Workshop on challenges, insights, and future directions for mouse and humanized models in cancer immunology and immunotherapy: a report from the associated programs of the 2016 annual meeting for the Society for Immunotherapy of cancer.

Understanding how murine models can elucidate the mechanisms underlying antitumor immune responses and advance immune-based drug development is essential to advancing the field of cancer immunotherapy. The Society for Immunotherapy of Cancer (SITC) convened a workshop titled, "Challenges, Insights, and Future Directions for Mouse and Humanized Models in Cancer Immunology and Immunotherapy" as part of the SITC 31st Annual Meeting and Associated Programs on November 10, 2016 in National Harbor, MD. The workshop focused on key issues in optimizing models for cancer immunotherapy research, with discussions on the strengths and weaknesses of current models, approaches to improve the predictive value of mouse models, and advances in cancer modeling that are anticipated in the near future. This full-day program provided an introduction to the most common immunocompetent and humanized models used in cancer immunology and immunotherapy research, and addressed the use of models to evaluate immune-targeting therapies. Here, we summarize the workshop presentations and subsequent panel discussion.

Antagonizing deactivating cytokines to enhance host defense and chemotherapy in experimental visceral leishmaniasis.

In experimental visceral leishmaniasis, inhibition of interleukin 10 (IL-10) signaling enhances Th1-cell-associated responses, promoting gamma interferon (IFN-gamma) secretion, granuloma assembly, macrophage activation with substantial liver parasite killing, and synergy with pentavalent antimony (Sb) chemotherapy. To determine if inhibiting other suppressive cytokines has similar therapeutic potential, Leishmania donovani-infected BALB/c mice were injected with anti-IL-4 monoclonal antibody or receptor fusion antagonists of IL-13 or transforming growth factor beta (TGF-beta). Targeting IL-13 or TGF-beta enabled inhibition of L. donovani replication but little parasite killing; anti-IL-4 had no effect. None of the three antagonists promoted IFN-gamma production, granuloma maturation, or Sb efficacy. Excess IL-13 and TGF-beta exacerbated liver infection; however, effects were transient. Among IL-10, IL-4, IL-13, and TGF-beta, cytokines capable of disabling Th1-cell mechanisms (including those which support chemotherapy), IL-10 appears to be the appropriate target for therapeutic inhibition in visceral L. donovani infection.

A monoclonal antibody to alpha1beta1 blocks antigen-induced airway responses in sheep.

The integrin alpha1beta1 (very late antigen-1; CD49a/CD29) is a major adhesion receptor for collagen I, IV, and VI, and its induced expression on activated monocytes and lymphocytes plays a central role in their retention and activation at inflammatory sites in autoimmune pathologies. However, the role of alpha1beta1 in allergic settings has not been explored. In this study, we show that a single 45-mg dose of aerosolized monoclonal antibody AQC2 to the alpha1 chain of human and sheep very late antigen-1, given 30 minutes before challenge, blocks both the allergen-induced late response and the associated airway hyperresponsiveness, functional indicators of allergen-induced inflammation, in sheep. AQC2 does not affect the early response. Consistent with these effects, AQC2 tended to reduce the cell response associated with local antigen instillation. An isotype-matched control antibody had no protective effects. Two humanized versions of AQC2, a wild-type IgG1 and an aglycosyl form of the same monoclonal antibody, which has reduced Fc receptor-mediated effector functions, are equally effective in blocking the antigen-induced late response and airway hyperresponsiveness in the sheep model. These data suggest that mononuclear leukocyte adhesion-dependent pathologies contribute to allergic lung disease and provide proof-of-concept that antagonists of alpha1 integrins may be useful in preventing these events.

Cell and molecular regulation of endothelin-1 production during hepatic wound healing.

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.

Transforming growth factor-beta-dependent events in vascular remodeling following arterial injury.

Constrictive remodeling has been identified as a major contributor to restenosis following angioplasty. Characterization of transforming growth factor-beta (TGF-beta)-mediated cellular events in the adventitia and their contribution to vascular remodeling, however, has not previously been studied in detail. The balloon catheter denudation model was performed on rat carotid artery, and groups of rats were treated with vehicle or a TGF-beta inhibitor, a soluble TGF-beta receptor type II (TGF-beta R:Fc). Adventitial cell proliferation, which peaked 4 days after injury, was characterized by the de novo formation of several cell layers surrounding the outer adventitia and this process was not dependent upon TGF-beta activity. These neoadventitial cells expressed an abundance of collagen type I and a fetal isoform of fibronectin containing the EIIIA domain, and the expression of both proteins was suppressed in the presence of TGF-beta R:Fc. Lumenal narrowing was apparent 14 days after injury. Inhibition of TGF-beta signaling promoted vessel enlargement. As a result, lumen size did not change despite neointima formation. In conclusion, adventitial fibrosis with abundant collagen matrix deposition but not adventitial cell proliferation is dependent upon endogenous TGF-beta activity. Furthermore, inhibition of TGF-beta signaling prevents injury-induced reduction in lumen area by promoting vessel enlargement.

Interference with TGF-beta1 and -beta3 in tumor stroma lowers tumor interstitial fluid pressure independently of growth in experimental carcinoma.

A high tumor interstitial fluid pressure (TIFP) is a pathologic characteristic distinguishing the stroma of carcinomas from normal interstitial loose connective tissues. The role of TGF-beta1 and -beta3 in generating a high TIFP was investigated in xenografted experimental anaplastic thyroid carcinoma (ATC) derived from the human ATC cell line KAT-4. A single intravenous injection of a soluble recombinant TGF-beta receptor type II-murine Fc:IgG(2A) chimeric protein that specifically inhibits TGF-beta1 and -beta3, significantly lowered TIFP in a time and concentration dependent manner but did not change total tissue water content in the tumors. Tumor growth rate was higher in tumors treated with the TGF-beta1 and -beta3 inhibitor compared to control tumors during the first 10 days after administration of the inhibitor. The apoptotic index of carcinoma cells, and expression of the cell cycle inhibitor p27(Kip1), were, however, increased in TGF-beta1 and -beta3 inhibitor-treated tumors. Prolonged treatment periods and administration of a second dose of the inhibitor decreased tumor growth rate. The TGF-beta1 and -beta3 inhibitor did not affect proliferation or expression of phosphorylated Smad2 protein in KAT-4 cells cultured in vitro. Our results indicate that members of the TGF-beta family are potential targets for novel anti-cancer treatment directed to the stroma. First by controlling TIFP and by that potentially the uptake of anticancer drugs into tumors and second by their suggested role in maintaining a supportive tumor stroma.

Effects of delayed treatment with transforming growth factor-beta soluble receptor in a three-dose bleomycin model of lung fibrosis in hamsters.

Transforming growth factor-beta (TGF-beta) plays a pivotal role in an exaggerated synthesis and accumulation of collagen in fibrotic disorders of many organs. We have previously demonstrated that repeated intratracheal (IT) instillation of TGF-beta soluble receptor (TR) in hamsters markedly decreased the bleomycin (BL)-induced lung fibrosis in response to a single dose. The present study was carried out in a 3-dose BL-hamster model of lung fibrosis to better evaluate the therapeutic potential of TR. Three doses of BL (2.5, 2.0, and 1.5 U/4 mL/kg) or an equivalent volume of isotonic saline was administered IT consecutively at weekly intervals, and phosphate-buffered saline (PBS) or TR (4 nmol/0.3 mL/hamster) by the same route twice a week, starting after the 2nd BL or 3rd BL dose. Twenty-one days after the 3rd dose of BL instillation, the hamsters were killed for bronchoalveolar lavage (BAL) and biochemical and histopathological analyses. The results showed that treatment with TR starting after either the 2nd or 3rd dose of BL caused significant reduction in BL-induced lung fibrosis, as demonstrated by marked decreases in the hydroxyproline level and prolyl hydroxylase activity of the lungs. Histopathological evaluation of the lungs also revealed that the hamsters in both BL+TR groups had markedly fewer fibrotic lesions than hamsters in BL+PBS group. These results demonstrate the beneficial effects of delayed treatment with TR in attenuating the progression of ongoing fibrotic process and suggest its potential therapeutic uses in the management of lung fibrosis in humans.

Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases.

TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

The EIIIA segment of fibronectin is a ligand for integrins alpha 9beta 1 and alpha 4beta 1 providing a novel mechanism for regulating cell adhesion by alternative splicing.

Alternative splicing of the fibronectin gene transcript gives rise to forms that include the EIIIA (or ED-A) segment. EIIIA-containing fibronectins are prominently expressed during embryogenesis and wound healing and appear to mediate changes in cell adhesion and gene expression. Nonetheless, integrins that bind the EIIIA segment have not been identified. We previously mapped the epitope for two function-blocking monoclonal antibodies to the C-C' loop region of the EIIIA segment (Liao, Y.-F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884). The sequence of this epitope ((39)PEDGIHELFP(48)) resembles the sequence within tenascin-C to which the integrin alpha(9)beta(1) binds. We now report that either integrin alpha(9)beta(1) or alpha(4)beta(1) can mediate cell adhesion to the EIIIA segment. Moreover, this interaction is blocked both by epitope-mapped EIIIA antibodies as well as by the respective anti-integrins. Deletion mutants of the EIIIA segment that include the C-C' loop and flanking sequence bind cells expressing either alpha(9)beta(1) or alpha(4)beta(1). Adhesion of alpha(4)beta(1)-containing MOLT-3 cells to the EIIIA segment stimulates phosphorylation of p44/42 MAP kinase. Our observation that two integrins bind the EIIIA segment establishes a novel mechanism by which cell adhesion to fibronectin is regulated by alternative splicing.