Accumulation Of CD8 Research Articles

Abstract 5571: Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine

Abstract Tryptophan oxidation and the ensuing production of Kynurenine (Kyn) and downstream products have been established to play an important role in immunosuppression within the tumor microenvironment (TME). The rate limiting step in Tryptophan oxidation is catalyzed by the IFN-γ inducible IDO-1 enzyme which is upregulated in numerous cancers and also, in some instances via the ectopic expression of TDO, which is normally expressed primarily in the liver. Currently, IDO-1 small molecule inhibitors in combination with checkpoint inhibitor antibodies are being evaluated in >12 clinical trials. However, whether the immune-suppressive effects of Tryptophan catabolism results from its depletion in the TME, or from the accumulation of Kynurenine, is not known. To distinguish between the effects of Trp depletion and Kyn accumulation in the TME we used an engineered Kynureninase (KYNase) enzyme that selectively degrades Kyn into immunologically inert and non-toxic metabolites. Peritumoral injection of PEGylated KYNase completely depletes serum and tumor Kyn levels for up to 72 hrs, while leaving Trp concentrations in both compartments unaffected. In the IDO-1 expressing CT26 colon carcinoma model KYNase treatment as monotherapy resulted in 16% complete and durable responses, accompanied by long-term immunity to tumor re-challenge. KYNase had no effect on tumor growth in IDO-/- mice, nor in RAG-/- or in CD8+ T cell depleted mice. Analysis of the TME demonstrated KYNase treatment resulted in increased accumulation of CD8+ cells, with a greater proportion expressing Granzyme B, undergoing proliferation and permeating the tumor interior. This effect was specific to the TME, as no detectable changes to the immune compartment occurred in other organs examined, nor overt signs of autoimmunity and toxicity. The increase in CD8+ cells was consistent with in vitro data showing that elevated concentration of Kyn directly induces apoptosis of activated CD8+ T cells partially through IL-2 suppression. No changes to the Treg percentages and phenotypes in the TME were observed, indicating that the Treg compartment is not impacted by treatment with KYNase. Cytokine analysis of tumor digests revealed increased IL-2 concentrations, as well as IFNγ and IL-9, in tumors after KYNase treatment. Furthermore, ex vivo stimulation of TIL after KYNase treatment demonstrated that the increase in cytokines in the TME is not solely due to an increase in TIL, but also an increased effector cytokine capacity on a per cell basis. Administration of KYNase together with either αCTLA4 or αPD1 synergistically elicited complete and durable regression of multiple established tumor models. In summary, our data support the hypothesis that Kyn accumulation in the TME rather than Trp depletion is the dominant IDO-mediated immune suppressive mechanism, and that enzyme-mediated Kyn depletion is a promising cancer immunotherapeutic approach. Citation Format: Todd A. Triplett, Kendra Triplett, Everett Stone, Michelle Zhang, Mark Manfredi, Candice Lamb, Yuri Tanno, Lauren Ehrlich, George Georgiou. Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2017-5571

Abstract 641: PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances tumor infiltrating CD8+ T cell accumulation and improves checkpoint inhibitor efficacy in murine syngeneic breast cancer models

Abstract Hyaluronan (HA) is an extracellular glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumors and is associated with rapid tumor progression and poor prognosis. In preclinical studies, enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the TME, reduces tumor interstitial fluid pressure, decompresses tumor blood vessels, and facilitates delivery of chemotherapeutics. We have previously shown that PEGPH20-mediated HA degradation enhances anti-PD-L1 and anti-PD1 efficacy in an HA accumulating murine pancreatic tumor model (Rosengren, AACR 2016 poster #4886). Accordingly, we aimed to extend these checkpoint blockade studies into orthotopic breast cancer models; while further elucidating the effect of PEGPH20-mediated HA degradation on modulating tumor infiltrating lymphocytes (TILs). The mammary fat pads of BALB/c mice were inoculated with either EMT-6 cells or 4T1/HAS3, a 4T1 cell line engineered to over-express hyaluronan synthase-3. During tumor progression, both EMT-6 and 4T1/HAS3 mammary carcinomas accumulate high levels of HA (average 730 ng/mg and 1408 ng/mg, respectively). When treated with PEGPH20 alone (0.0375mg/kg), EMT-6 tumor growth was inhibited by ≥30% (p=0.01). Additional EMT-6 studies were conducted to evaluate PEGPH20 in combination with anti-PD-L1. PEGPH20 increased anti-PD-L1 efficacy by 38% relative to anti-PD-L1 alone (86% vs 62.4% tumor growth inhibition (TGI), p=0.0024). Comparable tumor growth experiments were conducted in the 4T1/HAS3 model. TILs were evaluated by flow cytometry. PEGPH20-mediated HA removal enhanced both checkpoint efficacy and CD8+ T cell recruitment. Specifically, PEGPH20 alone (1mg/kg) increased anti-PD-L1 efficacy by 411% relative to anti-PD-L1 alone (93% vs 18.2% TGI, p<0.0001) and increased the accumulation of CD8+ TIL by 176% (p=0.0025). Taken together, the data suggest that tumor HA accumulation may act as a barrier to immune cell access and negatively modulate CD8+ TILs, and that PEGPH20-mediated HA reduction facilitates increased access of CD8+ T cells. This increased recruitment may contribute to the enhanced anti-PD-L1 efficacy observed when combined with PEGPH20. A Phase 1 trial is ongoing to evaluate PEGPH20 plus checkpoint blockade in patients with advanced or metastatic non-small cell lung cancer and in patients with locally advanced or metastatic gastric adenocarcinoma (NCT02563548). Citation Format: Renee Clift, Jisook Lee, Curtis B. Thompson, Yujun Huang. PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances tumor infiltrating CD8+ T cell accumulation and improves checkpoint inhibitor efficacy in murine syngeneic breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2017-641

CD4+ T cell receptor repertoire in sarcoidosis patients

Abstract Sarcoidosis is a multisystem disorder that most commonly affects the lungs and involves granuloma formation with the accumulation of CD4+ T cells at sites of disease. Considerable evidence indicates that CD4+ T cells are involved in the initiation and perpetuation of sarcoidosis. Particular T cell clones within the bronchoalveolar lavage (BAL) are preferentially expanded in patients with sarcoidosis, and these oligoclonal T cell populations accumulate within the lung and disappear with disease resolution. A link between the expansions of CD4+T cells expressing the T cell receptor (TCR) α-chain variable (V) region Vα2.3 and the presence of the HLA-DRB1*0301 (DR3) allele in sarcoidosis patients has been seen. We hypothesize that sarcoidosis-specific CD4+ T cells accumulate and expand in the lungs of sarcoidosis patients in response to a specific antigen. To determine whether these expanded CD4+T cell clones play a pathogenic role in disease progression, as well as to address their antigen specificity, we analyzed the TCR repertoire of CD4+ T cells isolated from the BAL of sarcoidosis patients utilizing iRepertoire, emulsion PCR, and single cell PCR analyses. We identified TCR Vα2.3/Jα42 CDR3 motifs that were found exclusively in HLA-DR3+ sarcoidosis patients (the motifs were not found in DR3− or DR3+ healthy control patients), suggesting a public TCR repertoire in this subset of patients. Importantly, these α-chains were paired with differing β-chains, suggesting a critical role of the α-chains in recognizing the HLA-DR3-peptide complex.

TACI-deficient macrophages alleviate metabolic disease symptoms in obese mice

Abstract TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) is one of receptors for the TNF superfamily cytokines BAFF (B cell activating factor) and APRIL (A Proliferation Inducing Ligand). In this study, we investigated the role of TACI in high fat diet induced obesity and metabolic disease. Compared to WT mice, TACI KO mice exhibited less weight gain, decreased body fat, enhanced glucose tolerance and insulin sensitivity. Improved metabolic changes in TACI KO mice were accompanied by significantly less accumulation of CD8+ T cells and F4/80+CD11c+ inflammatory macrophages in visceral fat pad. In addition, TACI KO mouse adipose tissue contained elevated numbers of ILC2, eosinophils and M2 macrophages. Underscoring the protective phenotype of M2-skewed macrophages, the transfer of TACI KO macrophages into obese WT mouse significantly improved insulin sensitivity and glucose tolerance in WT mouse. Interestingly, adoptively transferred TACI KO macrophages into WT mice also increased numbers of the host M2 macrophages and eosinophils in the adipose tissue. We identified IL-10 secreted by TACI KO macrophages was majorly responsible for the improved insulin sensitivity because not only TACI KO macrophage culture media contained high levels of IL-10 but also an IL-10 blocking antibody abolished the enhancement of insulin signaling provided by TACI KO macrophage culture media. These findings indicate that macrophage TACI mediated inflammatory signals promote obesity induced metabolic changes. Consequently, TACI blocking reagents may offer therapeutic benefits to obese patients suffering from metabolic complications.

IDO and galectin-3 hamper the ex vivo generation of clinical grade tumor-specific T cells for adoptive cell therapy in metastatic melanoma

Adoptive T cell transfer (ACT) with ex vivo-expanded tumor-reactive T cells proved to be successful for the treatment of metastatic melanoma patients. Mixed lymphocyte tumor cell cultures (MLTC) can be used to generate tumor-specific T cells for ACT; however, in a number of cases tumor-reactive T cell, expansion is far from optimal. We hypothesized that this is due to tumor intrinsic and extrinsic factors and aimed to identify and manipulate these factors so to optimize our clinical, GMP-compliant MLTC protocol. We found that the tumor cell produced IDO and/or galectin-3, and the accumulation of CD4+CD25hiFoxP3+ T cells suppressed the expansion of tumor-specific T cells in the MLTC. Strategies to eliminate CD4+CD25hiFoxP3+ T cells during culture required the depletion of the whole CD4+ T cell population and were found to be undesirable. Blocking of IDO and galectin-3 was feasible and resulted in improved efficiency of the MLTC. Implementation of these findings in clinical protocols for ex vivo expansion of tumor-reactive T cells holds promise for an increased therapeutic potential of adoptive cell transfer treatments with tumor-specific T cells.

Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

C–X–C-chemokine ligand 13 (CXCL13), the ligand for C–X–C chemokine receptor type 5 (CXCR5), is a major regulator of B-cell trafficking and plays an integral role in age-dependent clearance of hepatitis B virus (HBV) in the mouse model. However, the expression and function of CXCL13 in patients with chronic hepatitis B (CHB) remain unknown. By use of liver cell subpopulations isolated from CHB patients, we found that CXCL13 mRNA was abundantly expressed in Kupffer cells (KCs), but not in primary hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Interestingly, KC isolated from HBV-positive liver had much higher level of CXCL13 expression than non-HBV-infected controls. And its expression was induced by toll-like receptor 3 ligand poly I:C stimulation. Moreover, intense expression of CXCL13 protein and accumulation of CD4+ T and B cells were evident in follicular-like structures in the liver tissue of CHB patients, which indicated its chemotactic effect on CXCR5+ CD4+ cells and B cells. Consistently, the levels of serum CXCL13 were significantly higher in the CHB patients than in healthy controls. Furthermore, CXCL13 concentration was increased in the complete response (CR) group during weeks 0–12 and did not change significantly during the course of telbivudine treatment, compared with the patients who didn’t achieve CR. In conclusion, the HBV-related increase of CXCL13 production in KC and serum CXCL13 level during telbivudine treatment might be associated with immune control of chronic HBV infection.

Age-related changes in CD4+CD25+FOXP3+ regulatory T cells and their relationship with lung cancer.

ObjectivesCD4+CD25+FOXP3+ regulatory T cells (Treg) inhibit the anti-tumour immune response and reduce the effect of cancer immunotherapy. Although studies have demonstrated that the number and suppressive activity of Treg increase with age, it is not clear whether these changes correlate with a higher incidence of tumours in the elderly. This study was designed to explore the relationship between increase in CD4+CD25+FOXP3+ Treg and the higher risk of lung cancer in the elderly.MethodsSeventy lung cancer patients and 60 sex- and age-matched controls were recruited. Both groups were divided into three subgroups based on their age (young, middle-aged, or elderly). The proportion of CD4+CD25+FOXP3+ /CD4+ T cells was detected using flow cytometry, and the level of FOXP3 mRNA in the peripheral blood was examined with real-time RT-PCR.ResultsThe levels of CD4+CD25+FOXP3+/CD4+ T cells and FOXP3 mRNA were significantly higher in lung cancer patients than in healthy controls (t = 7.16, P < 0.01 and t = 3.65, P < 0.01, respectively). Within the healthy groups, the elderly group had larger proportion of CD4+CD25+FOXP3+ Treg (F = 32.54, P < 0.01) and higher FOXP3 mRNA expression (F = 4.76, P < 0.01) than their younger counterparts. Among the six subgroups, the elderly lung cancer patients exhibited the highest levels of both CD4+CD25+FOXP3+ Treg (11.81 ± 2.40%) and FOXP3 mRNA (3.14 ± 1.30).ConclusionsThe accumulation of CD4+CD25+FOXP3+ Treg with age correlates well with the increasing incidence of lung cancer in the elderly.

Amine oxidase activity regulates the development of pulmonary fibrosis.

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface-expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3-knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.

Demonstration of Tissue Resident Memory CD8 T Cells in Insulitic Lesions in Adult Patients with Recent-Onset Type 1 Diabetes

Subtypes of CD8+ T cells in insulitic lesions in biopsy specimens from six subjects with recent-onset type 1 diabetes (T1D) and six nondiabetic matched controls were analyzed using simultaneous multicolor immunofluorescence. Also, insulitic islets based on accumulation of CD3+ T cells were microdissected with laser-capture microscopy, and gene transcripts associated with inflammation and autoimmunity were analyzed. We found a substantial proportion, 43%, of the CD8+ T cells in the insulitic lesions to display a tissue resident memory T cell (TRM) (CD8+CD69+CD103+) phenotype in T1D subjects. Most TRM cells were located in the insulitic lesion in the endocrine-exocrine interface. TRM cells were also sporadically found in islets of control subjects. Moreover, gene expression analysis showed a lack of active transcription of genes associated with acute inflammatory or cytotoxic T-cell responses. We present evidence that a substantial proportion of T cells in insulitic lesions of recent-onset T1D patients are TRM cells and not classic cytotoxic CD8+ T cells. Our findings highlight the need for further analysis of the T cells involved in insulitis to elucidate their role in the etiology of T1D.

The effect of chronic kidney disease on T cell alloimmunity.

Altered differentiation and activation of T-cell subsets occur in patients with chronic kidney disease (CKD), but the impact on graft rejection and protective immunity during transplantation are not fully understood. Patients with CKD have decreased frequency of naïve T cells, accumulation of activated, terminally differentiated memory cells, and skewed regulatory versus T helper 17 ratio. Naïve and memory T-cell subsets do not appear to improve following kidney transplantation. Retained thymic output is associated with acute rejection, whereas naïve lymphopenia and accumulation of CD8 TEMRA cells correlate with long-term graft dysfunction. CD28 memory cells accumulate during CKD and appear to confer protection against acute rejection under standard immunosuppression and possibly costimulation blockade. T cells bearing CD57 are also increased in patients with CKD and may underlie rejection during costimulation blockade. The mechanisms by which CKD alters the differentiation and activation status of T-cell subsets is poorly understood. Further research is also needed to understand which cell populations mediate rejection under various immunosuppressive regimens. To date, there is little use of animal models of organ failure in transplant immunology research. CKD mouse models may help identify novel pathways and targets to better control alloimmunity in posttransplant.

The Deubiquitinating Enzyme Cylindromatosis Dampens CD8+ T Cell Responses and Is a Critical Factor for Experimental Cerebral Malaria and Blood-Brain Barrier Damage.

Cerebral malaria is a severe complication of human malaria and may lead to death of Plasmodium falciparum-infected individuals. Cerebral malaria is associated with sequestration of parasitized red blood cells within the cerebral microvasculature resulting in damage of the blood–brain barrier and brain pathology. Although CD8+ T cells have been implicated in the development of murine experimental cerebral malaria (ECM), several other studies have shown that CD8+ T cells confer protection against blood-stage infections. Since the role of host deubiquitinating enzymes (DUBs) in malaria is yet unknown, we investigated how the DUB cylindromatosis (CYLD), an important inhibitor of several cellular signaling pathways, influences the outcome of ECM. Upon infection with Plasmodium berghei ANKA (PbA) sporozoites or PbA-infected red blood cells, at least 90% of Cyld−/− mice survived the infection, whereas all congenic C57BL/6 mice displayed signatures of ECM, impaired parasite control, and disruption of the blood–brain barrier integrity. Cyld deficiency prevented brain pathology, including hemorrhagic lesions, enhanced activation of astrocytes and microglia, infiltration of CD8+ T cells, and apoptosis of endothelial cells. Furthermore, PbA-specific CD8+ T cell responses were augmented in the blood of Cyld−/− mice with increased production of interferon-γ and granzyme B and elevated activation of protein kinase C-θ and nuclear factor “kappa light-chain enhancer” of activated B cells. Importantly, accumulation of CD8+ T cells in the brain of Cyld−/− mice was significantly reduced compared to C57BL/6 mice. Bone marrow chimera experiments showed that the absence of ECM signatures in infected Cyld−/− mice could be attributed to hematopoietic and radioresistant parenchymal cells, most likely endothelial cells that did not undergo apoptosis. Together, we were able to show that host deubiqutinating enzymes play an important role in ECM and that CYLD promotes ECM supporting it as a potential therapeutic target for adjunct therapy to prevent cerebral complications of severe malaria.

Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity

Astrocytes are believed to bridge interactions between infiltrating lymphocytes and neurons during brain ischemia, but the mechanisms for this action are poorly understood. Here we found that interleukin-15 (IL-15) is dramatically up-regulated in astrocytes of postmortem brain tissues from patients with ischemic stroke and in a mouse model of transient focal brain ischemia. We generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15-expressing transgenic mouse (GFAP-IL-15tg) line and found enlarged brain infarcts, exacerbated neurodeficits after the induction of brain ischemia. In addition, knockdown of IL-15 in astrocytes attenuated ischemic brain injury. Interestingly, the accumulation of CD8+ T and natural killer (NK) cells was augmented in these GFAP-IL-15tg mice after brain ischemia. Of note, depletion of CD8+ T or NK cells attenuated ischemic brain injury in GFAP-IL-15tg mice. Furthermore, knockdown of the IL-15 receptor α or blockade of cell-to-cell contact diminished the activation and effector function of CD8+ T and NK cells in GFAP-IL-15tg mice, suggesting that astrocytic IL-15 is delivered in trans to target cells. Collectively, these findings indicate that astrocytic IL-15 could aggravate postischemic brain damage via propagation of CD8+ T and NK cell-mediated immunity.

Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells That Drive Alloimmune-Mediated Vascular Rejection.

IL-6 is an inflammatory cytokine that controls effector T cell responses but the mechanisms by which it controls allogeneic immune responses and vascular rejection that leads to transplant arteriosclerosis (TA) are poorly understood. We have examined the mechanism by which IL-6 contributes to the pathogenesis of vascular rejection and TA using a murine aortic interposition model of vascular rejection. The absence of IL-6 production from artery graft cells reduced the development of vascular rejection and arteriosclerotic thickening. There was no apparent effect of donor-derived IL-6 on endothelial cell integrity or on the intimal accumulation of smooth muscle cells, macrophages, and anti-donor antibodies. However, reduced vascular pathology in IL-6 artery grafts was accompanied by a significant reduction in the accumulation of CD4 and CD8 T cells. Further, the absence of graft-derived IL-6 resulted in a significant decrease in the activation and proliferation of alloreactive CD4 and CD8 T cells after transplantation as well as in a marked increase in cell death of effector T cells. Alloreactive effector T cells that expanded in the absence of IL-6 were also more susceptible to Fas-mediated activation-induced cell death in vitro. Finally, systemic neutralization of IL-6R did not reduce arteriosclerotic thickening but reduced endothelial integrity in allograft arteries, indicating differential effects of specific elimination of IL-6 in graft cells and systemic IL-6 neutralization. Donor-derived IL-6 amplifies the expansion of allogeneic T cell responses that cause vascular rejection and TA by increasing T cell proliferation and preventing Fas-mediated T cell death.

Abstract A119: CCL3 in the tumor microenvironment augments antitumor immune priming in the lymph node

Abstract Any therapeutic approach toward the eradication of metastatic tumor cells must involve targeting both the tumor's primary site of origin and those seeding in secondary tissues where metastasis has occurred. Lymph nodes represent an area of interest for targeting metastatic tumors because they play an essential role in tumor survival in non-native tissues. Interrogation of early cellular events in the draining lymph nodes (DLNs) during the initial priming of the adaptive T cell response can reveal new insights into how LN cells respond to a metastasizing tumor. The inflammatory chemokine CCL3 is important in orchestrating cellular contacts in vaccinated lymph nodes (LNs) and enhancing memory T cell generation. CCL3 has also been implicated in the modification and recruitment of natural killer (NK) cells and dendritic cells (DCs) to sites of epithelial insult and are important in establishing whether tumors will be tolerated or rejected. We hypothesize that by introducing a continuous supply of CCL3 into the microenvironment of a metastatic tumor, we can redirect a lymph node destined for tumor-tolerance toward the production of greater antitumor cellular responses. To interrogate our hypothesis, we subcutaneously inoculated naïve murine recipients with a Balb/c colon metastatic tumor (CT26) that is either the wild-type (WTTUs) or WTTUs transfected to secrete CCL3 (L3TUs). Immunocompetent mice injected with L3TUs resulted in a suppression of tumor growth compared to the WTTU group in a CD8+ T cell dependent manner. 1, 3, and 5-days after injection with WTTUs or L3TUs, there was an enhanced accumulation of endogenous DCs, NKs, and T cells in the DLNs and non-draining LNs (NDLNs). In vivo analysis of DCs in the DLN of L3TUs showed increased numbers of CD11c+ cells that upregulated the T cell costimulatory molecule, CD86+, while in vitro, SIINFEKL-pulsed BMDCs cultured with CCL3, showed an enhance capacity to induce proliferation of OT-I (CD8+) T cells. Examination of the day-5 DLN for direct signs of adaptive antitumor responses revealed an enhanced production of the antitumor cytokine, IFNγ, in the L3TU group, while the WTTU group showed a greater accumulation of CD4+ T-regulatory cells (Treg) over convention CD4+ T cells. Together these data suggest that CCL3 may enhance the overall immune response in the DLN in three ways. First, CCL3 attracts key cell-types such as NK cells to the DLN in larger quantities that can directly interact with DCs or T cells to enhance the development of IFNγ producing immune cells. Second, CCL3 directly influences DC maturation and indirectly enhances T cell proliferation. Third, CCL3 directly enhances the overall systemic accumulation of lymphocyte and myeloid cells in the DLN and NDLNs. Citation Format: Frederick Allen, Joseph M. Nthale, Saada K. Eid, Peter Rauhe, David Askew, Jay Myers, Alexander Tong, Alex Y. Huang. CCL3 in the tumor microenvironment augments antitumor immune priming in the lymph node [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A119.

Abstract A15: Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC), defined as tumors lacking expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), are especially difficult to treat effectively. While ER+ and HER2+ breast cancer subtypes can be treated with Tamoxifen and Herceptin, respectively, there are no targeted therapies for TNBC patients. Furthermore, while only 20-30% of TNBC patients respond to chemotherapy in the neoadjuvant setting, overall outcome remains poor for non-responding patients. However, mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be especially promising for TNBC patients. We and others have shown that the presence of CD8+ T cells, a crucial component of the cytotoxic arm of the adaptive immune response, is a sign of good clinical outcome in TNBC patients. However, good outcome only correlates with CD8+ T cell invasion of the tumor parenchyma. Here we show that some patients have an accumulation of CD8+ T cells in the surrounding tumor-associated stroma, but not the tumor epithelium, and these patients responded as poorly as patients with no CD8+ T cells at all. Yet how cancer associated fibroblasts (CAFs), a dominant cell type of the tumor-associated stroma, affects CD8+ T cell invasion into the tumor epithelium is still poorly understood. To identify potential stroma-dependent mechanisms that potentiate or inhibit CD8+ T cells invasion into the tumor epithelium, we performed gene expression profiling of laser-capture microdissected tumor-associated stroma (and matched epithelium) from 56 TNBC cases. Here we identify several key stromal features that may explain the accumulation of CD8+ T cells outside of the tumor epithelium. Preliminary data by immunohistochemistry and immunofluorescence validate some key stromal features and decipher the implication of other immune cell types in CD8+ T cells lack of tumor epithelium infiltration. These key stromal features that impair CD8+ T cell infiltration into the tumor in some patients might explain the relative low efficiency of immunotherapies in TNBC patients (20% of patients respond). One could speculate that targeting these key stromal features would allow a significant CD8+ T cell infiltration into the tumor and thus sensitize patients to immunotherapies. Citation Format: Tina Gruosso, Mathieu Gigoux, Nicholas Bertos, Sadiq Saleh, Atilla Omeroglu, Dongmei Zuo, Hong Zhao, Margarita Souleimanova, Valerie Weaver, Sarkis Meterissian, Michael Hallett, Morag Park. Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A15.

Abstract 3000: Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice

Abstract ONC201, a novel first-in-class, orally active anti-tumor agent that upregulates the cytotoxic ligand TRAIL (Allen et al., Sci. Trans. Med., 2013; Wagner et al., Oncotarget, 2014), activates the integrated stress response leading to tumor cell upregulation of TRAIL death receptor 5 (Kline et al., Sci. Sig., in press). ONC201 is active against bulk tumor and cancer stem cells (Prabhu et al., Can. Res., 2015). ONC201 is under clinical development by Oncoceutics, and is being evaluated in multiple phase I/II clinical trials. Results of the first-in-human ONC201 study presented at the 2015 AACR-NCI-EORTC meeting (Stein et al., Abstract C138) demonstrated ONC201 to be safe in humans, to exhibit predicted PK and sustained PD characteristics, and revealed a preliminary efficacy signal. As patients were dosed on an every 3-week schedule, based on supportive preclinical data, we investigated dose-intensification of ONC201 to determine whether a higher dose/frequency schedule might impact efficacy with limited toxicity. We hypothesized that ONC201 may be effective in dose-intensified schedule and may inhibit metastases. We tested a range of ONC201 doses including 25, 50, and 100 mg/kg and frequencies including every 4, 3, 2, 1 week as well as twice a week dosing. In colon and triple-negative breast cancer we observed that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo. Frequency effects were more pronounced at lower doses and dose-dependency was more impactful with less frequent schedules. We noted a potent anti-metastasis effect of ONC201 in vivo, not previously reported, as a function of both increased ONC201 dose and frequency of administration. ONC201 inhibits cancer cell migration and invasion in vitro in a TRAIL-dependent manner. We found ONC201 more potently suppresses Akt and ERK in tumors in vivo in a dose- and frequency-dependent manner, whereas its effect on TRAIL serum levels appeared to be impacted by frequency. We observed accumulation of CD3+/NK1.1+ cells within ONC201-treated tumors in athymic nude mice that lack T-cells. Accumulation of CD3+/NK1.1 cells within ONC201-treated tumors was more pronounced with dose intensification that correlated with superior efficacy. In summary, we have uncovered a potent anti-metastasis effect of ONC201 coupled with the appearance of CD3+/NK1.1+ cells within ONC201-treated tumors. We are further evaluating the biomarker characteristics and immune function of the CD3+/NK1.1+ cells and the relationship of their intra-tumoral accumulation to observed anti-tumor effects of ONC201, including in fully immunocompetent mice. Our results suggest that clinical investigation of both dose and frequency of ONC201 administration is warranted and is being evaluated in an ongoing clinical trial (NCT02609230). Citation Format: Jessica Wagner, Christina L.B. Kline, Marie Baumeister, Wafik S. El-Deiry. Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3000.

Abstract 4534: Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and a major cause of cancer related deaths in men in North America. The phosphatase and tensin homolog gene (PTEN) is frequently inactivated in PCa. Approximately 40% of primary and 70% of metastatic PCa's have genomic alterations in the PI3K-signaling, mostly through the loss of PTEN. Majority of metastatic PCa associated with PTEN loss are treated with docetaxel; an FDA approved therapy for recurrent PCa. However, in PTEN null PCa docetaxel treatment enhances senescence and does not significantly impact tumor volume. These findings were further confirmed in PCa patients with decreased levels of PTEN. In order to understand the global impact of docetaxel in PTEN null PCa we performed an unbiased RNA-seq of prostate tissues harvested from docetaxel treated prostate specific PTEN null mice. Data analysis showed strong enrichment of chromatin modifying enzymes reactome, mainly PRC2 as one of the top ranked categories. Among them EZH2, an oncogenic methyltransferase was found to be significantly upregulated. Quantitative PCR analysis confirmed statistically significant upregulation of EZH2 in docetaxel treated mice compared to vehicle control. EZH2 upregulation was independent of androgen levels as both androgen dependent and independent cells showed striking upregulation of EZH2 when treated with docetaxel. Next, we confirmed the nature of the upregulated EZH2 (catalytic or non-catalytic) by docetaxel. EZH2 methyltransferase activity assay data showed significant induction of methyltransferase activity by docetaxel in both androgen dependent and independent conditions and in PTEN null mouse tissues. Further, we measured other methyltransferase marks i.e. H3K27me1&amp;2, H3K4me3, H3K9me3 in the presence of docetaxel and found only specific upregulation of H3K27me3, confirming upregulation of only catalytic EZH2 by docetaxel. These data suggested a need for co-targeting EZH2 during docetaxel treatment in PTEN null PCa conditions. In order to provide a proof-of-principle we performed a tumor xenograft study in nude mice implanted with PTEN null PC3 cells. Combination of DZNeP (an EZH2 inhibitor) and docetaxel significantly inhibited tumor growth compared to each agent alone. Results obtained from the xenograft studies prompted us to perform a preclinical study in prostate specific PTEN null mice. Data obtained from this study clearly showed a significant reduction in number of high grade lesions in PTEN null tumors in combination group when compared to either docetaxel or DZNeP treated mice. We also observed clearance of senescent cells and accumulation of CD8+ T cells and classical dendritic (CD8+ Clec9a+ positive) cells. Our data suggests the necessity of designing a combination therapy of targeting EZH2 during docetaxel treatment specifically, which will likely increase the efficacy of docetaxel in PTEN null PCa. Citation Format: Mohammad Imran Khan, Abid Hamid, Vaqar Mustafa Adhami, Chloe Lang, Hasan Mukhtar. Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4534.

Overexpression of LMO2 causes aberrant human T-Cell development invivo by three potentially distinct cellular mechanisms.

Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development invivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoieticstem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development invivo; these mechanisms correlate with human T-ALL leukemogenesis.

The contribution of the HSCs niche to bone marrow failure in the IL-2 deficient model

Abstract Bone marrow failure (BMF) is a devastating disease where regeneration of peripheral blood lineages is compromised, resulting in anemia and mortality. In IL-2 KO BALB/c mice, the balance between early myeloid and lymphoid progenitor cell populations is skewed resulting in insufficient myelopoiesis with accumulation of CD8 T cells in the BM. It is widely accepted that crosstalk between cells of the bone microenvironment and hematopoietic cells affects each other’s behavior. However, it remains unknown how the BM niche in this mouse model contribute to the BMF. Multipotent stromal cells (MSCs), osteoblasts (OBs) and endothelial cells (ECs) are the three types of BM stromal cells (BMSCs) that may serve as the distinct functional niche to maintain HSCs. Repots have shown the interplay between CD8 T cells and MSCs cause a change in myelopoiesis and that MSCs promote the formation of Tregs. Our data also indicates that IL-2 KO CD8 T cells are important for the expansion of CD4 T cells in the bone. Since IL-2 KO bones are extremely brittle and CD4 T cells produce RANKL, the ligand needed for osteoclast (OC) differentiation, we evaluated the frequency of BMSCs and OCs. Our preliminary data shows clear differences in BMSCs profiles of IL-2 KO mice, namely elevated stromal cell numbers and a shift in Sca-1 expression in ECs and CD51 expression in OBs. Additionally, OC precursor frequency is elevated in the BM, indicating an increase in bone resorption. The ability of the different subsets of the BMSCs to support the differentiation of HSCs in vitro and the role of CD8 T cells in promoting these changes in vivo is being currently evaluated. Overall, our preliminary data suggests that the change in the bone microenvironment may contribute to the BMF observed in these mice.

Abstract 579: Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells that Drive Alloimmune-Mediated Vascular Rejection

Objective: IL-6 is an inflammatory cytokine that controls the development of effector T cell responses but the mechanisms by which it controls allogeneic immune responses and vascular rejection that leads to transplant arteriosclerosis (TA) is poorly understood. We have examined the effect of graft-derived IL-6. Methods: An aortic interposition model of vascular rejection and TA was performed using IL-6 +/+ and IL-6 -/- donors. Findings: The absence of IL-6 production from artery graft cells reduced the development of vascular rejection and arteriosclerotic thickening. There was no apparent effect of donor-derived IL-6 on endothelial cell integrity or on the intimal accumulation of smooth muscle cells and macrophages. However, reduced vascular pathology in IL-6 -/- artery grafts was accompanied by a significant reduction in the accumulation of CD4 and CD8 T cells. Further, the absence of graft-derived IL-6 resulted in a significant decrease in the activation and proliferation of alloreactive CD4 and CD8 T cells after transplantation as well as in a marked increase in cell death of effector T cells. Alloreactive effector T cells that developed in the absence of IL-6 were also more susceptible to Fas-mediated activation-induced cell death in vitro . Conclusions: Donor-derived IL-6 amplifies the expansion of allogeneic T cell responses that cause vascular rejection and TA by increasing T cell proliferation and preventing Fas-mediated T cell death.