected C57BL/6 mice with transplantable Vk*MYC cells
(Vk12598 cells)(18). The Vk*MYC transgenic mouse uniquely models the natural history
of human MM, with progression of disease from early pre-malignant stages to overt
MM(19). To specifically investigate the changes that occur overtime in the immune
microenvironment within the bone marrow, where the tumor actually grows, vs.
peripheral blood (PB), we used Mass Cytometry, Cytometry by Time of Flight
(CyTOF)(20) to analyze the immune cell profile of Vk*MYC-injected mice, both in the
BM and the PB, at two time points during disease progression compared to control mice
(n=3 per group, per time point, Figure 1A). The gating strategy for Tregs is described in
Supplementary Figures 1A and 1B. CyTOF data were validated by flow cytometry
analyses in independent experimental settings (n=5 per group).
We observed a significant increase in the proportion of Tregs (CD4+FOXP3+) in the PB
and BM of Vk*MYC-injected mice, compared to control mice. Interestingly, the
differences were observed earlier in the BM starting from the early time point (Figures
1B-C and Supplementary Figure 1B-F), but only became detectable in the PB at later time
point (Figure 1D), indicating that Treg regulation occurs early within the tumor
microenvironment, before these changes are reflected in the peripheral blood.
We then analyzed the effector T-cells (Teffs: CD4+CD44++CD62Llow and
CD8+CD44++CD62Llow)(21) (22)/Tregs ratio to assess the suppression of T-cell immunity
in the BM microenvironment. We observed a decrease in the Teffs/Tregs ratio in the BM
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microenvironment and PB at the late time point (Figure 1E, Supplementary Figure 2A),
suggesting the suppression of T-cell immunity at a more advanced stage of disease.
Previous reports have shown an increased frequency of functional FOXP3-expressing T-
cells in the PB and BM of myeloma patients(10, 23). Here, we sought to define whether
the increase in Tregs occurs in the bone marrow already at the precursor stage of multiple
myeloma, in smoldering multiple myeloma (SMM). For this, we analyzed the distribution
of Tregs in BM aspirates of SMM patients (n=17), and compared it to that of healthy BM
donors (n=11). CyTOF analyses of the CD138-negative BM fractions demonstrated a
significant increase of CD3+CD4+ T-cells (data not shown), activated
CD4+CD25+FOXP3+ T-cells as well as CD4+CD25+FOXP3+CD127-/low Tregs within
the CD45+CD3+ compartment in SMM BM compared to healthy controls (Figure 1F,
Supplementary Figure 2B). This data from the human samples is in agreement with the
murine transplantation model.
Immune checkpoint receptors are upregulated in Tregs present within the bone marrow
microenvironment of myeloma-injected mice.
Immune checkpoint receptors, such as PD1 (Programmed cell death 1), LAG3
(Lymphocyte activation gene 3) and TIM3 (T cell immunoglobulin mucin 3), inhibit Teffs
function in the presence of cognate ligands(24). Conversely, when these receptors are
expressed on Tregs, the function and/or proliferation of Tregs are enhanced(7). We
measured number of positive cells, as well as the mean expression of immune checkpoint
receptors (PD1, LAG3 and TIM3) on Tregs in the BM and PB of Vk*MYC-injected and
control mice by CyTOF. BM Tregs of myeloma-bearing mice showed significantly higher
expression of immune checkpoint receptors, compared to control BM Tregs (Figures 2A-
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C). In contrast, PB Tregs of Vk*MYC-injected mice did not show increased expression
of these receptors, compared to control PB Tregs (Figure 2D, Supplementary Figure 3A-
C), suggesting that tumor site affects Treg phenotype and enhances its suppressive
function. SPADE analysis, as depicted in Figure 2B, revealed a significant increase in
LAG3 expression on BM Tregs expressing PD1 and/or TIM3; LAG3 has been shown to
be a marker of Tregs expanding at the tumor site(25). Along with the increase in
proportion of Treg expressing immune checkpoint receptors in BM of myeloma-injected
mice, there is an elevated number of Tregs co-expressing PD1, LAG3 and TIM3 receptors
as compared to healthy mice (Supplementary Figure 3D). Interestingly, we also observed
significantly higher levels of expression of PD1, LAG3 and TIM3 on Vk*MYC-injected
BM CD4+ and CD8+ Teffs (CD44++CD62Llow), which represent markers of T-cell
exhaustion and suppressed effector functions (Figure 2E, Supplementary Figure 3E-G).
These results were further validated in a second mouse model of MM, C57BL/KalwRij,
that was injected with 5TGM1 murine myeloma cells. Similar to the results of the
Vk*MYC-injected mice, we observed a significant increase in the frequency and
activation of BM Tregs in the bone marrow microenvironment of 5TGM1-injected
C57BL/KalwRij mice, compared to control group (Supplementary Figure 4A-D).
In line with our murine data, we observed a significantly higher proportion of
CD4+CD25+FOXP3+ Tregs expressing immune checkpoint molecules in BM aspirates of
SMM patients (n=17) compared to those in healthy BM (n=11) (Figure 2F). Interestingly,
SMM patients have significantly higher numbers of C-C Chemokine Receptor type 7
(CCR7)-expressing cells in their CD4+CD25+FOXP3+ Treg cell compartment, which
indicates an upregulation of the migratory phenotype of FOXP3-expressing cells (Figure
2F). Examination of immune molecules co-expression on Treg cells revealed that mainly
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proportion of cells expressing PD1, TIM3 and CTLA4 were significantly increased in
SMM group (Supplementary Figure 4E). In addition, analysis of the mean expression fold
change of each immune checkpoint molecule indicates elevated levels of immune
checkpoint receptors on Treg cells in individual SMM patients as compared to healthy
donors (Figure 2G). Further analyses of the CyTOF profiles of immune checkpoint
molecules present on CD4+Teff and CD8+Teff cells in the BM of SMM patients,
compared to healthy BM, revealed individual patients with elevated expression of TIM3,
PD1, CTLA-4 and CCR7 as depicted in the heatmap data in Figure 2H. In 35.3% of the
SMM patients we observed at least a twofold increase in the levels of CD27 and CCR7
on CD4+Teff and TIGIT on CD8+Teff as compared to the expression of these molecules
in the BM of healthy donors. Together, these results indicate an upregulated expression
of immune checkpoint inhibitors on Tregs within the myeloma bone marrow
microenvironment as early as the smoldering myeloma stage.
Tregs contribute to Vk*MYC progression.
A CD38-expressing subset of Tregs from MM patients has been shown to exhibit
immunosuppressive activity in ex vivo co-cultures(17). In order to determine the
significance of Tregs in myeloma progression in vivo, we used a transgenic inducible
knock-out mouse model: “depletion of regulatory T cell” (DEREG) mice, expressing a
diphtheria toxin (DT) receptor-enhanced green fluorescent protein (GFP) fusion protein
under the control of the FOXP3 gene locus, that allows the selective depletion of FOXP3+
Tregs by DT injection(26). Survival of DEREG mice injected with Vk*MYC cells,
followed by 3 injections of DT, was compared to that of Vk*MYC-injected DEREG mice
injected with PBS and that of Vk*MYC-injected wild-type littermates injected with DT
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(Figure 3A). Effective depletion of BM Tregs in Vk*MYC-injected mice was observed
upon DT injection into DEREG mice (Figure 3B). A significant tumor reduction was
observed in the DT-treated DEREG mice, compared to control groups, as shown by the
number of CD138+B220- Vk*MYC cells obtained from their bone marrows
(Supplementary Figure 5A). More importantly, there was a significant difference in
survival, with the control group surviving for a median of 30 days, while the DT-treated
DEREG mice showed no evidence of myeloma progression for up to 60 days of
experimental setup (p<0.0001) (Figure 3C). Flow cytometry analysis of BM cells on day
3 after last DT treatment in Vk*MYC-injected mice demonstrates a significant decrease
in CD138-expressing plasma cells and an increased Teff/Treg ratio compared to control
group (Supplementary Figure 5B). Interestingly, CyTOF analysis of the cellular content
of bone marrow cells showed a significantly increased proportion of CD19-expressing B
cells and F4/80+ macrophages in mice with Treg depletion compared to control mice
(Supplementary Figure 5C).
To further confirm the critical importance of Tregs in regulating tumor progression in
MM, we performed a Treg transfer experiment, in an effort to demonstrate that the
adoptive transfer of Tregs will lead to rapid tumor progression in vivo. We injected sorted
C57BL/6 CD3+CD25- non-Tregs, with or without CD4+CD25+ Tregs, into RAG2 knock
out (ko) immune-deficient mice, which lack a mature T- and B-cell compartment,
followed by Vk*MYC transplantation, and measured their survival (Figure 3D). In the
short time period of about 30 days of survival of these mice, we did not observe weight
loss or signs of colitis in these mice. We did not observe diarrhea or GI symptoms in the
mice. Mice injected with Tregs and CD3+ non-Tregs had significantly shorter survival
compared to mice injected with CD3+ non-Tregs only (Figure 3E), indicating that the
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Treg compartment suppresses anti-myeloma T-cell immunity. Analysis of the BM of
moribund mice showed significant increase of Tregs in the group injected with
CD4+CD25+ Tregs (Figure 3F, Supplementary Figure 5D), confirming that transferred
Tregs proliferated in the recipient’s BM tumor microenvironment.
In parallel, to further validate our results, we specifically depleted Tregs in Vk*MYC-
injected mice using a CD25 antibody (250 Pg of anti-murine CD25 mAbs on day -4 and
day 10 post Vk*MYC cell injection, n=6); isotype antibody treatment was used in the
control group (n=6) as described in previous studies (27). Compared to isotype-treated
mice, the CD25-treated group demonstrated significantly longer survival (Supplementary
Figure 6A, 6B), further confirming the critical role of Tregs in myeloma progression.
Together, our data provide direct in vivo evidence of Tregs supporting multiple myeloma
disease progression.
Human and murine myeloma cells induce and expand Tregs in a contact-dependent
and independent manner
To elucidate mechanisms involved in Treg induction and expansion in MM, we performed
Treg induction and expansion assays by co-culturing Vk*MYC cells with CD4+CD25-
non-Tregs or CD4+CD25+ Tregs respectively, using direct co-culture assays. Both assays
showed significant increase in CD4+CD25+FOXP3+ Tregs when co-cultured with
Vk*MYC cells, compared to B-cells or “No cells” controls (Figure 4A, 4B). Similar
results were obtained when a transwell co-culture assay separating Vk*MYC cells from
T-cells was performed, indicating that it is a soluble factor produced from Vk*MYC cells
that leads to the induction and expansion of Tregs (Figure 4A, B and Supplementary
Figure 7A).
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We further explored a contact-independent Treg induction mechanism in human samples.
For this, we co-cultured human CD4+CD25- PB cells with MM1S and OPM2 myeloma
cell lines without cell-cell interactions. Consistent with our murine data, we observed a
significant induction of CD4+CD25+FOXP3+ cells by co-culture of CD4+CD25- PB cells
with myeloma cells, compared to CD19-expressing B-cells or “No cells” controls (Figure
4C).
Together, these data show that murine and human myeloma cells induce and promote
expansion of Tregs in a cell-cell contact-dependent and independent manner, leading to
the increased presence of Tregs in the tumor microenvironment.
Myeloma cells regulate Tregs in the BM microenvironment by secretion of type 1
interferons.
To further characterize mechanisms that lead to the activation of myeloma-associated
Tregs, we performed RNA sequencing of flow-sorted BM GFP-expressing Tregs from
Vk*MYC-injected DEREG mice and control DEREG mice. We observed a significant
difference in mRNA expression between Tregs derived from the bone marrow
microenvironment of myeloma-injected mice and those derived from control mice, as
depicted in the heat map in Figure 5A. The top genes that were significantly up- or
downregulated in Tregs from Vk*MYC-injected mice, compared to control, are shown in
Supplementary Table 1. Consistent with our CyTOF data at the protein level, expression
of checkpoint-related molecules, including PDCD1 (PD1), LAG3 and HAVCR2 (TIM3),
was significantly upregulated in Vk*MYC-associated Tregs, compared to control Tregs
(Figure 5B). Additionally, other checkpoint-related molecules, such as TNFRSF4
(OX40)(28) and TNFRSF18 (GITR)(29) were also found to be highly expressed in
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Vk*MYC-associated Tregs (Figure 5B).
Tregs use multiple effector molecules, such as IL-10, TGF-b, EBI3, FGL2, GZMB and
PRF1, to suppress the immune effector cells(30) (31) (32). In our study, Vk*MYC-
associated Tregs showed a significantly higher expression of EBI3, FGL2 and GZMB
(Figure 5C), indicating that these molecules may play a role in the suppressive activity of
myeloma-associated Tregs.
Chemokine receptors play a significant role in Treg migration and homing. We analyzed
the expression of CXCR3, CCR6 and CCR7, which are all well-known mediators of Treg
migration to inflammatory sites(33-37). CXCR3 was found to be significantly
overexpressed in Vk*MYC-associated Tregs, compared to control (Figure 5D),
suggesting a significant role for CXCR3 in Treg migration to the myeloma
microenvironment. Interestingly though, CCR7 levels were significantly downregulated
in Vk*MYC-associated Tregs, compared to control, indicating differences in the
regulation of Treg migration in the tumor setting.
To determine pathways that regulate Treg activation and expansion in the MM tumor
microenvironment, we performed Gene set enrichment analysis (GSEA) for the genes
that were found to be significantly upregulated in Vk*MYC-associated Tregs, compared
to control. Interestingly, GSEA analysis showed enrichment of type-1 interferon (IFN) -
related genes in Vk*MYC-associated Tregs, compared to control (Figure 6A). According
to the GSEA analyses, several differentially regulated proteins contributed to the
enrichment score for Interferon alpha beta signaling pathway. These include the type-1
interferon-induced proteins playing a critical role in cellular innate antiviral responses,
such as Interferon-induced 15 kDa protein (ISG-15), interferon-induced Mx1 and Mx2,
IFIT1 and IFIT3, OAS2, OAS3. Additionally, key transcriptional regulators of type I
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interferon (IFN)-dependent immune responses, such as Interferon Regulatory Factors 7
and 9 (IRF7, IRF9) contributed to the enrichment profile. Signal transducer and activator
of transcription STAT1 and STAT2 were enriched in the gene list as well. These results
indicate that type-1 IFN pathway is activated in the BM Tregs of Vk*MYC-injected mice
and may constitute a novel pathway of Treg regulation in the MM immune
microenvironment.
We then sought to examine whether myeloma cells secrete type-1 IFNs, potentially
leading to Treg activation. We observed a significant increase of IFN-beta in the
supernatants of Vk*MYC cells in culture, compared to the supernatant of control B-cells
obtained from C57BL/6 mice (Figure 6B), while IFN-alpha was below detection level in
both cell types (data not shown). Collectively, these results suggest that myeloma-cell-
secreted type-1 INF could be a mediator of Treg activation in MM.
INFA1 expression is associated with poor outcome in MM patients
We then investigated whether CD138-expressing plasma cells from MM patients showed
significant changes in pathways or the expression levels of genes related to type-1 IFN
signaling. GSEA analysis of two gene expression datasets generated from CD138-
enriched plasma cells, one from 73 newly diagnosed MM patients and one from 147 cases
of MGUS, SMM, MM and relapsed myeloma (GSE6477), showed significant enrichment
for type-1 IFN signaling compared to 15 healthy donors (Figure 6C). Notably,
upregulated expression of IFNA2, one of the INF�alpha homologous genes(38), and IFN
regulatory transcription factor 3 (IRF3)(39) contributed to core enrichment score in this
dataset. Further analysis showed significantly increased levels of IRF3 and other
members of the IFN regulatory factors, including IRF4, IRF5 and IRF9 (Figure 6D).
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Similarly, there was an increase in the expression of IFNA1 and IFNA2 isoforms, and
another key component of the IFN induction pathway, MAVS protein(40, 41), in MM
samples, compared to healthy controls. Interestingly, lower expression of TLR2 and
TLR8 receptors was also observed in MM patients, pointing to concurrent deregulation
of Toll-like receptor signaling in malignant plasma cells (Figure 6D).
We next sought to determine the prognostic role of genes regulating type-1 IFN in MM,
and therefore analyzed the survival probability of these genes in a large dataset of newly
diagnosed MM patients (n=542, GSE2658). Median modus was used for cutoff
determination, P values were corrected for multiple testing and R2 visualization platform
(http://r2.amc.nl) was used for calculation. We found a significant decrease in the overall
survival of patients with high levels of IFNA1 (Figure 6E), supporting the results of our
murine experiments and indicating that type-1 interferon is indeed an essential player in
MM progression. Interestingly, lower levels of endosomal Toll-like receptor TLR8(42),
were also associated with poor outcome in MM patients (Figure 6F), further suggesting a
high impact for deregulated type-1 IFN pathways on MM progression.
Blocking interferon alpha and beta receptor 1 (IFNAR1) suppresses inhibitory
function of Tregs and inhibits myeloma progression
In order to validate the effect of myeloma-cell-secreted type-1 IFN on Treg function, we
co-cultured Vk*MYC cells with sorted Tregs and treated them with either IFNAR1
(interferon alpha and beta receptor 1) antibody, which blocks ligand-induced intracellular
signaling and induction of type I IFN-induced biologic responses(43), or isotype control.
Treg proliferation, analyzed by Ki-67 expression, was significantly decreased in the
IFNAR1-treated group, compared to control. This led to a decrease in Treg expansion in
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IFNAR1-treated cells, compared to control (Figure 7A-D). This study was also performed
with 5TGM1 MM cells, which were capable of inducing expansion of C57BL/KaLwRij
Tregs in a transwell co-culture assay (Supplementary Figure 7B). Once more, IFNAR1-
blocking antibody treatment led to decreased Treg proliferation and expansion, compared
to treatment with isotype control (Supplementary Figure 7C). Collectively, our data
demonstrate that myeloma-cell-secreted Type-1 IFN induces proliferation and expansion
of Tregs when present in the myeloma microenvironment.
Finally, we sought to validate in vivo the significance of IFNAR1 as a critical regulator
of Treg function and expansion in MM. For this, we performed a T-cell transfer
experiment using IFNAR1 ko Tregs; we injected sorted wild-type (C57BL/6) CD3+CD25-
non-Tregs, wild-type Tregs or IFNAR1 ko Tregs into RAG2 ko mice, followed by
Vk*MYC transplantation, and then analyzed tumor growth and survival (Figure 7E).
Mice injected with IFNAR1 ko Tregs lacked the effect of wild-type Tregs in enhancing
progression of MM, with survival rates similar to those of mice that had been injected
with non-Tregs (CD3+ only) (Figure 7F). Namely, Tregs lacking IFNAR1 failed to
suppress MM-directed T-cell immunity, and that failure was further reflected in their
reduced proliferation and expansion in vivo. While an increase in the number of Tregs
was observed in mice that received wild-type Tregs, in accordance to the results of our
previous experiments, the expansion was abrogated in the IFNAR1 ko mice, confirming
that MM cells induce Treg expansion through type-1 IFN signaling (Figure 7G). As
expected, the BM Tregs of mice transferred with IFNAR1 ko Tregs had lower expression
of IFNAR1, compared to mice transferred with wild-type Tregs (Figure 7H). Moreover,
when comparing BM Tregs between mice injected with wild-type Tregs and those
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injected with IFNAR1 ko Tregs, we observed a significant decrease in the expression of
LAG3 in the latter group (Figure 7I), indicating that type-1 IFN signaling is also partly
responsible for checkpoint molecule expression on Tregs in the tumor microenvironment.
Thus, our data demonstrate that blocking IFNAR1 reduces myeloma-mediated Treg
immunosuppression, leading to better survival rates of myeloma-injected animals.
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DISCUSSION
Increased presence of Tregs in the tumor microenvironment of cancer patients has been
associated with both improved(44, 45) and poor prognosis(46, 47). A few studies
described an increase in the number of Tregs in the peripheral blood of patients with
myeloma, compared to healthy donors(10, 12, 13, 48), indicating a potential role for Tregs
in multiple myeloma in particular. However, the mechanisms of Treg expansion in
myeloma, as well as the role of Tregs in myeloma progression, have not been fully
elucidated(49). Understanding those mechanisms is critical, as it could be key in
developing new immune therapies in MM. In the present study, we identified a feedback
loop between myeloma cells and Tregs, wherein type-1 IFN secreted from myeloma cells
induces immunosuppression caused by Tregs, thus promoting myeloma progression.
Interrupting this loop by blocking IFNAR1 signaling relieves immunosuppression and
prevents/delays tumor progression.
First, we confirmed that Tregs increase in number and expand in the BM with disease
progression, a phenomenon that was detectable in the BM at an earlier time point,
compared to the PB, indicating that myeloma-promoting Treg deregulation first occurs in
the context of the tumor microenvironment. In addition, we showed that myeloma-
associated BM Tregs highly express immune checkpoint receptor molecules, like PD1,
LAG3 and TIM3. Again, in contrast to Teffs, where these molecules are markers of
exhaustion, in Tregs, they signify enhanced immunosuppressive function(7). Thus, our
data indicate that Tregs in the myeloma microenvironment are activated not only in terms
of quantity, but also quality.
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Next, we sought to determine the importance of Tregs in myeloma progression. Our in
vivo Treg depletion and Treg transfer experiments demonstrated a strong impact of Tregs
on the progression of multiple myeloma, as continuous Treg depletion in vivo led to tumor
remission and prolonged survival of Vk*MYC-injected mice. These results are consistent
with other tumor models where enhancement of anti-tumor immune responses occurs
with depletion of Tregs by targeting highly expressed surface receptors(17, 50-52).
Interestingly, we found that the cellular content of the bone marrow after Treg depletion
reflects an environment hostile for tumor growth with a significant increase in Teff/Treg
ratio as well as B cells and macrophages, indicating that these cells could be critical for
the reconstitution of the immune microenvironment and the prevention of tumor
progression in MM.
To further explore mechanisms by which myeloma-associated Tregs are activated, we
then conducted RNA sequencing, comparing BM Tregs of Vk*MYC-injected mice to BM
Tregs of control mice. In addition to checkpoint-related genes and Treg effector genes
being significantly upregulated, type-1 IFN pathway was found to be highly enriched in
the gene signature of myeloma-associated Tregs. Validating these results, expression data
from MM patients also demonstrated upregulation of well-known genes regulating type-
1 IFN induction pathways, such as IRF3, IRF4, IRF5, IRF9 and MAVS protein in
malignant plasma cells; these genes were associated with worse prognosis in the survival
of MM patients underscoring the impact of IFNA1 on survival outcome.
Type-I IFNs are known players in a variety of diseases, such as viral infection,
inflammatory bowel disease, systemic lupus erythematosus and type-I diabetes(53), but
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their role in myeloma remains poorly characterized. Recently, it was shown that type-1
IFN signaling is essential for maintenance of FOXP3 expression, as well as Treg
suppressive function under inflammatory conditions(54), while IFN-alpha/beta receptor
signaling was shown to promote Treg development and function under stress
conditions(55). In the cancer setting, IFNAR-mediated signaling was found to be
necessary for tumor-infiltrating Treg activation and production of IL10, as well as
suppression of T-helper 17 cells, in the tumor microenvironment of colon
adenocarcinoma(56). In our system, we demonstrated that type I IFN is critical for Tregs
function using in vitro and in vivo models.
Based on this, we thus hypothesized that suppression of the type-1 IFN pathway could
inhibit myeloma-mediated Treg expansion and hinder tumor progression. Indeed,
IFNAR1 antibody led to the suppression of the proliferation of myeloma-associated Tregs
in an in vitro co-culture assay, as well as extending the survival of myeloma-injected mice
in a T-cell transfer experiment with IFNAR1 ko Tregs.
In conclusion, we have unprecedented findings demonstrating that Treg function and
homeostasis is regulated by type-1 IFN secreted from myeloma cells in the BM
microenvironment, effectively evoking a feedback loop between Tregs and myeloma cells
that promotes disease progression and leads to inferior outcome. Prolonged survival in
MM mouse models was achieved by Tregs depletion and by blocking IFNAR1 signaling
and suppressing myeloma-mediated Treg induction and proliferation, indicating that
type-1 IFN signaling is a novel therapeutic target for regulating Treg-mediated
immunosuppression in multiple myeloma.
21
METHODS
Cells
The 5TGM1 cell line was a kind gift of Dr. David G. Roodman (Indiana University,
Indianapolis, Indiana, USA). The Vk*MYC derived transplantable Vk12598 cell line(18)
was kindly provided by Dr Marta Chesi and Dr P. Leif Bergsagel (Mayo Clinic, Scottsdale,
AZ) and was maintained and expanded by in vivo re-transplantation in Rag2 ko mice
similar to previous reports(5, 57). For in vitro experiments, these cell lines were cultured
in RPMI-1640 containing 10% fetal bovine serum/FBS (Sigma Chemical, St Louis, MO),
2mM L-glutamine, 100 U/mL penicillin, and 100μg/mL streptomycin (GIBCO, Grand
Island, NY). Human MM1S cells were purchased from ATCC. Human OPM2 myeloma
cell line was purchased from DMSZ.
Primary peripheral blood (PB) cells or bone marrow samples from patient/healthy donor
aspirates were collected from the Dana-Farber Cancer Institute. CD 138-negative bone
marrow cell fraction was isolated using MACS technology (Miltenyi Biotec).
Mice
C57BL/6, DEREG, Rag2 ko and IFNAR1 ko mice, all under C57BL/6 background, were
purchased from Jackson Laboratories (Bar Harbor, Maine). C57BL/KalwRij mice were
purchased from Harlan Laboratories (Horst, Netherlands).
2×106 Vk12598 cells were injected intravenously (IV) into each mouse for T-cell analysis
or survival studies. 3×106 5TGM1 cells were injected IV into C57BL/KalwRij mice for
T-cell analysis. BM cells and peripheral blood from mice were harvested for CyTOF
analysis by BM aspiration from the femur and by sub-mandibular bleeding, respectively.
For other experiments, BM cells were obtained through flushing of femurs with 1XPBS,
22
as previously described(58).
Treg depletion study
Treg depletion was performed during Vk*MYC progression on DEREG mice by injecting
intraperitoneally 10 ng/g/mouse of DT on day 5, 12 and 19 post Vk*MYC injection. For
control groups, same amount of DT or PBS were injected into C57BL/6 or DEREG mice,
respectively. Depletion of Tregs using CD25 antibody was performed as described in
previous reports (250 Pg of anti-murine CD25 mAbs)(27). Isotype antibody and anti-
CD25 antibody treatment was performed on day -4 and day 10 post tumor injection.
T-cell transfer study
Tregs were obtained from spleens of C57BL/6 or IFNAR1 ko mice by flow-sorting CD4+
CD25+ cells by FACSAria II flow cytometry system (BD Biosciences, San Jose, CA).
Similarly, CD3+ non-Tregs were obtained from C57BL/6 splenocytes by sorting CD3+
CD25– cells. 1×106 Tregs and 3×106 non-Tregs were injected IV into Rag2 ko mice,
followed by Vk12598 cell injection 5 days afterwards.
Analysis of cells by CyTOF
Sample staining and data analysis were performed as reported previously(57). Briefly,
cells were first stained with MaxPar Intercalator-Rh (Fluidgm Sciences Inc., Sunnyvale,
CA) for determination of viable cells. After blocking with mouse Fc Receptor Blocking
Reagent (Miltenyi Biotec Inc., San Diego, CA), cell surface marker antibodies were
added and stained. Following staining, cells were washed with MaxPar Cell staining
23
buffer (Fluidgm Sciences Inc.) and fixed/permeabilized with Fix/Perm buffer (PBS with
1.6% Paraformaldehyde and 0.3% Saponin). Cells were then washed with Cell staining
buffer containing 0.3% saponin, following Intra-cellular antibody staining. For mass
cytometry analysis, cells were stained with MaxPar Ir DNA intercalator (Fluidgm
Sciences Inc.) overnight at 4°C. Cells were then washed with Cell staining buffer and
then finally with MaxPar water (Fluidgm Sciences Inc.) alone. Cells were acquired on the
CyTOF2 mass cytometer (Fluidgm Sciences Inc.). The acquired data were analyzed in
Cytobank (Cytobank, Inc., Menlo Park, CA). The gating strategy of Tregs is described in
Supplementary Figure 1A.
The CD138-negative populations of SMM patients or healthy donors' bone marrow cells
were thawed, washed and barcoded using the Cell-ID™ 20-Plex Pd Barcoding Kit
(Fluidgm Sciences Inc.). After barcoding, cells were incubated with human surface
marker antibodies as a single multiplexed sample (TIGIT-143Nd, CD152 (CTLA4)-
152Sm, PD1-175Lu, TIM3-153Eu (Maxpar) and Maxpar® Human T-Cell Phenotyping
Panel Kit, 16 Marker). The cells were fixed with 4% Paraformaldehyde, permeabilized
with ice-cold Methanol and incubated with antibodies against intracellular proteins
(FOXP3 -clone PCH101, custom conjugation with 165Ho by Harvard Medical Area
CyTOF Antibody Resource and Core, Boston, USA). Then the cells were washed,
resuspended at the concentration of 1x106 cells/mL and acquired on a CyTOF Helios mass
cytometer (Fluidgm Sciences Inc.). Downstream analysis of the individual component
samples was performed after running the debarcoding application. Human T-cell
populations were gated according to the Maxpar Human T Cell Phenotyping Panel Kit.
24
Flow cytometric analysis
Anti-mouse antibodies against CD3, CD4, CD8, PD1 (Biolegend, San Diego, CA), CD25,
LAG3, TIM3, B220, FOXP3, Ki-67 (eBioscience, San Diego, CA), CD138 (BD
Biosciences, San Jose, CA) and anti-human antibodies against CD4, CD25, FOXP3
(eBioscience, San Diego, CA) were used for the FACS analyses. Cells were surface
stained, fixed/permeabilized and stained intracellularly using the FOXP3/Transcription
Factor Staining Buffer Set (eBioscience, San Diego, CA) according to the manufacturer’s
protocol. The data of the stained samples were acquired using FACS Canto II flow
cytometer (BD Biosciences). The acquired data were analyzed in Cytobank (Cytobank,
Inc.)
RNA Sequencing Analysis
Total RNA was isolated from flow-sorted CD4+FOXP3-GFP+ cells acquired from the BM
of Vk*MYC-injected and control mice using RNeasy Micro kit (QIAGEN, Venlo,
Netherlands). Whole RNA was subject to library preparation using NEBNext Ultra RNA
Library prep for Illumina kit (New England Biolabs, Ipswich, MA). Quality control of
the libraries was evaluated by Bioanalyzer analysis with High Sensitivity chips (Agilent
Technologies, Santa Clara, CA). Sequencing was performed on a HiSeq 2000 (Illumina,
San Diego, CA) by 2X50bp paired-end reads at the Biopolymers Facility of Harvard
Medical School. We used Picard and STAR to process and align the RNA-seq reads with
mouse reference genome (mm10) and to compute a series of quality control metrics.
RNA-seq transcript abundances were estimated using RSEM (Li B and Dewey CN, 2011).
Genes with low expression (RSEM expected counts) were filtered out and differential
gene expression between different samples was determined using DESeq2 (Love MI et
25
al., 2014). Differential expression was selected based on fold changes > 2.0 and a p-value
threshold of 0.05 (Sequencing data are submitted to GEO; reference number will be
provided). Gene set enrichment analysis (GSEA) was used to identify significantly
enriched pathways(59), with false discovery rate (FDR) <0.25 and p-value < 0.05. Gene
sets were downloaded from the Broad Institute’s MSigDB
(http://www.broadinstitute.org/gsea/index.jsp). The accession numbers for the human
data reported in this manuscript are GEO: GSE6477 and GSE2658.
ELISA
Murine myeloma cell lines' (C57BL/6 or C57BL/KalwRij mice) B-cells were seeded into
12-well plates at a concentration of 2x105 /mL and cultured for 24hrs. Murine IFN-alpha
and IFN-beta levels of cell culture supernatants were detected using VeriKine-HS Mouse
IFN ELISA Kits (PBL Assay Science, Piscataway, NJ) according to the manufacturer’s
protocol.
Treg induction and expansion assay
CD4+CD25- non-Tregs (induction assay) at 2×105 or CD4+CD25+ Tregs (expansion
assay), isolated from C57BL/6 or C57BL/KalwRij splenocytes were co-cultured with
1×105 myeloma cell lines in the presence of recombinant murine IL-2 (1000 IU/ml; R&D
Systems, Minneapolis, MN), anti–CD3-Ab (5 ug/ml; eBioscience) and anti–CD28-Ab (5
ug/ml; eBioscience) for 72 hrs. TGF-beta (10 ng/mL; R&D Systems) was added for the
induction assay. IFNAR1-Ab (Biolegend) or isotype control-Ab were added to the
appropriate wells at the concentration of 100 ug/mL.
26
Human CD4+CD25- non-Tregs or CD19-expressing B-cells were obtained from fresh PB
cells using CD4+CD25+ Regulatory T Cell Isolation Kit or CD19 Microbeads (Miltenyi
Biotech). Briefly, red blood cells were lysed (RBC lysis buffer, Biolegend), washed and
isolated according to manufacturer’s instructions. The CD4+CD25- non-Tregs at 2×105
were co-cultured with 1×105 myeloma cell lines in the presence of recombinant human
IL-2 (1000 IU/ml; R&D Systems, Minneapolis, MN) and the Treg Suppression Inspector
beads (Miltenyi Biotech) in 1:1 beads to cells ratio. Transwell permeable inserts with a
pore size 0.4 Pm were used to prevent cell-cell interactions (Corning).
Statistical analysis
One-way ANOVA was used when three or more independent groups were compared. The
unpaired Student t test was used to compare two independent groups with Bonferroni
correction for multiple comparisons. For survival data, Kaplan–Meier curves were plotted
and compared using a log-rank test. All tests were two-tailed. A p-value < 0.05 was
considered statistically significant. Analysis was performed with GraphPad Prism
Software (GraphPad Software Inc., La Jolla, CA).
Study approval
All mice were treated, monitored, and sacrificed in accordance with an approved protocol
of the Dana-Farber Cancer Institute Animal Care and Use Committee (Boston, USA).
Human studies were approved by the Dana-Farber Cancer Institute Institutional Review
Board (Boston, USA). Informed consent was obtained from all patients and healthy
volunteers in accordance with the Declaration of Helsinki protocol.
27
AUTHORS’CONTRIBUTIONS
Conception and design: Y. K., O. Z., J. A. and I. M. G.
Acquisition of data: Y. K., O. Z., M. M., K. K., T. M., N. M., M. R., Y. J. S., M. R., M.
B., P. L., E. S. K., S. T., S. M., J. S., S. T., M. R. R., D. H., A. S.
Analysis and interpretation of data: Y. K., O. Z., M.M., J. P., S. M., J. S., Y. T., M. C.,
P. L. B., A. M. R., J. A., I. M. G.
Writing, review, and/or revision of the manuscript: Y. K., O. Z., J. P., R. S. P., A. M.
R., J. A., I. M. G.
CONFLICT OF INTERESTS
The Authors declare no relevant conflict of interests.
Funding support: NIH R01 CA181683-01A1 and Leukemia and Lymphoma Society.
28
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0
3
6
9
Tregs
FOXP3 intensity
Control Vk*MYC
FIGURE 1
E a r ly L a te0
1
2
3
4
5
Te
ffs
/ T
reg
s
C o n tro l
V k *M Y C
Control
A Early Late
BM and PBcollection
BM and PBcollection
B
C
F
p=0.032
E a r ly L a te0
1 0
2 0T
reg
s (
%)
C on tro l
V k *M YC
p=0.0003
Peripheral blood
p=0.002 p=0.0003
E a r ly L a te0
1 0
2 0
3 0
Tre
gs
(%
)
C on tro l
V k *M YC
Bone marrow
Figure 1. Tregs are increased in the BM of Vk*MYC transplantable mouse model.(A) BM and PB were harvested from control (n=3) and Vk*MYC-injected mice (n=3) at early (day 14)and late (day 28) time points for CyTOF analysis. (B) Significant increase of Tregs frequency withinCD4+ T-cells in the BM of Vk*MYC-injected mice compared to control mice from early time points.Data validated by FACS (n=5). (C) Spanning-tree progression analysis of density-normalized events(SPADE) was conducted on late BM CD4+ T-cells of control and Vk*MYC injected mice. The size of thenodes indicates the frequency of each population, while the color indicates the expression of FOXP3.Increase of FOXP3+ cells within CD4+ T-cells was observed in Vk*MYC-injected BM. (D) Significantincrease of Tregs frequency within CD4+ T-cells in the PB of Vk*MYC-injected mice compared tocontrol mice at late time points. (E) Significant decrease of Teffs (CD4+CD44++CD62Llow andCD8+CD44++CD62Llow)/Tregs ratio was observed in BM of Vk*MYC injected mice at the late time pointas compared to BM of control mice. (F) Significant increase of CD4+CD25+FOXP3-expressing cells andCD4+CD25+FOXP3+CD127-/low Tregs in BM aspirates of SMM patients (n=17) compared to healthydonors (n=11). Data combined from 3 independent experiments. Cell numbers were normalized to thecell proportion in healthy BM. (t-test 2-tailed, error bars indicate SD)
E
Vk*MYC
D
Tre
gs(%
)
ControlVk*MYC
ControlVk*MYC
Tre
gs(%
)
Early Late
Early Late
Early Late
ControlVk*MYC
Tef
fs/T
regs
NBMSMMp=0.016
Cel
l rat
io (%
)
p=0.007
Human bone marrow
FIGURE 2A B
C
E
P D 1 L AG 3 T im 30
1 0
2 0
3 0
4 0
5 0
Ex
pre
ssio
n in
Tre
gs
(%
) C on tro l
V k *M YC
p=0.0001
p<0.0001
p=0.002
D
Control
Vk*MYC
Early Late
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
Fold change
Figure 2. Upregulation of immune checkpoint molecules in Tregs in the BM of Vk*MYC-injected mice andSMM patients. (A) BM Tregs of Vk*MYC-injected mice have significantly higher frequency of immune checkpointreceptor (PD1, LAG3 and TIM3) expressing Tregs compared to control BM Tregs at the late time point (n=3 pergroup, validated by FACS with n=5 per group). (B) SPADE tree was conducted on BM Tregs of control andVk*MYC-injected mice at the late time point. The nodes of the tree represent clusters of cells that are similar in markerexpression. The color indicates the expression of LAG3. Increase of LAG3+ cells within Tregs was observed inVk*MYC injected BM. Heatmap of immune checkpoint receptor (PD1, LAG3 and TIM3) expression of BM (C) andPB (D) Tregs at early and late time points. The color indicates the mean expression fold change of each molecule. BMTregs of Vk*MYC injected mice had higher expression of immune checkpoint receptors compared to control BMTregs, while PB Tregs of Vk*MYC injected mice did not show increased expression compared to control PB Tregsthroughout disease progression. (E) Increased number of Tregs expressing immune checkpoint receptors in BM ofSMM patients (n=17) as compared to healthy donors (n=11). Data combined from 3 independent experiments. (G)Heatmap of immune checkpoint molecules and CCR7-chemokine receptor expression of Tregs in BM of MM patientscompared to healthy donors. The color indicates the ratio of mean expression of each molecule. Tregs of MM patientsshow elevated expression levels of immune checkpoint molecules (H) CD4+ and CD8+ T effector cells in BM of MMpatients have higher expression of immune checkpoint molecules compared to healthy donors. The color indicates theratio of mean expression of each molecule. (t-test 2-tailed, error bars indicate SD)
Control Vk*MYCPD1+
LAG3+
Tim3+
LAG3 intensity
G
Early Late
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
Control
Vk*MYC
Fold change
CD4 Teffs
PD1
LAG
3
TIM
3
PD1
LAG
3TI
M3
CD8 Teffs
Control
Vk*MYC
Fold change
CD
27C
TLA
-4
CC
R7
TIM
3
PD1
TIG
IT
Tregs
NBM
SMM
Ratio of means
NBM
SMM
Ratio of means
CD
27C
TLA
-4
CC
R7
TIM
3
PD1
TIG
IT
CD4 Teffs
CD
27C
TLA
-4
CC
R7
TIM
3
PD1
TIG
IT
CD8 Teffs
ControlVk*MYC
PD1+TIM3+Control Vk*MYC
LAG3+
TIM3
LAG3 intensity
LAG3PD1
Exp
ress
ion
in T
regs
(%)
F
0
5
10Chart Title
NBM SMM
NBMSMM
Cel
l rat
io (%
)
p=0.032
p=0.018
p=0.022p=0.013
p=0.003
p=0.306
H
FIGURE 3
A B C
Figure 3. Treg depletion in DEREG mice leads to extended survival of Vk*MYC-injectedmice, while adoptive transfer of Tregs promotes tumor progression in vivo.(A) Diphteria toxin or PBS were injected 3 times (day 5, day 12 and day 19) post Vk*MYC-injection into DEREG mice or wild type mice (n=7/group). (B) Proportion of Vk*MYC cells(CD138+B220- cells) in the BM of each group at day 20 post Vk*MYC injection. 3 mice from eachgroup were sacrificed at day 20 for analysis of BM Vk*MYC frequency. Significant tumorreduction occurred in the DEREG mice treated with DT compared to control mice. (t-test 2-tailedwith Bonferroni correction) (C) Kaplan-Meier curve showing significant increase in survival ofDEREG mice treated with DT (DEREG + DT) compared to control groups (DEREG + PBS, Wildtype + DT) (n=7/group) (p<0.0001: Log-rank). (D) For Treg transfer experiments, 3×106 non Tregswere injected with or without 1×106 Tregs i.v into Rag2 ko mice followed by Vk*MYC cellinjection 5 days afterwards. (E) Kaplan-Meier curve showing mice injected with Tregs and CD3+
non Tregs (Tregs + CD3+ non Tregs: Median survival 21 days) had significantly shorter survivalcompared to mice injected only with CD3+ non Tregs (CD3+ non Tregs: Median survival 25.5 days)(n=6/group) (p<0.01: Log-rank). (F) FACS analysis of mice BM showing significant increase ofBM Tregs in the “Tregs + CD3+ non Tregs” group compared to “CD3+ non Tregs” group(n=3/group). T-test 2-tailed. (error bars indicate SD)
D E R E G -D T D E R E G -P B S W ild typ e -D T0
5
1 0
1 5
Vk
*MY
C c
ell
s i
n t
he
BM
(%) p<0.001
p<0.001
Vk*
MY
C c
ells
in th
e B
M (%
)
WT+DTDEREG DEREG+PBS
D E F
DEREG + DT
DEREG + PBS
DT
Wild type + DT
DT DT
DT DT DT
PBS PBS PBS
DEREG +DT
DEREG +PBS
Wild type +DT
DT DT DT
PBS PBS PBS
DT DT DT
p<0.001 (Log-rank)
DEREG+DT
Wildtype+PBSDEREG+PBS
Perc
ent s
urvi
val
Days post tumor injection
GDEREG+DTDEREG+PBSWild type+DT
p<0.001(Log-rank)
CD
3+ N
o n Tre
g s
T reg s +
CD
3+ N
o n Tre
g s0
1 0
2 0
3 0
Tre
gs
(%
)
p=0.001
Treg
s(%
)CD3+
non T
regs
Treg
s+CD3+
nonT
regs
p=0.001
0
20
40
60
80
100
15 20 25 30Days post tumor injection
Perc
ent s
urvi
val
p<0.01 (Log-rank)
Tregs+CD3+NonTregsCD3+nonTregs
Perc
ent s
urvi
val
Days post tumor injection
Tregs + CD3+ non TregsCD3+ non Tregs
p<0.01(Log-rank)
Tregs+
CD3+ non Tregs
CD3+ non Tregs
Tregs+
CD3+ non Tregs
CD3+
non Tregs
FIGURE 4
A B
C
B cells
OPM2 cells
No cells
MM1S cells
0
5
10
15
20
25
No cells B cells MM1S OPM2
Human Treg induction assay
Treg
s(%
)
No cells B cells MM1S OPM2
T r a n s -w e ll D ir e c t0
2 0
4 0
6 0
8 0
1 0 0
Tre
gs
(%
)
N o ce lls
B c e lls
V k *M Y C c e lls
p<0.001
p<0.001p<0.001
p<0.001
Treg expansion assay
No cellsB cellsVk*MYC cells
Trans-well Direct
Treg
s(%
)
T r a n s -w e ll D ir e c t0
1 0
2 0
3 0
4 0
5 0
Tre
gs
(%
)
N o ce lls
B c e lls
V k *M Y C c e lls
p<0.001
p<0.001p<0.001p<0.001
Treg induction assay
No cellsB cellsVk*MYC cells
Trans-well Direct
Treg
s(%
)
Figure 4. Myeloma cells induce and expand Tregs in vitro.(A) Treg induction assay. CD4+CD25- non-Tregs were co-cultured with C57BL/6 B cells or Vk*MYCcells for 72 hrs using trans-well or direct co-culture. Significant Treg induction was observed in theVk*MYC co-culture group either under trans-well or direct co-culture compared to “No cells” or “Bcells” groups. Average of experiments performed in triplicate is shown. The experiment was repeated 3times. (B) Treg expansion assay. CD4+CD25+ Tregs were co-cultured with C57BL/6 B cells orVk*MYC cells for 72 hrs using trans-well or direct co-culture. FOXP3+ cells within the CD4+ cells areshown. Significant Treg expansion was observed in the Vk*MYC co-culture group either under trans-well or direct co-culture compared to “No cells” or “B cells” groups. Average of experiments performedin triplicate is shown. The experiment was repeated 3 times. (C) Significant Treg induction wasobserved in co-culture of MM1S and OPM2 myeloma cells with human PB cells. Human CD4+CD25-
cells were co-cultured with MM1S or OPM2 myeloma cells for 72 hrs using trans-well co-culture.MM1S and OPM2 co-culture groups were compared to “No cells” or CD19-expressing “B cells”groups. Treg cells were assessed as CD4+CD25+FOXP3-expressing cells. Data from independentexperiments performed in triplicate of three different PB donors is shown. (t-test 2-tailed withBonferroni correction; *p<0.05, **p<0.01, error bars indicate SD)
***
****
FIGURE 5
A B
Figure 5. Differences in gene expression between myeloma-associated and control Tregs in the BMmicroenvironment.(A) mRNA expression profiling by RNA sequencing on total RNA isolated from Control (n=3) andVk*MYC-injected (n=3) BM Tregs. A heatmap was generated after supervised hierarchical clusteranalysis. Differential mRNA expression is shown in red (upregulation) versus blue (downregulation)intensity (Control versus Vk*MYC, 2-fold change, p<0.05). (B) Differences in checkpoint related geneexpression levels of Tregs isolated from Vk*MYC-injected and control mice. A number of checkpointrelated molecules were significantly up-regulated in Tregs from Vk*MYC-injected mice compared tocontrol Tregs. The gene expression fold change in Tregs from Vk*MYC-injected mice compared tothose from control Tregs is shown. (C) Differences in Treg effector gene expression levels betweenVk*MYC and control Tregs. The gene expression fold change of Tregs from Vk*MYC-injected micecompared to control Tregs is shown. (D) Differences in chemokine receptor gene expression levelsbetween Tregs from Vk*MYC-injected mice and control Tregs. (* p≤0.05, ** p<0.01, t-test 2-tailed,error bars indicate SD)
Control Vk*MYC
PD1OX40
CTLA-4
TIGIT
ICOS
GITR
LAG3TIM
30
1
2
3
4
Series1
Series2
ControlVk*MYC
Fold
cha
nge
*
***
** **
C
D
Fold
cha
nge
IL10
FGL2
GZMBPRF1
TGFB1EBI3
0
1
2
3
4
5
6
7
8
Series1
Series2
****
** ControlVk*MYCVk*MYCControl
CXCR3CCR6
CCR70
1
2
3
Series1
Series2
**
Fold
cha
nge
ControlVk*MYC
**
Vk*MYCControl
FIGURE 6
Bp=0.024
FDR 0.0p<0.01
FDR 0.0p<0.01
A
FDR 0.023p<0.01
FDR 0.02p<0.01
p=0.038
IFNA1
p=0.043
TLR8FE
C D
0
1
2
3
4
5
6
IRF3 IRF4 IRF5 IRF9 IFNA1 IFNA2 MAVS TLR8
Series1 Series2
HealthyPatients
Fold
cha
nge
** ** ***
**
** **
IRF3 IRF4 IRF5 IRF9 IFNA1 IFNA2 MAVS TLR8
Vk*MYCB cells
IFN
-bet
a (p
g/m
L)
Figure 6. Type-1 IFN signaling associated with Treg activation and overall patient survival.(A) Vk*MYC BM Tregs show an enrichment for Type-1 IFN-related mRNA signature compared to Control BMTregs, as presented by GSEA. The green curves show the enrichment score and reflect the degree to which eachgene (black vertical lines) is represented at the top or bottom of the ranked gene list. The heatmap indicates therelative abundance (red to blue) of the genes specifically enriched in the Vk*MYC Tregs as compared to controlTregs. FDR indicates false discovery rate and p indicates p values and are shown per each geneset analyzed. (B)IFN-beta secretion from Vk*MYC cells. IFN-beta secretion, detected by ELISA, in Vk*MYC cell culturesupernatants was significantly higher compared to the supernatants of control B cells obtained from C57BL/6 mice.Average of experiments performed in triplicate is shown. The experiment was repeated 3 times. (C) Myeloma cellsfrom MM patients show enrichment in type-1 IFN signaling pathway compared to cells from healthy donors. (D)Differential gene expression levels for genes involved in type-1 IFN production. (E) IFNA1 overexpression isassociated with worse overall survival in MM patients. (F) Expression of TLR8 receptor significantly correlates withworse outcome in MM patients. (t-test 2-talied, error bars indicate SD, * p<0.05, ** p<0.01)
E F
HG I
FIGURE 7
A
C D
p=0.0005
IFNAR1 AbIsotyp Ab
Ki-6
7+ T
regs
(%)
IFNAR1 Abki-67
FOXP
3
Isotype Ab IFNAR1 Ab
51.41 % 19.61 %
IFNAR1 AbIsotype Ab
Ki-67
FOX
P3
CD4
FOXP
3
Isotype Ab IFNAR1 Ab
70.37 % 55.63 %
IFNAR1 AbIsotype Ab
CD4
FOX
P3
p=0.0024p=0.0024
IFNAR1 AbIsotyp Ab
Ki-6
7+ T
regs
(%)
IFNAR1 Ab
WT Tregs+
CD3+ non Tregs
IFNAR1 ko Tregs+
CD3+ non Tregs
CD3+ non Tregs
WT Tregs+
CD3+ non Tregs
IFNAR1 ko Tregs+
CD3+ non Tregs
CD3+ non Tregs0
5 0
1 0 0
1 5 2 0 2 5 3 0
D a y s p o s t tu m o r in je c t io n
Per
cent
sur
viva
l p<0.01 (Log-rank)
WTTregs+CD3+NonTregsIFNAR1koTregs+CD3+NonTregsCD3+NonTregs
Perc
ent s
urvi
val
Days post tumor injection
p<0.01 (Log-rank)
WT Tregs + CD3+ non TregsIFNAR1 ko Tregs + CD3+ non TregsCD3+ non Tregs
P D 1 L AG 3 T im 30
2 0
4 0
6 0
Ex
pre
ssio
n in
Tre
gs
(%
) W ild ty p e
IF N A R 1 ko
p=0.018n.sn.s
P D 1 L AG 3 T im 30
2 0
4 0
6 0
Ex
pre
ssio
n in
Tre
gs
(%
) W ild ty p e
IF N A R 1 ko
p=0.018n.sn.s
Expr
essi
on in
Tre
gs(%
)
PD1 LAG3 TIM3
p=0.018
n.s n.s
Wild typeIFNAR1 ko
IFNAR1
60.22 % 9.52 %
Wild type IFNAR1 ko
IFNAR1
IFNAR1 ko
W ild typ e IF N AR 1 k o 0
5
1 0
1 5
2 0
2 5
Tre
gs
(%
)
p=0.0032
Treg
s(%
)
Wild type IFNAR1 ko
p=0.0032
B
Figure 7. Blocking IFNAR1 suppress Treg expansion and myeloma progression.(A) Representative FACS analysis of Ki-67 expression of Tregs expanded under co-culture with Vk*MYCcells and IFNAR1 antibody (IFNAR1 Ab) or isotype control (Isotype Ab). (B) Significant decrease of Ki-67expression in Tregs co-cultured with Vk*MYC and IFNAR1 Ab compared to those with Isotype Ab. Averageof experiments performed in triplicate is shown. The experiment was repeated 3 times. (C) RepresentativeFACS analysis of Treg expansion assay with IFNAR1 Ab or isotype Ab co-culture. Proportion of FOXP3+
cells in the CD4+ cells is shown. (D) Significant decrease of Treg proliferation by cells co-cultured withVk*MYC and IFNAR1 Ab compared to Isotype control. Average of experiments performed in triplicate isshown. The experiment was repeated 3 times. (E) 3×106 non Tregs were injected with or without 1×106
C57BL/6 Tregs or IFNAR1 ko Tregs i.v into Rag2 ko mice followed by Vk*MYC cell injection 5 daysafterwards. (F) Kaplan-Meier curve showing survival of mice injected with C57BL/6 Tregs and CD3+ nonTregs (WT Tregs + CD3+ non Tregs) compared to mice injected with IFNAR1 ko Tregs and CD3+ non Tregs(IFNAR1 ko Tregs + CD3+ non Tregs) and only with CD3+ non Tregs (CD3+ non Tregs) (n=6/group). (G)Significant decrease of BM Tregs in the “IFNAR1 ko” group (IFNAR1 ko Tregs + CD3+ non Tregs)compared to “Wild type” group (n=3/group) by FACS. (H) Representative FACS analysis of BM Tregs ofmice transferred with IFNAR1 ko Tregs compared to mice transferred with wild type Tregs. (I) FACSanalysis of checkpoint related molecules on BM Tregs obtained from mice injected with IFNAR1 ko Tregsand wild type Tregs. Significant decrease of LAG3 was observed in BM Tregs obtained from mice injectedwith IFNAR1 ko mice (n=3/group). (t-test 2 tailed, error bars indicate SD)
Wild type
Supplementary Figure 1
A
B
Supplementary Figure 1. Gating strategy for Treg analysis by CyTOF.(A) Gating strategy of CD4+ T cells by CyTOF. BM and PB CD4+ T cells were determined byfirst excluding non-viable cells. After initial gating on CD45+ cells, CD11b- CD11c- non myeloidcell population was gated, followed by selecting CD3+ B220- T cell population and finally gatedon CD4+ CD8- population. (B) Representative Treg plots by CyTOF. FOXP3+ cells weredetermined within CD4+ T cells. Control and Vk*MYC injected BM Tregs of late time points areshown. (C) Absolute numbers of Treg cells in BM of Vk*Myc-injected mice at late time point.(D) Number of Treg per 1 ml PB in Vk*Myc-injected mice at late time point. SPADE tree wasconducted on viable BM cells of control (E) and Vk*MYC-injected (F) mice at the early timepoint. The nodes of the tree represent clusters of cells that are similar in marker expression. Thecolor indicates level of marker expression. Size of the nodes represents number of cells.Increased numbers of CD3-expressing cells were observed in the BM of Vk*MYC-injected micecompared to control BM cells.
C D
B22
0
CD3
CD
8
CD4
CD
11c
CD11b
CD45+ CD11b- CD11c- CD3+ B220-
CD4
FOX
P3
Control Vk*MYC
30.56 %17.73 % p=0.014p=0.2
BM Tregs absolute numbers (/Femur)
PB Tregs absolute numbers (/mL)
ControlVk*MYC
Treg
coun
t (/F
emur
)
Control Vk*MYC
Treg
coun
t (/m
L)
Control BM (early) Vk*MYC–injected BM (early)E F
NBM
145N
d_C
D4
156Gd_CD25_IL2R 165Ho_FOXP3 176Yb_127_IL-7R
145N
d_C
D4
156Gd_CD25_IL2R 165Ho_FOXP3 176Yb_127_IL-7R
156Gd_CD25_IL2R 165Ho_FOXP3 176Yb_127_IL-7R
145N
d_C
D4
156Gd_CD25_IL2R 165Ho_FOXP3 176Yb_127_IL-7R
145N
d_C
D4
145N
d_C
D4
165Ho_FOXP3
1.61% 0.21% 0.06%
0.89% 0.15% 0.04%
0.94% 0.2% 0.08%
0.66% 0.09% 0.03%
0.61% 0.1% 0.04%
156Gd_CD25_IL2R 176Yb_127_IL-7R
SMM
Supplementary Figure 2
Peripheral bloodp=0.0068
Teff
s/Tre
gs
Early Late
A
B
Supplementary Figure 2. Decrease of Teff/ Tregs ratio in PB of Vk*MYC-injected miceand increased number of Tregs in BM of SMM patients.(A) Significant decrease of Teffs (CD4+ CD44++ CD62Llow and CD8+ CD44++ CD62Llow)/Tregsratio was observed in PB of Vk*MYC injected mice at the late time point as compared to BMof control mice. (B) Representative CyTOF profiles showing proportion of CD4+CD25+,CD4CD25FOXP3+ and CD4CD25FOXP3CD127-/low cells in CD45-expressing bone marrowfraction of 3 SMM patients compared to healthy donors. (t-test 2-tailed, error bars indicate SD)
p=0.002
p=0.097 p=0.002
p=0.006
Expr
essio
n in
Tre
gs(%
)
Supplementary Figure 3Ex
pres
sion
in T
regs
(%)
PD1 LAG3 Tim3
ControlVk*MYC
Peripheral blood (early)
p=0.036
PD1 LAG3 Tim3
Expr
essio
n in
Tre
gs(%
)
A
ControlVk*MYC
BPeripheral blood (late)
Expr
essio
n in
Tre
gs(%
)
ControlVk*MYC
PD1 LAG3 TIM3
Bone marrow (early)
Control
D
Vk*MYC
CD4+ Teffs
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
CD8+ Teffs
Fold change
Control
Vk*MYC
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
Fold change
Control
Vk*MYC
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
Fold change
Peripheral blood (early) Peripheral blood (late)
Bone marrow (early)
E F
G
CD4+ Teffs CD8+ Teffs CD4+ Teffs CD8+ Teffs
C
ControlVk*MYC
Bone marrow (late)
Supplementary Figure 3. Decrease of Teff/ Tregs ratio in PB of Vk*MYC-injected mice and expressionof immune checkpoint molecules in Tregs in the BM and PB at different time points post injection.Expression of immune checkpoint receptor (PD1, LAG3 and TIM3) on Tregs in PB of Vk*MYC-injectedmice (n=3) compared to control Tregs (n=3) at early (A), late (B) time point. (C) Expression of immunecheckpoint receptor (PD1, LAG3 and TIM3) on Tregs in BM of Vk*MYC-injected mice compared tocontrol Tregs at early time point. (D) Co-expression of checkpoint receptor (PD1, LAG3 and TIM3) onTregs in BM of Vk*MYC-injected mice compared to healthy controls at late time point. Heatmap ofimmune checkpoint receptor (PD1, LAG3 and TIM3) expression on PB CD4+ and CD8+ Teffs (CD44++
CD62Llow) at early (E) and late (F) time points. The color indicates the mean expression fold change of eachmolecule. (G) Heatmap of PD1, LAG3 and TIM3 expression on BM CD4+ and CD8+ Teffs (CD44++
CD62Llow) at early time point. (t-test 2-tailed, error bars indicate SD)
0
20
40
60
80
100
120
Supplementary Figure 4A B
C o n tr o l 5 T G M 10
2
4
6
8
1 0
Te
ffs
/ Tre
gs
C o n tr o l 5 T G M 10
1 0
2 0
3 0
4 0
Tre
gs
(%
)p=0.017 p=0.0257
C
Control
5TGM1
PD1
LAG
3
TIM
3
Fold change
D
Control
5TGM1
CD4+ Teffs
PD1
LAG
3
TIM
3
PD1
LAG
3
TIM
3
CD8+ Teffs
Fold change
Tregs
Supplementary Figure 4. Treg increased and activated in BM of 5TGM1-injected C57BL/KalwRij mice and co-expression of immune checkpoint molecules in BM of SMM patients.(A) Significant increase of Tregs frequency within CD4+ T cells in the BM of 5TGM1 injected mice(n=3) compared to control mice (n=3) at late time point (day 28). (B) Significant decrease of Teffs /Tregs ratio was observed at the late time point of 5TGM1 injected mice compared to control mice. (C)Heatmap of immune checkpoint receptor (PD1, LAG3 and TIM3) expression of BM Tregs at late timepoint. The color indicates the mean expression fold change of each molecule. BM Tregs of 5TGM1injected mice had higher expression of immune checkpoint receptors compared to control BM Tregs.(D) Heatmap of PD1, LAG3 and TIM3 expression of BM CD4+ and CD8+ Teffs at late time point.Higher expression of PD1, LAG3 and TIM3 on 5TGM1 injected BM Teffs. (E) Increased proportion ofTregs co-expressing PD1, TIM3 and CTLA4 in BM aspirates of SMM patients (n=17) compared tohealthy donors (n=11). Data combined from 3 independent experiments. (t-test 2-tailed, error barsindicate SD)
Control 5TGM1 Control 5TGM1
Treg
s, %
Teffs
/Tre
gs
NBMSMM
p=0.018
p=0.027 p=0.036
Expr
essio
n in
Tre
gs(%
)
Human bone marrowE
0
1
2
3
4
5
6
7
8
Co DT
Supplementary Figure 5
CD4
FOXP
3
DTPBS
27.12% 0.8%
A
D
Supplementary Figure 5. Proportion of Tregs after depletion by DT or adoptive transfer.(A) Effective depletion of BM Tregs in Vk*MYC-injected DEREG mice by DT injection comparedto PBS injection at day 20 post Vk*MYC injection. (B) Flow cytometry data of BM cells at day 3after last DT treatment in Vk*MYC-injected mice demonstrate a significantly increased Teff/Tregratio compared to PBS treated mice (4 weeks post Vk*MYC injection). (C) Phenotypic distributionof bone marrow cells in Vk*MYC-injected mice 4 weeks post Vk*Myc injection and DT treatmentcompared to Vk*MYC-injected, non-treated DEREG mice (n=5 per group). (D) RepresentativeFACS analysis of BM Tregs of mice injected with Tregs and CD3+ non Tregs (Tregs + CD3+ nonTregs) and mice injected only with CD3+ non Tregs (CD3+ non Tregs). (t-test 2-tailed, error barsindicate SD)
B
Non-treated CoDT-treated
Prop
ortio
n of
cel
ls in
BM
, %
p<0.05
p<0.05
C
CD4
FOX
P3
PBS DT
0
2
4
6
8
DEREG+PBS DEREG+DT
Teff
/Tre
gra
tio
p<0.001
CD4
FOXP
3 1.59 % 20.69 %
CD3+ non Tregs
Tregs+
CD3+ non Tregs
CD4
FOX
P3
CD3+ non Tregs
Tregs+
CD3+ non Tregs
20.69 %2.75 %
Supplementary Figure 6
Supplementary Figure 6. Blocking of CD25 receptor using CD25 antibody prolongs survival ofVk*MYC injected animals.(A) Treatment with anti-CD25 antibody leads to better survival of injected mice. Mice were treated withanti-CD25 antibody on day -4 and day 10 post Vk*MYC cell injection (n=6). Isotype antibody treatmentwas used in the control group (n=6). (B) Significantly lower numbers of Tregs were detected in PB of anti-CD25 antibody treated mice as compared to isotype controls. PB analysis was done on day 11 postVk*MYC injection. (t-test 2-tailed, error bars indicate SD)
A B
p=0.025 (Log-rank)
Days post tumor injection
Perc
ent s
urvi
val,
%
IsotypeCD25 Ab
p=0.026
Treg
s, %
Isotype CD25 Ab
Supplementary Figure 7
N o c e lls B c e lls 5 T G M 10
2 0
4 0
6 0
8 0
Tre
gs
(%
)
A
p<0.001
p<0.001
p<0.05
Is o typ e Ab IF N AR 1 Ab0
3 0
6 0
9 0
ki-
67+
Tre
gs
(%
)
BP=0.0062
Is o typ e Ab IF N AR 1 Ab0
2 0
4 0
6 0
8 0
Tre
gs
(%
)
CP=0.0007
Isotype AB IFNAR1 Ab Isotype AB IFNAR1 Ab
Ki-6
7+Tr
egs,
%K
i-67+
Treg
s, %
Treg
s, %
Treg
s, %
No cells B -cells 5TGM1
Supplementary Figure 7. Type-1 IFN pathway plays a role in Treg expansion by 5TGM1cells.(A) 5TGM1 cells are capable of expanding Tregs in vitro. CD4+ CD25+ Tregs were co-cultured with C57BL/KalwRij B cells or 5TGM1 cells for 72 hrs using trans-well. FOXP3+
cells within the CD4+ cells are shown. Significant Treg expansion was observed in the5TGM1 co-culture group under trans-well compared to “No cells” or “B cells” groups.Average of experiments performed in triplicate is shown. The experiment was repeated 3times. (B) Significant decrease of ki-67 expression of 5TGM1 expanded Tregs co-culturedwith IFNAR1 Ab was observed compared to Isotype Ab. Average of experiments performedin triplicate is shown. The experiment was repeated 3 times. (C) Significant decrease of Tregexpansion by 5TGM1 cells co-cultured with IFNAR1 Ab compared to Isotype Ab. Average ofexperiments performed in triplicate is shown. The experiment was repeated 3 times. (t-test 2-tailed, error bars indicate SD)
1
SUPPLEMENTARY TABLE 1
gene_name log2FoldChange pvalue CR2 -1.754105105 1.14E-07 GM38257 -1.663325205 5.07E-07 GM11175 -1.625350548 1.78E-08 H2-DMB2 -1.580643499 9.61E-07 NTRK3 -1.538300689 3.19E-06 RGS2 -1.535722917 2.48E-07 PPP1R15A -1.493961489 3.26E-06 DNAJB9 -1.489792391 2.98E-13 BAMBI -1.453324869 3.85E-06 TNFAIP3 -1.413637676 6.27E-06 SLC30A4 -1.363309794 5.53E-06 GM10698 -1.360612906 6.90E-07 FOS -1.312912328 5.00E-05 ALAS2 -1.311925596 2.46E-10 HBA-A1 -1.308836618 2.61E-16 CD22 -1.304986205 6.08E-06 SCD1 -1.299237228 6.01E-05 PTPRF -1.293199031 5.00E-05 ZFP354C -1.278876062 0.000119035 PAX5 -1.269783046 9.99E-05 RP23-4F16.12 -1.246683443 5.08E-06 BLK -1.232624802 0.000211979 JUN -1.229247208 0.000153806 ATP1B1 -1.198212543 1.09E-08 ZSCAN10 -1.196748707 0.000137553 CACNA2D2 -1.181106971 0.000334108 E430014B02RIK -1.180031188 0.000216646 VANGL2 -1.173394111 0.000122724 RASL11B -1.169355359 9.85E-05 MAFF -1.16763885 0.000336557 RYR2 -1.16547011 0.000458805 PIM3 -1.155030723 3.51E-08 C1QL3 -1.153808501 0.000493352 NR4A2 -1.147471441 5.88E-05 C230085N15RIK -1.145522682 0.000538342
2
TRP53INP2 -1.141599881 0.000274122 BTG2 -1.137583817 2.61E-15 LY6D -1.132680814 0.000252716 SPTY2D1 -1.116065657 2.57E-08 DAPK1 -1.103332552 4.56E-05 SIK1 -1.087969101 0.000940377 GFOD2 -1.07715986 0.000948759 TRIB1 -1.073847646 2.72E-09 CD55 -1.070912389 1.32E-13 GEM -1.068014009 0.000236276 CECR2 -1.067194084 0.001331875 GM37811 -1.065655387 0.00122256 ZFP36 -1.065106335 1.05E-12 DUSP1 -1.05100995 6.25E-11 GM30292 -1.047008312 0.001008288 IRS2 -1.043354899 3.16E-07 HBA-A2 -1.042529375 8.39E-05 SLC25A23 -1.041967417 7.54E-06 HBB-BS -1.036951957 0.000248255 TET1 -1.035944757 0.000285789 HBB-BT -1.026969464 9.51E-05 PRRT2 -1.011071105 0.000669147 CERS6 -1.009686382 3.13E-05 TOB1 -1.008933656 4.46E-07 IGHD -1.004497214 9.82E-05 GM20518 -1.002087894 0.002512408 SCAND1 1.001369748 0.002539504 EAF2 1.003642029 0.002477922 SH3BP2 1.008766975 5.94E-06 GM20559 1.013158536 4.92E-17 RGS12 1.018507433 0.000596453 MPP4 1.018949259 0.001831878 ICA1 1.024838143 0.001353931 HIP1 1.029539163 6.89E-07 BCL2A1A 1.034434774 0.000710139 GM3164 1.035517446 0.001872974 GM6637 1.038669623 0.000156508 ADGRE1 1.039700291 0.001723903 CP 1.041958608 0.001698374
3
TMEM158 1.04279845 8.42E-05 CHIL3 1.044581948 0.000156244 BC147527 1.047536484 2.42E-05 SOCS1 1.048840236 0.000789071 CASP4 1.049297949 6.17E-06 BASP1 1.05746133 0.001501014 NES 1.05757639 0.000452303 FCNA 1.068582796 0.001082541 EBI3 1.073064945 5.91E-07 SLC52A3 1.074613694 1.65E-06 GM6548 1.079941637 3.09E-07 CYP51 1.082781175 5.66E-08 IGKC 1.083198381 0.000733206 SH3PXD2B 1.087953986 0.001080636 MT-ND3 1.088637219 0.000786208 TCTEX1D2 1.08926198 0.000927467 BSPRY 1.091614041 8.79E-06 HMGN3 1.093532681 2.51E-05 STAT1 1.094043084 1.70E-20 LPCAT2 1.095535467 0.000645603 CD200R4 1.096444242 1.37E-05 5430427O19RIK 1.098073883 0.000707204 GM4841 1.098776209 0.000827399 FKBP11 1.10287262 0.000760344 H2-T24 1.105629551 1.11E-09 4930512H18RIK 1.108047937 0.000724009 BCL2A1B 1.110126289 5.95E-08 GM5970 1.110754461 0.000788536 GM5431 1.110866239 0.000777824 TOR3A 1.113582731 1.13E-13 GNGT2 1.113997466 0.000119205 GM18752 1.114592703 0.000795976 MPO 1.114824206 9.52E-05 RASA4 1.121610185 0.000575146 PHF11C 1.12277566 8.08E-12 CAMP 1.123186311 7.88E-05 GPR55 1.125583986 2.16E-08 RBMS3 1.128988564 0.00025565 TREX1 1.129992747 3.52E-10
4
ABCB1A 1.131277826 1.36E-16 CD63 1.133631118 0.000665654 CASP1 1.135809142 2.54E-07 TRIM30A 1.136887548 3.24E-20 CCR3 1.137533447 2.79E-06 PTAFR 1.139419711 5.59E-05 TMEM67 1.152764987 1.50E-05 PHF11D 1.152877254 4.99E-05 KLRG1 1.156370238 3.05E-13 GRN 1.156994471 0.000241286 GM15135 1.157079313 0.000211861 LDLR 1.159771627 1.07E-07 MAFB 1.167243015 0.000458463 APOBR 1.168616595 4.42E-05 IL1R2 1.173275516 0.00014506 TMCC3 1.174761286 9.20E-05 PON3 1.176099496 2.43E-05 CCRL2 1.187297139 1.95E-08 GM10175 1.187953348 2.99E-09 F830016B08RIK 1.190081222 0.000105571 MS4A7 1.195775093 0.000289061 GM3468 1.196550035 1.03E-05 CLIC4 1.197818519 1.91E-11 HERC6 1.199291097 4.61E-16 ERDR1 1.199785325 0.00030423 GZMA 1.199934318 0.00022304 SEMA7A 1.209890061 0.000208051 PYDC3 1.212671092 2.88E-14 MS4A4C 1.216484615 0.000223092 IGKV1-117 1.221325905 0.00021762 NR4A3 1.227888915 4.29E-10 IGTP 1.234096576 1.14E-31 CXCL11 1.248418621 4.89E-05 HOXB4 1.252410074 3.69E-06 NAMPT 1.252749973 1.59E-22 CD300A 1.26064519 0.000153922 CCL6 1.261792632 9.56E-05 GM26917 1.26385414 0.000108552 AW112010 1.266039911 3.26E-14
5
GM4955 1.271731042 3.13E-20 STAB1 1.281071179 2.40E-06 IFI47 1.282309228 1.86E-29 GM14005 1.286809507 4.65E-05 HAVCR2 1.287290048 6.65E-06 CITED4 1.295169111 8.08E-05 AW011738 1.306109907 5.61E-07 PPA1 1.320178061 2.15E-12 OAS1G 1.328872033 6.64E-05 GM43197 1.336949545 4.50E-11 SLC15A3 1.339568654 2.08E-06 GM12250 1.344169383 6.05E-14 OAS1B 1.347048356 2.30E-14 GBP6 1.353023467 3.13E-37 STAT2 1.354924947 1.24E-20 DLL1 1.378800133 2.31E-05 GM12185 1.38074827 7.18E-09 JCHAIN 1.380878605 1.39E-05 HOXB3 1.383021836 3.24E-05 PYDC4 1.385414901 2.56E-24 TGTP2 1.394913467 3.60E-35 PHF11B 1.40665229 2.04E-28 TRAFD1 1.410275673 1.17E-20 CLEC7A 1.413461174 2.18E-05 GM7609 1.420988286 5.86E-07 IFITM3 1.421733729 1.97E-05 CYBB 1.43884076 1.79E-15 TRIM30D 1.443810351 9.50E-27 A230005M16RIK 1.444853517 1.44E-05 GJB2 1.450925577 5.91E-06 PGAM2 1.459750164 1.03E-05 IGKV1-110 1.460227786 3.78E-06 TNFRSF8 1.464298932 2.29E-07 2310031A07RIK 1.478324191 1.81E-06 PARP9 1.483784389 2.75E-25 CTLA2B 1.4875031 6.37E-06 GM16464 1.489977611 8.97E-07 FGL2 1.491789641 2.09E-38 TDRD7 1.493025722 4.06E-12
6
GM43126 1.494357031 3.34E-06 IRGM1 1.496371348 3.41E-29 GBP6 1.511196014 1.70E-06 CAPN11 1.519456885 2.36E-06 DAXX 1.52137526 2.01E-21 PHF11A 1.532932568 1.68E-21 ASB2 1.536593957 2.46E-06 IFIH1 1.543091248 1.59E-22 TGTP1 1.547998408 9.61E-32 NBEA 1.555243044 3.01E-06 AI607873 1.569320377 2.31E-06 CLEC4A3 1.572916155 2.26E-06 ALOX15 1.598276628 1.10E-06 TRIM30C 1.60348943 1.48E-06 CCR5 1.610138097 1.91E-11 MLKL 1.629821429 1.30E-08 XAF1 1.645732679 7.43E-36 TBX21 1.648506268 2.38E-11 DDX60 1.654399355 1.55E-29 IFIT1BL1 1.669001668 9.40E-33 BC094916 1.684393885 5.76E-26 PLAC8 1.684564995 2.22E-11 LGALS3BP 1.694255968 3.50E-26 RNF213 1.711070519 2.19E-31 GM12338 1.722961279 1.23E-07 H2AFJ 1.730319213 2.54E-16 SERPINA3F 1.738391129 5.76E-10 NKG7 1.740084454 2.05E-11 DHX58 1.756183428 2.45E-28 CD86 1.778766006 3.41E-21 ZBP1 1.783705644 5.92E-37 FCGR4 1.88251529 1.60E-08 GBP10 1.905632856 2.57E-45 ISG20 1.935859627 1.97E-41 SDC3 1.949803332 8.09E-16 APOL9B 1.953308856 9.34E-11 USP18 2.008189107 2.41E-30 CCL5 2.01812883 1.89E-30 LY6A 2.023697966 3.07E-52
7
PARP12 2.030801403 2.54E-32 IIGP1 2.039347672 1.67E-38 CCL4 2.049569457 5.49E-10 IFI27L2A 2.050202143 1.16E-76 OAS1A 2.160719625 1.66E-33 CMPK2 2.19332456 5.27E-33 RTP4 2.193588902 4.17E-40 GM4951 2.212083177 1.50E-16 RSAD2 2.230319355 3.12E-36 BST2 2.274620521 3.44E-69 IRF7 2.33301622 7.37E-45 CXCL10 2.369495039 1.16E-13 OAS3 2.4292666 3.95E-39 OASL1 2.586914015 2.26E-29 IFIT1 2.645642235 4.44E-46 OASL2 2.68690895 4.73E-20 IFIT3 2.693997567 3.02E-20 IFIT3B 2.697812916 5.60E-19 GZMB 2.710077584 9.17E-71 MX2 2.735753401 1.50E-31 MX1 2.7380499 3.01E-22 MNDA 2.884359591 1.81E-25 IFI204 3.051636274 2.89E-27 OAS2 3.275260084 2.34E-53 ISG15 3.282058987 1.43E-83 IFI44 3.437057621 1.78E-43
Supplementary Table 1. Genes up-regulated and down-regulated in Vk*MYC
injected BM Tregs compared to control BM Tregs.
A list of the genes significantly up-regulated and down-regulated in BM Tregs obtained
from Vk*MYC injected mice (n=3) compared to BM Tregs from control mice (n=3).