Cancer Immunology, Immunotherapy

, Volume 61, Issue 12, pp 2273–2282 | Cite as

Mast cells impair the development of protective anti-tumor immunity

  • Anna Wasiuk
  • Dyana K. Dalton
  • William L. Schpero
  • Radu V. Stan
  • Jose R. Conejo-Garcia
  • Randolph J. Noelle
Original article

Abstract

Mast cells have emerged as critical intermediaries in the regulation of peripheral tolerance. Their presence in many precancerous lesions and tumors is associated with a poor prognosis, suggesting mast cells may promote an immunosuppressive tumor microenvironment and impede the development of protective anti-tumor immunity. The studies presented herein investigate how mast cells influence tumor-specific T cell responses. Male MB49 tumor cells, expressing HY antigens, induce anti-tumor IFN-γ+ T cell responses in female mice. However, normal female mice cannot control progressive MB49 tumor growth. In contrast, mast cell-deficient c-KitWsh (Wsh) female mice controlled tumor growth and exhibited enhanced survival. The role of mast cells in curtailing the development of protective immunity was shown by increased mortality in mast cell-reconstituted Wsh mice with tumors. Confirmation of enhanced immunity in female Wsh mice was provided by (1) higher frequency of tumor-specific IFN-γ+ CD8+ T cells in tumor-draining lymph nodes compared with WT females and (2) significantly increased ratios of intratumoral CD4+ and CD8+ T effector cells relative to tumor cells in Wsh mice compared to WT. These studies are the first to reveal that mast cells impair both regional adaptive immune responses and responses within the tumor microenvironment to diminish protective anti-tumor immunity.

Keywords

Cancer Tumor immunity Microenvironment T cells 

Introduction

Inflammatory immune cells contribute to the development of tumors by promoting neovascularization, tissue remodeling, and inflammation [1]. Mast cells (MCs) are among the first inflammatory cells to accumulate around tumors, infiltrating pre-malignant lesions and promoting tumor growth in murine models of pancreatic cancer, neurofibromas, colorectal cancer, and squamous cell carcinoma [2, 3, 4, 5]. MCs contribute to the growth of tumors in multiple ways. They are a rich source of pro-angiogenic molecules capable of promoting tumor vascularization and are associated with increased microvascular density in many tumors [2, 4, 6, 7]. MCs promote inflammation within the tumor microenvironment by recruiting inflammatory cells and also by switching regulatory T cells to a pro-inflammatory phenotype [3, 8, 9, 10]. Increased numbers of MCs infiltrating certain human tumors are associated with an unfavorable prognosis and decreased survival [11, 12, 13, 14].

Although high densities of MCs are associated with poor prognosis in human cancers, high densities of intratumoral T effector (Teff) cells, especially those with Th1 and cytotoxic gene signatures (such as IFN-γ, IRF-1, and granulysin) are associated with a favorable prognosis in many human cancers [15, 16, 17, 18]. However, the cells and molecules controlling the balance between protective and non-protective immune responses to tumors are poorly defined. In particular, the roles of MCs, which are immunomodulatory cells having both positive and negative effects on adaptive immune responses, are not well understood [19].

In this study, we investigated how MCs influence adaptive T cell responses to tumors. We used the Wsh mouse strain with a profound deficiency of MCs caused by a mutation in the c-kit receptor [20, 21]. The MB49 male bladder carcinoma line was used because this tumor line recruits MCs, which promote tumor angiogenesis [6]. Thus, comparing tumor growth in C57BL/6 wild-type (WT) and Wsh mice provides insight on the effect of angiogenesis on tumor growth. Also MB49 cells expressing HY antigens trigger anti-tumor T cell responses in females, but not males because they are centrally tolerant of HY antigens. Thus, comparing tumor growth in males and females provides insight on the effect of rapid T cell responses on tumor growth. However, even though WT females make rapid T cell responses, such responses are not sufficient to control the growth of this tumor [22].

Here, we present evidence that MC-deficient Wsh female mice were capable of controlling tumor growth and surviving systemic tumors significantly better than WT female mice. Enhanced resistance to tumors in Wsh female mice was T cell-mediated, as demonstrated by adoptive transfer of tumor immunity by T cell subsets, as well as loss of tumor resistance upon in vivo T cell depletion. Moreover, Wsh female mice had increased frequencies of IFN-γ-producing CD8+ T effector cells in tumor-draining lymph nodes (dLNs) compared with WT females. Additionally, Wsh female mice had significantly increased ratios of intratumoral CD4+ CD44hi and CD8+ CD44hi T cells relative to tumor cells compared to WT females. These studies are the first to reveal that MCs impair both regional adaptive immune responses and responses within the tumor microenvironment to diminish protective anti-tumor immunity, suggesting MCs are attractive therapeutic targets in promoting anti-tumor immunity.

Results

MCs accumulated around MB49 tumors and were associated with increased angiogenesis within tumors

MB49 tumor cells were injected intradermally (i.d.) on the right flanks. In WT mice, MCs accumulated abundantly at the growing edge of MB49 tumors at the boundary between tumor tissue and skin (Fig. 1a). To investigate angiogenesis in the tumors, sections were stained with anti-CD31 antibody to visualize the vasculature in and around the tumor mass and the staining was quantified (Fig. 1b, c). Microvascular density was similar in all groups on day 5 of tumor (not shown). However, the microvascular area of the tumor was reduced in both female and male Wsh mice as compared to female and male WT mice on day 12 (Fig. 1c). Both female and male WT mice have high microvascular density at the growing edge of tumor and within the tumor mass. In contrast, microvascular density was significantly reduced in both male and female Wsh mice and when vasculature was observed, it was present only at the growing edge of the tumor (Fig. 1b top panels). These data confirm that the c-Kit Wsh strain of MC-deficient mice, similar to the WBB6F1 WW-V strain of MC-deficient mice [6], exhibit reduced angiogenesis of MB49 tumors.
Fig. 1

Mast cells accumulate around MB49 tumors and contribute to increased microvascular density in tumors of WT mice. (a) Toluidine blue staining of MB49 tumor sections show mast cells surrounding the tumor. Two different sections from MB49 tumors (day 12) in WT female mice are shown. One representative normal skin section stained with Toluidine blue is shown. b Representative images of CD31 staining on tumor sections (day 12) from C57BL/6 female mice and Wsh female mice. The dotted lines on the top images show the boundary between tumor and normal tissues. c Quantification of immunohistochemical staining for CD31 on sections of C57BL/6 female and male and Wsh female and male i.d. tumor sections (n = 4 per group). Microvascular density was calculated by dividing number of pixels staining with CD31 over number of pixels in total skin section examined

Mast cell-deficient Wsh females, but not Wsh males controlled the growth of MB49 tumors

To determine whether the genetic loss of MCs influenced tumor growth, we compared the growth rates of MB49 tumors in WT and mast cell-deficient Wsh mice. Progressive tumor growth was observed in WT females, WT males, and Wsh males. In contrast, Wsh females controlled tumor growth significantly better than WT females (p < 0.0001) (Fig. 2a). Representative appearance of tumors on days 7 and 15 in WT and Wsh females and males are shown in Fig. 2b. On day 7, most Wsh female mice had developed central ulcers in their tumors, and by day 15, the tumors in most Wsh females regressed leaving a flat skin lesion containing few tumor cells. Some of the Wsh male and WT female mice also developed ulcers at the center of the tumors; however, the tumors continued to grow progressively around the apparently necrotic central core. In WT males, tumors grew progressively, forming large raised lesions. These data indicate that Wsh female mice controlled MB49 tumor progression better than WT females. Moreover, MC deficiency and the consequently diminished tumor angiogenesis in Wsh males were not sufficient to control the progressive growth of MB49 tumors.
Fig. 2

Mast cell-deficient Wsh female mice show superior regression of MB49 tumors. a MB49 tumor growth curves in C57BL/6 female and male mice and the genetically mast cell-deficient Wsh male and female mice. Mice were inoculated intradermally (i.d.) with 2.5 × 105 cells per mouse and perpendicular diameters of tumors were measured; shown are the mean and SEM for 6–8 mice per group. The graph shown is representative of 10 experiments. Tumor growth in female B6 compared with female Wsh mice was significantly different (2-way ANOVA). bTop panels are images of representative day 7 tumors. Bottom panels are images of representative day 15 tumors in WT males and female and Wsh males and females

Reconstitution of Wsh female mice with MCs abolished their enhanced resistance to systemic tumors

Systemic MB49 tumors were also used to investigate the role of MCs in anti-tumor immunity. Systemic challenge with MB49, resulting in lung tumors [23], led to the death of WT female and WT male mice and all male Wsh mice by day 80 (Fig. 3a). In contrast, 50 % of female Wsh mice survived systemic tumor challenge to day 80, corresponding to the end of the monitoring period. Such enhanced survival of Wsh female mice with systemic MB49 tumors was abolished by reconstitution of Wsh females with WT bone-marrow-derived MCs (BMMCs) (Fig. 3b). All of the female Wsh mice reconstituted with BMMCs died of lethal systemic tumor at the same rate as WT females. Mast cell reconstitution was validated by the enumeration of MCs in peritoneal lavage of reconstituted Wsh mice (Fig. 3c). These results demonstrate that MCs contribute to impaired resistance to tumors in the MB49 tumor model.
Fig. 3

Enhanced survival of Wsh females with systemic MB49 tumors was abolished by their reconstitution with bone-marrow-derived mast cells. a MB49 tumor was injected systemically through the tail vein, and mice were monitored for survival. Survival curve is a composite of four independent experiments. b Systemic reconstitution of Wsh female mice with C57BL/6 bone-marrow-derived mast cells (BMMC). Cells were inoculated i.v., i.d., intraperitoneally, and subcutaneously to ensure mast cell reconstitution in all anatomical locations. Survival graphs show data from three independent experiments. c FACS plots showing representative samples from C57BL/6, Wsh, and Wsh reconstituted with WT bone-marrow mast cells. Cells are from an intraperitoneal lavage stained for FcεRI and CD117 (c-kit), to identify mast cells

Enhanced tumor protection was T cell dependent in female Wsh mice

We next investigated the role of T cells in Wsh female mice with systemic tumors. In contrast to untreated Wsh female mice, all Wsh female mice depleted of CD4+ or CD8+ T cells succumbed to systemic tumors (Fig. 4a, b). Thus, the enhanced survival of Wsh female mice during systemic tumor challenge required both CD4+ and CD8+ T cells.
Fig. 4

Enhanced resistance of Wsh female mice to tumors is T cell mediated and can be adoptively transferred into mast cell-sufficient mice. Groups of mice were given MB49 tumors i.v. and monitored for survival. a and b Enhanced survival of Wsh female mice with systemic MB49 tumors was abolished by antibody-mediated depletion of CD8+ (α-CD8 Wsh F) or CD4+ T cells (α-CD4 Wsh F). Survival curves of Wsh mice depleted of CD4+ T cells and CD8+ T cells were significantly different from survival of intact Wsh mice *p < 0.04. c and d MB49 tumor was injected into groups of mice by i.d. route. Growth rates (perpendicular diameters) of i.d. MB49 tumor are shown with each point representing the mean and SEM of 6–8 mice. c Growth rate of MB49 tumor in Wsh females and B6 female and male controls shown for comparison to those in d. d Adoptive transfer of combined CD4+ and CD8+ T cells isolated from spleen and dLNs of Wsh female mice that had previously completely rejected i.d. MB49 (immune T cells), or from naïve Wsh female mice into naïve C57BL/6 female mice. Growth of MB49 tumor in recipients of immune T cells was significantly reduced compared with growth rate of MB49 tumors in naïve Wsh mice (p < 0.001). Data show composite results from 3 independent experiments. p values were calculated by the 2-way ANOVA

Additionally, T cell-mediated tumor immunity established in a mast cell-deficient environment was transferable to naïve WT female mice. “Immune” T cells were isolated from female Wsh mice 30–50 days after their complete rejection of tumors and were transferred into naïve female WT mice. In parallel, T cells were isolated from naïve Wsh female mice and transferred to naïve female WT mice. T cell recipients and male and female controls were then challenged with MB49 tumors. WT recipients of “immune” T cells from Wsh mice rejected tumor faster than naïve Wsh mice rejected primary tumor (Fig. 4c, d). Moreover, naïve WT mice that received T cells from naïve Wsh mice grew tumor with the same kinetics as naïve WT mice (Fig. 4c, d). Thus, immune T cells generated in a mast cell-deficient environment were protective upon transfer into a mast cell-sufficient environment.

Wsh female mice have increased frequencies of IFN-γ-secreting anti-tumor CD8+ T cells in tumor dLNs

The improved survival and enhanced tumor regression of Wsh female mice prompted us to compare their anti-tumor T cell responses with those of WT female mice. At 2 weeks after MB49 tumor inoculation, we purified CD8+ T cells from the tumor dLNs to assess IFN-γ production in response to male spleens and tumor antigens using a sensitive ELISPOT assay. CD8+ T cells from the tumor dLN of Wsh female mice had significantly higher frequencies of IFN-γ+ cells in response to irradiated male spleen cells (Fig. 5a) as well as to irradiated tumor cells compared with WT cells (Fig. 5b). Also, CD8+ T cells from the tumor dLNs of WT and Wsh males exhibited equally low IFN-γ responses to tumors, consistent with their failure to control tumor growth. Thus, in female Wsh mice lacking MCs, there were more IFN-γ-producing CD8+ T cells responding to both male antigens and tumor antigens as compared to WT female mice.
Fig. 5

Increased IFN-γ responses in tumor dLNs of Wsh female tumor-bearing mice. The frequencies of tumor-responsive CD8+ T cells were measured in an ELISPOT assay. Tumor-draining inguinal lymph nodes from four mice were pooled and CD8+ T cells were enriched to 90–95 % purity and plated into triplicate wells for each condition. Enriched CD8+ T cells were plated at 2 × 105 cells per well with a T-depleted male spleens as antigen-presenting cells (APCs) or b irradiated MB49 tumor cells. Bars show the mean and SEM of ELISPOTS per 2 × 105 input CD8+ T cells with 3–10 points per group measured in four independent experiments. On the X-axis, the following abbreviations were used: B6 F, WT females; Wsh F, Wsh females; B6M, WT males; Wsh M, Wsh males. Significance was determined by unpaired t test

Mast cell-deficient female mice have significantly increased ratios of CD4+ and CD8+ T effector cells relative to tumor cells in the tumor microenvironment

Since the majority of Wsh female mice rejected their tumors, we hypothesized that these mice may have increased infiltration of effector T cells within the tumor. We next measured the numbers of tumor cells and tumor-infiltrating T effector cells on days 7 and 12 after MB49 tumors were given. Tumors were excised and dissociated to single cell suspensions, stained with antibodies, and analyzed by flow cytometry with absolute count beads to determine the cellular composition. The time points were selected because all tumors were of similar size on day 7, but on day 12, the tumors on Wsh females had regressed. On day 7, the total numbers of tumor cells, CD8+CD44hi and CD4+CD44hi T cells, were similar in tumors of all groups. On day 12 after tumor implantation, the Wsh female mice had the lowest total numbers of tumor cells as well as the lowest total numbers of intratumoral CD8+CD44hi and CD4+CD44hi T cells compared with all other groups (Fig. 6a, b, c). In contrast, WT male mice with progressively growing tumors had significantly more total numbers of both tumor cells and intratumoral effector T cells compared with the other groups, indicating that the largest tumors contained the highest total numbers of T effector cells.
Fig. 6

Wsh female mice have significantly increased ratios of intratumoral CD4+ and CD8+ T effector cells relative to tumor cells compared with other groups. Mice were given MB49 tumors by i.d. route. Tumors were excised and analyzed by flow cytometry on days 7 and 12. Absolute numbers of a CD4+CD44hi T cells b CD8+CD44hi T cells c and tumor cells in tumors from WT and Wsh female and male mice. d Ratios of intratumoral CD4+CD44hi T cells per total numbers of tumor cells are shown for each group. Wsh female compared with WT females p = 0.0024**. e Ratios of intratumoral CD8+CD44hi T cells per total tumor cells are shown for each group. Ratios for Wsh females compared with WT females, p = 0.006**. Points represent the mean and SEM of 8–9 mice/group on day 7 and 10–16 mice/group on day 12, data points were obtained in two independent replicate experiments. The Mann–Whitney test was used to determine significance

We next compared the ratios of intratumoral T cells relative to tumor cells. Tumors from the Wsh female mice contained significantly higher ratios—on average 6–10-times more—of CD8+CD44hi T cells and CD4+CD44hi T cells, respectively, relative to tumor cells compared with tumors from WT female mice and other groups (Fig. 6d, e). Although increased absolute numbers of CD4+ and CD8+ T effector cells were present in WT males with progressively growing tumors, tumor cells outnumbered T effector cells by 100 to 1 in nine of twelve B6 male tumors investigated. Therefore, the ratio of T effector cells to tumor cells was low in B6 males. Hence, MCs greatly diminished the ratios of intratumoral T cells relative to tumor cells.

Discussion

The role of MCs in promoting malignancy is multifaceted, as they orchestrate the character of the tumor microenvironment promoting inflammation, angiogenesis, and tissue remodeling [24]. In addition, we now provide evidence that MCs impair the development of protective anti-tumor immunity resulting in diminished host survival. MCs in female mice impair effective anti-tumor IFN-γ-producing CD8+ effector T cells capable of controlling progressive tumor growth. We found that adoptive transfers of tumor-specific effector Wsh T cells were protective in MC-sufficient hosts suggesting that MCs likely influence the inductive, rather than effector, phase of anti-tumor immunity. Reconstitution of Wsh female mice with BMMCs abolished their resistance to tumors, implicating MCs in impairing anti-tumor immunity.

Insights into the relative importance of angiogenesis in the tumor microenvironment and adaptive immunity to host survival were provided by comparing tumor growth in male and female, WT and mast cell-deficient hosts. MC-deficient male and female Wsh mice both exhibited reduced tumor angiogenesis, confirming the role of MCs in tumor vascularization. The progressive growth of MB49 tumors in Wsh male mice indicates that the absence of MCs and their pro-angiogenic factors will not sufficiently impair tumor growth to promote enhanced host survival. Thus, the reduced angiogenesis in the Wsh tumor microenvironment had no overall impact on the growth of tumors in Wsh male mice. This is consistent with the minimal impact of anti-angiogenic therapeutics as monotherapies in oncology indications [25]. These findings are in striking contrast to tumor growth and host survival in female Wsh mice, where there was greatly impaired tumor growth and 50 % or more of Wsh female mice survived systemic tumors up to 80 days. In females, but not males, MB49 tumors induce anti-HY T cell responses. However, the adaptive immune response in WT female mice in the absence of tumor microenvironment disruption also was largely incapable of interfering with tumor growth. Our preliminary studies underway show that blockade of VEGF in female mice and not in male mice will similarly promote tumor rejection in female mice. Enhanced anti-tumor immunity to an immunogenic tumor with anti-VEGF has also been previously reported [26]. Taken together, these findings suggest that impaired angiogenesis synergizes with an adaptive T cell response to enhance protective anti-tumor immunity.

Mast cells are a source of a number of pro-angiogenic molecules including VEGF, bFGF, and IL-8 [27, 28]. Pro-angiogenic molecules not only play a role in the development of the tumor microenvironment, but also are powerful immunoregulatory molecules that control the development of tumor-specific immunity. For example, blockade of VEGF-induced angiogenesis has been shown to enhance infiltration of tumors with T effector cells during adoptive immunotherapy and tumor vaccination [29, 30]. Consistent with this, we found that in the absence of MCs, angiogenesis was reduced and tumors had increased ratios of CD4+ and CD8+ T effector cells relative to tumor cells.

A systemic impact of MCs on T cell responses may manifest through the ability of MCs to influence dendritic cells (DCs). MCs influence the early stages of DC migration and function [31, 32], and this may be the critical way in which MCs contribute to the development of aberrant tumor immunity. Mast cell mediators such as TNF-α, leukotrienes, histamine, and GM-CSF can dramatically modulate DC maturation and induce DC migration [31, 33, 34]. Many of the mast cell-mediated DC modifications reported must consequently be reflected in the development of particular T cell responses. In models of allergy for example, MCs induce a Th2 profile by the production of prostaglandin E2 and histamine, mast cell mediators that induce DCs to produce CCL17/22 [35]. The aforementioned mediators are Th2-cell recruitment factors and are known to suppress the frequency of Th1 allergen-specific cells both in vivo and in vitro [35, 36, 37]. In allergy, this augments inflammation and the pathology of the disease; yet in a different setting, such as that of the tumor microenvironment may contribute to impaired anti-tumor response.

Mast cells have been considered intermediaries in immune suppression in several immune contexts. They express MHC-II and MHC-I, and recent reports have shown MCs to be bona fide antigen-presenting cells in that they express other co-stimulatory molecules such as OX40L, CD30 ligand (CD30L), Fas, glucocorticoid-induced TNF receptor (GITR) as well as CD80, CD86, PD-L1, and PD-L2 [38, 39]. MCs expressing MHC class II are capable of expanding antigen-specific T regulatory cells [40]. MCs have the ability to skew naïve T cells into a Th2 phenotype by inducing production of IL-4, IL-10, and IL-13 and suppressing production of IFN-γ [41, 42]. Th2-mediated immunity in turn has been associated with the inhibition of anti-tumor immunity, both by promoting angiogenesis and by suppressing cell-mediated immunity and effective tumor clearance [43]. One example of mast cell-induced down-regulation of Ag-specific T cell proliferation is the IL-10-mediated suppression that is seen in the context of mosquito bites [44]. It is, therefore, plausible to hypothesize that MCs are directly contributing to the immune suppression seen in our model, albeit not necessarily in a contact-dependent manner. MCs also produce IL-10 and TGF-β and may as such be able to suppress T cell proliferation and even mediate in the generation of adaptive Tregs. It will be crucial to assess whether MCs and mast cell-derived VEGF, IL-10, or/and TGF-β could affect the number, phenotype, or function of Tregs in the tumor microenvironment.

Mast cells may impair the development of protective anti-tumor immunity by multiple mechanisms including direct MC interactions with T cells, MCs influencing DCs cytokines and functions, and by alterations in the tumor microenvironment such as increased tumor angiogenesis [45]. Studies are currently underway to understand the precise mechanisms by which MCs influence the adaptive immune responses to tumors.

Materials and methods

Mice

Male and female 6- to 8-week-old C57BL/6 mice were obtained from the National Cancer Institute (Bethesda, MD) and housed for 2–4 weeks in our specific pathogen-free animal facility prior to experiments. Mast cell-deficient mice KitW-sh/W-sh (Wsh) on the C57BL/6 background were bred in our animal facility. Experimental mice were used at 10–12 weeks of age except where noted. Experiments were approved by the Institutional Animal Care and Use Committee of Dartmouth College.

Antibodies, reagents and flow cytometry

Mouse monoclonal antibodies were purchased as follows: FceRI (MAR-1) from eBioscience, CD44 (IM7), CD45 (30-F11), CD117 (2B8) from Biolegend, CD8 (53-6.7) and CD4 (RM4-5) from BD Bioscience, CD31 (MEC7.46) from Abcam Inc. IL-3, and stem cell factor (SCF) were purchased from Peprotech.

Immunohistochemistry

Mast cells were detected by Toluidine Blue staining of formalin-fixed samples as described [20]. Microvascular density in frozen, acetone-fixed tumor sections was determined by staining with an antibody to CD31 (PECAM) as described [46].

Cell culture and tumor challenge

MB49 was maintained in RPMI complete medium with 10 % FBS. Mice were given 2.5 × 105 MB49 cells by intradermal (i.d.) route, and tumor diameters were measured with a caliper thrice weekly. Alternatively, mice were given tumor cells (2.5 × 105) intravenously (i.v.) in the tail vein and mice were monitored for survival.

Mast cell reconstitution

Bone-marrow-derived MCs were generated by culturing bone marrow cells with IL-3 (20 ng/ml) and SCF (50 ng/ml) for 5–8 weeks [47, 48]. Purity was assessed by CD117 (c-Kit) and FceRI staining. A total of 5 × 106 BMMCs were injected i.d., i.v., and intraperitoneally into Wsh recipients, which were rested 8 weeks before use.

In vivo depletion of T cell subsets

Hybridoma cell lines GK1.5 (anti-CD4) and 2.43 (anti-CD8) from ATCC were used to prepare depletion antibodies as described [49]. Antibodies were given on day −4 and 0 prior to tumor inoculation and weekly thereafter (250 μg/mouse). A 95 % reduction of targeted cells was confirmed by flow cytometry.

Enzyme-linked immunospot assay

IFN-γ enzyme-linked immunospot assay (ELISPOT; Mabtech) was performed as described previously [49]. Briefly, magnetic-bead purified CD8+ T cells from tumor dLN were plated at 2 × 105 per well with 2 × 105 irradiated MB49 tumor cells or 2 × 105 irradiated male T-depleted spleen cells for 12–16 h. ELISPOTs were detected using BD Biosciences reagents according to manufacturer’s protocol. Spots were counted using an Automated ELISPOT Reader System with KS 4.3 software (Carl Zeiss).

Isolation and characterization of tumor-infiltrating cells

Tumors were dissociated mechanically in PBS/5 mM EDTA, filtered (40 μm mesh), cells blocked in HBSS with 10 % serum, washed, and resuspended in PBS. Amine-reactive LIVE/DEAD near-IR fixable dye (Invitrogen) was included in mAb staining cocktails to stain dead cells, which were excluded from the analysis. CountBright beads (Invitrogen) were added to calculate absolute numbers of tumor cells (live CD45neg SSChi FSChi), CD4+CD44hi, and CD8+CD44hi T cells per tumor. The ratios of tumor and T cells were calculated from the total numbers of tumor cells and T effector cells in tumors.

Statistical analysis

Data were expressed as the mean ± SEM and differences between groups were analyzed by two-tailed ANOVA, the unpaired t test, and the Mann–Whitney test. To detect differences in survival, log-rank analyses of Kaplan–Meier data were conducted (GraphPad Software).

Notes

Acknowledgments

This work was supported by NIH grants CA123079 and CA123079-03S2 (R. J. N.), and CA124515 (J. R. C.) and HL 083249 (R. V. S).

Conflict of interest

The authors declare no competing financial interests.

References

  1. 1.
    Keibel A, Singh V, Sharma MC (2009) Inflammation, microenvironment, and the immune system in cancer progression. Curr Pharm Des 15(17):1949–1955PubMedCrossRefGoogle Scholar
  2. 2.
    Coussens LM, Raymond WW, Bergers G et al (1999) Inflammatory cells up-regualate angiogenesis during squamous epithelial carcinogenesis. Genes Dev 13:1382–1397PubMedCrossRefGoogle Scholar
  3. 3.
    Gounaris E, Erdman S, Restaino C et al (2007) Mast cells are an essential hematopoietic component for polyp development. Proc Natl Acad Sci USA 104:19977–19982PubMedCrossRefGoogle Scholar
  4. 4.
    Soucek L, Lawlor ER, Soto D, Shchors K, Swigart LB, Evan GI (2007) Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nat Med 13(10):1211–1218. doi:10.1038/nm1649 PubMedCrossRefGoogle Scholar
  5. 5.
    Yang FC, Ingram DA, Chen S, Zhu Y, Yuan J, Li X, Yang X, Knowles S, Horn W, Li Y, Zhang S, Yang Y, Vakili ST, Yu M, Burns D, Robertson K, Hutchins G, Parada LF, Clapp DW (2008) Nf1-dependent tumors require a microenvironment containing Nf1 ± and c-kit-dependent bone marrow. Cell 135(3):437–448. doi:10.1016/j.cell.2008.08.041 PubMedCrossRefGoogle Scholar
  6. 6.
    Dethlefsen SM, Matsuura N, Zetter BR (1994) Mast cell accumulation at sites of murine tumor implantation: implications for angiogenesis and tumor metastasis. Invasion Metastasis 14(1–6):395–408PubMedGoogle Scholar
  7. 7.
    Starkey JR, Crowle PK, Taubenberger S (1988) Mast-cell-deficient W/Wv mice exhibit a decreased rate of tumor angiogenesis. Int J Cancer 42(1):48–52PubMedCrossRefGoogle Scholar
  8. 8.
    Blatner NR, Bonertz A, Beckhove P, Cheon EC, Krantz SB, Strouch M, Weitz J, Koch M, Halverson AL, Bentrem DJ, Khazaie K (2010) In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction. Proc Natl Acad Sci USA 107(14):6430–6435. doi:10.1073/pnas.0913683107 PubMedCrossRefGoogle Scholar
  9. 9.
    Huang B, Lei Z, Zhang GM et al (2008) SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment. Blood 112(4):1269–1279PubMedCrossRefGoogle Scholar
  10. 10.
    Yang Z, Zhang B, Li D, Lv M, Huang C, Shen GX, Huang B (2010) Mast cells mobilize myeloid-derived suppressor cells and Treg cells in tumor microenvironment via IL-17 pathway in murine hepatocarcinoma model. PLoS ONE 5(1):e8922. doi:10.1371/journal.pone.0008922 PubMedCrossRefGoogle Scholar
  11. 11.
    Gulubova M, Vlaykova T (2009) Prognostic significance of mast cell number and microvascular density for the survival of patients with primary colorectal cancer. J Gastroenterol Hepatol 24(7):1265–1275. doi:10.1111/j.1440-1746.2007.05009.x PubMedCrossRefGoogle Scholar
  12. 12.
    Johansson A, Rudolfsson S, Hammarsten P, Halin S, Pietras K, Jones J, Stattin P, Egevad L, Granfors T, Wikstrom P, Bergh A (2010) Mast cells are novel independent prognostic markers in prostate cancer and represent a target for therapy. Am J Pathol 177(2):1031–1041. doi:10.2353/ajpath.2010.100070 PubMedCrossRefGoogle Scholar
  13. 13.
    Ribatti D, Ennas MG, Vacca A, Ferreli F, Nico B, Orru S, Sirigu P (2003) Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma. Eur J Clin Invest 33(5):420–425PubMedCrossRefGoogle Scholar
  14. 14.
    Yodavudh S, Tangjitgamol S, Puangsa-art S (2008) Prognostic significance of microvessel density and mast cell density for the survival of Thai patients with primary colorectal cancer. J Med Assoc Thai 91(5):723–732PubMedGoogle Scholar
  15. 15.
    Camus M, Tosolini M, Mlecnik B, Pages F, Kirilovsky A, Berger A, Costes A, Bindea G, Charoentong P, Bruneval P, Trajanoski Z, Fridman WH, Galon J (2009) Coordination of intratumoral immune reaction and human colorectal cancer recurrence. Cancer Res 69(6):2685–2693. doi:10.1158/0008-5472.CAN-08-2654 PubMedCrossRefGoogle Scholar
  16. 16.
    Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, Cascinelli N (1996) Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 77(7):1303–1310. doi:10.1002/(SICI)1097-0142(19960401)77:7<1303:AID-CNCR12>3.0.CO;2-5 PubMedCrossRefGoogle Scholar
  17. 17.
    Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P, Zinzindohoue F, Bruneval P, Cugnenc PH, Trajanoski Z, Fridman WH, Pages F (2006) Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313(5795):1960–1964. doi:10.1126/science.1129139 PubMedCrossRefGoogle Scholar
  18. 18.
    Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, Makrigiannakis A, Gray H, Schlienger K, Liebman MN, Rubin SC, Coukos G (2003) Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 348(3):203–213. doi:10.1056/NEJMoa020177 PubMedCrossRefGoogle Scholar
  19. 19.
    Galli SJ, Grimbaldeston M, Tsai M (2008) Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat Rev Immunol 8(6):478–486. doi:10.1038/nri2327 PubMedCrossRefGoogle Scholar
  20. 20.
    Grimbaldeston MA, Chen CC, Piliponsky AM et al (2005) Mast cell-deficient W-sash c-kit mutant KitW-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am J Pathol 167(3):835–848PubMedCrossRefGoogle Scholar
  21. 21.
    Wolters PJ, Mallen-St Clair J, Lewis CC, Villalta SA, Baluk P, Erle DJ, Caughey GH (2005) Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit(W-sh)/Kit(W-sh) sash mice. Clin Exp Allergy 35(1):82–88. doi:10.1111/j.1365-2222.2005.02136.x PubMedCrossRefGoogle Scholar
  22. 22.
    Melchionda F, McKirdy MK, Medeiros F, Fry TJ, Mackall CL (2004) Escape from immune surveillance does not result in tolerance to tumor-associated antigens. J Immunother 27(5):329–338PubMedCrossRefGoogle Scholar
  23. 23.
    Loskog A, Ninalga C, Hedlund T, Alimohammadi M, Malmstrom PU, Totterman TH (2005) Optimization of the MB49 mouse bladder cancer model for adenoviral gene therapy. Lab Anim 39(4):384–393. doi:10.1258/002367705774286475 PubMedCrossRefGoogle Scholar
  24. 24.
    Khazaie K, Blatner NR, Khan MW, Gounari F, Gounaris E, Dennis K, Bonertz A, Tsai FN, Strouch MJ, Cheon E, Phillips JD, Beckhove P, Bentrem DJ (2011) The significant role of mast cells in cancer. Cancer Metastasis Rev 30(1):45–60. doi:10.1007/s10555-011-9286-z PubMedCrossRefGoogle Scholar
  25. 25.
    Hsu JY, Wakelee HA (2009) Monoclonal antibodies targeting vascular endothelial growth factor: current status and future challenges in cancer therapy. BioDrugs 23(5):289–304. doi:10.2165/11317600-000000000-00000 PubMedCrossRefGoogle Scholar
  26. 26.
    Patel D, Bassi R, Hooper AT, Sun H, Huber J, Hicklin DJ, Kang X (2008) Enhanced suppression of melanoma tumor growth and metastasis by combined therapy with anti-VEGF receptor and anti-TYRP-1/gp75 monoclonal antibodies. Anticancer Res 28(5A):2679–2686PubMedGoogle Scholar
  27. 27.
    Ribatti D, Crivellato E (2010) Mast cells, angiogenesis, and tumour growth. Biochim Biophys Acta. doi:10.1016/j.bbadis.2010.11.010 PubMedGoogle Scholar
  28. 28.
    Aoki M, Pawankar R, Niimi Y, Kawana S (2003) Mast cells in basal cell carcinoma express VEGF, IL-8 and RANTES. Int Arch Allergy Immunol 130(3):216–223. doi:10.1159/000069515 PubMedCrossRefGoogle Scholar
  29. 29.
    Li B, Lalani AS, Harding TC, Luan B, Koprivnikar K, Huan TuG, Prell R, VanRoey MJ, Simmons AD, Jooss K (2006) Vascular endothelial growth factor blockade reduces intratumoral regulatory T cells and enhances the efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer Res 12(22):6808–6816. doi:10.1158/1078-0432.CCR-06-1558 PubMedCrossRefGoogle Scholar
  30. 30.
    Shrimali RK, Yu Z, Theoret MR, Chinnasamy D, Restifo NP, Rosenberg SA (2010) Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer. Cancer Res 70(15):6171–6180. doi:10.1158/0008-5472.CAN-10-0153 PubMedCrossRefGoogle Scholar
  31. 31.
    Suto H, Nakae S, Kakurai M et al (2006) Mast cell-associated TNF promotes dendritic cell migration. J Immunol 176:4102–4112PubMedGoogle Scholar
  32. 32.
    Sayed BA, Christy A, Quirion MR, Brown MA (2008) The master switch: the role of mast cells in autoimmunity and tolerance. Annu Rev Immunol 26:705–739. doi:10.1146/annurev.immunol.26.021607.090320 PubMedCrossRefGoogle Scholar
  33. 33.
    Grunig G, Banz A, de Waal Malefyt R (2005) Molecular regulation of Th2 immunity by dendritic cells. Pharmacol Ther 106(1):75–96. doi:10.1016/j.pharmthera.2004.11.004 PubMedCrossRefGoogle Scholar
  34. 34.
    Reuter S, Heinz A, Sieren M, Wiewrodt R, Gelfand EW, Stassen M, Buhl R, Taube C (2008) Mast cell-derived tumour necrosis factor is essential for allergic airway disease. Eur Respir J 31(4):773–782. doi:10.1183/09031936.00058907 PubMedCrossRefGoogle Scholar
  35. 35.
    McIlroy A, Caron G, Blanchard S, Fremaux I, Duluc D, Delneste Y, Chevailler A, Jeannin P (2006) Histamine and prostaglandin E up-regulate the production of Th2-attracting chemokines (CCL17 and CCL22) and down-regulate IFN-gamma-induced CXCL10 production by immature human dendritic cells. Immunology 117(4):507–516. doi:10.1111/j.1365-2567.2006.02326.x PubMedCrossRefGoogle Scholar
  36. 36.
    Mazzoni A, Siraganian RP, Leifer CA, Segal DM (2006) Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells. J Immunol 177(6):3577–3581PubMedGoogle Scholar
  37. 37.
    Theiner G, Gessner A, Lutz MB (2006) The mast cell mediator PGD2 suppresses IL-12 release by dendritic cells leading to Th2 polarized immune responses in vivo. Immunobiology 211(6–8):463–472. doi:10.1016/j.imbio.2006.05.020 PubMedCrossRefGoogle Scholar
  38. 38.
    Gaudenzio N, Espagnole N, Mars LT, Liblau R, Valitutti S, Espinosa E (2009) Cell-cell cooperation at the T helper cell/mast cell immunological synapse. Blood. doi:10.1182/blood-2009-02-202648 PubMedGoogle Scholar
  39. 39.
    Nakae S, Suto H, Iikura M et al (2006) Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF. J Immunol 176:2238–2248PubMedGoogle Scholar
  40. 40.
    Kambayashi T, Allenspach EJ, Chang JT, Zou T, Shoag JE, Reiner SL, Caton AJ, Koretzky GA (2009) Inducible MHC class II expression by mast cells supports effector and regulatory T cell activation. J Immunol 182(8):4686–4695. doi:10.4049/jimmunol.0803180 PubMedCrossRefGoogle Scholar
  41. 41.
    Kitawaki T, Kadowaki N, Sugimoto N, Kambe N, Hori T, Miyachi Y, Nakahata T, Uchiyama T (2006) IgE-activated mast cells in combination with pro-inflammatory factors induce Th2-promoting dendritic cells. Int Immunol 18(12):1789–1799. doi:10.1093/intimm/dxl113 PubMedCrossRefGoogle Scholar
  42. 42.
    Nakae S, Iikura M, Suto H, Akiba H, Umetsu DT, Dekruyff RH, Saito H, Galli SJ (2007) TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells. Blood 110(7):2565–2568. doi:10.1182/blood-2006-11-058800 PubMedCrossRefGoogle Scholar
  43. 43.
    DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, Coussens LM (2009) CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell 16(2):91–102. doi:10.1016/j.ccr.2009.06.018 PubMedCrossRefGoogle Scholar
  44. 44.
    Depinay N, Hacini F, Beghdadi W et al (2006) Mast cell-dependent down-regulation of antigen-specific immune responses by mosquito bites. J Immunol 176:4141–4146PubMedGoogle Scholar
  45. 45.
    Dalton DK, Noelle RJ (2012) The roles of mast cells in anticancer immunity. Cancer Immunol Immunother. doi:10.1007/S00262-012-1246-0
  46. 46.
    Benencia F, Courreges MC, Conejo-Garcia JR, Buckanovich RJ, Zhang L, Carroll RH, Morgan MA, Coukos G (2005) Oncolytic HSV exerts direct antiangiogenic activity in ovarian carcinoma. Hum Gene Ther 16(6):765–778. doi:10.1089/hum.2005.16.765 PubMedCrossRefGoogle Scholar
  47. 47.
    Razin E (1990) Culture of bone marrow-derived mast cells: a model for studying oxidative metabolism of arachidonic acid and synthesis of other molecules derived from membrane phospholipids. Methods Enzymol 187:514–520PubMedCrossRefGoogle Scholar
  48. 48.
    Saitoh S, Arudchandran R, Manetz TS, Zhang W, Sommers CL, Love PE, Rivera J, Samelson LE (2000) LAT is essential for Fc(epsilon)RI-mediated mast cell activation. Immunity 12(5):525–535PubMedCrossRefGoogle Scholar
  49. 49.
    Turk MJ, Guevara-Patino JA, Rizzuto GA, Engelhorn ME, Sakaguchi S, Houghton AN (2004) Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J Exp Med 200(6):771–782. doi:10.1084/jem.20041130 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Anna Wasiuk
    • 1
    • 2
  • Dyana K. Dalton
    • 1
  • William L. Schpero
    • 1
  • Radu V. Stan
    • 1
  • Jose R. Conejo-Garcia
    • 3
  • Randolph J. Noelle
    • 1
    • 4
  1. 1.Department of Microbiology and ImmunologyDartmouth Medical SchoolLebanonUSA
  2. 2.Cell and Developmental BiologyOregon Health & Science UniversityPortlandUSA
  3. 3.The Wistar InstitutePhiladelphiaUSA
  4. 4.King’s College London, King’s Health Partners, Medical Research Council (MRC) Centre for TransplantationGuy’s HospitalLondonUK

Personalised recommendations