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. 2012 Jul 8;62(1):87–99. doi: 10.1007/s00262-012-1313-6

B lymphocyte inhibition of anti-tumor response depends on expansion of Treg but is independent of B-cell IL-10 secretion

Yu Zhang 1, Yair Eliav 1, Seung-uon Shin 1, Taylor H Schreiber 2, Eckhard R Podack 2, Tamar Tadmor 3, Joseph D Rosenblatt 1,✉
PMCID: PMC11029618  PMID: 22772949

Abstract

The mechanisms by which B lymphocytes inhibit anti-tumor immunity remain poorly understood. Murine EMT-6 mammary tumors grow readily in immune competent mice (BALB/c), but poorly in B-cell-deficient μ−/− BALB/c mice (BCDM). T regulatory cell (Treg) expansion and function were impaired in BCDM compared with BALB/c. In this study, we compared tumor growth, Treg cell proliferation, tumor lymphocyte infiltration and cytolytic T cell activity in BALB/c, BCDM and BCDM partially reconstituted with B cells by adoptive transfer (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored EMT-6 tumor growth, which was independent of IL-10 secretion by B cells. Instead, high frequencies of intratumoral B cells were associated with increased recruitment and proliferation of Treg cells within the tumor microenvironment. The B-cell-dependent accumulation of Treg within the tumor microenvironment was associated with reduced tumor infiltration by CD49+ NK and CD8+ T cells and reduced cytotoxic T cell activity against EMT-6 targets. Our studies indicate that tumor-dependent immunosuppression of T-cell-mediated anti-tumor immunity is coordinated within the tumor microenvironment by B-cell-dependent cross talk with Treg cells, which does not require production of IL-10 by B cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-012-1313-6) contains supplementary material, which is available to authorized users.

Keywords: B-cell-deficient mice (BCDM), Immune competent mice (BALB/c), T regulatory cells (Treg), B lymphocyte, IL-10

Introduction

Mounting evidence suggests that B cells play an important role in the regulation of inflammatory responses as well as the immune response to tumors [1–7]. Although cancer patients often develop antibodies to tumor-associated antigens such as Her2/Neu, P53 and other tumor-associated antigens, these antibodies generally do not confer protection [8]. Paradoxically, in some studies, anti-tumor antibodies also correlate with poor prognosis [9]. Previous investigations in our laboratory demonstrated that T-cell-mediated immune responses to several primary tumors in mice genetically lacking B cells were enhanced relative to those in normal mice [6]. IgM μ-chain−/− B-cell-deficient mice (BCDM) exhibited enhanced resistance to several histologically diverse syngeneic tumors, including EL-4 thymoma, MC38 adenocarcinoma and B16 melanoma, which was reversed by adoptive transfer of B cells into BCDM [6]. Moreover, rejection of established tumors by therapeutic vaccination was enhanced in the absence of B cells [10]. Recent studies also documented a decreased response to the EMT-6 breast adenocarcinoma in wild-type mice compared with BCDM [11]. Increased tumor resistance in BCDM appeared to involve increased T-cell infiltration, an augmented TH1 cytokine response and increased cytolytic T cell (CTL) response [6]. Other investigators have also noted B-cell-mediated inhibition of anti-tumor responses in several animal models [5, 12]. The mechanisms by which B cells contribute to tumor-mediated T-cell immunosuppression remain unclear.

CD4+FoxP3+Treg cells (Treg) are a major T cell subset involved in regulation of anti-tumor immunity. A variety of mechanisms have been proposed by which Treg cells mediate inhibition of both CD4+ and CD8+ effector T cells, including soluble and membrane-bound cytokines such as TGF-β and IL-10 [13–15], Fas–Fas ligand interactions, downregulation of co-stimulatory receptor expression on NK and CD8+ T cell populations [15–23], and inhibition of dendritic cell maturation and function [24]. It is unclear whether the immunosuppressive effect of B cells is related to or coordinated with the immunosuppressive effect of Treg cells; however, preferential Treg cell proliferation is seen in vitro when B cells are used as antigen-presenting cells to facilitate allogeneic MLR, suggesting that coordination in vivo is possible [25]. In addition, Reichardt et al. [26] showed that naïve B cells could generate T regulatory cells in the presence of a mature immunologic synapse.

We studied the effects of B cells on Treg function in relation to anti-tumor immunity. Using a murine EMT-6 mammary tumor model in BALB/c mice lacking B cells (BCDM), we demonstrate marked inhibition of tumor growth in BCDM, which can be restored in the presence of B cells. We studied the effects of the presence or absence of B cells on Treg expansion in BCDM or BCDM reconstituted through adoptive transfer of B cells. Our studies indicate that the T cell anti-tumor response is enhanced in the absence of B cells, in part due to significant effects on Treg cell expansion and that these effects do not depend on the ability of B cells to secrete IL-10.

Materials and methods

Mice and tumor cell lines

Six- to eight-week-old BALB/c mice and C.129-Il10 tm1Cgn mice on BALB/c background (IL-10−/− mice) were purchased from Jackson Laboratories (Bar Harbor, ME); B-cell-deficient mice (IgM μ chain knockout mice, BCDM) on BALB/c background were a gift from Dr. Thomas Blankenstein (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany). All mice were maintained and bred in the University of Miami Vivarium under standard pathogen-free conditions and were cared for in accordance with the University of Miami institutional Animal Care and Use Committee guidelines. The EMT-6 mammary adenocarcinoma cell line was purchased from ATCC (Manassas, VA) and maintained in Iscove’s modification of Dulbecco’s medium containing 10 % FBS, 50 μM 2-mercaptoethanol, 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (C-IDMEM). For tumor implantation, mice were shaved on the right flank and subcutaneously injected with 106 EMT-6 tumor cells. The day of tumor implantation was designated as day 0. Tumor diameters were monitored twice a week using dial calipers, and tumor volume (mm3) was calculated as follows: 4/3 × 3.14 {(longest axis + shortest axis)/4}3 [27].

B-cell purification and adoptive transfer

Wild-type and IL-10−/− B cells were obtained from spleens of BALB/c and IL-10−/− mice. For B-cell purification, we used the BD IMag Mouse B Lymphocyte Enrichment Set-DM (BD Pharmingen, San Diego, CA, USA), following the manufacturer’s instructions. Briefly, single-cell splenocyte suspensions were obtained and Fc receptors blocked with anti-CD16/32 antibody and subsequently incubated with a biotin antibody cocktail, followed by incubation with BD IMag Streptavidin Particles Plus-DM and separation using a magnetic column. The extent of enrichment was measured using LSRII flow cytometry system (BD Biosciences) and staining for CD19 and B220. The purity of CD19+B220+ B cells following separation ranged from 95 to 99 %. Indicated numbers of B cells were injected intravenously into BCDM at indicated time points as described.

Intracellular cytokine staining

Single-cell suspensions from spleen and tumor-draining lymph nodes (TDLN) isolated on day 28–30 following tumor implantation were cultured in 24-well tissue culture plates in C-RPMI 1640 medium (1 × 106/ml) in the presence of 5/500 ng/ml PMA/ION for 6 h. BD Golgi Stop (2 μM) was added during the last 4 h to block protein secretion. The cells were washed and incubated with purified CD16/CD32, and stained for CD4 or CD8. Cells were then fixed and permeabilized using BD Biosciences Cytofix/Cytoperm Fixation/Permeabilization Solution kit following the manufacturer’s protocol, followed by IFN-γ mAb (XMG1.2, Rat IgG1,κ) staining and analysis using an LSRII flow cytometer to determine the percentage of cells expressing IFNγ.

Antibodies and flow cytometry

Anti-mouse antibodies CD16/CD32 (2.4G2), B220 (RA3-6B2), CD19 (1D3), CD8α (53–6.7), CD4 (RM4.5) and CD25 (3C7) were purchased from BD Pharmingen (San Diego, CA). FoxP3 (FJK-16s) and relevant isotype controls were obtained from eBiosciences (San Diego, CA, USA).

Single-cell suspensions were obtained from spleen, TDLN or excised tumor tissues. The cells were blocked with anti-CD16/CD32 antibody, followed by surface staining with the indicated antibodies at optimized concentration on ice for 15 min in the staining buffer (1 × PBS supplemented with 0.5 % BSA and 0.09 % Sodium Azide). For identification of Treg, cells were stained for CD4 and CD25 as above, and cells were subsequently fixed, permeabilized and stained with anti Foxp3 antibody or corresponding isotype control according to the manufacturer’s instructions (eBiosciences, San Diego, CA, USA). Stained cells were analyzed on an LSR II flow cytometer using Flowjo software (Tree Star Inc, Ashland, OR, USA). Anti-mouse CD25 antibody (PC61) or IgG1 isotype (ATCC, purified in the UM antibody core facility) were administered at day −7 and 0 (150 μg/mouse, i.v.). Treg depletion was assessed by staining with anti-CD4 (RM4-5) and anti-CD25 (3C7) antibody and intracellular staining with FoxP3 antibody.

CTL assays

A total of 5 × 106/ml splenocytes were harvested at the indicated time and co-cultured at 37 °C with mitomycin C–treated EMT-6 cells at a splenocyte : tumor cell ratio of 8:1 in C-RPMI 1640 supplemented with 50 U/ml huIL-2 for 7 days. Effector cells were harvested and live cells isolated by Lympholyte-M gradient (Cedarlane Laboratories) according to the manufacturer’s instructions. Mitomycin C–treated EMT-6 cells were labeled with 51Cr (Amersham Bioscience, Piscataway, NJ) at 100 μCi/106 cell/100 μl supplemented with equal amount of FBS and incubated for 1 h and extensively washed with C-RPMI 1640. Chromium labeled tumor cells were resuspended at 105/ml in C-RPMI-1640 and plated in 96-well U-bottom plates at 100 μl/well. Effector (100 μl/well) and target cell ratios were as indicated in the figure legends. Fresh naïve splenocytes were included as a negative control. Standard 4-h killing assays were performed. At the end of the assays, 100 μl of supernatant was harvested and 51Cr release measured using a gamma counter (Packard Instruments, Albertville, MN, USA). Spontaneous release was obtained from wells containing target cells alone, whereas maximum release was determined by lysing the labeled target cells with 2 % Triton-X 100 before harvesting.  % specific lysis calculated as follows: {experiment release − spontaneous release}/{maximum release − spontaneous release} × 100.

Allogeneic Treg proliferation in response to IL10−/− or WT B cells

MLR responder T cells were obtained from Foxp3-IRES-mRFP (FIR) mice developed on a C57BL/6 background [28]. Sorted CD4+, FIR+CD4+ or FIR−CD4+ cells were labeled with 5 uM CFSE and 105 cells/well cultured together with 106 purified IL-10−/− or WT B (BALB/c background) for 8 days in 48-well plates. T-cell proliferation was analyzed on a BD Fortessa flow cytometer. Baseline cell purity for the CD4+FIR+ or FIR− cells is indicated prior to initiation of MLR. The scatter plots are gated on CD4+ T cells and CFSE and FIR fluorescence levels are indicated.

Statistics

Statistical analysis of tumor growth was carried out with GraphPad Prism 4 (GraphPad Software) with two-way repeated measures (RM) analyses of variance (ANOVA), followed by Bonferroni post-test. Graphs were expressed as the mean values with 95 % CI. Differences were considered statistically significant at p < 0.05 values. For other statistical analyses, Student’s t test was used and labeled as: *p < 0.05; **p < 0.01; or ***p < 0.001.

Results

Tumor growth in B-cell-reconstituted BCDM

We previously noted decreased growth of EMT-6 mammary carcinoma in BCDM compared with BALB/c [11]. To determine whether this was due to the presence or absence of B cells or an intrinsic property of the mice, we reconstituted the B-cell compartment by serial adoptive transfer of purified B cells and then challenged the mice with EMT-6 tumor. As previously reported, minimal tumor growth was observed in BCDM (Fig. 1a). Increased tumor growth was noted in both BALB/c and BCDM, which received adoptively transferred B cells (BCDM+B) (Fig. 1a). Therefore, serial transfer of B cells into BCDM could restore EMT-6 tumor growth. Similar results were seen in multiple experiments (see Fig. 2a).

Fig. 1.

Fig. 1

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Tumor growth, IFN-γ-secreting CD8+ T cells and CTL activity in B-cell-reconstituted BCDM. 106 EMT-6 tumor cells were implanted subcutaneously at day 0; purified splenic B cells were adoptively transferred at day −7, day 0 and day +7. Thirty days post EMT-6 implantation, spleens were harvested and processed as described in the “Materials and methods” section. a EMT-6 tumor growth in B-cell-reconstituted BCDM: 5 mice/group, mean tumor volume ± SEM. *p < 0.05; **p < 0.01. b CTL assay: Splenocytes were co-cultured with mitomycin C–treated EMT-6 tumor cells at a splenocyte : tumor cell ratio of 8:1 for 7 days. Following co-cultivation, effector cells were obtained by Lympholite-M cell separation (Cedarland) and co-cultured with mitomycin C–treated Cr51-labeled EMT-6 at the indicated E:T ratio for 4 h. Cr51 release was measured as outlined in the “Materials and methods”, 4 mice/group, mean ± SD. c Frequency of IFN-γ+/CD8+ T cells on day 30 post-tumor implantation following stimulation in vitro with PMA/ION for 4 h. Mean ± SD, 4 mice/group from one representative experiment of 3 experiments. *p < 0.05; **p < 0.01(compared with BALB/c). d IFN-γ expression on CD8+ T cells: Whole splenocytes were harvested on day 30 post tumor implantation, stimulated with PMA/ION for 4 h in the presence of GolgiStop (BD Pharmingen), and stained for CD8 followed by intracellular staining for IFN-γ as outlined in “Materials and methods”. Representative mice from each group are shown

Fig. 2.

Fig. 2

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Tumor growth and Treg number in BCDM and following B-cell reconstitution. a EMT-6 Tumor growth in B-cell-reconstituted BCDM: purified B cells were adoptively transferred 7 days prior to tumor implantation (day −7), on the day of tumor implantation (day 0), and 7 days post tumor implantation (day 7). Tumor size was measured twice a week by caliper. 5 mice/group, mean tumor volume ± SEM. *p < 0.05; **p < 0.01. b CD4+Foxp3+ Treg cell number in spleen and TDLN 30 days post EMT-6 in BCDM or BCDM+B and BALB/c mice. 5 mice/group, mean ± SD. *p < 0.05; **p < 0.01

Decreased cytolytic T-cell activity and IFN-γ-secreting CD8+ T cells in B-cell-reconstituted BCDM

We next characterized the immune response to EMT-6 tumors in BCDM, BCDM +B and wild-type BALB/c mice. Analysis of CD8+ CTL activity against EMT-6 tumor cells in a 51Cr release assay demonstrated the highest levels of CTL activity in BCDM, intermediate levels in BCDM+B and the lowest level of CTL activity in either tumor-bearing wild-type BALB/c or naïve mice (Fig. 1b).

As demonstrated in Fig. 1c, BCDM showed the highest number of IFN-γ-secreting CD8+ T cells (3.71 ± 0.84 %). BCDM that had received B cells showed approximately half as many IFN-γ+CD8+ cells (2.36 ± 0.66 %, p < 0.05), with the least number of IFN-γ+CD8+ T cells observed in BALB/c (0.8 ± 0.09 %). Overall percentages of IFN-γ positive cells/CD8+ T cells were quantified and averaged over three groups of 5 mice each (Fig. 1c). Representative flow cytometry data are shown in Fig. 1d for BALB/c, BCDM and B-cell-reconstituted BCDM.

T regulatory cell (Treg) and T effector cell (Teff) expansion in B-cell-reconstituted BCDM

Treg cells suppress immune responses in the context of tumors and/or other pathogens, and other investigators have shown that naïve B cells can facilitate expansion of Treg cells [17, 20]. We examined whether significant changes were observed in the Treg compartment following adoptive B-cell transfer into BCDM and their relation to tumor growth. We serially transferred B cells into BCDM and measured tumor growth. Tumor growth was increased in BCDM that received B cells by adoptive transfer, compared with BCDM without B-cell transfer (Fig. 2a). Thirty days post-tumor implantation, the overall number of Treg cells was reduced in the spleens of BCDM relative to BALB/c mice (Fig. 2b). In contrast, following B-cell transfer into BCDM, the total number of Treg cells increased in both the spleen and TDLN (Fig. 2b).

Anti-CD25 antibody treatment inhibits tumor growth in B-cell-reconstituted BCDM

Since the adoptive transfer of B cells into BCDM resulted in a marked expansion of the Treg compartment, this suggested that increased numbers of Tregs might be responsible for the decreased anti-tumor response. We therefore tested whether depletion of CD25+ T cells using PC61 antibody, which is known to deplete a large proportion of CD4+CD25+FoxP3+Treg cells, would result in a decrease in tumor growth. As before, tumor growth was markedly diminished in BCDM compared with wild-type mice and was restored in BCDM following B-cell reconstitution (Fig. 3a). Treatment of B-cell-reconstituted BCDM with PC61 antibody reduced tumor growth to levels seen in non-reconstituted BCDM (Fig. 3a). CTL activity correlated inversely with tumor growth (Fig. 3b). We assessed total Treg cell numbers at day 30 in the spleen and TDLN of BCDM, BALB/c and BCDM+B with or without PC61 treatment. Levels of Treg cells were increased in BCDM+B group (Fig. 3c, d) and wild-type BALB/c mice. In contrast, levels of Treg cells in spleen and TDLN were lower in BCDM, or in BCDM+B treated with PC61 or BALB/c treated with PC61 (Fig. 3c, d).

Fig. 3.

Fig. 3

Fig. 3

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Anti-CD25 antibody treatment inhibits tumor growth in B-cell-reconstituted BCDM. 106 EMT-6 tumor cells were implanted subcutaneously at day 0; purified splenic B cells from wild-type mice were adoptively transferred at day −7, day 0 and day 7. Anti-CD25 antibody (PC61) and isotype control rat IgG1 were administered at day −7 and 0 at 150 μg/mouse, i.v. Thirty days post EMT-6 implantation, spleen and TDLN were harvested and processed as outlined in the “Materials and methods” section: a tumor growth in indicated groups, mean ± SEM, 8 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001. b CTL assays: at day 30, splenocytes were harvested from 3 mice per group, pooled and co-cultured as described in “Materials and methods”. Mean ± SD. c CD4+Foxp3+Treg number in spleen at day 30, 3 mice/group, each dot represents one mouse, bar represents mean value of each group, and error bar is SD. d CD4+Foxp3+Treg number in TDLN at day 30, 3 mice/group; each dot represents one mouse, mean value of each group ± SD. e Tumor-infiltrating lymphocytes in BCDM, BCDM+B and BALB/c with or without PC61 treatment: 30 days post EMT-6 tumor implantation in each group with or without PC61 treatment, tumors were excised and digested with Collagenase D/DNase, and dead cells or tumor cells and red blood cells were removed by using Histopaque-1077 (Sigma-Aldrich). Remaining mononuclear cells were stained as indicated in the “Materials and methods”. Lymphocytes were identified based on CD45 staining and analyzed for CD4, CD8, CD49b, CD19 and Foxp3 expression. Representative flow data for mice in each of the treatment groups are shown. (3f): Anti-tumor response in BCDM is dependent on CD8+ T cell and NK cells. GK1.5 (anti-CD4) or 2.43 (anti-CD8) or corresponding isotype controls were given at 200 μg/mouse, i.v. at day −7, 0 and 10. Anti-GM1 was given at 40 μl/mouse, i.v. twice a week for 3 weeks. EMT-6 tumors were implanted subcutaneously at day 0. Tumor growth was measured twice a week. 7–10 mice each group, data represent average tumor volume ± SEM

Analysis of lymphocyte infiltration into the tumor bed in the various treated groups revealed that large numbers of CD19+ B cells were seen infiltrating the tumor bed for both BALB/c and BCDM+B group as shown in representative mice in Fig. 3e. Both CD8+ and CD49b+ NK cell infiltrations were significantly increased in BCDM relative to either BCDM+B or BALB/c mice (Fig. 3e). Treatment with PC61 in BCDM+ B restored levels of CD8+ and CD49b+ NK infiltration to levels comparable to those seen in BCDM (Fig. 3e).We also noted markedly diminished CD19+ B-cell infiltration following PC61 treatment (Fig. 3e).

To determine whether CD8+ T cells or NK cells were important in tumor rejection in BCDM, we depleted CD4+, CD8+ or NK cells using antibodies to CD4, CD8 or asialo-GM-1, respectively. Depletion of CD4+ cells had little/no effect on tumor rejection, while depletion of CD8+ or GM-1+ cells restored tumor growth (Fig. 3f). Therefore, both NK and CD8+ cells appear to play a role in tumor rejection in BCDM.

These results suggest that B-cell reconstitution of BCDM increased Treg number and enhanced tumor growth and that anti-CD25 antibody treatment in B-cell-reconstituted BCDM restored anti-tumor immunity.

Anti-CD25 antibody treatment affects B-cell migration into tumor tissue

A novel B220+CD25hiCD69hiMHC-IIhi B cell subset with suppressive function in the murine tumor setting, which secreted TGF-β and IL-10 cytokines, was defined recently [12]. We assayed for CD19+CD25+ B cells following PC61 treatment to see how PC61 treatment affected Treg and B-cell infiltration into tumor tissue. As expected, CD25+Foxp3+CD4+ Treg numbers decreased following PC61 treatment in the spleen, TDLN (Supplemental figure 1b) and tumor tissue (Fig. 4a, p < 0.03). Administration of PC61 in wild-type mice did not alter B-cell frequency in spleen and TDLN as compared to control mice (Supplemental figure 1a). CD45+CD19+ B-cell infiltration into tumor tissue was markedly decreased in PC61-treated mice (Fig. 4a, p < 0.03). CD25 expression on CD19+ B cell and CD4+ T cells was examined at days 7, 14, 21 and 28 following tumor implantation. CD25-expressing CD4+ T cells were readily detected in the wild-type mice. Very low levels of CD19+CD25+ B cells were seen in spleen, in TDLNs and within the tumor bed on days 7, 14, 21 and 28 (generally below 1 % of CD45+ infiltrating cells) in wild-type mice (Supplemental figure 2).

Fig. 4.

Fig. 4

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Effects of PC61 depletion on B cell and CD4+CD25+Foxp3+Treg. 200 μg/mouse PC61 antibody or rat IgG1 was given at day −7 and day 0 intravenously. 106 EMT-6 tumor cells were injected subcutaneously at day 0. The percentage of CD4+CD25+Foxp3+Treg and CD19+B cells in spleen, TDLN and tumor tissue was measured at day 7, 14, 21 and 28 (gated on CD45+ lymphocytes in PC61 or rat IgG1–treated mice). a Kinetics of CD19+ B cell and CD4+CD25+Foxp3+Treg cell infiltration in tumor tissue. Tumors were harvested at indicated time points. CD19+ and CD4+/CD25+/FoxP3+ cells were assessed by flow cytometry as described in “Materials and methods”. 3 mice/group, mean ± SD. b BrdU uptake by CD19+-infiltrating cells in the tumor bed. 1 mg BrdU/mouse was injected daily into mice i.p. 3 days before killing on day +21 following tumor implantation. Tumor tissue was digested as described in Methods and intracellularly stained for BrdU using BD Pharmingen BrdU flow kit according to the manufacturer’s instructions. 3 mice/group, mean ± SD. Representative data are shown for mice analyzed on day 21 post-tumor implantation. Flow data are gated on CD45+ cells and then CD19+ versus BrdU+ cells. c BrdU uptake by CD4+Foxp3+ infiltrating cells in the tumor bed. As outlined above. Representative flow data are shown for mice analyzed on day 21 post-tumor implantation. Flow data gated on CD45+CD4+BrdU+ cells and then Foxp3+ versus BrdU+ cells. 3 mice/group, mean ± SD

To determine whether infiltrating T and B cells were actively proliferating, we assayed BrdU uptake in situ. BrdU staining on day 21 in wild-type or PC61-treated mice demonstrated very low levels of BrdU uptake within the infiltrating CD19+ B-cell population in the tumor bed in both groups(1.58 ± 0.46 % of CD19+ cells in PC61 vs 1.61 ± 1.05 % in control group) (Fig. 4b). In contrast, much higher levels of BrdU uptake were seen within the infiltrating CD4+FoxP3+ population in control group (36.22 ± 4.68 % of CD4+FoxP3+ cells) compared with PC61-treated group (18.58 ± 6.80 %, p = 0.02) as shown in Fig. 4c. (Note: anti-CD25 antibody treatment was performed at day −7 and day 0, and flow for CD4+Foxp3+ cells was performed at day 21.) Similar results were seen using Ki67 staining (data not shown). These results suggest active proliferation of Treg, but not of infiltrating B cells within the tumor bed. Taken together, these data indicate that PC61-antibody-mediated depletion of CD25+ cells decreases both B-cell recruitment to and Treg proliferation within the EMT-6 tumor microenvironment.

Both wild-type and IL-10−/−B cells rescue EMT-6 growth

B10 cells that produce interleukin-10 (IL-10) are known to play an important regulatory role in a variety of mouse models of inflammation [29]. To determine whether restoration of tumor growth in reconstituted BCDM was dependent on IL-10 secretion by adoptively transferred B cells, B cells from IL-10−/− or wild-type mice were adoptively transferred into BCDM. We confirmed the lack of IL10 secretion in IL10−/− mice using ELISA and genotype using PCR (Supplemental Fig. 3). Growth of EMT-6 tumor was similar in BALB/c and IL10−/− mice (Fig. 5a). Transfer of IL-10−/− B cells into BCDM restored tumor growth and resulted in decreased survival similar to what was seen with wild-type B-cell transfer (Fig. 5b, c). Overall levels of Treg cells in the spleen were increased to a similar degree in BCDM, which had received either IL-10−/− B cells or wild-type B cells (Fig. 5d). A vigorous CTL response was noted in BCDM implanted with EMT-6, while CTL responses were markedly diminished in wild-type mice or in BCDM that had received either wild-type or IL-10−/− B cells (Supplement Figure 4).

Fig. 5.

Fig. 5

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Wild-type B and IL-10−/− B cells rescue tumor growth in B-cell-reconstituted BCDM. 30 × 106 purified splenic B cells from wild-type and IL-10−/− mice were injected intravenously into BCDM at day −7, 0 and +7 relative to tumor implantation. 106 EMT-6 tumor cells were implanted on day 0 subcutaneously. 28 days post EMT-6 implantation, spleen and TDLN were harvested and processed. a Tumor growth in IL-10−/− mice and BALB/c mice, 5 mice/group, mean ± SEM. Data represent one of two experiments. b Tumor growth in BCDM, wild-type and IL-10−/− B-cell-reconstituted BCDM and wild-type BALB/c mice. 10 mice/group in B-cellreconstituted BCDM, 7 mice in BCDM and 5 mice in wild-type BALB/c group. Mean ± SEM. c Survival in each of the above groups. d Total CD4+Foxp3+ Treg cell number in spleen. 3 mice/group, mean ± SEM. Dots represent individual mice, bar represents mean value of 3 mice. e Allogeneic Treg proliferation in response to IL10−/− or WT B cells. MLR responder T cells were obtained from Foxp3-IRES-mRFP (FIR) mice developed on a C57BL/6 background. Sorted CD4+, FIR+CD4+ or FIR-CD4+ cells were labeled with 5 uM CFSE and 105 cells/well cultured together with 106 purified IL-10−/− or WT B (BALB/c background) for 8 days in 48-well plates. T cell proliferation was analyzed on a BD Fortessa flow cytometer. Baseline cell purity for the CD4+FIR+ or FIR− cells is indicated prior to initiation of MLR. The scatter plots are gated on CD4+ T cells and CFSE and FIR fluorescence levels are indicated

Since we observed proliferation of Treg in vivo following transfer of either IL10−/− or WT B cells, we tested whether IL10−/− B cells would support expansion of Treg in vitro in a mixed leukocyte reaction assay. Responder CD4+ T cells were obtained from Foxp3-IRES-mRFP (FIR) mice in which Foxp3+ cells are FIR+ [28]. Sorted CD4+, CD4+FIR+ or CD4+FIR− cells were labeled with CFSE and co-cultured with purified B cells from IL-10−/− or BALB/c mice at T:B ratio of 1:10 for 8 days. Proliferation of CD4+ T cells, Treg (CD4+FIR+) or CD4+FIR− cells was analyzed by assessment of CFSE dilution using flow cytometry. Prior to sorting, 12–18 % of CD4+ cells expressed FIR+. The sorted CD4+ population was >99 % pure, and FIR sorted populations were >98 % CD4+/FIR+ and CD4+/FIR−, respectively, prior to co-culture (Fig. 5e). At 8 days, using purified CD4+ cells as responders, FoxP3+CD4+FIR+ Treg represented the bulk of the dividing CD4+ T cell population in response to either IL10−/− or WT B cells (42.3 vs 64.0 % FIR+ in IL-10−/− B vs WT B, 11.2 % vs 20.0 % FIR- in IL-10−/− vs WT B)(Fig. 5e). Using purified CD4+FIR+ (FoxP3+) Treg as responders, a high percentage divided in vitro in response to co-culture with either WT B cells (47.8 %) or IL10−/− B cells (51.1 %, Fig. 5e). Therefore, IL-10 secretion by B cells does not appear to be required for the expansion of Treg in vitro or in vivo.

We also characterized tumor-infiltrating lymphocytes in the aforementioned treatment groups. As shown in representative mice in Fig. 6a, b, low frequencies of CD49b+ NK cells (2.81 ± 1. 0 %) and CD8+ T cells (5.88 ± 0.5 %) were noted within the tumor bed in BALB/c mice. In contrast, in BCDM, residual tumor tissue was markedly diminished in size and contained significant infiltrates of CD49b+ NK cells (17.8 ± 0.3 % gated on CD45+ lymphoid cells) and increased infiltration with CD8+ T cells (15.5 ± 1.32 %) relative to BALB/c mice (Fig. 6a, b). Levels of NK and T-cell infiltration in mice reconstituted with IL-10−/− B cells or WT B cells were modestly increased relative to BALB/c mice, but lower than levels seen in BCDM (Fig. 6b). These data demonstrate increased infiltration of CD8+ T cells as well as CD49b+ NK cells into tumors implanted in BCDM as compared to BALB/c or as compared to BCDM partially reconstituted with either WT or IL-10−/− B cells.

Fig. 6.

Fig. 6

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NK and CD8+ T-cell infiltration into tumor tissue 28 days post EMT-6 implantation. 30 × 106 purified splenic B cells from wild-type and IL-10−/− mice were injected intravenously into BCDM at day −7, 0 and +7. 106 EMT-6 tumor cells were implanted on day 0 subcutaneously. 28 days post EMT-6 implantation, tumor tissues were harvested and digested with collagenase D in the presence of DNase for 30 min at 37 °C, and then infiltrating lymphocytes were purified using Histopaque-1077 (Sigma-Aldrich) and stained for CD8+ and CD49+ cells and analyzed by flow cytometry as described in “Materials and methods”. a Lymphocyte infiltration into tumor tissue 28 days post-tumor implantation. Gated on CD45+ cells. Representative mice are shown. b Frequency of tumor infiltration by lymphocytes of indicated phenotype in tumor tissue at day 28 following tumor implantation. Gated on CD45+ cells, 4 mice/group, mean ± SEM

Discussion

In the present study, we examined the mechanism by which B cells contribute to tumor-mediated suppression of anti-tumor immunity. Reconstitution of BCDM by wild-type B cells abolished tumor resistance, suggesting that increased tumor resistance seen in BCDM was not due to intrinsic alterations in T cells or antigen-presenting cells, but was a significant consequence of the absence of B cells. These studies extend previous results to BALB/c mice, which have a significantly augmented TH2 response, and to yet another tumor model, the EMT-6 mammary carcinoma.

In these studies, we have defined a relationship between Treg cell proliferation within the tumor microenvironment and the presence of tumor-infiltrating B cells. Decreased tumor growth in BCDM was reversible by the addition of adoptively transferred B cells to the level seen in wild-type mice and correlated with the frequency of proliferating Treg cells within the tumor microenvironment. Further, restoration of tumor growth correlated with a significant increase in the number of Treg cells in both spleen and TDLN and with a decrease in IFN-γ secretion and CTL activity against tumor targets. The contribution of B cells to suppressing anti-tumor immunity was independent on B-cell secretion of IL-10, but significantly associated with B-cell-dependent accumulation of Treg cells within the tumor microenvironment and apparent cross talk between these two cell types.

Augmented Treg expansion in the presence of B cells may significantly contribute to suppression of anti-tumor immunity in this model. Mechanisms of Treg suppression of anti-tumor immunity are diverse and result in the suppression of effector cell function including, TH1 response, CD8+ T cell proliferation and/or CTL activity [15, 21]. B cells are known to function as antigen-presenting cells through presentation of antigens on MHC-II, antigen complexes through CD4+ T cells. B-cell-secreted cytokines may significantly influence CD4+ T-cell activation, proliferation and differentiation [30, 31]. B-cell stimulation has been demonstrated in several models to facilitate the expansion of Treg cells in vitro and in vivo [25]. Although antigen presentation by naïve B cells appears to induce tolerance [32], tolerance induction is not dependent upon IL-10 production by B cells, and other molecules and interactions, such as interactions involving LFA-1, CD154, CD40 and other molecules, may also play a role in induction of tolerance [33]. Thus, it is possible that the increased Treg proliferation observed following adoptive transfer of B cells was dependent upon accumulation of tumor-antigen-presenting B cells within the tumor microenvironment, which served as local antigen-presenting cells to tumor-infiltrating Treg.

IL-10-producing B cells or so-called “B10 cells” have been reported to play a major role in inhibition of autoimmune diseases and inflammatory responses in settings such as EAE, and systemic lupus erythematosis (SLE) [29]. We initially hypothesized that B10 cells may play a significant role in immune suppression seen in the EMT6 model. However, our adoptive transfer experiments using IL-10−/− derived B cells suggest that IL10 secretion by B cells may not be required for B-cell-mediated suppression of anti-tumor immunity, and other B-cell regulatory effects may have impacted anti-tumor immunity.

Despite the presence of B cells, depletion of CD25+ Treg cells following administration of PC61 in B-cell-reconstituted BCDM restored anti-tumor response. However, CD25 depletion changed the composition of the infiltrating lymphocyte subpopulations, and in the presence of PC61, fewer infiltrating B cells were seen in excised tumor tissue. Olkhanud et al. [12] described reduced 4T1 breast cancer metastasis and tumor growth in BCDM similar to results we have seen using the EMT-6 tumor model. They described a subpopulation of B220+CD69+CD25+ B cells generated in vitro which suppressed T-cell proliferative response and reduced anti-tumor response in vivo. We therefore assayed for CD25 expression on CD19+ B and CD4+ T cells at day 7, 14, 21 and 28 post-tumor implantation. In contrast to their reported findings in the 4T1 tumor model, we observed minimal CD25 expression on CD19+ B cells in spleen, TDLN and EMT-6 tumor tissue (Supplemental figure 2). Therefore, we believe the effects of PC61 in restoring anti-tumor response are primarily due to a reduction in the number of Treg cells rather than depletion of CD25+CD19+ B cells.

Our findings demonstrate that Treg expansion is limited in B-cell-deficient mice and adoptive transfer of B cells results in enhanced Treg expansion and inhibition of overall anti-tumor response that is not dependent on B-cell secretion of IL10. Whether similar B-cell-mediated suppression of anti-tumor responses occurs in human tumors is not known. Significant B-cell infiltration has been observed in some breast cancers, ovarian and other solid tumors [34, 35]. Our results suggest that depletion of intratumoral Treg may occur as a result of depletion of B cells in vivo and may facilitate anti-tumor immunity with clinically available B-cell-depleting antibodies. Investigation of the immunomodulatory effects of B-cell-depleting antibodies in humans could be used to develop novel preclinical and clinical immunotherapeutic strategies.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 35 kb) (35KB, doc)
Supplementary material 2 (PPTX 206 kb) (206.4KB, pptx)

Acknowledgments

We are grateful to Dr. Dietlinde Wolf for kindly providing the PC61 antibody and the staff of the SCCC flow laboratory for excellent technical support. This work was financially supported by National Institution of Health (Grant 5-P01-CA-109094-04) and the Arnall Family Foundation.

Conflict of interest

The authors declare that they have no conflict of interest.

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Supplementary Materials

Supplementary material 1 (DOC 35 kb) (35KB, doc)
Supplementary material 2 (PPTX 206 kb) (206.4KB, pptx)

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