Soluble IL-6R Expressed by Myeloid Cells Reduces Tumor-Specific Th1 Differentation

Author Manuscript Published OnlineFirst on February 24, 2017; DOI: 10.1158/0008-5472.CAN-16-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Soluble IL-6R expressed by myeloid cells reduces tumor-specific Th1 differentation

and drives tumor progression

Hirotake Tsukamoto1, 6, *, Koji Fujieda1, 2, 6, Masatoshi Hirayama3, Tokunori Ikeda4, Akira Yuno3, Keiko

Matsumura1, Daiki Fukuma3, Kimi Araki5, Hiroshi Mizuta2, Hideki Nakayama3, Satoru Senju1, and

Yasuharu Nishimura1, *

Authors’ Affiliation: 1 Department of Immunogenetics, Graduate School of Medical Sciences, 2

Department of Orthopaedic Surgery, 3 Department of Oral and Maxillofacial Surgery, 4 Department of

Clinical Research Center, Faculty of Life Sciences, 5 Division of Developmental Genetics, Institute of

Resource Development and Analysis, Kumamoto University, Honjo 1-1-1, Chuo-ku, Kumamoto

860-8556, Japan. 6 These authors equally contributed to this work.

Running title: Myeloid cell-derived sIL-6R dampens anti-tumor Th1 responses.

Key words: IL-6, sIL-6R, immunosuppression, Th1, cancer immunotherapy

Financial support: This work was supported by JSPS KAKENHI No. 26430165 to H. Tsukamoto, and

JSPS KAKENHI No. 15H04311 to Y. Nishimura. H. Tsukamoto was also supported by The Takeda

Science Foundation, and the Kumamoto University Advance Research Project A “International Research

Center for Cancer and Metabolism”.

* Correspondence to Hirotake Tsukamoto and Yasuharu Nishimura, Department of Immunogenetics, 1

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 24, 2017; DOI: 10.1158/0008-5472.CAN-16-2446 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Graduate School of Medical Sciences, Kumamoto University. Honjo 1-1-1, Chuo-Ku, Kumamoto

860-8556, Japan; Phone: +81-96-373-5313; Fax: +81-96-373-5313; E-mail:

[email protected] and [email protected]

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed.

Word count: Abstract: 248 words; Main body of text: 5,364 words; Figure legends: 1,077 words

Total number of figures: 6 figures and 1 Table (+ 4 supplementary figures and 4 supplementary tables)

Abstract

IL-6 produced by tumor cells promotes their survival, conferring a poor prognosis in cancer patients.

IL-6 also contributes to immunosuppression of CD4+ T cell-mediated antitumor effects. In this study, we focused on the impact of IL-6 trans-signaling mediated by soluble IL-6 receptors (sIL-6R) expressed in tumor-bearing hosts. Higher levels of sIL-6R circulating in blood was observed in tumor-bearing mice, whereas the systemic increase of sIL-6R was not prominent in tumor-bearing mice with myeloid cell-specific conditional deletion of IL-6R even when tumor cells produced sIL-6R. Abundant sIL-6R was released by CD11b+ cells from tumor-bearing mice but not tumor-free mice. Notably,

IL-6-mediated defects in Th1 differentiation, T cell helper activity for tumor-specific CD8+ T cells and downstream antitumor effects were rescued by myeloid-specific deletion of sIL-6R. Expression of the T cell transcription factor c-Maf was upregulated in CD4+ T cells primed in tumor-bearing mice in an

IL-6-dependent manner. Investigations with c-Maf loss-of-function T cells revealed that c-Maf activity was responsible for IL-6/sIL-6R-induced Th1 suppression and defective T cell-mediated antitumor responses. In cancer patients, myeloid cell-derived sIL-6R was also possibly associated with Th1 suppression and c-Maf expression. Our results argued that increased expression of sIL-6R from myeloid cells and subsequent c-Maf induction were adverse events for counteracting tumor-specific Th1 generation. Overall, this work provides a mechanistic rationale for sIL-6R targeting to improve the efficacy of T cell-mediated cancer immunotherapy. 3

Introduction

The usage of T-cell-mediated cancer immunotherapies has gained momentum for achieving successes in advanced malignancies, and is currently being hailed as a promising therapeutic approach (1, 2).

While the induction of tumor-specific CD8+ T cells have been the main focus in cancer immunotherapy, recent studies have suggested that the better prognosis is correlated with higher levels of

IFN-γ-expressing CD4+ T (Th1) cells in cancer patients, implying an important role of Th1 cells in controlling tumor regression (3-5). However, Th1 responses tend to be abolished in cancer patients (3, 5) and tumor-bearing animals (6, 7). An induction of tolerance against immunosurveillance in tumor microenvironment is suggested to be the cause for failure of tumor-specific T cells to eradicate malignant cells (1, 3, 6). It has been proposed that immunosuppression occur through complex interactions among tumor cells and a variety of immune cells such as T cells, macrophages, dendritic cells (DC), and other myeloid-lineage cells (1, 8, 9). Therefore the identification of pathways involved in these interactions and an understanding of the mechanism underlying Th1 suppression will be beneficial in predicting patient’s prognosis and augmenting anti-tumor responses with immunotherapy.

The role of interleukin (IL)-6 signal in promoting tumor-cell survival has been demonstrated in vitro and in vivo (10-13). Importantly, a higher level of IL-6 was significantly associated with unfavorable prognosis in cancer patients (10, 14, 15). Canonical IL-6 signaling is transmitted through 4

membrane-bound IL-6 receptor (IL-6R) ligation and gp130 transmembrane receptor dimerization, whereas an alternative way of IL-6 signal transmission “IL-6 trans-signaling” is mediated by soluble

IL-6 receptor (sIL-6R) that forms a complex with IL-6 and directly engages the gp130 even in cells without membrane-bound IL-6R (16, 17). The importance of sIL-6R is highlighted by the fact that approximately 70% of the secreted IL-6 binds the sIL-6R in blood (18). The sIL-6R functions as a carrier molecule for IL-6, thereby markedly prolonging its half-life in vivo, and stabilizing IL-6 signaling (17). In addition to IL-6, a higher level of sIL-6R is associated with a pathological grade of various cancers (12, 15, 16), and thus may have a diagnostic and prognostic significance in cancer patients (19). The tumor-promoting effects of IL-6, sIL-6R, and STAT3 activation, a critical signaling component activated by IL-6 (10), demonstrated in mouse models also supports their usefulness as rational therapeutic targets for controlling cancer progression (12, 13). In fact, the chimeric anti-IL-6 antibody (Ab) has been used in clinical trials for multiple myeloma, metastatic renal cell carcinoma, prostate cancer, and other cancers (10, 14, 20) with an intention to abrogate its direct effect on tumor growth. A better understanding of the biological effect of IL-6/sIL-6R in cancer patients will lead to improvement of their clinical management.

We have previously shown that priming of tumor-specific CD4+ T cells in combination with a treatment of anti-IL-6 blocking Ab enhanced the anti-tumor CD4+ T-cell responses (7). However, the mechanistic action by which IL-6 attenuates the T-cell-mediated anti-tumor responses remains to be 5

elucidated. In this study, we demonstrated that activation of tumor-specific CD4+ T cells did not promote the tumor elimination due to an early skewing into the dysfunctional cells in an IL-6/sIL-6R-dependent manner. Furthermore, myeloid lineage-specific deletion of IL-6R in tumor-bearing mice revealed that myeloid-cell-derived sIL-6R functioned as a dominant factor in IL-6-mediated attenuation of Th1 differentiation, which subsequently dampened their anti-tumor activity. Our study proposes a possibility that monitoring and manipulating the IL-6/sIL-6R signaling are novel approaches to improve the

T-cell-mediated immunity against malignant tumors.

Materials and Methods

Mice

C57BL/6 mice were purchased from Nihon Clea. CD45.1+B6.SJL-PtprcaPep3b/BoyJ mice and

IL-6Rα-flox/flox (IL-6Rfl/fl) mice were purchased from The Jackson Laboratory. The mice with ubiquitous deficiency of IL-6Rα were generated by crossing of IL-6Rfl/fl mice with the mice expressing

CAG promoter-driven Cre transgene (21). To generate the IL-6R-deficient ovalbumin (OVA)-specific

CD4+ T cells, CAG-Cre/IL-6Rfl/fl mice were crossed with CD45.1+ OT-II T cell receptor (TCR)

transgenic mice. Myeloid-cell-specific IL-6R conditional knock-out (KO) mice were generated by

crossing the IL-6Rfl/fl mice with the mice expressing Lysozyme M promoter-driven Cre recombinase

(kindly provided by Dr. Irmgard Foester). C3H-background MafOfl mice with a mutant c-Maf allele that

lacks the ability to bind to its target genes (22) were provided by the Medical Research Council. MafOfl/+

OT-II transgenic mice were generated by backcrossing MafOfl/+ mice with CD45.1+ OT-II transgenic

mice for 10 generations. All the mice including IFN-γ-deficient (23) and IL-6-deficient mice were

housed at the Center for Animal Resources and Development, Kumamoto University. All the

experimental procedures were approved by the Institutional Animal Committee of Kumamoto

University and performed in accordance with the guidelines.

Blood samples from healthy donors (HD) and head and neck malignant tumor (HNT) patients

Blood samples were obtained from patients with HNT enrolled in a randomized phase II trial (24) before and after receiving the peptides vaccine. This trial was approved by the Institutional Review

Board of Kumamoto University, and registered in the University Hospital Medical Information Network

Clinical Trials Registry (UMIN-CTR; 000008379). Written informed consents were obtained before enrollment. HD samples were obtained as well. Peripheral blood mononuclear cells (PBMC) from

Ficoll-separated blood and plasma were cryopreserved until experimental use.

Tumor inoculation and Ab treatment

Mice were inoculated subcutaneously with 5 × 105 OVA-expressing MCA205, MCA-OVA (7),

OVA-expressing EL4 thymoma (EG7), or 2.5 × 104 Rauscher murine leukemia virus (MuLV)-induced lymphoma, RMA (25). Tumor size is expressed as tumor index, which is the square root of (length × width) as described previously (7). In the pulmonary metastatic model, mice were intravenously injected with 5 × 105 OVA/firefly luciferase-expressing melanoma, MO4-Luc (7). To monitor their lung metastasis, luminescent images were analyzed using NightOWL (Berthold Technologies).

Two-hundred-and-fifty µg anti-IL-6R, anti-IL-6 Ab (BioXCell), or control IgG Ab (Millipore) was injected 1 day before and after immunization. For in vivo cell depletion, mice were injected with anti-CD4 Ab (50 µg) before and after immunization or RMA inoculation. Two-hundred-and-fifty ng 8

recombinant sgp130 (R&D Systems) was intravenously injected.

Adoptive transfer and immunization

CD4+ T cells (8 x 105) isolated from CD45.1+ OT-II transgenic mice using naïve CD4+ T-cell isolation

kit (Miltenyi Biotec) were injected intravenously into tumor-free or tumor-bearing CD45.2+ mice. Eight hours later, mice were immunized with an intravenous injection of 4 x 105 bone marrow-derived DC

pulsed with peptide ISQAVHAAHAEINEAGR (referred to OVA-IIp) that is recognized by

I-Ab-restricted OT-II cells, or with the MuLV Env-gp70 H13.3 peptide SLTPRCNTAWNRL (EnvH13.3)

that is recognized by I-Ab-restricted CD4+ T cells (25). The peptide SIINFEKL (OVA-Ip) recognized by

H2-Kb-restricted OVA-specific CD8+ T cells was also utilized for immunization.

Flow cytometric analysis and cytokine measurement

Cells from spleen and lymph nodes (LNs) were stained with the following Abs for flow cytometric analyses: anti-CD11b, anti-Vβ5, anti-CD4 Abs and PerCP-streptavidin (BD Biosciences), anti-Ly6C,

anti-Gr-1, anti-CD45.1, anti-Ly6G, anti-CXCR3 Abs (eBioscience), anti-human HLA-DR, anti-CD16

(BD Biosciences), anti-CD14, anti-CD33, anti-CD4 Abs (BioLegend), and anti-IL-6R Ab (Beckman

coulter). The H-2Kb/SIINFEKL-tetramer-PE was from MBL. For cytokine staining, CD4+ T cells

isolated using anti-CD4 beads (Miltenyi Biotec) or from in vitro cultures were re-stimulated with 9

OVA-IIp-pulsed DC or PMA/ionomycin, and stained with anti-IL-2, anti-IL-17A, anti-IL-10

(eBioscience), or anti-IFN-γ (TONBO), anti-TNF-α Abs (Biolegend) as described previously (7). For human samples, anti-human IL-2, anti-IFN-γ, or anti-TNF-α Abs (BD Bioscineces) was used. Staining with anti-phospho-STAT3 Ab (BD Bioscience) was performed as described previously (26). Staining with anti-human/mouse T-bet, anti-RORγt, anti-c-Maf (eBiosciences), or anti-GATA3 Abs (BD

Biosciences) was performed using the Transcription Factor Buffer (BD Biosciences).

Immunofluorescent images were analyzed using the FACSVerse or FACSCalibur (Becton Dickinson).

Data were analyzed using FlowJo software (Tree star). IL-6 and soluble IL-6R (sIL-6R) levels were

measured by ELISA (R&D systems). For the ELISPOT assay (BD Biosciences), 1 x 105 draining LN

cells and 3 x 104 DC pulsed with H13.3, Db-binding MuLV GagL (LCCLCLTVFL) or Kb-binding Env peptides (SSWDFIT) (25) were mixed and incubated for 12 hours. IFN-γ spots were visualized and analyzed as previously described (27).

In vitro T-cell differentiation

Mouse naïve T cells were stimulated with plate-coated anti-CD3 and anti-CD28 Abs (both TONBO) in the presence of IL-12 (4 ng/ml; Wako) with or without IL-6 (Peprotech) and sIL-6R (Peprotech).

After the culture for 6-8 days, T cells were analyzed. For human samples, naïve CD4+ T cells (5×104) were isolated with the naïve CD4+ T-cell isolation kit (Miltenyi Biotec) from PBMC, and stimulated 10

with anti-CD3/CD28-coated beads (Gibco) for 4 days in the presence of IL-12 (20 ng/ml; R&D systems)

together with or without IL-6 (20 ng/ml; R&D systems), sIL-6R (Peprotech) or sgp130 (60 ng/ml; R&D

systems). Cells were re-stimulated for 2 days, and then following one day of resting culture, cells were

analyzed. CD14+ cells were isolated from PBMC using CD14 microbeads (Miltenyi Biotec), and their

culture supernatant was added into T-cell culture simultaneously with IL-6 (10 ng/ml).

siRNA transfection

siRNAs against Stat3 and negative control duplexes were purchased from Applied Biosystems.

Transfection of 100 pmol siRNA into anti-CD3/CD28 Abs-stimulated T cells (1x106) were conducted

using HVJ-E vector (GenomeONE™, Ihsihara Sangyo Ltd.) according to manufacturer’s instruction.

After 72 hours of transfection, cells were analyzed for cytokine production by flow-cytometry.

Real-time PCR

RNA was extracted with the RNeasy Plus Mini Kit (QIAGEN), and reverse-transcribed with

ReverTra Ace (TOYOBO). Real-time PCR was performed on ViiA7 Real-Time PCR System with

TaqMan probes and Master Mix reagents (Applied Biosystems). Each gene expression was normalized

to the Gapdh expression with the comparative 2[-∆∆CT] method.

Microarray

The mice were transferred with OT-II cells and immunized with OVA-IIp-pulsed DC. Four days later,

CD4+ T cells were isolated, and total RNA was extracted. Microarray analysis using Agilent SurePrint

G3 Mouse Gene Expression 8X60K Microarray Kit and data processing were performed by MBL. Data analysis was performed using GeneSpring GX (Agilent Technology). Background corrected intensity values between arrays were normalized using the 75th percentile shift method. Normalized

log2-transformed probe fluorescence intensities from independent samples showing a median fold-change in expression > 2.0 or < -2.0 was considered as differentially expressed genes. Microarray data are available under GEO accession number GSE93105.

Statistical analysis

Multiple comparisons were performed by one-way ANOVA followed by Tukey-Kramer post-hoc tests.

A Kruskal-Wallis test was the nonparametric alternative to ANOVA. Data were also analyzed using unpaired Student’s t-test when comparing two experimental groups. These analyses were performed using the Prism 4.0 (GraphPad).

For path analysis based on structural equation models, hypothetical pathways predicted from the mouse model were examined using 48 samples that were collected from 22 HNT patients along with the vaccination, and 25 HD samples. For modeling the pathway, the concentration of sIL-6R and IL-6 in 12

plasma, the frequencies of CD14+CD16- cells and CD4+ T cells in PBMC, and c-Maf+CD4+ T cells were included. Correlations induced by multiple samples from each patient as well as effects of age and a number of peptides vaccinations were adjusted. After visual investigation of the distribution of measurements, some variables were log-transformed to approximate normal distributions. STATA

(version 14.1) was used to fit the above models. P values less than 0.05 were considered significant.

Results

IL-6-mediated inhibition of Th1 differentiation attenuates anti-tumor immune responses

We previously demonstrated that differentiation of CD4+ T cells expressing OVA-specific TCR (OT-II

cells) into IFN-γ-producing Th1 cells was attenuated in mice with tumors expressing the surrogate

tumor-associated antigen, OVA, and that the defect was reversed by anti-IL-6 Ab treatment (Fig.1A and

(7)). This finding was confirmed by the fact that the defective Th1 differentiation of OT-II cells primed

with OVA-IIp-pulsed DC was observed in tumor-bearing wild-type (WT) but not in IL-6-deficient

counterparts, regardless of the type of tumor cells such as MCA-OVA or EG7 (Fig.1B and C). IL-6 did

not affect the priming or expansion of donor T cells, or the proportion of immunosuppressive

components such as Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) or Foxp3+ regulatory T cells (Fig.1B-D and (7)). We also found an IL-6-dependent decrease of GM-CSF-producing cells and a modest but significant increase of IL-10-producing cells in tumor-bearing mice (Fig. 1E). This

IL-6-dependent Th1 suppression was mediated through the direct action of IL-6 on CD4+ T cells because

a substantial STAT3 activation was detected in donor OT-II cells in tumor-bearing mice, which was

abrogated by in vivo administration of anti-IL-6R Ab (Fig. 1F).

In terms of anti-tumor responses triggered by CD4+ T cells, the treatment with anti-IL-6R Ab

augmented the anti-tumor effect of WT OT-II cells in MCA-OVA-bearing WT host mice (7) or even in 14

IFN-γ KO hosts (Fig. 1G). However, the effect of IL-6R blockade was not observed in tumor-bearing mice that were transferred with IFN-γ KO OT-II cells, suggesting that the beneficial effect of IL-6(R) blockade on CD4+ T-cell-mediated anti-tumor immunity was exerted through their Th1 responses. These results were consistent with the observation that when WT OT-II cells were transferred, their helper activity for endogenous tumor (OVA)-specific CD8+ T cells was recovered by IL-6 blockade in tumor-bearing mice, which was not observed in IFN-γ-deficient OT-II cells-transferred mice (Fig. 1H).

Given that CD8+ T cells were essential for tumor rejection in this model (7), these data suggest an indispensable role of IFN-γ derived from CD4+ T cells (Th1 responses) to provide help for the activation of tumor-specific CD8+ T cells, which was abrogated by IL-6 signaling in CD4+ T cells.

IL-6 trans-signaling via sIL-6R is responsible for the defective Th1 response

We found that CD4+ T cells temporally lost the surface IL-6R expression in response to antigenic stimulation in vivo (Fig. 2A), regardless of the presence of a tumor or IL-6 activity (Fig. 2B), confirming the previous study (28). However, despite the lack of surface IL-6R at priming phase, the IL-6 signal could be transmitted in OT-II cells activated in tumor-bearing mice as indicated by STAT3 activation

(Fig. 1E). This discrepancy might be explained by “IL-6 trans-signaling” mediated through IL-6/sIL-6R complex (16, 17). To address this possibility, we analyzed the Th1 differentiation of IL-6R-deficient

CD4+ T cells under Th1-skewed condition in the presence of sIL-6R in vitro. IL-6-dependent inhibition 15

of Th1 differentiation was recapitulated in vitro (Fig. 2C). Although an additive effect of sIL-6R on

attenuating the ability to produce IFN-γ was observed in IL-6-stimulated WT cells, sIL-6R-dependent

Th1 inhibition was more obvious in IL-6R-deficient OT-II cells, although the dependency of IL-6

trans-signaling in IL-6R+ cells are still unclear. The synergistic effect of sIL-6R on IL-6-mediated

attenuation of Th1 differentiation was also confirmed by human polyclonal CD4+ T cells (Fig. 2D).

Furthermore, IL-6-dependent Th1 inhibition was partially rescued by Stat3 knockdown (Fig. 2E),

confirming the involvement of STAT3 in this event.

Next, we directly tested the involvement of IL-6 trans-signaling in defective Th1 development in

tumor-bearing mice using recombinant soluble gp130 (sgp130), which selectively captures the

IL-6/sIL-6R complex and competitively antagonizes its ligation with membrane-anchored gp130 (16,

29). Th1 differentiation in both IL-6R-sufficient and -deficient donor OT-II cells, but not their expansion,

was significantly augmented by an administration of sgp130 in tumor-bearing mice (Fig. 2F). These

results suggest that the surface expression of IL-6R on T cells is not essential, and that T-cell-extrinsic

sIL-6R contributed to the Th1 inhibition in tumor-bearing mice. This was supported by the systemic

increase of sIL-6R in tumor-bearing mice, which was abolished in IL-6R-deficient hosts (Fig. 2G),

despite comparable tumor sizes in both IL-6R-/- and IL-6R+/+ mice (Fig. 2H). The sIL-6R-dependent

attenuation of Th1 differentiation was further demonstrated in ubiquitous IL-6R-deficient mice that were

utilized as tumor-bearing hosts, where a subtle decrease in IFN-γ-producing cells was observed at early 16

phase (within 8 days) during tumor progression (Fig. 2I). Collectively, these results suggest that IL-6 trans-signaling via sIL-6R, rather than classical IL-6 signaling, attenuates Th1 differentiation of tumor-specific CD4+ T cells.

Myeloid-lineage cells from tumor-bearing mice produce sIL-6R

The sIL-6R can be released through a cleavage of membrane-bound IL-6R via TNF-converting enzymes (TACE) such as a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 (16, 28).

In fact, PMA/ionomycin-stimulated sIL-6R production from splenocytes of tumor-bearing mice was abrogated with the treatment of TACE inhibitor, TAPI-0 (Supplementary Fig. S1A). To determine potential sources of sIL-6R in tumor-bearing mice, TACE activity and sIL-6R production in isolated cell fractions were examined. To this end, Gr-1+ cells and CD11b+ cells exhibited higher TACE activity

(Supplementary Fig. S1B). CD11b+Gr-1- macrophages and CD11b+Gr-1+Ly6C+ MDSC cells from tumor-bearing mice exhibited an abundant production of sIL-6R and higher expression of Adam10/17

(Supplementary Fig. S1C-E). Consistent with these results, in vivo depletion of MDSC or macrophages with anti-Ly6G Ab, anti-Gr-1 Ab, or clodronate, respectively significantly reduced the sIL-6R concentration in tumor-bearing mice, but did not lead to its complete abrogation (Supplementary Fig.

S1F and G).

The systemic increase of sIL-6R and subsequent Th1 suppression were not prominent in 17

tumor-bearing IL-6R-/- hosts even when sIL-6R was produced by tumor cells (Fig. 2F, G and

Supplementary Fig. S2A). Indeed, despite a higher TACE activity and ADAM-dependent sIL-6R

production in tumor cells (Supplementary Fig. S1B and S2A), Th1 inhibition was not restored in mice

with ADAM10/17-knockdown tumor cells that reduced the ability to produce sIL-6R (Supplementary

Fig. S2A-C), suggesting that tumor-derived sIL-6R did not have a significant impact on Th1 inhibition

during the early phase of tumor progression.

Myeloid cells-derived sIL-6R leads to defective CD4+ T-cell-mediated anti-tumor responses

To further assess the involvement of myeloid cell-derived sIL-6R, we generated mice with

myelomonocytic lineage-specific deletion of IL-6R (LysM-Cre x IL-6Rfl/fl; referred to as IL-6R mKO),

resulting in a failure to produce sIL-6R in myeloid cells. As shown in Fig. 3A, during the early phase of

tumor expansion, the level of sIL-6R in serum rapidly increased in WT mice. In contrast, conditional

deletion of sIL-6R in myeloid cells resulted in a significant delay of sIL-6R induction, despite the

comparable increase of IL-6 concentration and tumor size in both mice when the mice did not receive

any treatments (Fig. 3A and B).

Next, IL-6R-deficient OT-II cells were primed in tumor-bearing IL-6R mKO mice, and their ability to

produce IFN-γ and GM-CSF were assessed. As a result, the detrimental effect of tumor-bearing mice on

Th1 differentiation was abrogated in IL-6R mKO mice (Fig. 3C). Consistent with this, we observed a 18

marked increase of CXCR3 expression, a marker for Th1 cells when OT-II cells were primed in IL-6R

mKO mice, whereas the expression levels of co-inhibitory or co-stimulatory molecules such as LAG3,

PD-1, and ICOS, were comparable in both IL-6R mKO and control mice (Supplementary Fig. S3A and

B). Furthermore, tumor-specific CD8+ T cells in tumor draining LNs were dramatically increased by the

helper activity of OT-II cells primed in IL-6R mKO mice (Fig. 3D).

We next examined the anti-tumor effect when IL-6R-deficient OT-II cells were transferred into litter

control or IL-6R mKO mice with more aggressive OVA-expressing melanoma, MO4. As evidenced by

the metastatic burden in the lung, significant therapeutic effect of tumor-specific CD4+ T cells was

observed in IL-6R mKO but not in WT mice (Fig. 3E). This effect was comparable to that of IL-6

blockade in vivo. In line with this, recruitment and infiltration of tumor-specific CD8+ T cells into tumor-draining LNs and lung were augmented in IL-6R mKO mice (Supplementary Fig. S3C and D).

Conversely, immunization with OVA-Ip-pulsed DC to directly prime tumor-specific CD8+ T cells

elicited comparable anti-tumor effects in both IL-6R mKO and WT mice, circumventing help from

cognate CD4+ T cells (Fig. 3F). This was consistent with that the response of tumor-specific CD8+ T

cells was not altered by sIL-6R blockade when they were directly primed (Fig. 3G and Supplementary

Fig. S3E).

We also investigated the suppressive effect of sIL-6R on more physiological responses of endogenous

tumor-specific CD4+ T cells in mice inoculated with MuLV-induced lymphoma, RMA (25). Only in 19

conjunction with an administration of sgp130, immunization with tumor-specific MuLV peptide

EnvH13.3 presented by I-Ab (25) induced IFN-γ-producing endogenous tumor-specific CD4+ T cells, and tumor (MuLV)-specific CD8+ T cells (Fig. 4A). Abrogation of specific CD8+ T-cell responses by depletion of CD4+ T cells revealed that sgp130 administration improved CD4+ T-cell-mediated helper

activity toward CD8+ T cells rather than having a direct effect on CD8+ T-cell activation. Furthermore,

CD4+ T-cell-mediated anti-tumor effects primed with H13.3 peptide-pulsed DC was profound in

RMA-bearing IL-6R mKO mice as compared to those in WT counterparts, which was also abolished by

CD4 depletion (Fig. 4B). These results demonstrated that the immunosuppressive effect of sIL-6R was

mediated through CD4+ T cells. Collectively these data suggest that myeloid cell-derived sIL-6R

represents a critical factor for the attenuation of Th1 responses, and subsequent impairment of CD4+

T-cell-mediated anti-tumor immunity.

IL-6-dependent c-Maf expression in CD4+ T cells promotes the immunosuppression in tumor-bearing

mice

To explore the molecular mechanism dictating the IL-6/sIL-6R-mediated Th1 inhibition, we examined

the gene expression profile of CD4+ T cells isolated from tumor-free or -bearing mice (Fig. 5A). We

found that Il4, Ccr4, and c-maf, which was Th2-associated gene signature, were substantially increased

when the tumor existed, and their up-regulation were reversed by anti-IL-6R Ab treatment. However, the 20

other Th2-related genes such as Gata3, Irf4, Il5, and Il13, or key transcription factors that regulate the commitment to other effector lineages such as Tbx21(T-bet), Rorc, Bcl6, or Foxp3 (30) was not dramatically altered. In contrast, Ccl3(MIP-1α) and Wnt5a expression were decreased in tumor-bearing mice in an IL-6-dependent manner. IL-6-dependent alterations of these genes were recapitulated by in vitro TCR stimulation of naïve CD4+ T cells in the presence of IL-6/sIL-6R under Th1-skewed conditions (Fig. 5B). Importantly, consistent with previous in vitro studies (27, 31), IL-6/sIL-6R stimulation induced robust up-regulation of c-maf, which was one of the most prominently altered transcription factors (Fig. 5A and B), and was abrogated by Stat3 knockdown (Supplementary Fig. S3F and G). Intracellular staining of transcription factors in OT-II cells primed in anti-IL-6R Ab-treated mice confirmed the IL-6-mediated c-Maf expression in tumor-bearing mice (Fig. 5C and D).

To further investigate the role of c-Maf, we generated OT-II cells with c-Maf mutation (designated as

Ofl) that was located in DNA binding domain (22) and thereby resulted in its dysfunction. In vitro stimulation of Ofl cells revealed that c-Maf function affected the expression of many genes that were identified as IL-6-dependent altered genes (Fig. 5B). Actually, Il4, Il21, IL10 and Gata3 were greatly down-regulated by c-Maf inactivation. When Ofl OT-II cells were transferred and primed in tumor-bearing mice, their ability to produce IFN-γ and GM-CSF was partially rescued as compared to those in WT OT-II cells (Fig. 5E), suggesting the requirement of IL-6-induced c-Maf activity for Th1 suppression. In addition, c-Maf mutant OT-II cells significantly retarded the growth of OVA-expressing 21

melanoma, which was not observed in WT OT-II cells (Fig. 5F). These results suggest that

IL-6/sIL-6R-mediated signaling intrinsically inhibited Th1 differentiation through c-Maf induction,

resulting in a dysfunction of CD4+ T-cell-mediated anti-tumor activity.

The sIL-6R serves as an immunosuppressive marker in cancer patients

As well as other type of cancers (10, 12, 15), the levels of IL-6 and sIL-6R in plasma were significantly higher in HNT patients as compared with those in HD (Fig. 6A). The detailed subject information is summarized in Supplementary Table S1 and S2. Next we assessed the expression of surface IL-6R on myeloid cells from HNT patients and HD because the decreased level of surface IL-6R expression indicates the shedding of membrane-bound IL-6R in sIL-6R-producing cells (16, 28). A

profound decrease of IL-6R expression was observed in CD14+CD16- classical monocytes from HNT patients (Fig. 6B and E), whereas no difference was detected in CD4+ T cells as compared with those

from HD (Supplementary Fig. S4A), suggesting that myeloid population was a likely source of sIL-6R

in cancer patients.

Based on the mouse model, we hypothesized that the shedding and the release of sIL-6R from

myeloid cells contributed to a systemic increase of sIL-6R in cancer patients, resulting in the

up-regulation of c-Maf in CD4+ T cells. Therefore we analyzed c-Maf expression in CD4+ T cells from

HNT and HD. Although CD4+ T-cell population was decreased in NHT significantly, a higher frequency 22

of c-Maf+CD4+ T cells was observed in HNT as compared with HD group (Fig. 6C). To investigate the

directed dependency and correlation among a set of these variables, and to examine the difference and

significance of this pathway between HNT and HD cohorts, we performed a path analysis based on

structural equation modeling. As shown in Fig. 6D, there was a substantial positive correlation between

the frequency of IL-6RlowCD14+ cells in PBMC and the plasma levels of sIL-6R/IL-6 in HNT but not in

HD. Furthermore, in the HD group, the frequency of c-Maf+ cells decreased along with an increase in

the CD4+ T-cell population, however, such relationship was not observed in the HNT group (Fig. 6D and

Table). The comparative analysis of this pathway suggest that there is an obvious difference in the

regulatory pathway inducing c-Maf+ cells in CD4+ T cells between HNT and HD cohorts

(Supplementary Table S3, p < 0.002). These results imply a functional linkage between

myeloid-cell-derived sIL-6R and c-Maf induction in CD4+ T cells only in cancer patients but not in HD.

An adjustment and integration of the age and the frequency of peptides vaccination as additional factors

in the structural equation modeling did not attenuate each association among the variables

(Supplemental Fig. S4 and Table S4).

To confirm the functional relevance of sIL-6R derived from cancer-associated myeloid cells in c-Maf

up-regulation and Th1 inhibition, we analyzed sIL-6R production in CD14+ cells from cancer patients, and its ability to attenuate Th1 differentiation in the presence or absence of sgp130. Consistent with the hypothesis, lower IL-6R expression and higher sIL-6R production in HNT-derived CD14+ cells were 23

reversed by the inhibition of IL-6R shedding with TAPI-0 treatment in vitro (Fig. 6E and F).

Furthermore, culture supernatant of CD14+ cells from HNT but not HD suppressed the Th1 differentiation, which was rescued by sIL-6R neutralization with sgp130 treatment (Fig. 6G). Conversely, the culture supernatant of HNT-derived CD14+ cells specifically up-regulated c-Maf expression (Fig.

6H). As with the case of Th1 inhibition, c-Maf expression was inhibited by sgp130, substantiating the sIL-6R-dependent c-Maf induction. Collectively, the path diagrams and in vitro results suggest that

CD14+ cell-derived sIL-6R leads to c-Maf up-regulation and defective Th1 differentiation in cancer patients.

Discussion

The surface IL-6R as a source of sIL-6R is mainly expressed by monocytes/macrophages, hepatocytes, neutrophil, and lymphocytes in addition to tumor cells (12, 13, 16). Our study demonstrated that while either impaired release of sIL-6R from tumor cells or genetic ablation of IL-6R in T cells had only a marginal effect in Th1 suppression in tumor-bearing mice, depletion of myeloid cells or myeloid cell-specific deletion of IL-6R diminished the systemic increase of sIL-6R in tumor-bearing mice, leading to improved Th1 development and subsequent anti-tumor responses. These findings are consistent with that the presence of myeloid cells, including macrophages and MDSC, is associated with enhanced inflammatory status and poor clinical outcome in cancer patients (1, 8). Preferential interaction between tumor-specific CD4+ T cells and myeloid cells rather than tumor cells because of their MHC class II expression may provide the dominant effect of myeloid cell-derived sIL-6R on Th1 inhibition at the priming in the microenvironment such as tumor-draining LNs. Thus, it is plausible that myeloid cells promote tumor progression partly through sIL-6R-mediated dysregulation of tumor-specific Th1 cells.

Contrary to Th1, Th2-biased differentiation of CD4+ T cells may in turn enhance the skewing toward the tumorigenic myeloid cells through IL-4 production (9, 32). In addition, cancer burden is likely to initiate qualitative changes in myeloid cells, as indicated by an increase in Adam10/17 expression and subsequent vigorous release of sIL-6R. Collectively, sIL-6R appears to serve as one of the key 25

molecules that mediate a mutual activation loop among T cells, myeloid cells and tumor cells to

exacerbate tumor progression. This proof-of-concept based on the mouse model was supported by the

results that sIL-6R released from CD14+ cells in cancer patients exerted an immunosuppressive function

that modified the human CD4+ T-cell property.

An imbalance in quantity and quality of tumor-associated CD4+ T cells including regulatory T cells are highly relevant to the magnitude of anti-tumor immune responses and clinical outcomes (3, 5, 32,

33). We also demonstrated that IL-6/sIL-6R signaling in tumor-specific CD4+ T cells is uniquely

predisposed to redirect Ccr4, Il4, Il21-expressing non-classical Th2-like c-Maf+ cells rather than

functional Th1-skewed responses, which was possible cause for defective T cell-mediated tumor

regression, as demonstrated using IFN-γ-deficient CD4+ T cells. In cancer patients, dominant Th2

cytokine profiles resulted in unfavorable outcomes (3, 5, 33), while inhibition of IL-4 in conjunction

with immunotherapy improved the survival rate of tumor-bearing hosts (9, 34). Although little is known

about the detailed transcriptional targets of IL-6/sIL-6R that dictate such unfavorable T-cell

differentiation, in vitro and in vivo experiments elucidated that loss-of-function in c-Maf conferred

resistance to IL-6/sIL-6R-mediated Th1 inhibition, and significantly abrogated Il4 and Il21 expression,

which were partly responsible for Th1 inhibition (27, 31). These findings provided the evidence for an

immunosuppressive role of IL-6/sIL-6R-STAT3-c-Maf axis in intrinsically dictating the biased

differentiation of tumor-specific CD4+ T cells into tolerogenic Th2 rather than the beneficial Th1 cells in 26

tumor microenvironment, resulting in improved anti-tumor effects. However, Th1 differentiation

restored by c-Maf inactivation was less than that in IL-6-targeted mice, suggesting the additional target

molecules or cells of IL-6/sIL-6R for dampening Th1 differentiation. Actually, the expression of Ccr4,

Ccl3, Wn5a, or Bcl11a, was affected by IL-6/sIL-6R signaling but not by c-Maf inactivation.

It has been shown that the immunosuppression in tumor microenvironment hinders the development

of de novo CTL responses to tumor-associated (neo-)antigens (1). Considering the inherent ability of

CD4+ T cells to potentiate CTLs (1, 4, 35, 36), a part of defective CD8+ T-cell activation appear to be a consequence of the IL-6/sIL6R-induced failure in CD4+ T cells to provide a help for CD8+ T cells

because the IL-6/sIL-6R blockade in tumor-bearing hosts allowed CD4+ T cells to promote the

recruitment of tumor-specific CD8+ T cells into draining LN and tumor site. These ideas are supported

by the clinical finding in cancer patients that the presence of CD8+ T-cell response and/or

IFN-γ-producing CD4+ T cells specific for NY-ESO-1 was associated with favorable anti-tumor

responses and prolonged survival, while the presence of IL-4-producing CD4+ T cells was not associated with a survival benefit and even abolished the favorable effect of CTL responses (33).

Our findings provide not only a plausible explanation for the failure of CD4+ T cells to regress tumors,

but also may have direct implications for practical applications in cancer immunotherapy. Taking into

account the role of sIL-6R in preventing a clearance of relatively lower level of IL-6 and in amplifying

its bioactivity (16, 17), it is postulated that the proceeding increase of sIL-6R in the early phase of tumor 27

progression is the rate-limiting step for the attenuation of the anti-tumor T-cell responses, and thereby

may provide an earlier prognostic value over that of IL-6 for monitoring the immunosuppressive status

in cancer patients to predict their susceptibility to T-cell-mediated immunotherapies. However, more

intensive investigations with a larger number and longitudinal follow-ups of patients will be required to

determine their prognostic values in cancer progression, because the significant correlation between

overall survival of HNT patients and their levels of sIL-6R was not obtained due to the small size and

advanced tumor stages of our current cohort.

Human CD4+ T cells still exhibit the plasticity with flexible genetic programs rather than irreversibly differentiated status even after antigen experiences, and can acquire different properties in the context of secondary responses (37, 38). This supports a possibility that IL-6/sIL-6R converts the transferred or in vivo activated CD4+ T cells into dysfunctional T cells in cancer patients when T-cell-mediated

immunotherapy such as adoptive T-cell transfer including chimeric antigen receptor-expressing T cells

(CAR-T cells) therapy, vaccination with tumor-associated antigenic peptides, or immune-checkpoint

blockade, were given. Indeed, recent trials demonstrated that the level of IL-6 correlated with overall

survival rate of patients treated with immunotherapies (39-41). It is particularly intriguing that following

CAR-T-cell infusion, the humanized anti-IL-6R Ab Tocilizumab is widely used to lessen cytokine

release syndrome (CRS)-related toxicities (2, 42). The patients with CRS exhibited higher levels of

sIL-6R/IL-6, and the profiles that mirrors macrophage activation syndrome (42). Although whether 28

Tocilizumab improves the Th1 responses of CD4+ T cells within the CAR-T cells remains to be investigated, a combined IL-6/sIL-6R blockade in immunotherapeutic regimens reduces not only toxicity risks such as CRS, but also may be a promising strategy for further enhancement of their therapeutic efficacy through improving the quality of tumor-specific T cells, in addition to the approaches that compensate for their quantitative decrease.

Acknowledgements

We thank Drs. Youichiro Iwakura, Shigeo Koyasu, Irmgard Foester, and Akira Shibuya for the generous supply of IFN-γ-deficient embryos, OT-II TCR transgenic embryos, Lysozyme M-Cre knock-in embryos, and RMA cells, respectively.

References

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Table

Result of multilevel linear models without covariates. (Related to Fig. 6D) Endogenous Exogenous Estimate SE Z p value 95% CI variable variable HD (n = 25) Log (sIL-6R) Intercept 5.614 0.072 78.51 < 0.001 (5.474, 5.755) % IL-6Rlow 0.211 0.027 0.77 0.439 (-0.324, 0.075) CD14+CD16- Log (IL-6) Intercept -0.941 2.742 -0.34 0.732 (-6.315, 4.434) Log (sIL-6R) 0.204 0.483 0.42 0.673 (-0.742, 1.150) Log (CD4) Intercept 3.108 0.067 46.25 < 0.001 (2.976, 3.24) Log (IL-6) 0.236 0.104 2.26 0.024 (0.032, 0.44) % c-Maf+ Intercept 28.54 5.419 5.27 < 0.001 (17.92, 39.16) Log (CD4) -5.908 1.617 -3.65 < 0.001 (-9.077, -2.74) HNT (n = 44)a Log (sIL-6R) Intercept 5.904 0.051 116.3 < 0.001 (5.805, 6.004) % IL-6Rlow + - 0.028 0.007 3.69 < 0.001 (-0.032, 0.075) CD14 CD16 Log (IL-6) Intercept -3.547 1.928 -1.84 0.066 (-7.325, 0.231) Log (sIL-6R) 0.77 0.315 2.44 0.015 (0.152, 1.388) Log (CD4) Intercept 2.274 0.341 6.67 < 0.001 (1.606, 2.943) Log (IL-6) -0.005 0.218 -0.02 0.983 (-0.431, 0.422) % c-Maf+ Intercept 12.02 2.683 4.48 < 0.001 (6.756, 17.27) Log (CD4) 0.009 1.07 0.01 0.994 (-2.089, 2.107)

HD: healthy donors, HNT: head and neck malignant tumor patients, SE: standard error; CI: confidence

interval.

a Forty-eight samples were collected from 22 HNT patients before and after peptide vaccination. In some

cases, sample collection was performed several times along with the vaccination.

Figure legends

Figure 1. IL-6 directly acts on tumor-specific CD4+ T cells, resulting in defective Th1 differentiation.

A-E, Ten days after inoculation of MCA-OVA (A, B and E) or EG7 (C and D), IL-6+/+ or IL-6-/- mice

were transferred with OT-II cells and immunized by OVA-IIp-pulsed DC. Control or anti-IL-6R Ab were

injected in A. The number of OT-II cells, the frequencies of cytokine-positive cells (A-C, and E), and the

proportion of Gr-1+CD11b+MDSC (D) were determined. F, Three days after Ab injection, and transfer of OT-II and DC into MCA-OVA-bearing mice, phospho-STAT3 in OT-II cells from draining LNs were determined. G and H, MCA-OVA-bearing IFN-γ-deficient (γKO) mice were transferred with wild-type

(WT) or γKO OT-II T cells, and immunized together with Ab injection. Tumor outgrowth (G) and

OVA-Tetramer+CD44hiCD8+ T cells in tumor-draining LNs were analyzed (H). The values represent

mean ± SEM with n = 4-7 /group; * p < 0.05, ** p < 0.01, *** p < 0.001. The data are representative of

3 or more independent experiments.

Figure 2. IL-6 trans-signaling attenuates Th1 differentiation in tumor-bearing mice. A and B, As in Fig.

1, Ab injection, transfer of OT-II cells and OVA-IIp-pulsed DC were performed. Representative IL-6R expression on OT-II cells (A, left) and percentages of IL-6R+ OT-II cells on day 3 (B) or their kinetic changes (A, right) are shown. C-E, IL-6R+/+ or IL-6R-/- OT-II cells (C), human (D), or Stat3

siRNA-transfected mouse (E) polyclonal CD4+ T cells were stimulated with anti-CD3/CD28 Abs and

IL-12 in the presence or absence of sIL-6R and IL-6 in vitro. IFN-γ production upon re-stimulation was assessed. F, MCA-OVA-bearing mice were transferred with IL-6R+/+ or IL-6R-/- OT-II cells, and injected

with sgp130. The number of OT-II cells in spleen and LNs (lower), and IFN-γ-producing cells (upper)

were analyzed. G and H, Ten days after MCA-OVA inoculation in CAG-Cre/IL-6Rfl/fl (IL-6R-/-) or

Cre-IL-6R+/+ mice, the concentration of sIL-6R in serum (G) and the tumor size (H) were measured. I,

At the indicated time (Day 0, 8 or 16) after MCA-OVA inoculation, mice were transferred with OT-II cells, and immunized. The frequencies of IFN-γ-producing cells were determined. n = 4-6. * p < 0.05,

** p < 0.01. The data are representative of 3 independent experiments.

Figure 3. Myeloid cell-derived sIL-6R contributes to attenuated Th1 differentiation in tumor-bearing

mice. A and B, The concentration of sIL-6R (left) and IL-6 (right) in serum from IL-6Rfl/fl/LysM-Cre+/-

(IL-6R mKO) or IL-6Rfl/fl/Cre-/- (WT) mice were monitored after MCA-OVA inoculation (A). Tumor

sizes are also shown (B). C and D, MCA-OVA-bearing mice were transferred with IL-6R-deficient

OT-II cells, and immunized. Cytokine-producing cells in spleen (C), or OVA-tetramer+CD44hiCD8+ (D, left) or OT-II cells (right) in tumor-draining LNs were assessed. E, After intravenous injection of

MO4-Luc, IL-6R-deficient OT-II-cell transfer and immunization were performed. Luminescent images

(upper), lung tumor (lower left) at day 27, and the kinetics of their photon-counts (lower right; mean ±

SEM with n = 8-10 mice/group) are shown. F, MO4-Luc-bearing mice were immunized with OVA-Ip- 37

or OVA-IIp-pulsed DC as in E. Tumor growth (photon-counts) on day 19 are shown. G, IFN-γ production from OVA-specific CD8+ T cells in MO4-Luc-bearing mice primed with OVA-Ip-pulsed DC and sgp130 was assessed by ELISPOT. * p < 0.05, ** p < 0.01, *** p < 0.001. The data are representative out of 3 independent experiments.

Figure 4. The sIL-6R dampens CD4+ T-cell-mediated anti-tumor responses. A and B, WT and/or IL-6 mKO mice were treated with control or anti-CD4 Ab two days before and after RMA inoculation, and injected with H13.3-pulsed DC and sgp130. IFN-γ production from tumor-draining LN cells re-stimulated with H13.3-, Env/Kb- or GagL/Db-pulsed DC were assessed by ELISPOT (A). The kinetics of tumor outgrowth (upper), and tumor size at the endpoint (lower) are shown (B). The values represent mean ± SEM with n = 6-9. ** p < 0.01, *** p < 0.001. NS, not significant. The data are representative of

2 or more independent experiments.

Figure 5. IL-6-dependent c-Maf induction was responsible for the defective Th1 response in tumor-bearing mice. A, C, and D, MCA-OVA-bearing, anti-IL-6R Ab-treated WT mice were transferred with OT-II cells, and immunized with OVA-IIp-pulsed DC. Heatmap depicts the fold-changes of genes expressed in CD4+ T cells from indicated conditions with two independent experiments. * Probes showing a median fold-change in expression > 2.0 or < -2.0 were considered as altered gene expressions 38

(control Ab-treated tumor-bearing versus tumor-free and anti-IL-6R Ab-treated tumor-bearing) (A).

Indicated protein expression in donor OT-II cells (C and D) was analyzed. Representative histograms

anti-CD3/CD28 Abs and IL-12 in vitro. Three days later, the indicated mRNA expression was assessed

by RT-PCR. Fold-changes (Log10-transformed) in IL-6/sIL-6R-stimulated WT or Ofl/+ cells relative to

WT cells without IL-6/sIL-6R stimulation are shown (n = 3). E and F, MO4-Luc-bearing mice were

transferred with WT or Ofl OT-II cells and immunized with OVA-IIp-pulsed DC. Cytokine-positive

OT-II T cells were analyzed (E). Pulmonary tumor progression was monitored via their luciferase

activity (F). The values represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. The data are

representative of 2 or more independent experiments.

Figure 6. Functional association between myeloid cell-derived sIL-6R and c-Maf expression in cancer

patients. A-C, The levels of IL-6 and sIL-6R in plasma, IL-6RlowCD14+CD16- cells and c-Maf+CD4+ T cells in PBMC from HNT patients without vaccination or with minimal times of vaccine received (n =

22) or HD older than 50 years (n = 12) were analyzed. D, The structural equation models with these variables (A-C) represented by path diagram shown here are results of multilevel linear models without covariates. Twenty-five (from HD) and 48 samples from 22 HNT patients were used in this analysis.

Each estimate and p value (HNT; left underlined, HD; right) are shown. E and F, CD14+ cells from 39

HNT or HD were cultured in vitro with or without TAPI-0. The level of surface IL-6R (E) and the

concentration of sIL-6R in the supernatants (F) were determined. G and H, HD-derived CD4+ T cells

were stimulated with anti-CD3/CD28 Abs in the presence of IL-12 and suboptimal IL-6 together with the HNT- or HD-derived CD14+ cell-culture supernatant. The sgp130 was added in differentiation

culture. Cytokine production (G), and c-Maf expression (H) in re-stimulated T cells were assessed. The

representative plots and individual values along with the mean are shown. * p < 0.05, ** p < 0.01, *** p

< 0.001.

Soluble IL-6R expressed by myeloid cells reduces tumor-specific Th1 differentation and drives tumor progression

Hirotake Tsukamoto, Koji Fujieda, Masatoshi Hirayama, et al.

Cancer Res Published OnlineFirst February 24, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2446

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