Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8 lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells (DCs), from the thymic tumor microenvironment (TME) support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in two distinct mouse models of T-ALL, and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of anti-tumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

The provision of T cell co-stimulation via members of the TNFR super-family, including OX40 (CD134) and 4-1BB (CD137), provides critical signals that promote T cell survival and differentiation. Recent studies have demonstrated that ligation of OX40 can augment T cell-mediated anti-tumor immunity in pre-clinical models and more importantly, OX40 agonists are under clinical development for cancer immunotherapy. OX40 is of particular interest as a therapeutic target as it is not expressed on naïve T cells but rather, is transiently up-regulated following TCR stimulation. Although TCR engagement is necessary for inducing OX40 expression, the downstream signals that regulate OX40 itself remain unclear. In this study, we demonstrate that OX40 expression is regulated through a TCR and common gamma chain cytokine-dependent signaling cascade that requires JAK3-mediated activation of the downstream transcription factors STAT3 and STAT5. Furthermore, combined treatment with an agonist anti-OX40 mAb and IL-2 augmented tumor immunotherapy against multiple tumor types. Dual therapy was also able to restore the function of anergic tumor-reactive CD8 T cells in mice with long-term well-established (>5 wks) tumors, leading to increased survival of the tumor-bearing hosts. Together, these data reveal the ability of TCR/common gamma chain cytokine signaling to regulate OX40 expression and demonstrate a novel means of augmenting cancer immunotherapy by providing dual anti-OX40/common gamma chain cytokine-directed therapy.

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Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.

Keywords: CD4 r CD25 r Co-stimulation r IL-2 r Immunotherapy Supporting Information available online IntroductionT-cell survival and effector function are sensitive to the availability of essential cytokines during development, homeostasis, and activation. Interleukin-2 (IL-2) is a 15.5 kDa α-helical protein discovered for its ability to culture T cells long term in vitro [1]. IL-2 has broad effects on T lymphocytes, including survival, Correspondence: Dr. Andrew D. Weinberg e-mail: Andrew.Weinberg@providence.org proliferation, activation-induced cell death (AICD), T-cell differentiation, cytokine production, and immune tolerance [2][3][4]. The high-affinity receptor for IL-2 (IL-2R) is composed of three subunits, the α-subunit (CD25), β-subunit (CD122), and the common γ-chain (CD132). CD122 and CD132 are also subunits for other cytokine receptors, whereas CD25 is specific to the IL-2 receptor. IL-2 signaling occurs exclusively through the cytoplasmic tails of CD122 and CD132; CD25 has a short cytoplasmic tail and is not involved in IL-2 signaling. Instead, CD25 has the highest affinity for IL-2 among the individual subunits and acts as an affinity converter [2]. At high concentrations, IL-2 can signal in the absence of CD25 through CD122 and CD132, which form the C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu1894 Todd A. Triplett et al. Eur. J. Immunol. 2012. 42: 1893-1905 intermediate-affinity IL-2R. However, CD25 in addition to CD122 and CD132 is required to respond to low concentrations of IL-2 by forming the high-affinity IL-2 receptor [2]. Once formed, the IL-2/CD25/CD122/CD132 quaternary complex is short-lived (t 1/2 10-20 min) on the cell surface [5]. Upon internalization, IL-2, CD122, and CD132 are targeted for lysosomal degradation, whereas CD25 is recycled to the cell surface [6,7]. Though CD25 has been shown to influence effector function of lymphocytes, CD25 is thought to play a greater role in immune tolerance in mice [2,8]. Initially, it was found that depletion of CD4 + CD25 + T cells from adoptive cell transfer experiments into nude mice resulted in systemic autoimmune disease [9]. These CD4 + CD25 + cells were later shown to express the transcription factor Foxp3 (FOXP3 in humans) and are now termed regulatory T (Treg) cells that comprise 5-15% of CD4 + T cells in humans [10].Treg cells depend on IL-2 signaling for their survival in vitro and in vivo [11][12][13]. Therefore, constitutive expression of CD25 on Treg cells is thought to be crucial to their survival and maintenance of immune homeostasis. This idea is supported by studies of mice deficient in CD25 or IL-2, which have low numbers of Treg cells and develop severe systemic autoimmune disease as they age [14,15]. Despite the positive effects of IL-2 on effector and memory T cells, CD25/IL-2 deficiency in mice does not appear to greatly hinder T-cell immunity, reviewed elsewhere [8]. Therefore, it is thought that in mice, CD25/IL-2 plays a dominant role in immune tolerance and less for adaptive immunity, perhaps b...

Recent developments in pre-clinical screening tools, that more reliably predict the clinical effects and adverse events of candidate therapeutic agents, has ushered in a new era of drug development and screening. However, given the rapid pace with which these models have emerged, the individual merits of these translational research tools warrant careful evaluation in order to furnish clinical researchers with appropriate information to conduct pre-clinical screening in an accelerated and rational manner. This review assesses the predictive utility of both well-established and emerging pre-clinical methods in terms of their suitability as a screening platform for treatment response, ability to represent pharmacodynamic and pharmacokinetic drug properties, and lastly debates the translational limitations and benefits of these models. To this end, we will describe the current literature on cell culture, organoids, in vivo mouse models, and in silico computational approaches. Particular focus will be devoted to discussing gaps and unmet needs in the literature as well as current advancements and innovations achieved in the field, such as co-clinical trials and future avenues for refinement.

In preclinical tumor models, aOX40 therapy is often successful at treating small tumors, but is less effective once the tumors become large. For a tumor immunotherapy to be successful to cure large tumors, it will most likely require not only an agonist to boost effector T-cell function but also inhibitors of T-cell suppression. In this study, we show that combining aOX40 antibodies with an inhibitor of the TGFb receptor (SM16) synergizes to elicit complete regression of large established MCA205 and CT26 tumors. Evaluation of tumor-infiltrating T cells showed that SM16/aOX40 dual therapy resulted in an increase in proliferating granzyme B þ CD8 T cells, which produced higher levels of IFNg, compared with treatment with either agent alone. We also found that the dual treatment increased pSTAT3 expression in both CD4 and CD8 T cells isolated from tumors. Because others have published that STAT3 signaling is detrimental to T-cell function within the tumor microenvironment, we explored whether deletion of STAT3 in OX40-expressing cells would affect this potent combination therapy. Surprisingly, we found that deletion of STAT3 in OX40-expressing cells decreased the efficacy of this combination therapy, showing that the full therapeutic potential of this treatment depends on STAT3 signaling, most likely in the T cells of tumor-bearing mice. Cancer Immunol Res; 3(5); 526-35. Ó2015 AACR.