Mast cells impair the development of protective anti-tumor immunity
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.
KeywordsCancer Tumor immunity Microenvironment T cells
Inflammatory immune cells contribute to the development of tumors by promoting neovascularization, tissue remodeling, and inflammation . 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 .
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 . 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 .
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.
MCs accumulated around MB49 tumors and were associated with increased angiogenesis within tumors
Mast cell-deficient Wsh females, but not Wsh males controlled the growth of MB49 tumors
Reconstitution of Wsh female mice with MCs abolished their enhanced resistance to systemic tumors
Enhanced tumor protection was T cell dependent in female Wsh mice
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
Mast cell-deficient female mice have significantly increased ratios of CD4+ and CD8+ T effector cells relative to tumor cells in the tumor microenvironment
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.
The role of MCs in promoting malignancy is multifaceted, as they orchestrate the character of the tumor microenvironment promoting inflammation, angiogenesis, and tissue remodeling . 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 . 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 . 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 . 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 . 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 . 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 . 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 . Studies are currently underway to understand the precise mechanisms by which MCs influence the adaptive immune responses to tumors.
Materials and methods
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.
Mast cells were detected by Toluidine Blue staining of formalin-fixed samples as described . Microvascular density in frozen, acetone-fixed tumor sections was determined by staining with an antibody to CD31 (PECAM) as described .
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 . 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 . 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.
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).
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.
- 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
- 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
- 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
- 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.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.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.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
- 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
- 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.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
- 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
- 45.Dalton DK, Noelle RJ (2012) The roles of mast cells in anticancer immunity. Cancer Immunol Immunother. doi:10.1007/S00262-012-1246-0