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Recombinant human macrophage colony-stimulating factor (rhM-CSF) was given to cynomolgus monkeys by continuous intravenous infusion or subcutaneous injection, at a dose of 50 to 100 micrograms/kg/d in repetitive 14-day cycles. Starting within 24 to 48 hours of initiation of rhM-CSF, there was a progressive increase in the number of circulating monocytes, from a baseline of 811 +/- 253 cells/microL to a peak of 3,495 +/- 712 cells/microL on day 5 to 7. Many of these cells were large, granular, and extensively vacuolated. The expanded cell population expressed HLA-DR, LFA3, CD11b (904), and CD14 (MY4), and was 77% CD16 (FcRIII) positive by two-color cytofluorometry. In functional assays, fresh monocytes showed little cytotoxicity against cultured human melanoma cells (SKMel-1), with or without prior rhM-CSF treatment. However, after 3 days of in vitro culture in rhM-CSF, monocytes from treated animals mediated efficient antibody-dependent cytotoxicity (ADCC) against SKMel-1 using the murine monoclonal antibody 3F8 (IgG3, anti-ganglioside GD2). Under the same conditions, monocytes from control animals showed little ADCC (17% versus 82%, P less than .05). Antitumor cytotoxicity in the absence of antibody was less efficient and was not significantly different between the two groups. There was a mild decrease in platelet count during rhM-CSF treatment, without clinical symptoms. No abnormalities of serum biochemical parameters were seen. We conclude that parenteral rhM-CSF increases the number of circulating monocytes in nonhuman primates, and that these monocytes mediate increased antitumor ADCC after a brief period of in vitro differentiation. This study has implications for the design of possible future clinical trials combining antitumor monoclonal antibodies and rhM-CSF.

Natural regulatory mechanisms prevent inappropriate immune activation to self and innocuous foreign antigens. Here, we adapt the notion of immune privilege, which was originally applied to transplanted tissues, to consider how antigenic tumour cells and chronic pathogens might exploit natural regulatory mechanisms to become non-immunogenic. This conceptual approach reveals new mechanistic perspectives that may help to explain the paradoxical persistence of tumours and chronic pathogens, and suggests new opportunities to improve immunotherapy to treat these chronic inflammatory diseases.

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.

Regulatory T-cells (Tregs) play a critical role in preventing autoimmune and alloimmune reactions, including graft-versus-host disease (GVHD). Two recent clinical trials demonstrated that in patients undergoing hematopoietic stem cell transplantation, adoptive transfer of Tregs significantly reduced the incidence of grades II-IV GVHD. While Tregs significantly reduced GVHD severity, they did not eliminate GVHD. One potential way to augment Treg-mediated inhibition of GVHD is to increase Treg suppressive potency. We showed previously that Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function (Science 328:372, 2010). However, it is unclear whether PKC-θ inhibition can boost Treg function in a systemic inflammatory condition like GVHD. Furthermore, the mechanism by which PKC-θ inhibition augments Treg function is unknown. In this study, we address these unanswered questions. Using a mouse MHC class I/II disparate acute GVHD model, we found that freshly isolated Tregs treated for 30 minutes with 10uM of the clinically available PKC-θ inhibitor AEB071 suppressed GVHD mortality (Fig 1A) and severity significantly better than DMSO treated Tregs. As Tregs exert much of their protective effect against GVHD early in the course of the disease, we analyzed proliferation of GVHD-causing conventional T-cells (Tcon) on D4 after transplant. We observed a significant reduction in Tcon proliferation in mice given AEB071 treated Tregs compared to DMSO treated Tregs. We then performed multi-photon microscopy on D4 after transplant using TEα-GFP Tcon, CD11c-eYFP antigen presenting cells (APCs) and wild-type Tregs. Compared to DMSO, AEB071 treated Tregs significantly increased Tcon velocity and displacement from APCs. Increased velocity and displacement are indicative of decreased Tcon-APC interactions, suggesting reduced priming when AEB071 Tregs are present. Mechanistically, AEB071 vs DMSO treatment of Tregs resulted in augmented expression of the suppressive molecules Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3) after in vitro activation (Fig 1B, C) and in Tregs isolated from acute GVHD mice. Antibody blockade of Nrp1 and Lag3 in in vitro transwell suppression assays reduced the effect of AEB071 treatment, suggesting that these molecules may play a role in enhancing Treg function after PKC-θ inhibition. Flow cytometry analysis of phosphorylated proteins in activated Tregs revealed that PKC-θ inhibition resulted in reduced phosphorylation of the mTORC2 target FoxO3a, but not mTORC1 targets S6 and 4E-BP1. In addition, the mTORC2-specific phosphorylation site on Akt, serine 473, was reduced, whereas the mTORC1-specific site, threonine 308, was unaltered. Together, these data suggest reduced mTORC2 activity. Reduced phosphorylation increases Foxo3a nuclear translocation, which may result in increased Nrp1 and Lag3 expression, since Foxo3a has binding sites in both gene promoters. As both mTORC1 and 2 are involved in T-cell metabolism, we investigated the effect of AEB071 treatment on Treg oxygen consumption rate (OCR). Compared to DMSO, AEB071 treatment significantly increased Treg baseline and maximal OCRs after activation (Fig 1D). Increased OCR has been associated with increased Treg function. To identify additional alterations in phosphorylated proteins after PKC-θ inhibition, we performed a phosphoproteomic screen using in vitro expanded human Tregs treated with AEB701 or DMSO. We identified significant alterations in phosphorylation sites on 72 proteins, including reduced phosphorylation of an adaptor molecule that links PKC-θ to the intermediate filament vimentin. We found that vimentin is highly upregulated in Tregs compared to Tcon and that in Tregs, vimentin interacts with PKC-θ after activation. AEB071 treatment reduced the interaction between vimentin and PKC-θ. As with AEB071 treatment, Vimentin siRNA significantly increased Treg suppression in vitro compared to control transfected Tregs (Fig 1E), and augmented expression of Nrp1 and Lag3. AEB071 treatment of vimentin siRNA transfected Tregs did not further augment Treg function, suggesting an overlapping mechanism. In summary, our data demonstrate that PKC-θ interacts with mTORC2 and vimentin to modulate multiple aspects of Treg function, and that a brief incubation of Tregs with a PKC-θ inhibitor may be a viable method to enhance the efficacy of Treg therapeutics. Disclosures No relevant conflicts of interest to declare.

The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD. PD-L1 expression was up-regulated on donor CD4 and CD8 T cells during GVHD. We considered the possibility that PD-L1 expression on activated donor T cells might inhibit GVHD by down regulating donor anti-host T cell responses, consistent with PD-L1 co-inhibitory activity when expressed on host parenchymal cells during GVHD. Surprisingly, T cell mediated GVHD lethality was markedly reduced in recipients of PD-L1-/- compared to WT donor T cells in both B6 to BALB/c model of GVHD(P Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P < 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Figure 1. PD-L1-/- vs. WT donor T cells lessen GVHD lethality, independent of donor Treg function. (A) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells. (B) Survival of B10.BR mice with WT B6 or PD-L1-/- T cells. (C) Survival of BALB/c mice with WT B6 or PD-L1-/- T cells, or with WT B6 or PD-L1-/- CD25 depleted T cells (recipients of WT T cells vs. WT CD25-depleted T cells, P = 0.0003; recipients of PD-L1-/- T cells vs. PD-L1-/- CD25-depleted T cells, P = 0.2306; recipients of WT vs. PD-L1-/- T cells, P < 0.0001). (D) BALB/c mice transplanted with WT B6 or PD-L1-/- T cells. Mice were killed on d7 post-BMT and splenocytes were analyzed for LPAM-1, CCR9, and CXCR3 expression (not shown) on donor T cells. (E-F) BALB/c mice were transplanted with B6 Ly5.2 T cells plus PD-L1-/- T cells. Mice were killed on d3 post-BMT and splenocytes were analyzed for CTLA-4 and Lag-3 expression on donor T cells. Disclosures Aoyama: CHUGAI PHAMACEUTICAL CO.,LTD: Honoraria; Mochida Pharmaceutical Co.,Ltd: Honoraria; Kyowa Hakko Kirin Company,Limited: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Miller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Promote Suppressive cytokines • IDO1-expressing cells regulate inflammation and immune response to foreign as well as non pathological antigens • Several cancers induce the IDO1 pathway in tumor and host immune cells, permitting immune escape and the attainment of immunologic tolerance • GDC-0919 (RO 7077339; previously NLG919) is a small molecule investigational immunotherapy designed to inhibit IDO1 for the treatment of immune tolerance associated with cancer (Mautino et al.; 2013), intended as combination therapy

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060

Tryptophan catabolism via the enzyme indoleamine 2,3-dioxygenase (IDO) allows human monocyte-derived macrophages (MDM) and other APC to suppress T cell proliferation. IDO helps protect murine fetuses from rejection by the maternal immune system and can promote tolerance and immunosuppression. For tryptophan to be catabolized by IDO, it must first enter the APC via transmembrane transport. It has been shown that MDM in vitro readily deplete tryptophan present in the extracellular medium to nanomolar levels via IDO activity; yet, no currently known amino acid transport system displays high affinity and specificity sufficiently to permit efficient uptake of tryptophan at these low concentrations. Here, we provide biochemical characterization of a novel transport system with nanomolar affinity and high selectivity for tryptophan. Tryptophan transport in MDM was predominantly sodium-independent and occurred via two distinct systems: one consistent with the known system L transporter and a second system with 100-fold higher affinity for tryptophan (Km

By ligating CD80/CD86 (B7) molecules, the synthetic immunomodulatory reagent CTLA4-Ig (soluble synthetic CTLA4 fusion protein) induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO) in some dendritic cells (DCs), which acquire potent T cell regulatory functions as a consequence. Here we show that this response occurred exclusively in a population of splenic DCs co-expressing the marker CD19. B7 ligation induced activation of the transcription factor signal transducer and activator of transcription (STAT1) in sorted CD19+, but not CD19(NEG), DCs. STAT1 activation occurred even when DCs lacked receptors for type II IFN (IFNgamma); however, STAT1 activation and IDO up-regulation were not observed when DCs lacked receptors for type I IFN (IFNalphabeta). Thus, IFNalpha, but not IFNgamma, signaling was essential for STAT1 activation and IDO up-regulation in CD19+ DCs following B7 ligation. Consistent with these findings, B7 ligation also induced sorted CD19+, but not CD19(NEG), DCs to express IFNalpha. Moreover, recombinant IFNalpha induced CD19+, but not CD19(NEG), DCs to mediate IDO-dependent T cell suppression, showing that IFNalpha signaling could substitute for upstream signals from B7. These data reveal that a minor population of splenic DCs expressing the CD19 marker is uniquely responsive to B7 ligation, and that IFNalpha-mediated STAT1 activation is an essential intermediary signaling pathway that promotes IDO induction in these DCs. Thus, CD19+ DCs may be a target for regulatory T cells expressing surface CTLA4, and may suppress T cell responses via induction of IDO.

Effector T cells must be kept under control to suppress autoimmunity and immunity to innocuous antigens encountered at mucosal surfaces. In mice, T cells with regulatory functions are potent suppressors of T-cell responses and can protect tissues from T-cell-mediated destruction. The aim of this research focus is to summarize recent reports suggesting that regulatory T cells (Tregs) might be key components that protect mammalian fetuses from potentially lethal maternal adaptive immunity. Hence, defective generation or function of Tregs might explain some cases of human pregnancy failure.

The accumulation of macrophage foam cells in atherosclerotic lesions is associated with both initiation and progression of this disease. Scavenger receptors CD36 and SRA are the primary receptors responsible for conversion of macrophages into foam cells. Integrin alphaVbeta3 plays a role in the differentiation of several cell types, but its involvement in the transition of macrophages into foam cells and the potential role of this receptor in atherosclerosis have not been examined. Using an in vitro model of single surface receptor activation by binding with an immobilized monoclonal antibody specific to alphaVbeta3 integrin we show that ligation of alphaVbeta3 integrin prevents differentiation of blood monocytes and macrophages into the foam cell phenotype via coordinate down-regulation of CD36 and SRA. This effect of alphaVbeta3 integrin ligation can be reproduced by contact with endothelial cells, whereas the inhibition of alphaVbeta3 receptor ligation restores the uptake of oxidized low-density lipoprotein. Moreover, we found that alphaVbeta3 integrin is readily detected in situ on macrophages in early and advanced atherosclerotic lesions and that in vitro exposure to oxidized low-density lipoprotein up-regulates alphaVbeta3 integrin expression. We hypothesize that alphaVbeta3 integrin regulates macrophage functional maturation into foam cells in a persistent manner, and therefore, by targeting alphaVbeta3 receptor it could potentially be possible to regulate progression of atherosclerosis in humans.

Human monocyte-derived dendritic cells (DCs) are capable of expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which allows them to suppress Ag-driven proliferation of T cells in vitro. In DCs that express IDO, the activity of the enzyme is tightly regulated, with the protein being constitutively expressed, but functional activity requiring an additional set of triggering signals supplied during Ag presentation. We now show that triggering of functional IDO obligately requires ligation of B7-1/B7-2 molecules on the DCs by CTLA4/CD28 expressed on T cells. When this interaction was disrupted, IDO remained in the inactive state, and the DCs were unable to inhibit T cell proliferation. Inhibition could be fully restored by direct Ab-mediated cross-linking of B7-1/B7-2. Although both CD4+ and CD8+ T cells were susceptible to inhibition once IDO was induced, the ability to trigger functionally active IDO was strictly confined to the CD4+ subset. Thus, the ability of CD4+ T cells to induce IDO activity in DCs allowed the CD4+ population to dominantly inhibit proliferation of the CD8+ population via the bridge of a conditioned DC. We hypothesize that IDO activation via engagement of B7-1/B7-2 molecules on DCs, specifically, engagement by CTLA4 expressed on regulatory CD4+ T cells, may function as a physiologic regulator of T cell responses in vivo.

Tumors create a pathological state of tolerance towards tumor-associated antigens. The mechanisms by which this occurs are still largely unknown. Recent studies suggest that one mechanism contributing to this phenomenon could be tryptophan catabolism carried out by the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO can be expressed by tumor cells themselves, and we propose that the recruitment of antigen-presenting cells expressing IDO to tumor-draining lymph nodes might constitute another potent mechanism for inducing tolerance to tumor-derived antigens. Therapeutic strategies based on blocking this pathway might represent a novel class of adjuvants for tumor immunotherapy.

The mechanism by which the immune system of a tumor-bearing host acquires tolerance toward tumor antigens is still elusive. Antigen-presenting cells (APCs) are critical regulators of the decision between immune response and tolerance. APCs that express the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) have been found to inhibit T-cell responses both in vitro and in vivo. We hypothesized that malignant tumors exploit this mechanism by recruiting IDO-expressing APCs to the tumor-draining lymph nodes. To test this hypothesis, archival tissues and records of 26 cases of lymph node dissection for invasive cutaneous melanoma were obtained. IDO immunohistochemistry was performed on 14 cutaneous tumors and 328 regional lymph nodes. Abnormal accumulations of IDO-positive cells with a monocytoid or plasmacytoid morphology were identified in the perisinusoidal regions of draining lymph nodes in 45% of nodes studied. Recruitment of IDO-positive cells was seen in nodes with and without malignancy. We hypothesize that these IDO-positive APCs may contribute mechanistically to acquired tolerance to tumor antigens. Immunostaining of tumor-draining lymph nodes for abnormal accumulation of IDO-expressing cells might thus constitute an adverse prognostic factor and could contribute to the decision process and the appropriate care of patients with this deadly disease.

Macrophages are professional scavengers of apoptotic and necrotic cells, and hence constantly take up self antigens. Paradoxically, macrophages are also professional antigen-presenting cells, which would seem to invite autoimmune disorders. Moreover, macrophages are effector cells in the tissue-destruction phase of autoimmune disorders, where they encounter additional self antigens in the stimulatory context of chronic inflammation. This review examines the array of immunosuppressive mechanisms which may help macrophages suppress unwanted T cell responses, and considers the consequences of a breakdown in these negative-regulatory systems in autoimmunity.

Cells expressing indoleamine 2,3 dioxygenase (IDO) play key roles in regulating adaptive immune responses orchestrated by T cells. In this report we discuss our working model, the tryptophan depletion hypothesis, to explain links between IDO expression and inhibition of T cell responses. We posit that IDO+ cells, particularly professional antigen presenting cells (APCs) promote T cell entry but block cell cycle progression due to tryptophan catabolism. We discuss experimental evidence supporting predictions from the tryptophan depletion hypothesis and the implications that this model has for understanding the origin of tolerant states that explain immunological paradoxes, such as fetal survival, tumor persistence and failure to eradicate pathogens like HIV that cause persistent infections.

The priming of an appropriate anti-tumor T cell response rarely results in the rejection of established tumors. The characteristics of tumors that allow them to evade a T cell-mediated rejection are unknown for many tumors. We report on evidence that the expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO) by mononuclear cells that invade tumors and tumor-draining lymph nodes, is 1 mechanism that may account for this observation. Lewis lung carcinoma (LLC) cells stimulated a more robust allogeneic T cell response in vitro in the presence of a competitive inhibitor of IDO, 1-methyl tryptophan. When administered in vivo this inhibitor also resulted in delayed LLC tumor growth in syngeneic mice. Our study provides evidence for a novel mechanism whereby tumors evade rejection by the immune system, and suggests the possibility that inhibiting IDO may be developed as an anti-cancer immunotherapeutic strategy.

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8+ dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.

HIV-1 encephalitis (HIVE) and its associated dementia can occur in up to 20% of infected individuals, usually when productive viral replication in brain mononuclear phagocytes (macrophages and microglia) and depletion of CD4+ T lymphocytes are most significant. T cells control viral replication through much of HIV-1 disease, but how this occurs remains incompletely understood. With this in mind, we studied HIV-1-specific CTL responses in a nonobese diabetic (NOD)-SCID mouse model of HIVE. HIV-1-infected monocyte-derived macrophages (MDM) were injected into the basal ganglia after syngeneic immune reconstitution by HLA-A*0201-positive human PBL to generate a human PBL-NOD-SCID HIVE mouse. Engrafted T lymphocytes produced HIV-1gag- and HIV-1pol-specific CTL against virus-infected brain MDM within 7 days. This was demonstrated by tetramer staining of human PBL in mouse spleens and by IFN-γ ELISPOT. CD8, granzyme B, HLA-DR, and CD45R0 Ag-reactive T cells and CD79α-positive B cells migrated to and were in contact with human MDM in brain areas where infected macrophages were abundant. The numbers of productively infected MDM were markedly reduced (>85%) during 2 wk of observation. The human PBL-NOD-SCID HIVE mouse provides a new tool for studies of cellular immune responses against HIV-1-infected brain mononuclear phagocytes during natural disease and after vaccination.

Meeting abstracts Clinical Trial Registration Number: [NCT02048709][1] The enzyme Indoleamine 2,3-dioxygenase (IDO1) catalyzes the cleavage of L-tryptophan, resulting in the production of kynurenine. Tryptophan depletion and kynurenine metabolites enhance the number and function of Tregs (

Combination immunotherapy regimens incorporating agents that target more than a single regulatory pathway or immune inhibitory checkpoint can confer marked enhancement of anti-tumor immune responses when combined with active immunotherapy. We have tested the antitumor effect of the IDO pathway inhibitor NLG-919 (currently in Phase I clinical trial) in combination with indoximod (a different IDO pathway inhibitor currently in Phase II clinical trials), chemotherapy, vaccination and/or PD-1/PD-L1/PD-L2 blockade. Here we show that the highly potent, orally-bioavailable IDO pathway inhibitor NLG-919 is synergistic with blockade of the PD-1/PD-L1/PD-L2 pathway in mouse models of large established tumors. The combination of NLG-919 plus PD-1/PD-L1/PD-L2 blockade showed significantly enhanced anti-tumor effect compared to either agent alone. This synergy was particularly evident in the setting of large established tumors treated with normally ineffective immunotherapy regimens (e.g., a single dose of chemotherapy plus a vaccine against a poorly immunogenic shared-self antigen). In vivo, administration of NLG-919 enhanced anti-tumor vaccine responses against B16F10 melanoma treated with hgp100 vaccine plus resting, non-activated pmel-1 T cells NLG-919 also reduced Treg-mediated suppression in tumor-bearing hosts, and enhanced dendritic cell activation in tumors and TDLNs. In combination with chemotherapy, treatment with NLG-919 allowed effector T cell responses against endogenous tumor antigens released by chemotherapy. By each of the preceding readouts, the in vivo biologic effect of NLG-919 was qualitatively identical to that of indoximod, but the same immunologic effects could be achieved at lower plasma concentrations in vivo. In multiple models, a combination of oral NLG-919 with oral indoximod produced synergistic anti-tumor effects, which was further enhanced by blockade of the PD 1/PD L1/PD-L2 pathway. In a more stringent B16F10 melanoma tumor model that did not involve adoptive transfer of pmel-1 cells, a combination of immune checkpoint inhibition involving NLG-919, indoximod and anti-PD1/PD-L1/PD-L2 antibodies with chemotherapy and hgp100 peptide vaccine was able to achieve a significant antitumor effect. The current preclinical studies suggest a mechanistic rationale for a combining NLG919 with indoximod and with agents targeting the PD 1/PD L1/PD-L2 pathway. Citation Format: Mario R. Mautino, Charles J. Link, Nicholas N. Vahanian, James T. Adams, Clarissa Van Allen, Madhav D. Sharma, Theodore S. Johnson, David Munn. Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2014-5023

Chronic graft versus host disease (cGVHD) is a life threatening impediment to the otherwise curative potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Intensive study has unveiled the lymphoid subsets that drive multifaceted autoimmune and fibrotic cascades which disrupt healthy tissues. Clinical and in-vivo data implicate B-cells and certain subsets of CD4 helper T-cells in the development and progression of cGVHD. These specific immune subsets are unique in their singular dependence upon the TEC-family kinases BTK or ITK for receptor driven activation. Ibrutinib is an irreversible inhibitor of BTK and ITK that has produced durable remissions in B-cell malignancies with minimal toxicity. We sought to examine ibrutinib's potential to attenuate B-cell and CD4 T-cell driven anti-host immunity in the setting of cGVHD. We utilized two complementary murine models independently focused on interrogating sclerodermatous T-cell driven cGVHD (LPJ→C57BL/6) or alloantibody driven bronchiolar obliterans (BO) and multi-organ system disease without skin involvement (C57BL/6→B10.BR). We examined therapeutic administration of 25mg/kg/day ibrutinib initiated when mice first develop signs of cGVHD (25-28 days after allo-HSCT). In the minor-MHC mismatch LPJ→C57BL/6 model, ibrutinib significantly extended median time to cGVHD progression by 14 days and 33% (6 of 18) of ibrutinib treated mice remained progression free as compared to 12% (2 of 18) of mice receiving vehicle and 10% (1 of 11) of mice receiving cyclosporine 10mg/kg/day (p Citation Format: Jason A. Dubovsky, Ryan Flynn, Jing Du, Bonnie K. Harrington, Yiming Zhong, Carrie Yang, William Towns, Amy Lehman, Amy Johnson, Steven Devine, Samantha Jaglowski, Jonathan S. Serody, William J. Murphy, David H. Munn, Leo Luznik, Geoffrey Hill, Kelli K.P. MacDonald, Ivan Maillard, John Koreth, Corey Cutler, Robert J. Soiffer, Joseph H. Antin, Jerome Ritz, Angela Panoskaltsis-Mortari, John C. Byrd, Bruce R. Blazar. Ibrutinib can reverse established chronic graft-versus-host disease, which is dependent upon IL-2 inducible T-cell kinase (ITK) and Bruton's tyrosine kinase (BTK)-driven lymphocyte activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2014-2591

Background Indoleamine 2,3-dioxygenase (IDO) is an enzyme with immune-suppressive properties that is commonly exploited by tumors to evade immune destruction. Anti-tumor T cell responses can be initiated in solid tumors, but are immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade. We hypothesized that IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction. Methods To test this hypothesis, we utilized a syngeneic orthotopic glioblastoma model in which GL261 glioblastoma tumor cells were stereotactically implanted into the right frontal lobes of syngeneic mice. These mice were treated with IDO-blocking drugs in combination with chemotherapy and radiation therapy. Results Pharmacologic inhibition of IDO synergized with chemo-radiation therapy to prolong survival in mice bearing intracranial glioblastoma tumors. We now show that pharmacologic or genetic inhibition of IDO allowed chemo-radiation to trigger widespread complement deposition at sites of tumor growth. Chemotherapy treatment alone resulted in collections of perivascular leukocytes within tumors, but no complement deposition. Adding IDO-blockade led to upregulation of VCAM-1 on vascular endothelium within the tumor microenvironment, and further adding radiation in the presence of IDO-blockade led to widespread deposition of complement. Mice genetically deficient in complement component C3 lost all of the synergistic effects of IDO-blockade on chemo-radiation-induced survival. Conclusions Together these findings identify a novel mechanistic link between IDO and complement, and implicate complement as a major downstream effector mechanism for the beneficial effect of IDO-blockade after chemo-radiation therapy. We speculate that this represents a fundamental pathway by which the tumor regulates intratumoral vascular activation and protects itself from immune-mediated tumor destruction.

TPS3121 Background: IDO is an enzyme that catalyzes the initial and rate limiting step in the conversion of tryptophan to kynurenine. Tryptophan depletion enhances the number and function of the Tr...

TPS2107 Background: Indoleamine 2, 3-dioxygenase (IDO) is a key immune-modulatory endogenous enzyme that inhibits CD8+ T cells and enhances the suppressor activity of Tregs. IDO is expressed in a l...

3110 Background: A majority of patients with advanced cancer expire from uncontrolled disseminated disease due to lack of effective systemic treatment. Radiation (RT) serves a limited role as palli...

The 28th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held November 8–10, 2013 in National Harbor MD, and was organized by Paolo Ascierto (Instituto Nazionale Tumori – Fondazione 'G. Pascale’), David Munn (Georgia Regent’s University), A. Karolina Palucka (Baylor Institute for Immunology Research) and Paul Sondel (University of Wisconsin). This meeting included over 1100 registered participants from 32 separate countries, making it the largest SITC meeting held to date. It highlighted significant worldwide progress in the development and application of cancer immunology to the practice of clinical oncology, including advances in diagnosis, prognosis and therapy, utilizing a variety of immunological pathways and mechanisms for a variety of oncologic conditions. Presentations and posters demonstrated that many concepts that had been pursued preclinically in the past are now being translated into clinical practice, with clear benefits for patients. One month after this 28th annual meeting, the Journal Science selected the field of Cancer Immunotherapy as the overall #1 scientific “breakthrough” for 2013.

The human MHC class Ib antigen HLA-G is thought to regulate maternal immune responses during pregnancy. Here we show that expression of HLA-G in transgenic mice diminished cellular immunity by inhibiting maturation of myelomonocytic cells into functional antigen-presenting cells (APC). Skin allografts applied to HLA-G transgenic mice survived longer and resultant T cell responses were less potent compared to control mice. T cells from HLA-G mice responded normally to allogeneic APC and immunohistological analyses of spleen revealed no marked abnormalities. However, spontaneous outgrowths of myeloid cells were observed when bone marrow or splenocytes from HLA-G mice were cultured in vitro, but functionally competent APC did not develop spontaneously in bone marrow cultures supplemented with granulocyte macrophage colony stimulating factor (GM-CSF). Addition of lipopolysaccharide (LPS) to GM-CSF-derived bone marrow cultures rescued APC maturation. Studies using HLA-G tetrameric reagents revealed that HLA-G-specific binding activity was associated with CD11c(+) myelomonocytic cells, while binding to lymphoid and NK cell subsets was undetectable. These data show that spontaneous maturation of functionally competent dendritic cells (DC) is compromised in HLA-G mice. We hypothesize that HLA-G inhibits maturation of DC via receptor-mediated interactions with myelomonocytic precursors, which render immature DC precursors unable to receive signals from activated T cells.

In 1953 Medawar pointed out that survival of the genetically disparate (allogeneic) mammalian conceptus contradicts the laws of tissue transplantation. Rapid T cell–induced rejection of all allogeneic concepti occurred when pregnant mice were treated with a pharmacologic inhibitor of indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme expressed by trophoblasts and macrophages. Thus, by catabolizing tryptophan, the mammalian conceptus suppresses T cell activity and defends itself against rejection.

Publisher Summary Macrophage colony-stimulating factor (M-CSF)—also known as CSF-1—is a hematopoietic growth factor that supports the survival, proliferation, and differentiation of cells of the monocyte lineage. M-CSF is produced in various cells—including monocytes, smooth-muscle cells, endothelial cells, and fibroblasts—as a disulfide-linked homodimer. The molecular mass of M-CSF varies because of the synthesis of two distinct M-CSF precursor proteins and the post-transcriptional addition of glycosyl residues and chondroitin sulfate to the molecule. It is one of the few cytokine/growth factors found in measurable concentrations in various tissues including plasma, urine, cerebrospinal fluid, and placenta. Human M-CSF is encoded by a single 20 kb gene containing 10 exons. Its two precursor proteins share amino acid (aa) residues: -32 to +149. M-CSF gene expression is constitutive or inducible in various cell types including fibroblasts, smooth-muscle cells, vascular endothelial cells, bone marrow stromal cells, T cells, uterine cells, hepatocytes, and cells of the monocyte/macrophage lineage. M-CSF is also expressed in malignant cell lines, and gene expression can be induced by endotoxin, IL-1α tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), phorbol esters, and progesterone/estrogen. M-CSF is also found in detectable concentrations in blood, urine, cerebrospinal fluid, and fetal amniotic fluid.

Comment on Tan et al, page [2936][1] Dendritic cells with low expression of B7 molecules tend to induce tolerance, but it has been difficult to show that the observations are related. A new study suggests that they may be. Recently, it has become clear that “resting” (unactivated or immature

Introduction One obstacle that continues to pose a major risk for hematopoietic stem cell transplantation is the occurrence of chronic graft-versus-host disease (cGVHD). Although the exact cause of cGVHD is unknown, preclinical findings have demonstrated a significant increase in class-switched anti-host reactive antibody deposition and fibrosis in target organs. We have previously demonstrated the dependence of cGVHD on donor bone marrow (BM)-derived B cells and splenic-derived T cells in a model of cGVHD that develops bronchiolitis obliterans (BO), a pathognomonic symptom of lung cGVHD (Blood 119:1570-80, 2012). T:B cell pathways responsible for cGVHD have yet to be elucidated. Germinal Center (GC) reactions are dependent on T follicular helper (TFH) cell stimulation and controlled by T follicular regulatory cells (TFR). TFH cells are located in the B-cell zones, express Bcl6, CXCR5, ICOS, and high levels of PD1, and provide stimulation through IL-21 production and ICOS and CD40L expression. TFR are located in the GC and express FoxP3. In this study we define a previously unknown role for TFH cells in the development of cGVHD. Methods B10.BR (H2k) mice were conditioned with cyclophosphamide and irradiation and transplanted with B6 (H2b) BM and mature splenic-derived T cells. After 8 weeks, pulmonary function of the mice was measured during mechanical ventilation using whole body plethymography. BO was defined by low compliance and high elastance and resistance in association with collagen deposition leading to obliterated bronchioles. Target organs (lung, liver, spleen) were analyzed for collagen accumulation, immunoglobulin deposition, size of GCs, frequency and of TFH, GC B cells and TFR. Interventions included use of knockout (KO) donors or administration of monoclonal antibody (mAb) from days 28-56 to treat cGVHD. Results cGVHD is associated with high GC number, TFH cell number and low TFR (Figure 1 A-D). To determine whether TFH cells were required for cGVHD, we transplanted mice with wild-type (WT) BM and WT T cells or T cells deficient for the TFH master regulator Bcl-6. In contrast to WT T cells, recipients of BCL6 KO T cells had no pathogenic antibody deposited in the lung and no evidence of BO (Figure 1 E-H). Recipients of TFH deficient T cells had a significant decrease in the collagen surrounding the bronchioles and in frequency of GC B cells and TFH compared to cGVHD mice. Consistent with an essential role for TFH cells, TFH deficient, CXCR5 KO donor T cells were unable to induce cGVHD. To determine whether the TFH-produced cytokine IL-21 was necessary for cGVHD, donor IL-21 KO T cells with WT BM or WT T cells with IL-21R KO BM-derived B cells did not develop cGVHD. Importantly, therapeutic administration of anti-IL-21 neutralizing mAb (kindly provided by Novo Nordisk, Copenhagen, Denmark) was able to reverse established cGVHD in this model. Studies using TFH-deficient, ICOS KO donor T cells or anti-ICOS blocking mAb beginning day 28 as cGVHD therapy confirmed the requirement for ICOS:ICOS ligand signaling in cGVHD generation and maintenance. Moreover, blocking CD40L:CD40 interactions in mice with established cGVHD using anti-CD40L blocking mAb was effective in reversing cGVHD. Lastly, studies using a highly B cell depleting anti-CD20 mAb for cGVHD therapy suggested that plasmablasts and plasma cells derived from GC B cells were critical for continued pathogenic Ab production as cGVHD was not reversed by anti-CD20 mAb in this model system. However, it is unknown if prophylactic treatment with anti-CD20 mAb is effective for preventing onset of disease. Conclusions These data provide novel insights into cGVHD pathogenesis, indicating the essential role of TFH in these processes and suggest new lines of therapy using mAb that target TFH cells, GC B cells, and their interactions, with the potential to reverse established pulmonary cGVHD. Disclosures: Browning: Biogen Idec: Employment.

Meeting abstracts The CTLA-4 blocking antibody ipilimumab results in durable responses in metastatic melanoma, though therapeutic benefit has been limited to a fraction of patients. This calls for identification of resistance mechanisms and development of combinatorial strategies. We examine the

The IDO pathway mediates immunosuppressive effects through the metabolization of tryptophan (Trp) to kynurenine (Kyn), triggering downstream signaling through GCN2, mTOR and AHR that can affect differentiation and proliferation of T cells. Expression of the IDO1 gene by tumor cells or host APCs can inhibit tumor-specific effector CD8+ T cells and enhance the suppressor activity of Tregs, and high expression of IDO correlates with worse clinical prognosis in patients with a variety of malignancies. Therefore, targeting the IDO pathway via inhibition of the IDO enzyme or blocking its downstream signaling effects is a prime target for small-molecule immunomodulatory drugs in cancer. Here we describe the pharmacological and biological properties of NLG919, a novel small-molecule IDO-pathway inhibitor. NLG919 potently inhibits this pathway in vitro and in cell based assays (Ki=7 nM; EC50 =75 nM). It is orally bioavailable (F>70%); and has a favorable pharmacokinetic and toxicity profile. In mice, a single oral administration of NLG919 reduces the concentration of plasma and tissue Kyn by ∼ 50%. Using IDO-expressing human monocyte-derived DCs in allogeneic MLR reactions, NLG919 potently blocked IDO-induced T cell suppression and restored robust T cell responses with an ED50=80 nM. Similarly, using IDO-expressing mouse DCs from tumor-draining lymph nodes, NLG919 abrogated IDO-induced suppression of antigen-specific T cells (OT-I) in vitro, with ED50=120 nM. In vivo, in mice bearing large established B16F10 tumors, administration of NLG919 markedly enhanced the anti-tumor responses of naïve, resting pmel-1 cells to vaccination with cognate hgp100 peptide plus CpG-1826 in IFA. In this stringent established-tumor model, NLG919 plus pmel 1/vaccine produced a dramatic collapse of tumor size within 4 days of vaccination (∼95% reduction in tumor volume compared to control animals receiving pmel-1/vaccine alone without NLG919). In conclusion, NLG919 is a potent IDO pathway inhibitor with desirable pharmacological properties, suitable for the treatment of immunosuppression associated with cancer. Citation Format: Mario R. Mautino, Firoz A. Jaipuri, Jesse Waldo, Sanjeev Kumar, James Adams, Clarissa Van Allen, Agnieszka Marcinowicz-Flick, David Munn, Nicholas Vahanian, Charles J. Link. NLG919, a novel indoleamine-2,3-dioxygenase (IDO)-pathway inhibitor drug candidate for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2013-491

Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell–induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by N G -nitro- l -arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200×10 3 per well)–dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin- and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1α. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1β neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous l -arginine failed to improve NO release, suggesting that oxidant stress–induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-α. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.

We investigated the effects of monocytes on endothelial cell (EC) ectoenzyme activity. Coculture of human aortic ECs with human monocytes (2×10 5 monocytes per 2-cm 2 well) led to a decrease in EC angiotensin-converting enzyme (ACE) activity (64.5±3.5% of control) but not aminopeptidase N, aminopeptidase P, and 5′-nucleotidase activities. Similar results were obtained using human umbilical vein EC–human monocyte and porcine aortic EC–porcine monocyte cocultures. The decrease in ACE activity was monocyte concentration and coculture time dependent, reaching a maximum of 65% decrease in activity at 120 hours. Monocyte-mediated reduction in ACE activity did not require cell to cell contact, since exposure of ECs to conditioned medium from cocultures (CCCM) or from monocyte cultures (MCM) produced a decrease in ACE activity similar to that observed in EC-monocyte cocultures. Exogenously added tumor necrosis factor (TNF)-α and interleukin (IL)-1α, two known secretory products of monocytes, simulated the effects of monocytes on ACE activity. Western blot analysis revealed a decrease in the amount of ACE protein in TNF-α–treated and CCCM-treated ECs compared with control ECs. Both TNF-α and IL-1α were present in CCCM and MCM but not EC-conditioned medium. Incubation of the cocultures with a mixture of neutralizing antibodies against TNF-α and IL-1 totally abolished the monocyte-induced decrease in ACE activity. In conclusion, monocytes decrease ACE activity in cultured ECs through the release of cytokines such as TNF-α and IL-1.

Human monocytes exposed in vitro to recombinant macrophage-colony-stimulating factor (rhMCSF) differentiate into monocyte-derived macrophages (MDM), which mediate efficient antibody-dependent cytotoxicity (ADCC) against tumor cells. We and others have shown that this form of ADCC is unusual in that phagocytosis, rather than extracellular lysis, appears to play the major role in target cell killing. In this study, we asked whether the phagocytic form of cytotoxicity seen with ADCC could occur in the absence of an opsonizing antibody. We now report that, whereas cell lines derived from solid tumors are often resistant to antibody-independent cytotoxicity, malignant cells of lymphoid origin appear particularly susceptible to such antibody-independent killing. We found that all of nine lymphocytic leukemia and lymphoma cell lines tested in a total of 35 experiments, plus all four samples of fresh leukemic blasts, were consistently susceptible to antibody-independent MDM cytotoxicity. Antibody-independent cytotoxicity against these cells was efficient (40%-63% killing) at effector: target (E:T) ratios as low as 2:1. Like ADCC, antibody-independent cytotoxicity involved phagocytosis of target cells, as demonstrated by ingestion of fluorescently labeled targets and analysis by flow cytometry. At the time of phagocytosis, the majority of target cells retained membrane integrity, as indicated by the direct transfer of intracellular [51Cr]chromate from radiolabeled targets to phagocytosing MDM, without release of the label into the medium. However, in contrast to ADCC, we found that the degree of antibody-independent cytotoxicity was not a function of the E:T ratio. Instead, a constant proportion of the available target cells were killed regardless of the E:T ratio, suggesting that target cell recognition, rather than effector cell potency, might be the limiting factor in determining cytotoxicity. In additional experiments, we have also identified a second tumor cell type, nueroblastoma, as being susceptible to antibody-independent phagocytosis (all of five cell lines tested, cytotoxicity 40%-93%, E:T = 3:1). Our data thus indicate that the cytotoxicity induced by rhMCSF is not confined to antibody-mediated killing, and that phagocytosis can play a significant role in target cell destruction even in the absence of opsonizing antibody.

Abstract 3504 Programmed Death-1 (PD-1) is an inhibitory cell surface receptor of the immunoglobulin superfamily expressed on activated lymphocytes, monocytes and dendritic cells. Although PD-1 function is best characterized in T-cells, it is known that PD-1 also suppresses the immune response of B lymphocytes through protein phosphatase recruitment and dephosphorylation of signaling molecules downstream of the B-cell receptor (BCR). Recent studies have found that PD-1 expression is elevated at the mRNA as well as the protein levels in B cells obtained from chronic lymphocytic leukemia (CLL) patients compared to those from healthy controls. Using genome-wide DNA methylation sequencing, we identified PD-1 as one of the significantly hypomethylated genes in CLL compared to normal B-cell samples. Three differentially methylated regions (DMRs) were discovered in the first intron, proximal promoter and up-stream enhancer regions. We validated these DMRs in 43 CLL and 7 normal control samples using bisulfite pyrosequencing. The pyrosequencing analysis further confirmed that all three regions were significantly hypomethylated in CLL patient samples (p Disclosures: No relevant conflicts of interest to declare.

Abstract 526 Chronic lymphocytic leukemia (CLL) is a biologically and clinically heterogeneous disease. The somatic hypermutation status of the immunoglobulin heavy chain variable (IGHV) genes has been identified as one of the most robust prognostic markers in CLL. Patients with unmutated IGHV status (U-CLL) typically experience an inferior outcome compared to those whose clones express mutated IGHV genes (M-CLL). We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from a group of 43 CLL patients using reduced representation bisulfite sequencing (RRBS). Using base-pair resolution methylation sequencing, 2323 differentially methylated regions between CLL and normal B cells (CLL-specific DMRs) and 569 between M-CLL and U-CLL samples (IGHV-specific DMRs) were identified in the CLL genomes. The IGHV-specific DMRs are mostly unique when compared to the CLL-specific DMRs. Less than 10% of the IGHV-specific DMRs are located in promoter regions; however, more than half of these overlap with known DNase I hypersensitive sites, enhancer regions marked by histone modification (H3K4Me1 and H3K27Ac), and transcription factor binding sites in the ENCODE datasets, which indicates that these DMRs contain regulatory sequences. Distinctive DNA methylation patterns were observed in M-CLL and U-CLL samples. Overall, U-CLL was found to contain 50% more hypermethylated regions than M-CLL samples. The hypermethylated loci observed in the U-CLL samples also appear to be hypermethylated in normal naïve B cells as compared memory B cells, suggesting that M-CLL and U-CLL differ in differentiation status corresponding to normal B cell differentiation stages. RNA-seq analysis performed using matched samples (n=34), in which both DNA methylation and gene expression data were available, demonstrated excellent correlation between DNA methylation and gene expression. Several genes whose expression status was previously shown to be associated with CLL prognosis such as ZAP70, CRY1, LDOC1, SEPT10, LAG3, and LPL were differentially methylated in the promoter regions between M-CLL and U-CLL samples indicating that DNA methylation plays an important role in defining the gene expression patterns of these prognostic genes. We further validated 9 genes with IGHV-specific DMRs in the promoter regions using bisulfite pyrosequencing, and the results demonstrated excellent correlation between differential methylation and IGHV mutation status. These novel differentially methylated genes could be developed into biomarkers for CLL prognosis. In addition, DNA hypomethylation was observed in a significant number of genes involved in lymphocyte activation such as PDCD1, NFATc1, and CD5. DNA hypomethylation was observed in the proximal promoter and far up-stream enhancer regions of CD5, an important cell surface marker that uniquely identifies CLL. Overall, the DNA methylation landscape in CLL patients indicates that CLL B cells possess an active B-cell phenotype; at the same time, U-CLL and M-CLL are faithfully committed to their lineage resembling either naïve or memory B cells. In summary, this comprehensive DNA methylation analysis has identified a large number of novel epigenetic changes in CLL patients. The results from this study will further advance our understanding of the epigenetic contribution to molecular subtypes in CLL. Disclosures: No relevant conflicts of interest to declare.

2501 Background: The indoleamine 2, 3 dioxygenase pathway (IDO) can create immune suppression and unresponsiveness to tumor antigens in tumor-bearing hosts. 1-methyl-D-tryptophan (1-MT) is an oral inhibitor of the IDO pathway, currently in development as an immune adjuvant for anti-tumor vaccines and chemotherapy. The primary goal of this trial is to determine the MTD and toxicity for oral 1-MT. Secondary endpoints include response rates, PK, serum tryptophan catabolites, circulating T-reg cells, and C reactive protein (CRP) levels. Methods: This phase I study utilizes a 3+3 design comprised of ten dose levels (200, 300,400,600,800,mg QD, 600, 800,1200,1600,2000mg BID). Inclusion criteria: patients with measurable metastatic solid malignancy, age ≥18, life expectancy >4 months, and adequate organ/marrow function. Exclusion criteria: chemotherapy in the past 3 weeks, untreated brain metastases, active autoimmune disease, or malabsorptive GI disease. Continuous toxicity monitoring early stopping rules were used. Results: At submission, 48 out of the planned 50 patients were accrued. MTD was not reached at 2000mg BID. At 200mg QD dose, 3 patients previously treated with active immunotherapy (ipilimumab, n=2, CD40-mAb, n=1) developed an autoimmune hypophysitis. Since IDO is closely linked to both CTLA-4 and CD40/CD40L pathways this was considered on-target effect. Subsequently, patients with prior immunotherapy were excluded and no additional treatment related G3-5 adverse events were observed. Five patients showed SD>6 months (2 melanoma, 2 sarcoma 1 colon) as well as some mixed responses were seen in some patients including regression of a liver visceral metastases. 1-MT plasma PK AUC and Cmax were proportional with dose. Cmax (~40 μM at 2000mg BID) is at 2.9 hours and the t1/2 is at 10 hours. Elevations in CRP levels and declines in T-reg cell counts were seen across multiple dose levels. Additional correlative data analysis is ongoing. Conclusions: The treatment was well tolerated and orally bioavailable. Biologic activity in terms of prolonged disease stabilization, induction of hypophysitis, and changes in T-reg levels and CRP were observed. Trials combining 1-MT with docetaxel and a dendritic cell vaccine are ongoing.

Background Plasmacytoid dendritic cells (pDC) are crucial cells implicated in anti-viral immune responses. On recognizing HIV, they become activated, secreting high amounts of IFNα and inflammatory cytokines, thereby potentiating anti-viral innate and adaptive immune responses. However, the role of pDC in adaptive immunity is still debated. Several studies have documented a role for activated pDC in the induction of CD4+ or CD8+ regulatory T cells (Treg), both in vitro and in vivo. A direct correlation between CD8+ T cell activation levels and disease progression levels has been confirmed in many studies. We investigated here whether HIV-stimulated pDC can regulate the levels of immune activation by promoting the differentiation of regulatory CD4+ T cells.

Best characterized as initiators of immunity, dendritic cells (DCs) also play an integral role in immune modulation. Immature DCs, for example, process self-antigens to induce and maintain tolerance. The immunoregulatory effects of DCs, however, are not limited to immature subtypes. Immunogenic mature DCs can also induce T regs to curb immune responses. We have found that human monocyte-derived DCs (moDCs) upregulate the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) with maturation and expand functionally active, naturally occurring as well as inducible regulatory T cells (T regs) in an IDO-dependent manner. Priming of resting bulk T cells with autologous, IDO-expressing, mature moDCs in the absence of exogenous cytokines results in up to 10-fold expansion of CD4+CD25hiFoxp3+CD127neg T cells that mediate significant dose-dependent suppression of both allogeneic and autologous T cells stimulated de novo by DCs. The expansion of T regs by IDO-expressing moDCs involves cell-to-cell contact, CD80/CD86 ligation, and IL-2. Autologous priming in the presence of a competitive inhibitor of IDO, 1-methyl-tryptophan, diminishes T reg expansion. Candidate T regs were further characterized after cytofluorographic sorting primed bulk T cells into CD4+CD25hi, CD4+CD25int, and CD4+CD25neg subpopulations. Post-sort analysis showed that >60% of the CD4+CD25hi cells coexpressed Foxp3, which was not present in the CD4+CD25neg cells. CD4+CD25hi T regs exerted dose-dependent inhibition of DC-stimulated allogeneic T cell proliferation, with >90% inhibition at a suppressor to responder T cell ratio of 1:1 and ~50% inhibition at a ratio of 1:25. CD4+CD25int cells produced intermediate suppression depending on dose, and CD4+CD25neg cells were not inhibitory. CD4+CD25hi T regs mediated similar suppression of autologous T cell responses to stimulation de novo by DCs. CD4+CD25hi T regs also inhibited the generation of cytotoxic T lymphocytes (CTLs) specific for the Wilms’ tumor gene product (WT-1). The addition of CD4+CD25hi T regs to CTL-priming cultures resulted in a >80% decrease in specific target cell lysis of a WT-1-expressing cell line. Separate studies showed that T reg-mediated suppression is contact dependent and also requires TGF-beta, suggesting inhibition by naturally occurring and inducible T regs, respectively. Depletion of CD4+CD25hi T cells from bulk T cells by negative immunoselection with anti-CD25 magnetic beads at the outset of autologous priming significantly blunts T reg expansion, indicating a requirement for pre-existing T regs in the bulk T cell population. T reg expansion also occurs in priming cultures using cytofluorographically-sorted CD4+CD25neg T cells, indicating de novo generation of T regs from CD4+CD25neg precursors. In summary, our results demonstrate a mechanism by which mature, IDO-expressing, human moDCs expand autologous, naturally occurring as well as inducible T regs that functionally suppress the proliferation of both autologous and allogeneic T cells. Inhibition of this counter-regulatory pathway should result in more sustained benefit from active DC-based immunotherapy.

Macrophages and cultured human monocytes can mediate efficient antibody-dependent cytotoxicity (ADCC) against human tumor cells using monoclonal antibodies (mAbs). The mechanism of this killing is usually assumed to involve secreted factors (reactive oxygen intermediates, tumor necrosis factor, or other cytotoxic factors) leading to target cell lysis. In this study, we present evidence that phagocytosis of intact target cells is the principal mechanism of antitumor cytotoxicity in our in vitro model of ADCC by cultured monocytes. Human monocytes cultured in recombinant human macrophage colony-stimulating factor ingested up to 100% of fluorochrome-labeled melanoma and neuroblastoma target cells, in the presence of an appropriate antitumor mAb. Electron microscopy demonstrated phagocytosis of intact tumor cells by cultured monocytes during ADCC. All of the radionuclide in radiolabeled target cells was taken up by monocytes during phagocytosis. By preventing the release of radioisotope tracers, phagocytosis thus prevents the detection of this very efficient form of cytotoxicity by most conventional assays.

Graft versus host disease (GVHD), the major cause of morbidity and mortality after bone marrow transplant (BMT), is initiated following activation of donor T cells by host antigen presenting cells (APCs). The immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by APCs and parenchymal cells and is further inducible by inflammation. We investigated whether lethal conditioning and GVHD induce IDO and if IDO prevents tissue injury by suppressing immune responses at the induction site. Using a fully MHC-mismatched murine GVHD model, we determined that IDO is a critical regulator of GVHD, most strikingly in the colon, where epithelial cells dramatically upregulated IDO expression during GVHD. No IDO upregulation was detected in mice that received irradiation only or irradiation and T cell-depleted BM without added alloreactive T cells. When mice with a genetic ablation of the IDO gene (IDO−/−) were used as recipients of BM and T cells, they suffered accelerated GVHD lethality compared to wild-type recipients, displaying increased colonic inflammation and T cell infiltration. This was true using grafts of either CD4+ T cells or combined CD4+ and CD8+ T cells. In addition, the IDO inhibitor 1-methyltryptophan caused accelerated GVHD in wild-type recipients. Lethality was not due to increased radiation sensitivity, as sublethally irradiated IDO−/− recipients showed no morbidity, and syngeneic BM was capable of rescuing lethally irradiated IDO−/− recipients. GVHD protection was not mediated by early control of T cell proliferation or apoptosis in lymph nodes or spleen. Furthermore, IDO did not affect expression of T cell effector molecules in lymphoid organs. Though studies have suggested a link between IDO and T regulatory cell (Treg) function, Treg depletion of donor grafts did not abrogate the IDO effect, indicating donor Tregs are not required for the suppressive function of IDO. Conversely, cultured Tregs added to the graft delayed GVHD in IDO−/− recipients, proving Tregs do not require host IDO for their function in this model. Instead, colons from IDO−/− hosts contained more proliferating CD4+ cells than wild-type colons after transplant. Together, this evidence suggests that in GVHD IDO acts primarily at the site of its expression to decrease effector T cell proliferation, diminish colonic inflammation, and reduce disease severity. These studies are the first to identify a function for IDO in GVHD lethality and suggest that modulation of the IDO pathway may be an effective strategy for treatment of this disease.