Andrew J. Gunderson

B cells regulate macrophage phenotype and response to chemotherapy in squamous carcinomas.

B cells foster squamous cell carcinoma (SCC) development through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγ receptor-dependent activation of myeloid cells. Because human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced support for SCC growth. Although ineffective as a single agent, treatment of mice bearing preexisting SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and Taxol-based chemotherapy. Improved chemoresponsiveness was dependent on altered chemokine expression by macrophages that promoted tumor infiltration of activated CD8(+) lymphocytes via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anticancer therapy in select tumors.

Bruton Tyrosine Kinase–Dependent Immune Cell Cross-talk Drives Pancreas Cancer

UNLABELLED Pancreas ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here, we report that targeting Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, restores T cell-dependent antitumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy. We report that PDAC tumor growth depends on cross-talk between B cells and FcRγ(+) tumor-associated macrophages, resulting in T(H)2-type macrophage programming via BTK activation in a PI3Kγ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a T(H)1 phenotype that fostered CD8(+) T-cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacologic inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. SIGNIFICANCE We report that BTK regulates B-cell and macrophage-mediated T-cell suppression in pancreas adenocarcinomas. Inhibition of BTK with the FDA-approved inhibitor ibrutinib restores T cell-dependent antitumor immune responses to inhibit PDAC growth and improves responsiveness to chemotherapy, presenting a new therapeutic modality for pancreas cancer.

B cells and their Mediators as Targets for Therapy in Solid Tumors

B cells have recently been appreciated as paracrine mediators of solid tumor development. Their ability to influence various hallmarks of cancer development, aside from antigen presentation, can be attributed to the diversity of soluble mediators they express, including cytokines and immunoglobulins, that can act directly and indirectly on the diversity of leukocyte subsets that infiltrate developing tumors, evolving neoplastic cells, as well as select T cell populations in secondary lymphoid organs and within tumor stroma. Herein, we review the literature supporting these interactions and discuss novel approaches to ameliorate protumoral B cell effects for anti-cancer therapy.

CD8+ T Cells Mediate RAS-Induced Psoriasis-Like Skin Inflammation through IFN-γ

The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RASV12G in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses and increased Th1/Th17 and Tc1/Tc17 skin infiltration. The majority of skin infiltrating CD8+ T cells co-expressed IFN-γ and IL-17A. When RAS was expressed on a Rag1−/− background, microabscess formation, iNOS expression and keratinocyte hyperproliferation were suppressed. Depletion of CD8+ but not CD4+ T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17+ γΔ T cells, and epidermal hyperproliferation to levels similar to a Rag1−/− background. Reconstitution of Rag1−/− inducible RAS mice with purified CD8+ T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ but not IL-17A in CD8+ T cell reconstituted Rag1−/− mice expressing RAS blocked CD8-mediated skin inflammation, iNOS expression and keratinocyte hyperproliferation. These results show for that CD8+ T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.

TGFβ1-Induced Inflammation in Premalignant Epidermal Squamous Lesions Requires IL-17

Overexpression of transforming growth factor-beta1 (TGFbeta1) in the normal epidermis can provoke an inflammatory response, but whether this occurs within a developing tumor is not clear. To test this, we used an inducible transgenic mouse to overexpress TGFbeta1 in premalignant squamous lesions. Within 48 hours of TGFbeta1 induction, there was an increase in IL-17 production by both CD4(+) and gammadelta(+) T cells, together with increased expression of T-helper-17 (Th17)-polarizing cytokines. Induction of TGFbeta1 in premalignant primary keratinocytes elevated the expression of proinflammatory and Th17-polarizing cytokines, and the keratinocyte-conditioned media caused IL-17 production by naive T cells that was dependent on T-cell TGFbeta1 signaling. Microarray analysis showed significant upregulation of proinflammatory genes 2 days after TGFbeta1 induction, and this was followed by increased MPO(+), F4/80(+), and CD8(+) cells in tumors, increased CD8(+) effectors and IFNgamma(+) cells in skin-draining LNs, and tumor regression. In parallel, the percentage of tumor CD11b(+)Ly6G(+) neutrophils was reduced. Neutralization of IL-17 blocked TGFbeta1-induced CD11b(+) Ly6G(-) tumor infiltration but did not alter the reduction of neutrophils or tumor regression. Thus, TGFbeta1 overexpression causes IL-17-dependent and IL-17-independent changes in the premalignant tumor inflammatory microenvironment.

TGFβ1 Overexpression by Keratinocytes Alters Skin Dendritic Cell Homeostasis and Enhances Contact Hypersensitivity

Overexpression of Transforming Growth Factor Beta1 (TGFβ1) in mouse epidermis causes cutaneous inflammation and keratinocyte hyperproliferation. Here, we examined acute effects of TGFβ1 overproduction by keratinocytes on skin dendritic cells (DCs). TGFβ1 induction for 2 and 4 days increased numbers and CD86 expression of B220+ plasmacytoid DCs (pDCs) and CD207+CD103+, CD207−CD103−CD11b+ and CD207−CD103−CD11b− dermal DCs (dDCs) in skin draining lymph nodes (SDLN). The dermis of TGFβ1-overexpressing mice had significantly more pDCs, CD207+CD103+ dDCs and CD207-CD11b+ dDCs in the absence of increased dermal proliferation. Application of dye, TRITC, in dibutylpthalate (DBP) solution after TGFβ1 induction increased the numbers of TRITC+CD207− dDCs in SDLN, and augmented TRITC/DBP-induced Langerhans cell (LC) migration 72 hrs post-TRITC treatment. Consistent with this, LC migration was increased in vitro by TGFβ1 overexpression in skin explants and by exogenous TGFβ1 in culture media. Transient TGFβ1 induction during DNFB sensitization increased contact hypersensitivity responses by 1.5-fold. Thus, elevated epidermal TGFβ1 alone is sufficient to alter homeostasis of multiple cutaneous DC subsets and enhance DC migration and immune responses to contact sensitizers. These results highlight a role for keratinocyte-derived TGFβ1 in DC trafficking and the initiation of skin inflammation.

Tumor-promoting role of TGFβ1 signaling in ultraviolet B-induced skin carcinogenesis is associated with cutaneous inflammation and lymph node migration of dermal dendritic cells

Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine in the skin that can function both as a tumor promoter and suppressor in chemically induced skin carcinogenesis, but the function in ultraviolet B (UVB) carcinogenesis is not well understood. Treatment of SKH1 hairless mice with the activin-like kinase 5 (ALK5) inhibitor SB431542 to block UVB-induced activation of cutaneous TGFβ1 signaling suppressed skin tumor formation but did not alter tumor size or tumor cell proliferation. Tumors that arose in SB-treated mice after 30 weeks had significantly reduced percentage of IFNγ(+) tumor-infiltrating lymphocytes compared with control mice. SB431542 blocked acute and chronic UVB-induced skin inflammation and T-cell activation in the skin-draining lymph node (SDLN) and skin but did not alter UVB-induced epidermal proliferation. We tested the effect of SB431542 on migration of skin dendritic cell (DC) populations because DCs are critical mediators of T-cell activation and cutaneous inflammation. SB431542 blocked (i) UVB-induced Smad2 phosphorylation in dermal DC (dDC) and (ii) SDLN and ear explant migration of CD103(+) CD207(+) and CD207(-) skin DC subsets but did not affect basal or UV-induced migration of Langerhans cells. Mice expressing a dominant-negative TGFβ type II receptor in CD11c(+) cells had reduced basal and UVB-induced SDLN migration of CD103(+) CD207(+) and CD207(-) DC subsets and a reduced percentage of CD86(high) dDC following UVB irradiation. Together, these suggest that TGFβ1 signaling has a tumor-promoting role in UVB-induced skin carcinogenesis and this is mediated in part through its role in UVB-induced migration of dDC and cutaneous inflammation.

Multiplex immunohistochemistry for immune profiling of HPV-associated head and neck cancer

Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically distinct from HPV-negative HNSCC, and as such requires differential therapeutic approaches. Accumulating evidence indicates a significant linkage between the immune response within the tissue and pathogenesis of HPV-associated HNSCC. To further elucidate immune-related signatures in HPV-associated HNSCC, we performed multiplex histological analysis in de-identified tissue microarray sections including HPV-positive (n = 21), HPV-negative (n = 17), and normal oropharynx (n = 8). Following immunohistochemistry (IHC) for CD45, CD3, CD8, Foxp3, T-bet, GATA-3, RORgT, CD20, CD56, CD68, MHC class II, CSF1R, CD66b, tryptase, CD83, DC-SIGN, PD-1, and PD-L1, the cell intensity per mm2 ratio/composition, localization were quantitatively evaluated. The HPV-status was confirmed by HPV16/18 polymerase chain reaction and IHC for p16INK4a. IHC for p16 or EpCAM were utilized for defining tumor region. Infiltration of T cell populations including CD45+CD3+CD8+ T cells (P < 0.01), CD45+CD3+CD8−Foxp3+ regulatory T cells (P < 0.05) and CD45+CD3+CD8−Foxp3+T-bet+ Th1 cells (P < 0.01), CD45+CD20+CD3−B cells (P < 0.05), CD45+CD68+CD163− macrophages (P < 0.001), and CD45+Tryptase+ mast cells (P < 0.01) was significantly higher in the HPV-positive group than in the HPV-negative group. CD8/CD68 ratio of HPV-positive tumor was higher than that of HPV-negative tumor (P < 0.05), and the highest CD163−CD68+/CD163+CD68+ ratio was observed in the intra-tumor region of HPV-positive tumors. High PD-L1 expression on CD68+CD163+ macrophages and MHC class II+CD83+ dendritic cells was intensively observed in the intra-tumor region of the HPV-positive group while maturation of dendritic cells assessed by CD83/DC-SIGN ratio was significantly higher in the peritumoral stroma than intra-tumor regions (P < 0.05), indicating distinct immune regulatory mechanisms between intra and peritumoral regions. These signatures provide further evidence of anti-tumor immunity against HPV-positive head and neck cancer, and potentially lead to personalized treatment including immunomodulatory therapeutic targets specialized for the HPV/immune status.

Differentiated state of initiating tumor cells is key to distinctive immune responses seen in H-RasG12V-induced squamous tumors

Immune therapies are usually successful in a fraction of patients. Squamous cell carcinomas originate in a stratified epithelium, and distinctly different immune responses were found to be driven by the state of differentiation of the tumor-initiating cell. Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-RasG12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1−/− background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1+/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198–210. ©2017 AACR.

Abstract 2673: Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. We used doxycycline-inducible epidermal squamous cancer models driven by oncogenic Ras to test whether the origin of the tumor-initiating cell influenced the tumor immune response. Threshold doxycycline induced expression of H-RasG12V in either the basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), caused distinct immune responses despite similar levels of Ras, tumor latency, and tumor numbers. End stage K14Ras tumors had an immunosuppressed microenvironment with abundant Tregs and Bregs which evolved from an initially Th1 dominant environment in the earliest detectable lesions. In contrast InvRas tumors had a Th2 pro-inflammatory microenvironment, with significantly fewer Tregs or Bregs at any time point. This difference in immune microenvironment between the tumor models was also found in hyperplastic skin after 5 days of maximal Ras expression, indicating that it was not simply a property of the end stage tumor. Surprisingly, adaptive immunity had opposite roles in tumor development. InvRasRag1-/- mice developed fewer and smaller tumors that regressed while K14RasRag1-/- mice developed more tumors with shorter latency than Rag1+/+ controls. In both models adoptive transfer and depletion studies showed that cooperation between B and CD4 T cells drove the opposing tumor responses, lymphocyte polarization, and tumor immune phenotype. Co-culture of tumor-conditioned but not splenic B cells from each model with naive CD4 T cells showed that direct contact and CD40-CD40L ligation was required for opposite polarization towards either a Th2 or Treg phenotype. Importantly, we found that anti-CD40L mAb caused regression of preexisting InvRas tumors but enhanced growth of K14Ras tumors. In contrast, CD40 agonist mAb enhanced growth of preexisting InvRas tumors, and suppressed growth of K14Ras tumors. Thus in an in vivo setting the type of tumor immune microenvironment and opposing role of CD40-CD40L in tumor development generates distinct responses to therapeutic antibodies. Together these data show that the position of a tumor initiating cell within the stem cell hierarchy in a stratified squamous epithelia has important consequences for the type of tumor immune microenvironment and response to checkpoint therapy. Citation Format: Adam B. Glick, Jacob T. Bailey, Andrew Gunderson, Kyle Breech, Michael Podolsky. Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2673. doi:10.1158/1538-7445.AM2017-2673