6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10.

TLR2/6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10 Ileana S. Mauldin 1,Ena Wang 2, Donna H. Deacon 1, Walter C. Olson 1, Yongde Bao 3,and Craig L. Slingluff Jr. 1* 1

2

Department of Surgery, University of Virginia, Charlottesville, VA 22908 Department of Transfusion Medicine, National Institutes of Health, Bethesda, MD 20892, and 3 Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA 22908

* Corresponding author: Craig L. Slingluff, Jr. P.O. Box 800709 Charlottesville, VA 22908 Phone: 434-924-1730 FAX: 434-243-6844 Email: [email protected] Short Title: TLR2/6 agonists and IFNγ induce CXCL10 from melanoma Key words: Melanoma, Immune therapy, TLR, Migration, Tumor infiltration Abstract: 199 words Manuscript: 4914 words Figures: 6/6

Financial Support:

This work was supported by the UVA Cancer Center Grant P30

CA044579, the Farrow Fellowship (ISM), and Rebecca Harris Fellowship (ISM).

Disclosure of conflicts: The authors declare no competing financial interests.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.29515

This article is protected by copyright. All rights reserved.

International Journal of Cancer

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TLR2/6 agonists and IFNγ induce CXCL10 from melanoma

Novelty and Impact We have identified a novel synergy of TLR2/6 agonists and IFNγ for inducing CXCL10 production directly from melanoma cells.

TLR2/6 agonists +IFNγ treatment significantly

enhances CXCL10 production from melanoma cells when compared to IFNγ treatment alone. Since CXCL10 promotes the migration of CD4+ and CD8+ T-cells, these findings suggest a possible new therapeutic approach for enhancing CXCL10 production and thus immune cell infiltration of melanoma.

2 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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Abstract Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME.

Here we show that toll-like

receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferongamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.



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Introduction Immune therapies have emerged as the most effective therapies for inducing durable regressions and control of melanoma, mediating clinical benefit through inducing, expanding, or re-activating antitumor T-cells (1-8). Tumors lacking T-cells commonly fail to respond to immune therapies and are associated with shorter overall patient survival (8-15). Metastases with immune cell infiltrates can be differentiated from those without infiltrates, in part, by their expression of immune cell-attracting chemokines, including CXCL10 (11;15). High CXCL10 expression in the TME is associated with better T-cell infiltration, tumor control, and diseasefree survival (14;16;17). CXCL10 has also been implicated mechanistically in T-cell infiltration associated with PD-1 blockade (18). Thus, approaches to increase CXCL10 production in melanoma metastases may lead to enhanced T-cell infiltration and improve the overall efficacy of immune therapies. TLR agonists induce expression of cytokines and chemokines from various cells. Also, interferons specifically induce CXCL9, CXCL10, and CXCL11 (19). Thus, both TLR agonists and interferons are candidate agents for inducing chemokines in melanoma metastases. Immune cells are commonly considered the dominant source of chemokines, but others and we have reported that cancer cells including melanoma can produce CXCL10 in response to IFNγ (11;20).

However, it is not known whether TLR agonists induce melanoma cells to

produce CXCL10 or other T-cell-attracting chemokines (CCL2-5, CXCL9-10 and CXCL12 (15)). We hypothesized that TLR ligation would induce T-cell-attracting chemokines, including CXCL10, directly from melanoma cells, alone or in combination with IFNγ. We find that melanoma cells produce few immune cell-attracting chemokines constitutively.

Melanomas express multiple TLRs; however, stimulation with TLR agonists

poly-ICLC (TLR3), LPS (TLR4), MALP-2 (TLR2/6), FSL-1 (TLR2/6), imiquimod (TLR7), 4 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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resiquimod (TLR7&8), or CpG (TLR9) alone induced little chemokine production. In contrast, treatment with TLR2/6 agonists (MALP-2 or FSL-1) +IFNγ increased CXCL10 production, compared to either treatment alone. These findings are corroborated on surgical specimens of human melanoma metastases.

Furthermore, TLR2 and TLR6 are expressed by most

melanoma lines tested. Collectively, these data support investigation of combined therapy with TLR2/6 agonists plus IFNγ as a novel approach to enhance CXCL10 production directly from melanoma cells, which may facilitate T-cell infiltration into melanoma metastases. Experimental Procedures Cell lines and culture.

Melanoma cell lines were obtained from Duke University (DM6,

DM13, DM93, DM122) or established from patients at the University of Virginia (VMM1, VMM5A, VMM12, VMM14, VMM15, VMM17, VMM18, VMM39, VMM86, VMM150, VMM273, VMM389) with IRB approval (protocols 5202, 6346, and 10598).

Melanoma lines A375,

Malme3M, SLM2, SKMEL2, and SKMEL24, and human lymphoma line Ramos were obtained from the American Type Culture Collection (Manassas, VA); HEK293 (human embryonic kidney cells) parent and TLR7-transfected lines from Imgenex (San Diego, CA); HUVEC and HMVECad endothelial cell lines from Lonza (Walkersville, MD); and Leukopak human peripheral blood leukocytes (PBMC) from BRT Laboratories Inc. (Baltimore, MD). Melanoma cells were cultured in RPMI 1640 media supplemented with 5% fetal bovine serum, 1% Pen/Strep and 1% L-glutamine; hereafter referred to as RPMI (Gibco, Grand Island, NY). TLR staining. Melanoma cell lines were seeded into 6-well plates at 1x106 cells/well in RPMI and cultured overnight in the presence or absence of recombinant human IFNγ (rh-IFNγ, 1000 IU/mL; PeproTech, Rocky Hill, NJ). Then, cells were detached using Accutase (Millipore, Billerica, MA), stained with indicated antibodies and fixed using Cytofix (BD Biosciences, San 5 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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Jose, CA). Surface staining antibodies: TLR2-Alexa647, Streptavidin-PE (BD Pharmigen, San Jose, CA), TLR6–Biotin (eBioscience, San Diego, CA), TLR4-APC (R&D Systems, Minneapolis, MN).

Intracellular staining: cells were fixed and permeabilized using the BD

Cytofix/Cytoperm kit (BD Biosciences) and stained with appropriate antibodies: TLR7-CFS, IgG2α-CFS (R&D Systems), TLR3-PE, TLR9-APC (eBioscience), TLR8-Alexa488 (Dendritics, San Diego, CA), IgG1-PE, and IgG2α-PE (Caltag, Towcester, UK).

Flow cytometric analysis. Stained cells were collected on the FACSCalibur instrument (BD Biosciences) and analyzed with FlowJo 8.8.6 software (TreeStar, Ashland, OR). Analyses were conducted on live cells (95%) as defined by forward and side-angle scatter. Gates were set based on isotype-matched (ISO) controls or fluorescence-minus-one (FMO) controls (Supplemental Figure 1). TLR stimulation. For intracellular cytokine staining, melanoma cell lines were seeded into 6well plates at 1x106 cells/well in RPMI and individually stimulated with TLR agonists or PMA and Ionomycin overnight in the presence of GolgiStop (1µL/mL, BD Biosciences) at 37°C, at these concentrations: LPS (10µg/mL), CpG ODN (5µg/mL), resiquimod (5µg/mL), imiquimod (25µg/mL), MALP-2 (100ng/mL; Imgenex), poly-ICLC (20µg/mL), FSL-1 (5µg/mL; InvivoGen, San Diego, CA), PMA (60ng/mL) and Ionomycin (1mg/mL; Sigma-Aldrich, St. Louis, MO). These were used with or without rh-IFNγ (1000 IU/mL; PeproTech). After stimulation, cells were detached using Accutase. Intracellular chemokine staining.

Cells were fixed and permeablilized using the BD

Cytofix/Cytoperm kit and then stained with the indicated antibodies. Staining antibodies were: CCL2-PE, CCL3-PE, CCL4-PE, CCL5-PE, CXCL10-PE (BD Pharmigen), CXCL9-PE,


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CXCL12-APC (R&D Systems), IgG1-PE, and IgG2α-PE (Caltag). Flow cytometric analyses were conducted as described. ELISA assays. Melanoma cell lines or cells from patient samples were stimulated with TLR agonists as described, but without GolgiStop. Supernatants were collected and centrifuged to remove cell debris, then assayed for secreted chemokines. CXCL10 was measured using the BD OptEIA human IP-10 ELISA Set with BD OptEIA buffers (BD Biosciences). CCL22 (MDC) was measured using primary and secondary α-MCD antibodies, and rMDC was used for standards (PeproTech). Detection was done using Streptavidin-horseradish peroxidase conjugate and ABTS Substrate Solution (Sigma-Aldrich). CFSE assay. Melanoma cells were stained with the CellTrace CFSE Cell proliferation kit 5µm/1x106 cells (Life Technologies, Carlsbad, CA), prior to treatment with TLR agonists and flow cytometric analysis. Apoptosis assay. Melanoma cells were treated with TLR agonists as described then stained with CellEvent Caspase-3/7 Green Flow Cytometry kit according to manufacturer instructions (Life Technologies). Apoptosis was assessed by flow cytometric analysis. MHC staining. Cells were stained with HLADR-Percp (Biolegend, San Diego, CA), HLA-ABCFITC (eBioscience) antibodies prior to intracellular staining for CXCL10-PE (BD Pharmigen) and flow cytometric analyses were conducted as described. Immunohistochemistry staining. Stimulated cells were pelleted by centrifugation, paraffinembedded and sectioned. Sections were deparaffinized and hydrated using xylene and a graded alcohol series. Antigen retrieval was performed in Vector’s Target Retrieval Solution. Sections were individually stained with the following antibodies: CXCL9, CXCL10 (Peprotech), 7 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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Melan-A/MART-1, HMB-45 (Dako, Carpinteria, CA).

Expression was detected using

ImmPRESS Peroxidase kits (MP-7401 and MP-7402, Vector Laboratories, Burlingame, CA). Peroxidase staining was visualized with DAB substrate (SK-4105) and counterstained with Gill’s hematoxylin (Vector Laboratories). Statistical analysis. p values were calculated using the paired Student's t-test. p values less than 0.05 were considered significant. For analysis of synergy:

levels of CXCL10 induced by TLR stimulation alone and IFNγ

stimulation alone were added together and compared to the induction of CXCL10 after the combined treatment TLR +IFNγ by the paired student’s t-test. p values less than 0.05 were considered significant for synergistic upregulation. Additional methods are located in Supplemental Experimental Procedures. Results Melanoma cells produce little chemokine in response to treatment with TLR3, TLR4, TLR7, TLR8 or TLR9 agonists. Gene expression profiling of four human melanoma cell lines VMM1, DM13, DM93 and DM122 revealed expression of TLRs 1, 3, 4, and 6, when compared to HEK293 cells which lack TLR expression (Figure 1A). Effects of TLR agonists on gene expression profiles were assessed for the following: the four melanoma cell lines; 3 melanoma metastasis biopsies (TPF15529, 15100, and 15289); and a limited assessment of a 5th melanoma line VMM39. As controls HEK293 cells were analyzed since they lack TLR expression; TLR7 transfected HEK293 cells (TLR7-HEK293) as TLR7 responsive controls; endothelial cell lines (HUVEC and HMVECad), which express most TLRs and Ramos cells, which express most TLRs. Principal component analysis indicated that TLR stimulation had


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only modest effects on each melanoma cell line, and that the melanoma lines clustered together, and separately from endothelial, Ramos, and HEK lines (Supplemental Figure 3A-B). Chemokines CCL2-5, CXCL9-10, and CXCL12 support T-cell recruitment to tissues (15); we assessed whether melanoma cells could produce them constitutively or after TLR stimulation. Changes in expression of genes encoding those chemokines suggested possible effects of TLR3 and TLR4 agonists on individual cell lines (Supplemental Figures 3C and 4AB), but when analyzed across all 4 cell lines, no effects on those chemokine genes were significant (Figure 1B). TLRs 2-4, 6, 7, and 9 were detected on several or all 4 cell lines and on PBMC (Figure 1C). Therefore, we tested effects of the same TLR agonists evaluated in the gene array, plus two combinations (imiquimod and poly-ICLC; LPS and CpG) on chemokine production. Since melanoma cells expressed TLR6 genes (Figure 1A), TLR2/6 agonists (MALP-2 and FSL-1) were also tested.

TLR6 interacts with TLR2 to form a functional receptor that binds the

bacterial lipoprotein MALP-2 and its synthetic homologue FSL-1 (21).

Less than 10% of

melanoma cells produced CCL2, CCL4-5, CXCL9, and CXCL12, constitutively (untreated cells); however, greater than 50% produced CCL3 (Figure 1D).

TLR agonists did not

significantly alter production of CCL2, CCL4-5, CXCL9, or CXCL12; however TLR2/6 agonists increased CCL3 production, compared to untreated cells (Figure 1D). Melanoma cells upregulate CXCL10 production upon stimulation with TLR2/6 agonists and IFNγγ. Chemokines CXCL9-10 support T-cell recruitment to tissues (15), and these chemokines are induced by IFNγ (11). Thus, we tested whether TLR ligation given in combination with IFNγ would augment CXCL9 and CXCL10 chemokine production by melanoma cells, as well as CCL2-5, and CXCL12 (11). There was no effect on CCL2, CCL49 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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5, CXCL9, or CXCL12 (Supplemental Figure 5A-B), but CCL3 production was enhanced from melanoma cells treated with TLR2/6 agonists +IFNγ compared to IFNγ stimulation alone (Supplemental Figure 5A), but this effect was similar to that with TLR2/6 agonists alone (Figure 1D). However, the proportion of melanoma cells producing CXCL10 increased strikingly after stimulation with TLR2/6 agonists and IFNγ, compared to either treatment alone or untreated cells, for the four melanoma cells lines (Figure 2A-B). When data were averaged across the four melanoma lines, the percent of cells producing CXCL10 constitutively was 1.6% (untreated cells), while 7% of cells produced CXCL10 after IFNγ treatment alone (Figure 2B). However, TLR2/6 agonists and IFNγ induced CXCL10 production from a significantly higher percentage of melanoma cells (41% MALP-2/IFNγ, and 38% FSL-1/IFNγ) than either agent alone, or any other tested TLR agonist (Figure 2B and Supplemental Figure 5C). LPS +IFNγ treatment also increased the percentage of CXCL10 producing cells in two melanoma cell lines (VMM1 and DM13), however the TLR2/6 agonists and IFNγ induced greater increases in CXCL10 in these lines than LPS +IFNγ (Figure 2A-B). The mean fluorescence intensity (MFI) of CXCL10 for melanoma cells treated with TLR2/6 agonists +IFNγ were also increased, compared to treatment with either agent alone, indicating greater CXCL10 production on a per-cell basis (Figure 2C). Additionally, there was a robust increase in CXCL10 staining after MALP-2 or FSL-1 and IFNγ treatment as assessed by immunohistochemistry in tested VMM1 and DM13 cell lines (Figure 3A). CXCL10 protein levels were also significantly increased in the supernatants of ten melanoma cell lines, and one freshly resected patient melanoma, after stimulation with MALP-2 or FSL-1 and IFNγ, (average


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of 29091pg/ml, 28552pg/ml respectively) as compared to untreated (15pg/ml) or IFNγ treated cells (3551pg/ml) (Figure 3B). We sought to determine whether CXCL10 induction by TLR2/6 agonist stimulation of melanoma was dose dependent.

Dose-response assays assessing CXCL10 production

induced in DM13 and VMM1 cells by TLR2/6 agonists, with a constant concentration of IFNγ, showed that MALP-2 at 100ng/ml and FSL-1 at 5µg/ml induced maximal levels of CXCL10 (Figure 3C). Increasing the dose of other TLR agonists in the presence of IFNγ had modest effects on CXCL10 induction highlighting the uniqueness of the TLR2/6 pathway. However a high concentration of LPS (100µg/ml), given in combination with IFNγ, induced CXCL10 at levels comparable to much lower doses of MALP-2 or FSL-1 and IFNγ (Supplemental Figure 5D). TLR2 and TLR6 are widely expressed among melanoma. We had found that TLR2 and TLR6 were expressed among 4 melanoma cell lines; however, the potential future clinical application of TLR2/6 agonists depends on broad expression across a larger population. Thus, we assessed TLR2 and TLR6 expression for 21 melanoma cell lines, using Ramos cells and PBMC (Leukopak) as positive controls (Figure 4A and Supplemental Figure 1D-E). Staining intensities (MFI) equal to or greater than those of Ramos cells, which express all TLRs, were considered positive.

We found that 20 of the 21 screened melanoma lines

expressed TLR2 (Figure 4B and Supplemental Figure 1F), while 17 of the 21 expressed TLR6 at levels comparable to or greater than Ramos cells (Figure 4C and Supplemental Figure 1G). Slightly lower level expression may also be significant; so these serve as minimum estimates. TLR2 and TLR6 were widely expressed among the 21 tested melanoma cell lines, with 17 cell lines (81%) expressing both TLRs at levels higher than Ramos cells. Interferons modulate 11 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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expression of TLRs on a variety of cells, and have been reported to enhance signaling downstream of TLR ligation (22). Thus, we assessed whether IFNγ modulated expression of TLR2 or TLR6 on the same 21 melanoma cell lines, but found that neither was impacted by IFNγ stimulation in these studies (data not shown). TLR2/6 agonists and IFNγγ impact CXCL10 gene expression. To assess the global effects of TLR2/6 agonist stimulation on melanoma cells, gene expression profiling was performed on four human melanoma cell lines VMM1, DM13, DM93 and VMM39. The analysis revealed that treatment of melanoma cell lines with MALP-2 or FSL-1 alone induced increases in IL-8 gene expression, while treatment of melanoma cells with IFNγ alone increased expression of CXCL9 and CXCL10 genes as well as others (Supplemental Figure 6). Treatment of melanoma cells with IFNγ and either MALP-2 or FSL-1 , both induced expression of IL-8, CXCL11, IL-32 and cathepsin S genes when compared to IFNγ treatment alone, as well as CXCL10 (Supplemental Figure 6 and Figure 5A). CXCL9 gene expression was upregulated in FSL-1+IFNγ treated cells (Supplemental Figure 6). Expression of CXCL9 and CXCL11 protein were assessed by immunohistochemistry for VMM1 and DM13 cell lines.

There was a modest induction of

CXCL9 when compared to treatment with either agent alone (Figure 5B) for both lines. However, CXCL11 protein was not detected in melanoma cells, and its expression was unaffected by TLR2/6 agonist +IFNγ treatment (data not shown). CCL22 chemokine attracts T regulatory cells (Tregs) to the TME which can limit immune activation (17;23).

Although, CCL22 genes were not upregulated in our gene arrays, we

evaluated whether TLR2/6 agonists impacted CCL22 protein production from ten melanoma cell lines. We found that many of the cell lines produced CCL22 basally (untreated cells),


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however treatment of melanoma cells with TLR2/6 agonists +IFNγ did not increase CCL22 production (Figure 5C). Effects of TLR agonists and IFNγ are not expected to be limited to chemokine induction. Other effects that could impact clinical outcomes of cancer patients might include antiapoptotic signaling downstream of NF-κB, alteration in the proliferative capacity of melanoma cells, or alterations in expression of tumor antigens or MHC which could impact the ability of immune cells to recognize and kill melanoma. We examined whether TLR2/6 ligation would impact the proliferation or apoptosis of melanoma cells. We found that TLR2/6 ligation given with or without IFNγ, did not hinder apoptosis, or significantly impact proliferation, in nine evaluated cell lines (Figure 5D-E and Supplemental Figure 7A-B). Furthermore, we found that TLR2/6 agonists +IFNγ slightly enhanced expression of MHC Class II with no significant change in MHC Class I (Supplemental Figure 8A). By immunohistochemistry, we assessed expression of gp100 and Melan-A by melanoma cells and found that Melan-A expression was slightly downregulated after TLR2/6 agonists +IFNγ treatment while gp100 expression was unchanged (Supplemental Figure 8B). Surgically excised human melanoma cells produce CXCL10 in response to TLR2/6 agonists and IFNγγ stimulation. Having observed production of CXCL10 by cultured human melanoma cell lines in vitro, we asked whether the same would be observed from melanoma cells isolated from surgically-excised human metastases. TLR2 and TLR6 are expressed on some immune cell subsets (24;25), thus we also assessed whether TLR2/6 agonists and IFNγ may impact CXCL10 production from immune cell subsets in patient tumor specimens as well as the melanoma cells.

Resected melanoma specimens were studied from patients who

required surgery for isolated regional or distant melanoma metastases. 13 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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Among the melanoma cells (S100+CD45neg) assessed from 12 patient samples, stimulation with TLR2/6 agonists alone did not increase in the percentage of CXCL10 producing cells above untreated cells. However, combinatorial treatment of melanoma cells with FSL-1 and IFNγ markedly enhanced the percentage of cells making CXCL10, in terms of both the percentage of CXCL10+ cells and the MFI of CXCL10, when compared to untreated cells or IFNγ treated cells (Figure 6A-B). Tumor associated B-cells also upregulated CXCL10 production after treatment with FSL-1 +IFNγ, compared to treatment with IFNγ alone or TLR2/6 agonists alone (Figure 6C). Both melanoma cells and B-cells treated with MALP-2 +IFNγ upregulated CXCL10 production when compared to untreated cells, and showed trends for an effect (p = 0.0098 and p = 0.0244 respectively) when compared to IFNγ treated cells (Figure 6A-C). Production of CXCL10 by dendritic cells, macrophages, natural killer cells (NK) and Tcell subsets was not significantly impacted by TLR2/6 agonist and IFNγ treatment, although there were trends for an effect of IFNγ on macrophage, and NK cell subsets from patient tumors (Supplemental Figure 9A-D). Discussion Favorable clinical outcomes for melanoma patients, and responses to immune therapy, are closely associated with immune cell infiltration of metastatic lesions (11-14;26).

Gene

expression profiling analyses of melanoma metastases containing high numbers of tumorinfiltrating lymphocytes reveal that these tumors express chemokines known to recruit immune cells (CCL2-5, CXCL9-10) (15). However, these studies are limited by their inability to define which cells in melanoma tumors produce the chemokines. Furthermore, most melanomas either have no infiltrating immune cells or contain immune cells limited to perivascular spaces (13). To recruit antitumor T-cells to sites where they can mediate tumor clearance, it may be 14 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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ideal if melanoma cells produced T-cell-attracting chemokines themselves. We have found, that treatment of melanoma cells with TLR2/6 agonists and IFNγ induces high levels of CXCL10 production directly from melanoma cells, and that the observed effect is much greater than treatment with either agent alone (Figure 2A-C, Figure 3A-B, and Figure 6A-B). Limited prior data support our finding that melanomas express TLRs -2, -3, -4, -7 and -9 (27-29). However, our study may be the first to report expression of TLR6 by melanoma cells (Figure 1C and Figure 4A&C). The melanoma cell lines investigated here expressed varied levels of TLR2 and TLR6 (Figure 4A-C). However, our data suggest that low levels of TLR2/6 expression may be sufficient to allow responses to MALP-2 and FSL-1 +IFNγ, since DM13 cells which have relatively low TLR2 expression are responsive to treatment (Figure 4B). As our data cannot rule out the possibility that MALP-2 or FSL-1 may be able to signal through additional TLRs, further studies will need to be conducted to clarify the specificity of these ligands in melanoma. The dominance of TLR2/6 agonists +IFNγ in promoting CXCL10 over other TLR agonists was unexpected, as was the striking selectively in the chemokines produced by melanoma cells themselves (Figure 2B and Supplemental Figure 5A-C). Furthermore, we found that even when higher doses of the other TLR agonists were used in combination with IFNγ, TLR2/6 agonists were more potent inducers of CXCL10, with only high concentrations of LPS (≥100µg) inducing comparable levels of CXCL10 (Supplemental Figure 5D). These data suggest that in contrast to immune cells, which produce a wide range of cytokines and chemokines in response to TLR ligation (30;31), melanoma cells may have altered or mutated signaling pathways which may limit the chemokines they are able to produce, and their responsiveness to TLR stimulation (32). Future studies will need to be performed to determine 15 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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the mechanism of action of TLR2/6 agonists and IFNγ towards promoting selective CXCL10 production in melanoma. Gene profiling of melanoma cells lines treated with TLR2/6 agonists and IFNγ demonstrate that a selective profile of genes are induced after combination treatment, which include CXCL10, and may be overall favorable towards promoting immune cell infiltration (Figure 5A and Supplemental Figure 6). Treatment of melanoma cell lines with TLR2/6 agonists +IFNγ had little impact on the genes of chemokines or other common targets which would induce Treg generation or promote their migration (such as CCL22), or on genes which would promote other suppressive immune cells (Supplemental Figure 6). By ELISA assays we also did not see significant changes in CCL22 production from melanoma cells treated with TLR2/6 agonists +IFNγ (Figure 5C).

Furthermore, genes that would promote

survival or proliferation of melanoma cells were also largely unaffected in melanoma cells treated with TLR2/6 agonists +IFNγ (Supplemental Figure 6). In follow up assays, we also did not see significant change in the apoptotic ability of melanoma cells or proliferation after TLR2/6 agonists +IFNγ treatment (Figure 5D-E). Interestingly IFNγ treatment alone did induce some negative modulators of immune activity such as indoleamine-2,3-dioxygenase (IDO) and PD-L1 (Supplemental Figure 6); therefore, it may be beneficial to use tumor-directed TLR2/6 agonists +IFNγ in combination with systemic therapy to block PD-1 and/or IDO. Previous studies have shown that IFNγ, or IL-12 through induction of IFNγ from immune cells can induce CXCL10 production from tumors (11;20).

We have found that TLR2/6

agonists can dramatically augment the response to IFNγ. In prior melanoma studies, CXCL10 has reduced tumor cell proliferation, metastasis, and angiogenesis, and has promoted immune cell recruitment and cell-mediated immunity (14;16). CXCL10 promotes migration of CD4+ and 16 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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CD8+ T-cells (19;33;34). Thus, these findings suggest that there may be therapeutic value for enhancing T-cell migration. While FSL-1 has not been tested in clinical trials, MALP-2 has been tested for the treatment of pancreatic cancer in phase I/II clinical trials and was administered intratumourally safely up to 20µg; MALP-2 was also tested in a phase I trial for wound healing (35;36).

IFNγ has been tested as a treatment for melanoma and was

administered intratumorally at a dose of 2 million IU of IFNγ (NCT00977145). Extrapolating from our dose response assays (Figure 3C), we anticipate that for a 5cm diameter metastasis, doses of MALP-2, FSL-1, and IFNγ expected to cause CXCL10 production would need to be 6.5µg, 325µg, and 65,000 IU, respectively. Since MALP-2 and IFNγ have been administered in clinical trials at doses higher than these, it is possible that these agents may be safely administered at doses expected to induce CXCL10 in the TME. Many immune therapies appear to be more effective in tumors that contain CD8+ T-cell infiltrates and have immune signatures; thus, it is reasonable to explore the possibility of intratumoral administration of a TLR2/6 agonist and IFNγ, in combination with other immune therapies such as vaccination, adoptive cell transfer, or blockade of CTLA-4, PD-1, or IDO (10-12;14;37). Newer approaches to target selective delivery of such agents to tumor may also enable application for patients with disseminated melanoma.

Acknowledgments The authors thank the staff of the Human Immune Therapy Center (Nadia Galeassi, Kelly Smith and Cheryl Murphy), the Biorepository and Tissue Research Facility for technical assistance with assays, and the Beirne B. Carter Center for use of flow cytometers. We thank Mark Smolkin, Dr. Nolan Andrews, and Dr. Gina Petroni for helping with the statistical analysis, and Dr. Timothy Bullock for critical review of this manuscript. 17 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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TLR2/6 agonists and IFNγ induce CXCL10 from melanoma Reference List

(1) Alexandrescu DT, Ichim TE, Riordan NH, Marincola FM, Di NA, Kabigting FD, Dasanu CA. Immunotherapy for melanoma: current status and perspectives. J Immunother 2010 Jul;33(6):570-90. (2) Shapira-Frommer R, Schachter J. Adoptive immunotherapy of advanced melanoma. Curr Treat Options Oncol 2012 Sep;13(3):340-53. (3) Lee S, Margolin K. Tumor-infiltrating lymphocytes in melanoma. Curr Oncol Rep 2012 Oct;14(5):468-74. (4) Korman AJ, Peggs KS, Allison JP. Checkpoint blockade in cancer immunotherapy. Adv Immunol 2006;90:297-339. (5) Menaa F. Latest approved therapies for metastatic melanoma: what comes next? J Skin Cancer 2013;2013:735282. (6) Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, Dronca R, Gangadhar TC, et al. Safety and Tumor Responses with Lambrolizumab (Anti-PD-1) in Melanoma. N Engl J Med 2013 Jun 2. (7) Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K, Burke MM, Caldwell A, et al. Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med 2013 Jun 2. (8) Powderly JD, Koeppen H, Hodi FS, Sosman JA, Gettinger SN, Desai R, Tabernero J, Soria J-C, Hamid O, Fine GD, Xiao Y, Mokatrin A, et al. Biomarkers and associations with the clinical activity of PD-L1 blockade in a MPDL3280A study., 31 ed 2013. p. Abstract #3001. (9) Weiss GR, Grosh WW, Chianese-Bullock KA, Zhao Y, Liu H, Slingluff CL, Jr., Marincola FM, Wang E. Molecular insights on the peripheral and intratumoral effects of systemic high-dose rIL-2 (aldesleukin) administration for the treatment of metastatic melanoma. Clin Cancer Res 2011 Dec 1;17(23):7440-50. (10) Gough M, Crittenden M, Thanarajasingam U, Sanchez-Perez L, Thompson J, Jevremovic D, Vile R. Gene therapy to manipulate effector T cell trafficking to tumors for immunotherapy. J Immunol 2005 May 1;174(9):5766-73. (11) Dengel LT, Norrod AG, Gregory BL, Clancy-Thompson E, Burdick MD, Strieter RM, Slingluff CL, Jr., Mullins DW. Interferons induce CXCR3-cognate chemokine production by human metastatic melanoma. J Immunother 2010 Nov;33(9):965-74. (12) Wu R, Forget MA, Chacon J, Bernatchez C, Haymaker C, Chen JQ, Hwu P, Radvanyi LG. Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J 2012 Mar;18(2):160-75. (13) Erdag G, Schaefer JT, Smolkin ME, Deacon DH, Shea SM, Dengel LT, Patterson JW, Slingluff CL, Jr. Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma. Cancer Res 2012 Mar 1;72(5):1070-80.


Page 19 of 29

MAULDIN et al.



(14) Hong M, Puaux AL, Huang C, Loumagne L, Tow C, Mackay C, Kato M, Prevost-Blondel A, Avril MF, Nardin A, Abastado JP. Chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring T-cell infiltration and tumor control. Cancer Res 2011 Nov 15;71(22):6997-7009. (15) Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, McKee M, Gajewski TF. Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res 2009 Apr 1;69(7):3077-85. (16) Tanese K, Grimm EA, Ekmekcioglu S. The role of melanoma tumor-derived nitric oxide in the tumor inflammatory microenvironment: its impact on the chemokine expression profile, including suppression of CXCL10. Int J Cancer 2012 Aug 15;131(4):891-901. (17) Muthuswamy R, Berk E, Junecko BF, Zeh HJ, Zureikat AH, Normolle D, Luong TM, Reinhart TA, Bartlett DL, Kalinski P. NF-kappaB hyperactivation in tumor tissues allows tumor-selective reprogramming of the chemokine microenvironment to enhance the recruitment of cytolytic T effector cells. Cancer Res 2012 Aug 1;72(15):3735-43. (18) Peng W, Liu C, Xu C, Lou Y, Chen J, Yang Y, Yagita H, Overwijk WW, Lizee G, Radvanyi L, Hwu P. PD-1 blockade enhances T-cell migration to tumors by elevating IFN-gamma inducible chemokines. Cancer Res 2012 Oct 15;72(20):5209-18. (19) Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD. IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 2002 Apr 1;168(7):3195-204. (20) Tannenbaum CS, Tubbs R, Armstrong D, Finke JH, Bukowski RM, Hamilton TA. The CXC chemokines IP-10 and Mig are necessary for IL-12-mediated regression of the mouse RENCA tumor. J Immunol 1998 Jul 15;161(2):927-32. (21) Kang JY, Lee JO. Structural biology of the Toll-like receptor family. Annu Rev Biochem 2011 Jun 7;80:91741. (22) Schroder K, Sweet MJ, Hume DA. Signal integration between IFNgamma and TLR signalling pathways in macrophages. Immunobiology 2006;211(6-8):511-24. (23) Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer 2010 Aug 15;127(4):759-67. (24) Nakao Y, Funami K, Kikkawa S, Taniguchi M, Nishiguchi M, Fukumori Y, Seya T, Matsumoto M. Surfaceexpressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells. J Immunol 2005 Feb 1;174(3):1566-73. (25) Ochoa MT, Legaspi AJ, Hatziris Z, Godowski PJ, Modlin RL, Sieling PA. Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue. Immunology 2003 Jan;108(1):10-5. (26) Crittenden M, Gough M, Chester J, Kottke T, Thompson J, Ruchatz A, Clackson T, Cosset FL, Chong H, Diaz RM, Harrington K, Alvarez VL, et al. Pharmacologically regulated production of targeted retrovirus from T cells for systemic antitumor gene therapy. Cancer Res 2003 Jun 15;63(12):3173-80. (27) Goto Y, Arigami T, Kitago M, Nguyen SL, Narita N, Ferrone S, Morton DL, Irie RF, Hoon DS. Activation of Toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors. Mol Cancer Ther 2008 Nov;7(11):3642-53. 19 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


MAULDIN et al.

Page 20 of 29


(28) Saint-Jean M, Knol AC, Nguyen JM, Khammari A, Dreno B. TLR expression in human melanoma cells. Eur J Dermatol 2011 Nov;21(6):899-905. (29) Yu N, Zhang S, Zuo F, Kang K, Guan M, Xiang L. Cultured human melanocytes express functional toll-like receptors 2-4, 7 and 9. J Dermatol Sci 2009 Nov;56(2):113-20. (30) Makela SM, Strengell M, Pietila TE, Osterlund P, Julkunen I. Multiple signaling pathways contribute to synergistic TLR ligand-dependent cytokine gene expression in human monocyte-derived macrophages and dendritic cells. J Leukoc Biol 2009 Apr;85(4):664-72. (31) Barksby HE, Nile CJ, Jaedicke KM, Taylor JJ, Preshaw PM. Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide. Clin Exp Immunol 2009 Jun;156(3):479-87. (32) Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004 Oct;5(10):987-95. (33) Roth SJ, Carr MW, Springer TA. C-C chemokines, but not the C-X-C chemokines interleukin-8 and interferon-gamma inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur J Immunol 1995 Dec;25(12):3482-8. (34) Franciszkiewicz K, Boissonnas A, Boutet M, Combadiere C, Mami-Chouaib F. Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response. Cancer Res 2012 Dec 15;72(24):6325-32. (35) Schmidt J, Welsch T, Jager D, Muhlradt PF, Buchler MW, Marten A. Intratumoural injection of the tolllike receptor-2/6 agonist 'macrophage-activating lipopeptide-2' in patients with pancreatic carcinoma: a phase I/II trial. Br J Cancer 2007 Sep 3;97(5):598-604. (36) Niebuhr M, Muhlradt PF, Wittmann M, Kapp A, Werfel T. Intracutaneous injection of the macrophageactivating lipopeptide-2 (MALP-2) which accelerates wound healing in mice--a phase I trial in 12 patients. Exp Dermatol 2008 Dec;17(12):1052-6. (37) Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012 Jun 28;366(26):2443-54.


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Figure Legends Figure 1. Melanoma cells express several TLRs, but TLR stimulation does not impact CCL2, CCL4, CCL5, CXCL9 and CXCL12 chemokine production from melanoma. A.

Relative expression levels of TLR transcripts represented as normalized hybridization

intensity data. B. Relative fold changes in gene expression for the indicated chemokines, TLR stimulated cells were compared to unstimulated cells (mean +SD, pooled data from melanoma cell lines VMM1, DM13, DM93 and DM122).

Data in A-B are from a single array.

C.

Representative expression of TLRs expressed by melanoma cell lines and PBMC (leukopak); graphed data are the MFI of TLR expression assessed by flow cytometric analysis.

D.

Melanoma cells were analyzed by flow cytometry for chemokine production after overnight stimulation with the indicated TLR agonists.

Graph of the percentage of melanoma cells

expressing chemokines CCL2-5, CXCL9 or CXCL12 after stimulation with the indicated TLR agonists or no treatment. Data shown are pooled from 4 melanoma cell lines VMM1, DM13, DM93 and DM122 and represent the mean +SD for the percent of melanoma cells that expressed the indicated chemokine. Data are from 3 or more independent experiments for each cell line. Figure 2.

Melanoma cells upregulate CXCL10 production in response to TLR2/6

agonists and IFNγγ treatment.

VMM1, DM13, DM93 and DM122 melanoma cells were

analyzed by for CXCL10 production after overnight stimulation with TLR agonists LPS, MALP2, and FSL-1 individually, or in combination with IFNγ (+IFNγ); as controls untreated, IFNγ treated and PMA and Ionomycin treated cells were also assessed. A. Bar graphs for each cell line represent the mean percentage of melanoma cells (+/- SD) producing CXCL10 after stimulation with the indicated TLR agonists. B. Graph of the percentage of melanoma cells expressing CXCL10 (mean of the 4 melanoma cell lines +SD) assessed by flow cytometry. C. 21 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.


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Representative flow cytometric histogram overlays demonstrating the MFI for CXCL10. Data are from 3 independent experiments per cell line. Figure 3. Melanoma cells upregulate CXCL10 after treatment with TLR2/6 agonists and IFNγγ. Melanoma cells were analyzed for CXCL10 production after overnight stimulation with MALP-2, or FSL-1 individually, or in combination with IFNγ.

A. Representative image of

immunohistochemistry staining for CXCL10 in stimulated DM13 (top) and VMM1 (bottom) cell lines; Scale bars represent 100µm. Data are representative of 2 experiments. B. ELISA assay to detect CXCL10 from supernatants of stimulated melanoma cell lines (n = 10) and one patient melanoma specimen. Data are from 2-3 independent experiments per cell line. C. TLR2/6 agonists dose response assay. DM13 and VMM1 melanoma cell lines were treated with MALP-2 (left) or FSL-1 (right) and IFNγ; TLR2/6 agonists were tested over a broad range of concentrations, as indicated. Graph shows the percent of CXCL10 producing cells (mean +/- SD of the 2 cell lines), as assessed by flow cytometry. Data are from 2 independent experiments per cell line. Figure 4.

TLR2 and TLR6 are broadly expressed on melanoma.

TLR2 and TLR6

expression was assessed on 21 melanoma cell lines by flow cytometry. A. Bar graphs depict data of the percent of melanoma cells expressing TLR2 and TLR6 for 21 assayed melanoma cell lines. B-C. The MFI for TLR2 (B) and TLR6 (C) for melanoma cell lines, Ramos cells and PBMC cells (leukopak) are graphed. Data are representative of at least 2 experiments. Figure 5. Melanoma cells upregulate CXCL10 gene expression after TLR2/6 agonist and IFNγγ treatment. Melanoma cells were analyzed by gene expression profiling after overnight stimulation with MALP-2, or FSL-1 individually, or in combination with IFNγ. A. Graph of CXCL10 gene expression for VMM1, DM13, DM93 and VMM39 melanoma cells lines; relative 22 John Wiley & Sons, Inc. This article is protected by copyright. All rights reserved.

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to untreated cells. Data are from a single gene array.

B.

Representative image of

immunohistochemistry staining for CXCL9 in DM13 (top) and VMM1 (bottom) cells; Scale bars represent 100µm. C. ELISA assay to detect CCL22 from supernatants of stimulated melanoma cell lines (n = 10). D. Graph of the percentage of cell apoptosis (mean of 9 cell lines +SD), as assessed by Caspase3/7 and viability staining, and flow cytometry. E. Graph of the percentage of divided melanoma cells (mean of 9 cell lines +SD) assessed by CFSE staining and flow cytometry 24 hours (left panel) and 48 hours (right panel) post treatment. Data are from 2 or more independent experiments. Figure 6. Patient melanoma cells upregulate CXCL10 production after treatment with TLR2/6 agonists and IFNγ. Cells from resected human melanoma metastases (n = 12) were analyzed for CXCL10 production after stimulation with MALP-2 or FSL-1 +/-IFNγ by flow cytometry, untreated cells (UN) and IFNγ treated cells were also evaluated. Cell subsets were defined as:

melanoma (CD45negS100+), and B-cell (CD20+).

A.

Representative flow

cytometric analysis showing the percent of CXCL10+ melanoma cells (top panel), and MFI for CXCL10 expression of melanoma cells (bottom panel), from patient four’s tumor.

B-C.

Graph of the percent of CXCL10+ cells from melanoma cells (B), and B-cells (C) patient 4 not evaluable for B-cells, in patient tumors (n = 12); treatment conditions are indicated.



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Human B cells produce chemokine CXCL10 in the presence of Mycobacterium tuberculosis specific T cells.

Human CD141+ dendritic cells induce CD4+ T cells to produce type 2 cytokines.

Kappa- and delta-opioid agonists synergize to produce potent analgesia.

Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα.

Cytokine-stimulated human dermal microvascular endothelial cells produce interleukin 6--inhibition by hydrocortisone, dexamethasone, and calcitriol.

Melanocytes are more responsive to IFN-γ and produce higher amounts of kynurenine than melanoma cells.

Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.

Filarial excretory-secretory products induce human monocytes to produce lymphangiogenic mediators.

Interleukins 1, 4 and 6 induce the colony formation of human bone marrow B lineage cells.

Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells.

IL-12-polarized Th1 cells produce GM-CSF and induce EAE independent of IL-23.

Splenic stromal cells from aged mice produce higher levels of IL-6 compared to young mice.

[The ability of Agrobacterium to induce tumors and to produce phage PS8].

Endogenous gene and amplifiable cDNA construct both produce unstable t-PA mRNA in Bowes melanoma cells.

Bioactive proanthocyanidins inhibit growth and induce apoptosis in human melanoma cells by decreasing the accumulation of β-catenin.

CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells.

Extracts of centipede Scolopendra subspinipes mutilans induce cell cycle arrest and apoptosis in A375 human melanoma cells.

Synergistic up-regulation of CXCL10 by virus and IFN γ in human airway epithelial cells.

Effect of JAK Inhibitors on Release of CXCL9, CXCL10 and CXCL11 from Human Airway Epithelial Cells.

LPS-stimulated bovine aortic endothelial cells produce IL-1 and IL-6 like activities.

Various peroxisome proliferator-activated receptor (PPAR)-γ agonists differently induce differentiation of cultured human keratinocytes.

B cells produce less IL-10, IL-6 and TNF-α in myasthenia gravis.

Leukemia cells induce changes in human bone marrow stromal cells.

or -6.