Abstract
Chronic lymphocytic leukemia (CLL) patients progressively develop an immunosuppressive state. CLL patients have more plasma IL-10, an anti-inflammatory cytokine, than healthy controls. In vitro human CLL cells produce IL-10 in response to BCR cross-linking. We used the transgenic Eμ–T cell leukemia oncogene-1 (TCL1) mouse CLL model to study the role of IL-10 in CLL associated immunosuppression. Eμ-TCL mice spontaneously develop CLL because of a B cell–specific expression of the oncogene, TCL1. Eμ-TCL1 mouse CLL cells constitutively produce IL-10, which is further enhanced by BCR cross-linking, CLL-derived IL-10 did not directly affect survival of murine or human CLL cells in vitro. We tested the hypothesis that the CLL-derived IL-10 has a critical role in CLL disease in part by suppressing the host immune response to the CLL cells. In IL-10R−/− mice, wherein the host immune cells are unresponsive to IL-10–mediated suppressive effects, there was a significant reduction in CLL cell growth compared with wild type mice. IL-10 reduced the generation of effector CD4 and CD8 T cells. We also found that activation of BCR signaling regulated the production of IL-10 by both murine and human CLL cells. We identified the transcription factor, Sp1, as a novel regulator of IL-10 production by CLL cells and that it is regulated by BCR signaling via the Syk/MAPK pathway. Our results suggest that incorporation of IL-10 blocking agents may enhance current therapeutic regimens for CLL by potentiating host antitumor immune response.
This article is featured in In This Issue, p.3869
Introduction
Chronic lymphocytic leukemia (CLL), the most common human leukemia in the Western world, is defined by accumulation of malignant CD5+CD19+ B lymphocytes in the peripheral blood (PB), bone marrow, and secondary lymphoid organs (1). CLL cells express BCR as well as CD5, a molecule characteristic of T lymphocytes, similar to the B-1 B cell subset (2, 3). Systemic immunosuppression is associated with a more aggressive CLL disease resulting in infections and secondary cancers such as melanoma, head and neck cancer, and lymphoblastic leukemia (4–6).
Many mechanisms of immunosuppression have been described in CLL. Interestingly, like normal B-1 cells, CLL cells themselves constitutively produce IL-10, an immunosuppressive factor (7, 8). IL-10, a Th2-type cytokine, was identified by its ability to suppress macrophage cytokine production (9, 10). In addition, IL-10 regulates T cell responses indirectly through its effects on APCs, inhibiting their MHC class II expression and limiting their production of proinflammatory mediators (11). IL-10 can also directly inhibit T cell proliferation and production of cytokines, such as IL-2 and IFN-γ (12, 13). IL-10 from CLL cells was shown to inhibit macrophage TNF production, but its importance for CLL disease was not tested (14). In this study, we investigated the direct effects IL-10 has on T cell responses in CLL using the Eμ–T cell leukemia oncogene-1 (TCL1) mice. These mice serve as an excellent model of CLL as they overexpress TCL1 under the control of the B cell–specific μ-enhancer and the IGHV promoter leading to a progressive CLL-like disease at 8–11 mo of age (15). In this study, we showed that IL-10 failed to affect the survival of CLL cells in vitro but that it played an important role in inhibiting T cell antitumor immunity in vivo. This could play a significant role in affecting antitumor responses to current immunotherapy strategies. For example, the efficacy of chimeric Ag receptor (CAR) T cell therapies is much less (26% complete response) for CLL than for other B cell leukemias (78% complete response) (16), which could be owing to the inhibition of CD19-specific CAR T cells by IL-10. Another study implicated IL-10 as one of the adaptive resistance mechanisms that undermine the efficacy of Abs to programmed cell death protein-1 (PD-1) monotherapies in ovarian cancer (17). Indeed, the combination of PD-1 blockade and IL-10 neutralization improved survival and delayed tumor growth in tumor bearing mice, which was in part a result of increased T and B cell responses (17).
Despite several studies on molecular basis of IL-10 expression by various cell types (18), the mechanisms involved in IL-10 production by CLL cells remain understudied. BCR-derived signals are crucial for CLL survival. In this study, we found that BCR-dependent constitutive activation of Src, Btk, and Syk family kinases is required for constitutive IL-10 production by murine and human CLL cells. In addition, our results indicate a novel role for the transcription factor Sp1 in BCR-dependent production of IL-10 by CLL cells.
Materials and Methods
Mice and cells
C57BL/6J, B6.129S2-IL10rbtm1Agt/J (IL-10R−/−), B6.129S7-Rag1tm1Mom/J (B6.Rag1−/−), and NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ (NSG) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Eμ-TCL1 mice on C57/BL6J background were provided by Dr. J. Byrd (The Ohio State University, Columbus, OH) and bred in house. The University of Kentucky’s Institutional Animal Care and Use Committee protocol number for this study is 2011-0904. Cohorts of Eμ-TCL1 mice were maintained and monitored regularly for CLL cells in blood by flow cytometry. Mice were bled by submandibular bleeding using a lancet. Blood was collected in K2 EDTA Microtainer tubes (no. 365974; BD Biosciences, San Diego, CA). Most Eμ-TCL1 mice developed CLL between 6 and 9 mo of age (at least 30% CD5+CD19+ B cells in the blood). Mice were euthanized when CLL cells were 80–90% or when their body condition score was ≤2 (19). In addition to using CLL cells from primary Eμ-TCL1 mice, we adoptively transfer CLL cells from spleens of euthanized Eμ-TCL1 mice with 80–90% CLL into syngeneic mice, which leads to a reliable and consistent development of the disease in the recipient mice within weeks of injection. Actual kinetics of disease development in the adoptive transfer model vary depending on the individual Eμ-TCL1 mouse donor of the CLL cells used for the transfer. Hence, in any given adoptive transfer experiment, CLL cells from a single Eμ-TCL1 donor mouse were used. Spleens from mice with CLL lost their follicular architecture and had proliferative centers shown by histology as suggested by Bichi et al. (15). Normal splenic cells were isolated from spleens of C57BL/6J mice and CD19+
Human samples
Patients with CLL were recruited from the University of Kentucky Markey Cancer Center (Table I). All patients gave informed consent according to protocols approved by the University of Kentucky Institutional Review Board. For healthy controls, leukopak units were obtained from the Kentucky blood bank. Normal human plasma samples were provided by the Sanders Brown Center on Aging at the University of Kentucky. B cells were enriched using Ficoll–Paque PLUS density gradients (GE Healthcare, Pittsburgh, PA) and the Miltenyi Biotec Microbeads (San Diego, CA) as described previously by Ramsay et al. (20). CLL preparations were always >90% CD5+CD19+ B cells.
Reagents
Phorbol 12-myristate 13-acetate (PMA), ionomycin, LPS, and Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Millipore Sigma-Aldrich (St. Louis, MO). Purified anti-mouse IL-10 and anti-mouse IL-10R Abs were from BD Biosciences Pharmingen (San Diego, CA). AffiniPure F(ab′)2 fragment goat anti-mouse IgM and AffiniPure F(ab′)2 fragment goat anti-human IgM were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Magnetic beads to isolate mouse and human B cells and mouse CD8a and CD4+
CLL and T cell adoptive transfer
Adoptive transfers were performed by transferring 1–10 × 106 Eμ-TCL1 splenic cells into C57BL/6J, IL-10R−/−, BL/6.Rag−/−, or NSG mice (without any preconditioning) i.v. via retro-orbital injections. CLL disease was monitored by periodic submandibular bleeding and CD5+CD19+ cells were quantified by flow cytometry (21). For T cell adoptive transfer experiments, CD8+ T cells were purified as noted above. Refer to Supplemental Fig. 1 for additional experimental schematics.
ELISA
For cytokine analysis, normal B-1, murine CLL, and human CLL cells were cultured in triplicate (2 × 106
In vitro cell survival, proliferation, and differentiation assays
Eμ-TCL1 CD5+CD19+ splenic cells were stimulated with LPS in the presence or absence of anti–IL-10R or anti–IL-10 Abs for 48 h, and cell survival was determined by MTT assay (22). For T cell proliferation and differentiation studies, CD8+ cells were purified using Miltenyi Microbeads according to the methods described by Keir et al. (23) and cultured (1–2 × 105 cells) with irradiated (25 Gy) Eμ-TCL1 cells (2 × 105 cells) for 72 h. The cultures were pulsed with 1 μCi of tritiated thymidine for 4 h. The incorporated radioactivity was measured using a TopCount microplate scintillation counter from Packard/PerkinElmer (Waltham, MA). For cytokine analysis, cells were cultured in triplicate (2 × 105 cells) for 24 h with or without various treatments. Secreted IL-10 and IFN-γ levels were quantified using ELISA.
Immunofluorescence analysis and cell sorting
Single-cell lymphocyte suspensions from mouse tissues were prepared by pressing tissues through a 40-μm strainer using the plunger end of a syringe in HBSS (Millipore Sigma-Aldrich). Tibiae and femora were flushed with a 26G syringe in HBSS. Peritoneal cells were obtained by peritoneal lavage with HBSS. Cells were resuspended in RPMI 1640 medium (Corning, New York, NY) supplemented with 10% FBS. For multicolor immunofluorescence analysis, single-cell suspensions of mononuclear cells were first incubated with normal rat IgG (10 μg/106 cells) at 4°C for 15 min to block Fcγ receptors. The cells were then labeled with fluorochrome-conjugated anti-mouse Abs for 30 min on ice. Stained cells were analyzed using BD LSR II flow cytometer and CellQuest Pro software. Anti-CD19, anti-CD11b, and anti-CD5 were used to identify and sort B-1a (CD19+CD5+CD11b+), B-1b (CD19+CD5−CD11b+), and B-2 (CD19+CD5−CD11b−) cells from the peritoneum of C57BL/6J mice using iCyt Synergy sorter system from (Sony Biotechnology, San Jose, CA). Anti-CD19 and anti-CD90.2 Abs were used to identify and sort T cells (CD19−CD90.2+
Immunoblotting
Cells were lysed in Cell Signaling lysis buffer containing protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE and processed for Western blotting. The blots were developed with HyGLO chemiluminescence reagent and exposed to HyBlot CL autoradiography film (Denville Scientific, Holliston, MA). Band densitometry analysis was performed using the National Institutes of Health ImageJ program. Phospho-protein levels were normalized to total protein. Total protein levels were normalized to either GAPDH or β-actin expression.
Short hairpin RNA sequence and cell infection
6 cells/ml in a six-well assay plate, infected with 25–50 μl of concentrated shRNA lentiviral supernatants with the addition of polybrene (10 μg/ml), and centrifuged for 90 min at 2800 rpm and 10°C. Virus and cells were incubated for 24 h at 37°C and then fresh media was replenished. Puromycin antibiotic selection began at day 3 and remained in culture for the entire period of experimentation after proper titration. Lyn shRNA Clone number TRCN0000010101 is represented in this paper.
Quantitative real-time PCR
Total RNA was isolated from Eμ-TCL1 cells using TRI reagent (Sigma-Aldrich). RNA was used to make cDNA using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD) according to the manufacturer’s protocol. iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) was used to perform the RT-PCR reaction. RT-PCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). Primer sequences used were as follows: IL-10 forward, 5′-ACTGGCATGAGGATCAGCAG-3′; IL-10 reverse, 5′-AGAAATCGATGACAGCGCCT-3′; SP1 forward, 5′-TGCCACCATGAGCGACCAAGATCA-3′; SP1 reverse, 5′-TGCTGCTGCTTCGAGTCTGAGAAA-3′; 18S forward, 5′-CGCCGCTAGAGGTGAAATTCT-3′; and 18S reverse, 5′-CGAACCTCCGACTTTCGTTCT-3′. The 18S-specific primers were used for loading control. All primers were obtained from Integrated DNA Technologies (Coralville, IA).
Chromatin immunoprecipitation for quantitative chromatin immunoprecipitation analysis
Chromatin immunoprecipitation (ChIP) analysis was performed using Cell Signaling Technology’s SimpleChIP Enzymatic Chromatin IP kit following manufacturer’s protocol. Proteins linked to the DNA were immunoprecipitated with anti-Sp1 Ab. For quantitative ChIP, RT-PCR was performed on the eluted DNA using SYBR Green Reaction Mix. Specific primers used to amplify a region of the IL-10 promoter described to contain an Sp1 binding site (24) were as follows; forward, 5′-GCAGAAGTTCATTCCGACCA-3′; reverse, 5′-GGCTCCTCCTCCCTCTTCTA-3′.
Human CLL IGHV sequencing
For each patient, total RNA from PBMCs was reverse transcribed, and Ig sequences were amplified from cDNA with IGH family–specific forward primers and isotype-specific reverse primers, as previously described (25, 26). The resulting PCR products were unidirectionally Sanger sequenced and aligned via the online IgBLAST tool. Sequence reads covered the entire variable domain and included the first 38–124 nt of the C region. For each patient, only one productive sequence was obtained and thus was termed the major clone. Patients with ≥98% sequence homology to the germline variable domain were categorized as unmutated, as others have done previously (25–28).
Tissue histology and disease scoring
Colons were dissected from euthanized mice and fixed in 10%-buffered formalin (no. SF93-4; Fisher Scientific, Fair Lawn, NJ), embedded in paraffin, and stained with H&E by the University of Kentucky histology services. Histological scoring was based on the method previously described (29).
Statistical analysis
GraphPad Prism 7 was used for statistical analyses. Statistical significance of differences between groups was evaluated by Student t test, and p values <0.05 were considered significant. Normality testing was conducted and, wherever appropriate, nonparametric tests were employed.
Results
Blocking of IL-10 signaling does not affect in vitro survival of the Eμ-TCL1 CLL cells
Eμ-TCL1 CD5+CD19+ CLL cells secrete IL-10 constitutively, similar to the peritoneal CD5+CD11b+ B-1 cells, although the amount produced is variable (Fig. 1A) (30). This constitutive production is a property of CLL B cells because highly purified CD19+ CLL B cells and unpurified Eμ-TCL1 splenic cells from mice with de novo disease or from adoptive transfer models produced similar amounts of IL-10, whereas normal spleen cells do not produce IL-10 (Fig. 1A). Neutralization of CLL-induced IL-10 using anti–IL-10 Abs or anti–IL-10 receptor (IL-10R) Abs did not affect the survival of Eμ-TCL1 cells with or without TLR4 stimulation (∼1000 pg/ml IL-10 produced upon LPS treatment) (Fig. 1B), in agreement with our previous observations (30). Flow cytometry analysis confirmed the presence of IL-10R on the, surface of Eμ-TCL1 splenic CLL cells (Fig. 1C, left), and it appeared to be functional because treatment of CLL cells with exogenous IL-10 induced phosphorylation of STAT3 transcription factor, a key-signaling molecule downstream of IL-10R (Fig. 1C, right). Thus, Eμ-TCL1 cells appear to be distinct from normal B-1 cells in not responding to IL-10–mediated suppressive effects in vitro.
Constitutive IL-10 production by CLL cells and its role in CLL cell survival. (A) CLL cells were harvested from spleens of Eμ-TCL1 (n = 23) or adoptive transfer mice (n = 9). B-1a cells were isolated from the peritoneal cavity (n = 3) and normal CD19+ cells were isolated from spleens of C57BL/6J mice (n = 5). Cells were cultured for 24 h and IL-10 was measured in the supernatants by ELISA. Bars represent mean ± SE. (B) Splenic Eμ-TCL1 cells were cultured with αIL-10 or αIL-10R Abs ± LPS (5 μg/ml) for 48 h. Survival was measured by MTT. Values represent mean ± SD of triplicate cultures. (C) Flow cytometric analysis of IL-10R expression by Eμ-TCL1 cells (left) and protein lysates of Eμ-TCL1 CLL cells stimulated with exogenous IL-10 were analyzed for STAT3 activation by immunoblot (right). β-Actin is used for loading control. *p < 0.05.
CLL cell growth is reduced in IL-10R−/− mice
The lack of direct effects of CLL-derived IL-10 on their survival led us to hypothesize that IL-10 may contribute to immunosuppression in CLL by affecting adaptive immune responses to CLL. First, we evaluated if there is an adaptive tumor immune response against CLL in the Eμ-TCL1 mouse model, using an adoptive transfer model by injecting CLL cells from the de novo Eμ-TCL1 mouse into syngeneic C57BL/6J wild type (WT) or immunodeficient mice (NSG or B6.Rag1−/−). NSG mice lack mature B and T cells as well as NK cells, whereas B6.Rag1−/− mice do not have an adaptive immune response because of a total lack of B and T cells. CLL cells in B6.Rag1−/− mice were detectable at an earlier time point than WT mice and had to be euthanized at day 17 because of poor body condition, with an average of 75% CD5+CD19+ cells in PB (Fig. 2A), whereas WT mice had only ∼10% CD5+CD19+ cells in PB at this time (Fig. 2A). Similarly, in NSG mice, CLL cells grew at a faster rate than WT mice (Fig. 2B). Overall, C57BL/6J mice survived longer than NSG mice (Fig. 2C). Despite the fact that NSG mice were on a NOD background, no significant difference was observed between CLL growth kinetics in NSG and B6.Rag1−/− mice (Fig. 2D).
Lack of B, T, and NK cells leads to an acceleration of CLL growth kinetics. (A–D) Eμ-TCL1 cells (4 × 106) were adoptively transferred into WT, B6.Rag1−/−, or NSG mice by retro-orbital injection. Leukemic status is determined by weekly submandibular bleeding. Graph shows the percentage of CD5+CD19+ cells in the PB at indicated time points (A, B, and D). Kaplan–Meier plot represents the survival of C57BL/6J and NSG mice during the course of the experiment (n = 6) (C). Similar results were obtained in another experiment. *p < 0.05, ***p < 0.001, determined by Student t test for (A), (B), and (D) and by log-rank (Mantel–Cox) test for (C).
The difference in CLL growth kinetics between immunodeficient and C57BL/6J mice could be due to adaptive immunity and/or other differences in the microenvironment. Hence, we investigated if murine CLL-derived IL-10 could inhibit anti-CLL immune responses. CLL cells were injected into WT and C57BL/6.IL-10R−/− mice. We hypothesized that the lack of IL-10R would eliminate any inhibitory effects of IL-10 in the CLL microenvironment of the recipient mice including its effects on T cells, allowing more effective anti-CLL immune response and, hence, a delay in CLL development. Accordingly, we found that CLL cells grew at a slower rate in IL-10R−/− mice than in WT mice (Fig. 3A). After euthanization, CLL tumor burden in spleen, bone marrow, and peritoneal cavity was higher in WT mice than in IL-10R−/− mice (Fig. 3B, Supplemental Fig. 2A, 2B). There was no significant difference in plasma IL-10 levels between WT and IL-10R−/− recipient mice at the end of the experiment (Supplemental Fig. 2C). We used 3-wk-old mice as recipients, because IL-10R−/− mice are known to develop colitis by 12 wk of age. To be certain that the death of recipient mice in this experiment is not due to colitis, we performed colon histology. Colon sections from both WT and IL-10R−/− recipients at the time of euthanization showed no significant difference in inflammation by histopathology (Supplemental Fig. 2D).
CLL cell growth is reduced and T cell function is enhanced in IL-10R−/− mice. (A and B) Eμ-TCL1 CLL cells (4 × 106) were i.v. transferred into WT and IL-10R−/− mice (n = 5). Leukemia status was monitored by weekly bleeding and is shown as %CD5+CD19+ cells after gating on CD45+ cells in PB (A), and tumor burden was expressed as total number of CD5+CD19+ cells per spleen (B). Values represent arithmetic mean ± SD (n = 5). (C and D) Magnetic bead–purified CD8+ cells (105) from WT and IL-10R−/− mice 20 d post–CLL injection were cultured with irradiated CLL cells for 72 h. Proliferation, measured by [3H] incorporation (C), and IFN-γ secretion by CD8+ T cells (D) are shown. Values represent mean ± SD of triplicate cultures. (E) Intracellular staining of IFN-γ was performed after 4 h of stimulation with PMA and ionomycin. Levels of cytoplasmic IFN-γ+ CD8+ (left) and CD4+ (right) T cells from WT and IL-10R−/− mice at different times post–CLL injection are shown. Values represent arithmetic mean ± SE (n = 4). *p < 0.05, comparing WT and IL-10R−/− groups.
Decrease in T cell function in WT compared with IL-10R−/− mice injected with CLL
To determine whether the reduced CLL growth in IL-10R−/− mice is because of the inhibitory effects of IL-10 on host anti-CLL immune response, we investigated the function of T cells in the adoptive transfer recipients. In agreement with such a notion, upon restimulation with CLL cells, CD8+ T cells from IL-10R−/− recipients proliferated better than CD8+ T cells from WT mice at day 20 postinjection (Fig. 3C). Similar results were found at other time points (data not shown). Also, CD8+ T cells isolated from IL-10R−/− recipients secreted more IFN-γ than CD8+ T cells from WT recipients (Fig. 3D). In addition, intracellular staining of IFN-γ was performed after 4 h stimulation with PMA and ionomycin. Cytoplasmic IFN-γ+ CD4+ and CD8+ T cells were higher in IL-10R−/− than WT mice injected with CLL cells (Fig. 3E). These data suggest a role for IL-10 in inhibiting T cell responses against Eμ-TCL1 CLL cells in vivo.
IL-10R−/− T cells controlled CLL growth better than WT T cells
To determine whether the functional differences between WT and IL-10R−/− T cells generated after CLL injection affects their ability to control CLL growth in vivo, we performed adoptive transfer experiments using NSG mice as recipients (Supplemental Fig. 1A). Overall, injection of primed WT or IL-10R−/− T cells along with CLL cells delayed onset of disease in PB by a significant amount of time (Supplemental Fig. 3A). At day 65, when recipients were euthanized, 60% of WT T cell recipients, but none of the IL-10R−/− T cell recipients, developed CLL disease (Supplemental Fig. 3B). Thus, IL-10R−/− T cells that cannot respond to IL-10 were more effective in controlling CLL disease than IL-10–responsive WT T cells. To determine whether the anti-CLL response of T cells is due to CD8+ T cells, we injected purified CD8+ T cells from WT and IL-10R−/− mice primed with CLL along with CLL cells at a T cell/CLL cell ratio of 1:32 into NSG mice (Supplemental Fig. 1B). Interestingly, CD8+ T cells were sufficient to delay CLL growth, and CD8+ T cells from IL-10R−/− mice were better at controlling disease development than CD8+ T cells from WT mice (Fig. 4A). Upon euthanization, spleens of mice receiving IL-10R−/− CD8+ T cells had fewer CLL cells than mice receiving WT CD8+ T cells; however, no significant difference was observed between groups receiving CD8+ T cells from IL-10R−/− mice and PBS control mice (Fig. 4B). At day 30 postinjection, 100% of the mice that received no T cells or WT CD8+ T cells but only 16.7% of IL-10R−/− CD8 T cell recipients developed disease (Fig. 4C).
Adoptive transfer of CLL-primed CD8+ T cells effectively delayed CLL growth. (A) Purified CD8+ cells from WT and IL-10R−/− mice primed with CLL cells 14 d before they were injected into NSG mice along with CLL cells at a T cell/CLL ratio of 1:32. Leukemic status was monitored by CD5+CD19+ cell accumulation in PB. Values represent mean ± SE (n = 6). (B) CD5+CD19+ cell numbers in the spleens of recipients at the time of euthanization. (C) Percentage of mice that developed CLL from each group. *p < 0.05, **p < 0.01, ***p < 0.001, comparing disease in recipients of WT and IL-10R−/− CD8+ T cells.
Novel role of BCR signaling and Sp1 in constitutive and induced IL-10 production by CLL cells
Establishing a role for IL-10 in CLL growth led us to investigate the possible mechanisms by which IL-10 is produced by CLL cells. Previously, we reported our preliminary studies about the novel role of BCR signaling in IL-10 production by normal B-1 and malignant Eμ-TCL1 CLL cells (30). Inhibition of Src, Btk, or Syk family kinases that are essential for signal transduction via BCR reduced both constitutive and anti-IgM–induced IL-10 production by Eμ-TCL1 cells in a dose-dependent manner (IC50 ≈ 0.3 μM) (Fig. 5A, 5B). BCR cross-linking increases IL-10 mRNA levels, which were reduced by the Syk inhibitor (Fig. 5C), suggesting that IL-10 production is controlled at the transcript level. Intracellular staining for IL-10 showed that 60–80% of CLL cells were IL-10 producers, which was reduced by Syk inhibition (Supplemental Fig. 4A, 4B). MEC1, a human CLL cell line, also secretes IL-10 constitutively, which is diminished by inhibition of Src family kinase (SFK), Syk, or Btk (Fig. 6A). Moreover, BCR cross-linking in MEC1 cells increases IL-10 production, which is also diminished by the BCR inhibitors (Fig. 6B). In addition, we used a shRNA to knock down Lyn, an SFK, and one of the earliest enzymes activated with BCR cross-linking. MEC1 cells with 50% Lyn knockdown produced fewer IL-10 than control shRNA-treated cells (Fig. 6C, 6D).
The role of BCR signaling in IL-10 production by Eμ-TCL1 CLL cells. (A and B) Eμ-TCL1 cells were cultured without (A) or with (B) αIgM ± indicated doses of dasatinib (an SFK inhibitor), Syk inhibitor IV, or Btk inhibitor (ibrutinib). IL-10 levels in supernatants were measured after 24 h (at which time the cell viability was 75–80%). Values are normalized to the no-drug control. IL-10 range for untreated Eμ-TCL1 cells is 193–349 pg/ml. (C) IL-10 mRNA levels (normalized to mouse 18S RNA) are determined by quantitative real-time PCR (qRT-PCR) in Eμ-TCL1 CLL cells treated with or without αIgM ± Syk inhibitor IV. Values represent mean ± SD of triplicates. Results are representative of three to eight experiments. *p < 0.05, comparing untreated to αIgM ± Syk inhibitor–treated cells.
The role of BCR signaling in IL-10 production by MEC1 cells. (A) MEC1 cells were cultured without (A) or with (B) αIgM ± dasatinib, Syk inhibitor IV, or Btk inhibitor. IL-10 levels in supernatants were measured after 24 h. For (B), 2 μM of indicated drugs is used. (C) Immunoblot showing a reduction in Lyn in MEC1 cells expressing Lyn-specific shRNA compared to control (Ctl) shRNA. Lyn protein values were normalized to β-actin. (D) IL-10 levels were measured in the supernatants of MEC1 cells expressing either control or Lyn shRNA. Values represent mean ± SD of triplicates. Results are representative of two to five experiments. *p < 0.05.
The importance of p38-MAPK and STAT3 was tested because they are known to be involved in IL-10 production by myeloid cells. Surprisingly, phosphorylation of the p38-MAPK or STAT3 was not affected by Syk inhibition (Fig. 7A). In contrast, phosphorylation of ERK1/2 was reduced after Syk inhibition (Fig. 7A). Similar results are obtained with ibrutinib (data not shown).
IL-10 production by Eμ-TCL1 CLL cells is dependent on ERK1/2 MAPK and the transcription factor Sp1 but not on p38MAPK or STAT3. (A) Eμ-TCL1 cells were treated with Syk inhibitor IV (5 μM) for indicated time periods. Levels of key molecules downstream of BCR signaling were quantified by immunoblotting. Band densitometry analysis was performed using the National Institutes of Health ImageJ program. Phosphoprotein levels were normalized to total protein. Results are representative of three experiments. (B) Sp1 mRNA levels were quantified by quantitative real-time PCR (qRT-PCR) after treatment of Eμ-TCL1 cells with αIgM ± Syk inhibitor IV. Fold change was normalized to the no-treatment group. Values represent mean ± SD of triplicate determinations. (C) Eμ-TCL1 cells were treated with various doses of mithramycin A for 24 h and IL-10 in the supernatant was quantified by ELISA. (D) Immunoblot analysis of IL-10 protein levels in CLL cells after treatment with mithramycin A (5 μM). (E) ChIP with anti-Sp1 or control IgG was carried out as described in the Materials and Methods. qRT-PCR was performed on the ChIP DNA product using primers specific for the consensus Sp1 binding site sequence in the IL-10 promoter. Results are calculated using the Fold Enrichment Method. *p < 0.05, ***p < 0.001, comparing untreated to αIgM ± Syk inhibitor–treated cells.
To find a possible downstream transcription factor, which is involved in BCR-induced IL-10 production, we tested the transcript levels of multiple transcription factors (SMAD4, GATA3, CREB, ATF1, and Sp1) known to be required for IL-10 transcription in various immune cells (Supplemental Fig. 4C) (18). Of these, Sp1 was the only transcription factor enhanced by BCR signaling and reduced by Syk inhibition (Fig. 7B). Mithramycin A, a well-known inhibitor of Sp1 (31), reduced IL-10 protein levels in a dose-dependent manner (Fig. 7C, 7D). Sp1 is proposed to bind and transactivate IL10 gene in macrophages and T cells (32). To further establish its role in IL-10 transcription in Eμ-TCL1 cells, we used ChIP to test if Sp1 binds to the IL-10 promoter in CLL cells. RT-PCR of the ChIP product revealed 8-fold enrichment in binding of Sp1 to IL-10 promoter over the input sample (Fig. 7E). Taken together, these results indicate that IL-10 production by murine CD5+CD19+ CLL cells is dependent on the transcription factor Sp1. Because ERK1/2 activation is regulated by BCR signaling (Fig. 7A), we hypothesized that ERK1/2 activation is the link between BCR and Sp1, based on previous studies (33). Accordingly, treatment of CLL cells with SCH772984, a specific ERK1/2 inhibitor, decreased IL-10 in a dose-dependent manner (Fig. 8A). Interestingly, Sp1 protein and transcript levels were reduced upon ERK1/2 inhibition (Fig. 8B, 8C). Also, consistent with data using Syk inhibitor, ERK1/2 inhibitor did not decrease activation of STAT3 (Fig. 8B). This suggests that IL-10 production by CLL cells is regulated by ERK1/2 dependent activation of Sp1.
Effect of ERK1/2 inhibition on Sp1 and IL-10 levels. Eμ-TCL1 CLL cells were cultured with the ERK1/2 inhibitor (SCH772984) (2 μM). (A) IL-10 levels in 24 h supernatants were measured by ELISA. Values represent mean ± SD of triplicates. (B) Levels of p-ERK1/2, total ERK1, p-STAT3, total STAT3, and Sp1 were quantified by immunoblot analysis. Results are representative of three experiments. (C) Sp1 mRNA levels were quantified by quantitative real-time PCR (qRT-PCR). Fold change was normalized to the no-treatment group. Values represent mean ± SD of triplicates ***p < 0.001, comparing groups with and without ERK1/2 inhibitor.
BCR signaling regulates IL-10 production by human CLL cells
Human CLL PBMCs (Table I) produced a significant amount of IL-10 after BCR cross-linking in comparison with normal human PBMCs with very little constitutive production (Fig. 9A). There was a significant amount of IL-10 in the plasma of CLL patients in comparison with age-matched controls (Fig. 9B), suggesting that in vivo CLL cells may be receiving activation signals via BCR. Neutralization of anti-IgM–induced IL-10 did not affect the survival of human CLL cells (Fig. 9C). Inhibition of Src, Syk family kinases, Btk, and ERK1/2 led to the complete abrogation of anti-IgM–induced IL-10 production by human CLL cells (Fig. 9D). To confirm that CLL cells were the source of IL-10, we show that B cells purified from the PBMCs of CLL patients secrete IL-10, which was also diminished by BCR inhibitors (Fig. 9D). Finally, inhibition of BCR signaling in human CLL reduced ERK1/2 activation and Sp1 and IL-10 levels (Fig. 9E).
Human CLL cells use BCR signaling for IL-10 production. (A and B) IL-10 levels in 24-h supernatants of human CLL cells ± αIgM (A) or in the plasma of human CLL patients (n = 15) and normal donors (n = 28) (B). (C) Human CLL cells were cultured with αIL-10 or αIL-10R Abs ± αIgM for 48 h. Survival of CLL cells was measured by MTT. (D) Human total CLL cells or purified CLL B cells were stimulated with αIgM ± inhibitors of Btk, Syk, SFK, or ERK1/2 for 24 h (2 μM). IL-10 levels in the supernatants were measured by ELISA. Human B cell CLL cells were purified by negative selection with MojoSort Human B cells (CD43−
E) Human CLL cells were treated with Syk inhibitor IV (2 μM). Protein levels indicated were quantified by immunoblot analysis. For (A), (C), and (D), values represent mean ± SD of triplicate cultures. *p < 0.05, ***p < 0.0001.Discussion
A variety of functional defects in T cells from patients with CLL as well as in Eμ-TCL1 mice with CLL have been reported, including reduction in activation-induced CD40L expression, immunologic synapse formation with APCs, and alteration in gene expression profiles (34, 35). In this study, for the first time, to our knowledge, we show a clear link between CLL-derived IL-10 and dysfunction of T cell responses to CLL cells. We demonstrated that T cells from IL-10R−/− mice primed with CLL proliferate and differentiate better than T cells isolated from mice with intact IL-10 signaling. Furthermore, primed T cells and purified CD8+ T cells reduced incidence and progression of the disease in immunodeficient mice injected with CLL cells. T cells from IL-10R−/− mice were better at controlling CLL growth.
IL-10 has been found to have a dual function of immune stimulation and immune suppression in different types of cancer (36), which explains the complexity of studying the effects of IL-10 on cancer. For example, previous reports have shown contradictory results regarding the requirement of IL-10 for the survival and proliferation of CLL cells. Fluckiger et al. (37) reported that exogenous IL-10 inhibited the proliferation of human CLL cells and decreased the survival of CLL cells in culture by inducing apoptosis, similar to normal B-1 cell regulation by IL-10, as we showed previously that normal B-1 cell–derived IL-10 inhibited their proliferation responses to TLR or BCR ligation (8, 30). However, Kitabayashi et al. (38) reported that IL-10 prevents apoptotic cell death of CLL cells. In our study, both mouse and human CLL cells appear to be unique in that their survival/proliferation is not suppressed directly by IL-10 in vitro but that IL-10 affects CLL growth indirectly by suppressing anti-CLL T cells.
The question then remains of why T cells in the de novo CLL disease do not control disease development. T cells isolated from CLL patients have higher expression of checkpoint molecules such as cytotoxic T lymphocyte–associated protein-4 and PD-1 (39, 40). This possibility of multiple pathways of immunosuppression in CLL raises the need for combination therapy to target multiple modulators of immune suppression. The combination of different immunotherapies is widely studied and is demonstrating tremendous promise for the treatment of diverse tumor types. In an interesting study, it was found that PD-1high tumor Ag–specific CD8+ T cells in patients with advanced melanoma upregulate IL-10R expression (41). When IL-10 directly acts on these CD8+ T cells, it limits their proliferation and survival (41). The authors found that PD-1 blockade increases the expression of IL-10R by CD8+ T cells, increasing their sensitivity to the immunosuppressive effects of endogenous IL-10 (41). However, when they used combination therapy of IL-10 and PD-1 inhibition, IL-10 blockade strengthened the effects of PD-1 blockade in expanding tumor-specific CD8+ T cells (41). Another study found that treatment of ovarian tumor–bearing mice with anti–PD-1 Ab caused an increase in IL-10 levels, which contributed to the resistance seen with anti–PD-1 monotherapy (17). When the combination of PD-1 blockade and IL-10 neutralization was used, improved survival and delayed tumor growth were seen in these mice (17).
Future studies will need to identify the CLL Ags recognized by the T cells, which could be used in developing new CAR T cell therapy for CLL. A novel category of tumor-associated T cell Ags were identified in a recent study that analyzed the landscape of naturally presented HLA class I and II ligands of primary CLL (42). Specific expression of these HLA ligands exclusively in CLL patients correlated with the frequencies of immune recognition by patient T cells (42). Interestingly, patients displaying immune responses to multiple Ags exhibited better survival than those with responses to one or fewer Ags (42). Variable efficacy of CAR T cells in CLL could be because of this suppression by CLL-derived IL-10, which can be targeted to make CAR T cells more effective.
Understanding the specific molecular events that regulate the production of IL-10 will help in designing new strategies of immune intervention. It was found that BAFF stimulation via TACI receptor–enhanced IL-10 production by leukemic B cells in CLL patients and Eμ-TCL1 mice (43). Another study demonstrated an epigenetic control of IL-10 production, in which differential IL10 gene methylation was responsible for the variability of IL-10 production by human CLL cells (44). Our studies demonstrated a novel role of BCR signaling in the constitutive production of IL-10. A novel role for Sp1 in BCR signaling–dependent IL-10 production by CLL cells is described in this study. Sp1 bound to the IL-10 promoter in CLL cells, and inhibition of Sp1 led to a decrease in IL-10 production. Sp1 activity required activation of ERK1/2, which is consistent with regulation of Sp1 by ERK1/2 in other cell types (45, 46). Our results introduce a new rationale to therapeutics targeting the ERK–Sp1 pathway.
In conclusion, we demonstrated that BCR signaling, a key mechanism for the survival of normal and CLL B cells, leads to IL-10 production by CLL B cells. The CLL-derived IL-10 suppresses anti-CLL immune response. Hence, inhibition of IL-10 production by small molecules, such as mithramycin or its analogues, with less toxicity, could be used in combination with current CLL therapies, such as BCR signaling inhibitors and combination chemotherapy as well as the new therapeutic modalities like checkpoint inhibitor or CAR T cells, to induce better anti-CLL immunity.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Dr. Greg Bauman and Jennifer Strange for help with flow cytometry and Beth Gachuki for technical support. We thank Dr. Donna Wilcock for help with normal human plasma samples.
Footnotes
This work was supported by National Institutes of Health Grant CA 165469, the Flow Cytometry and Biospecimen Procurement and Translational Pathology shared resource facilities of the Markey Cancer Center (Grant P30CA177558), and Alzheimer’s Disease Center Grant P30-AG028383 at the University of Kentucky.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- CAR
- chimeric Ag receptor
- ChIP
- chromatin immunoprecipitation
- CLL
- chronic lymphocytic leukemia
- IL-10R
- IL-10 receptor
- PB
- peripheral blood
- PD-1
- programmed cell death protein-1
- PMA
- phorbol 12-myristate 13-acetate
- SFK
- Src family kinase
- shRNA
- short hairpin RNA
- TCL1
- T cell leukemia oncogene-1
- WT
- wild type.
- Received February 20, 2018.
- Accepted April 6, 2018.
- Copyright © 2018 by The American Association of Immunologists, Inc.