Immune Suppressive Cell Populations Research Articles

Abstract CT016: Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab

Abstract Background The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDA) is characterized by a paucity of cytotoxic tumor-infiltrating lymphocytes, an abundance of immune-suppressive cell populations, and a general resistance to immune checkpoint inhibitor (ICI) therapy. In preclinical models of PDA, the HDAC inhibitor entinostat modulated the transcriptional programming of myeloid cells, reducing their capacity for immunosuppression and sensitizing tumors to ICI therapy. Methods We conducted a single-center, open-label, Simon two-stage, phase II study of entinostat in combination with the PD1 inhibitor nivolumab in patients with advanced PDA. Patients received oral entinostat 5 mg once a week. After a 14-day lead-in with entinostat monotherapy, patients concurrently receive entinostat 5 mg orally once a week plus nivolumab 240 mg intravenously every 2 weeks. After 4 months, therapy was continued with entinostat 5 mg weekly plus nivolumab at a dose of 480 mg fixed dose every 4 weeks until the time of progression or unacceptable toxicities. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Peripheral immune profiles at baseline and on treatment were assessed by high-dimensional mass cytometry by time of flight (CyTOF) and by Luminex of chemokines in the plasma, and changes in the TME were performed on paired baseline and on treatment biopsies by multiplexed immunohistochemistry (mIHC), image cytometry-based quantification, and bulk RNA-seq analysis. Results From November 2017 to November 2020, 27 evaluable patients were enrolled. Three patients had a partial response (PR) by RECIST v1.1 criteria (objective response rate (ORR)) of 11%, 95% CI, 0.024-0.292), with a median duration of response of 10.2 months. Grade ≥3 treatment-related adverse events (TRAEs) were observed in 19 (63%) patients. The most common grade ≥3 TRAEs were decreased lymphocyte count, anemia hypoalbuminemia, and hyponatremia. Dendritic cell (DC) activation, maturation, migration, antigen processing, and presentation were increased in the periphery after entinostat treatment, consistent with HDAC inhibitor mediated myeloid reprogramming. Gene expression analysis of paired baseline and on treatment tumors demonstrated an enrichment in inflammatory response signaling pathways with combination treatment. Conclusions Entinostat and nivolumab demonstrated durable radiological responses in a subset of patients with PDA. Paired tissue and peripheral analysis showed immunomodulation of myeloid cell populations, consistent with the preclinical hypothesis supporting the trial. This study creates a roadmap for this strategy with future combinatorial therapeutic approaches to enhance the clinical benefit in PDA patients further. Citation Format: Marina Baretti, Ludmila Danilova, Jennifer N. Durham, Courtney B. Betts, Leslie Cope, Dimitrios N. Sidiropoulos, Joseph A. Tandurella, Soren Charmsaz, Nicole Gross, Alexei Hernandez, Won J. HO, Chris Thoburn, Rosalind Walker, James Leatherman Leatherman, Sarah Mitchell, Brian Christmas, Daria A. Gaykalova, Srinivasan Yegnasubramanian, Elana J. Fertig, Lisa M. Coussens, Mark Yarchoan, Elizabeth Jaffee, Nilofer S. Azad. Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT016.

Phenotypic immune characterization of gastric and esophageal adenocarcinomas reveals profound immune suppression in esophageal tumor locations.

Tumors in the distal esophagus (EAC), gastro-esophageal junction including cardia (GEJAC), and stomach (GAC) develop in close proximity and show strong similarities on a molecular and cellular level. However, recent clinical data showed that the effectiveness of chemo-immunotherapy is limited to a subset of GEAC patients and that EACs and GEJACs generally benefit less from checkpoint inhibition compared to GACs. As the composition of the tumor immune microenvironment drives response to (immuno)therapy we here performed a detailed immune analysis of a large series of GEACs to facilitate the development of a more individualized immunomodulatory strategy. Extensive immunophenotyping was performed by 14-color flow cytometry in a prospective study to detail the immune composition of untreated gastro-esophageal cancers (n=104) using fresh tumor biopsies of 35 EACs, 38 GEJACs and 31 GACs. The immune cell composition of GEACs was characterized and correlated with clinicopathologic features such as tumor location, MSI and HER2 status. The spatial immune architecture of a subset of tumors (n=30) was evaluated using multiplex immunohistochemistry (mIHC) which allowed us to determine the tumor infiltration status of CD3+, CD8+, FoxP3+, CD163+ and Ki67+ cells. Immunophenotyping revealed that the tumor immune microenvironment of GEACs is heterogeneous and that immune suppressive cell populations such as monocytic myeloid-derived suppressor cells (mMDSC) are more abundant in EACs compared to GACs (p<0.001). In contrast, GACs indicated a proinflammatory microenvironment with elevated frequencies of proliferating (Ki67+) CD4 Th cells (p<0.001), Ki67+ CD8 T cells (p=0.002), and CD8 effector memory-T cells (p=0.024). Differences between EACs and GACs were confirmed by mIHC analyses showing lower densities of tumor- and stroma-infiltrating Ki67+ CD8 T cells in EAC compared to GAC (both p=0.021). This comprehensive immune phenotype study of a large series of untreated GEACs, identified that tumors with an esophageal tumor location have more immune suppressive features compared to tumors in the gastro-esophageal junction or stomach which might explain the location-specific responses to checkpoint inhibitors in this disease. These findings provide an important rationale for stratification according to tumor location in clinical studies and the development of location-dependent immunomodulatory treatment approaches.

Abstract 1353: CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow

Abstract Tumors emerge and evolve in complex and dynamic microenvironments that influence their growth, invasion, and metastasis. In the tumor microenvironment, the immune system plays a critical role in the prevention and elimination of tumors. Despite immune surveillance, tumors still manage to develop through evolution facilitating the establishment of an immune suppressive microenvironment. There is emerging evidence showing that the antitumor immunity of immune effector cells can be obstructed by immune suppressive cell populations such as MDSCs or Tregs. Hematologic malignancies associated with FGFR1 translocations, also known as Stem Cell Leukemia/Lymphoma (SCLL) syndrome, show a complex presentation including myeloproliferative neoplasm, AML, and T- or B-cell lineage lymphoblastic leukemia/lymphoma, providing an ideal, homogeneous model to investigate mechanisms underlying leukemogenesis. Our recent investigation in mouse models of SCLL revealed that mice xenografted with SCLL cells showed increased CD11b+Ly6CHigh M-MDSC and CD11b+Ly6CInt PMN-MDSC levels in the peripheral blood (PB) during leukemia development, accompanied by significantly decreased levels of T cells. To further confirm this MDSC-mediated suppression of T cell function, MDSC depletion experiments were performed in the SCLL cell engraftment model using either the anti-Ly6G antibody (for PMN-MDSCs) or the anti-Gr-1 (aka Ly6G/Ly6C; for total MDSCs) antibody. In both cases, significantly increased levels of CD4+ and CD8+ cells were observed, along with significantly prolonged mouse survival. These observations were consistent in multiple SCLL cell line models, supporting the idea that SCLL cells promote the accumulation of MDSCs, which in turn suppresses the antitumor activity of cytotoxic T cells, allowing SCLL progression. To further characterize these leukemia cell induced MDSCs, CyTOF analysis was performed with the PBMC and bone marrow (BM) samples from leukemic and naïve mice. In peripheral blood leukemia cell induced M-MDSCs also express macrophage markers, including F4-80 and CD64, implying a potential overlap of MDSC and tumor-associated macrophage (TAM). These TAMs also express high CD71 and CD184. Most importantly, we can also identify a CD64/CD71/CD184 expressing cluster in the BM samples. Different from the PB, these cells in the BM express stem cell marker CD117, suggesting there are potential progenitor cells of TAMs. Taken together, our studies demonstrated that SCLL cells establish a tumor favorable microenvironment by reshaping hematopoiesis toward MDSC generation in the BM. A comprehensive understanding of these immune modulation processes holds the promise of improving patient outcomes by reversing the generation of MDSC/TAM in the BM and overturning the establishment of the immunosuppressive microenvironment at the outset. Citation Format: Xiaocui Lu, Ting Zhang, Atsuko Matsunaga, Xuexiu Fang, Stephanie F. Mori, John Cowell, Tianxiang Hu. CD71 and CD117 identifies an early cancer associated macrophage progenitor population in leukemic mouse bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1353.

Runx2 Deficiency in Committed Osteoblasts Promotes Myeloma Cell Homing to and Progression in New Bone Sites

Multiple myeloma (MM) is a plasma cell malignancy that arises and preferentially grows in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that primary MM cells secrete soluble factors that orchestrate changes in distant bone sites prior to the arrival of metastatic tumor cells. One such alteration is the simultaneous suppression of osteoblastogenesis and bone formation via inhibition of the critical Runt-related transcription factor 2 (Runx2) in osteoblast (OB) progenitors and committed OBs. While the mechanisms regulating MM-induced Runx2 suppression in these cells have been described, how this change in distant sites provides feedback to regulate MM cell homing and survival has not been determined.To study the effect of OB-Runx2 suppression at distant bone sites on MM dissemination and progression to these sites, we generated a syngeneic mouse model of MM in which Runx2 is specifically deleted in the committed OBs of C57BL6/KaLwRij mice (OB-Runx2-/- mice). Bone-homing and growth of luciferase-expressing 5TGM1 (5TGM1-Luc) MM cells was tested by tail vein injection in 5-week-old OB-Runx2+/+ (controls) and OB-Runx2-/- mice (n=7 per group). Compared with controls, OB-Runx2-/- mice showed significantly enhanced bone homing of MM cells and increased tumor growth in bone, which was confirmed by weekly luciferin imaging and ELISA for serum levels of IgG2bκ (a soluble marker of 5TGM1 MM cells). These results demonstrate that Runx2 deficiency in committed OBs in new bone sites promotes MM metastasis to and progression in these areas.To investigate the molecular mechanism by which OB-Runx2 deficiency promotes MM bone-homing and tumor progression, we harvested BM from 5-week-old OB-Runx2+/+ controls and OB-Runx2-/- mice with 1 ml PBS (n=3-4 per group) and centrifuged it to collect BM cells and supernatants. Measurement of 48 cytokines related to tumor cell bone-homing and bone remodeling revealed substantially increased levels of pro-metastatic cytokines in the BM supernatants of OB-Runx2-/- mice, including EGF, GM-CSF, M-CSF, ICAM-1, IGF-1, MMP-3, OPN, SDF-1, TGF- β, RANKL, VCAM-1, and IL-10. Flow cytometric analysis of the immune cell profile showed significantly more immune inhibitory cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells, in the BM of OB-Runx2-/- mice. These data indicate that Runx2 deficiency in committed OBs induces an inflammatory and immunosuppressive pre-condition in the BM microenvironment, which attracts MM cells and allows their invasion into the bone tissue.Next, we evaluated the survival and proliferation status of MM cells and the immune cell response to MM cells in the BM under the condition of OB-Runx2 suppression. We injected 2 x 106 5TGM1-Luc cells into 5-week-old OB-Runx2+/+ control and OB-Runx2-/- mice via tail vein (n=5 per group). After 4 weeks, MM and BM cells isolated from the BM of these mice were analyzed by immunostaining and flow cytometry with antibodies specific for CD138, a marker of MM cells; Ki-67, a marker of cell proliferation; and Bcl-2, an anti-apoptotic protein. Results showed significantly more proliferating and non-apoptotic MM cells in the BM of Runx2 OB-/- mice. Additional flow cytometry analysis of isolated BM cells revealed a striking increase in immune-suppressive cell populations, significantly higher expression of CD8+ T cell inhibitory molecules (Arginase 1, iNOS, IL-10) in MDSCs, and dramatically decreased production of tumor-killing factors (Perforin, Granzyme B, and IFN-γ) in T cells in OB-Runx2-/- mice. Together, these findings indicate that Runx2 deficiency in committed OBs impairs antitumor immunity, leading to tumor proliferation and progression in bone.In conclusion, this study demonstrated that Runx2 inhibition in committed OBs in distant bone sites, induced by primary MM, can feed back to invite MM cell metastasis to and progression in these new bone areas. The increase in pro-metastatic cytokines and immunosuppressive cells induced by OB-Runx2 deficiency in new bone sites play a critical role in this process. These novel discoveries provide insights into the mechanisms of MM dissemination and progression and may inform the prognosis of outcomes in patients with MM. DisclosuresNo relevant conflicts of interest to declare.

P3.04-008 A Phase 1b/2 Study of Atezolizumab With or Without Daratumumab in Advanced or Metastatic Non-small Cell Lung Cancer (NSCLC)

Daratumumab (DARA), a human CD38 monoclonal antibody, is approved for the treatment of patients with relapsed/refractory multiple myeloma (RRMM). DARA produces deep clinical responses in RRMM and induces T-cell expansion through the reduction of immune suppressive cell populations, such as CD38+ myeloid-derived suppressor cells and regulatory T and B cells. Atezolizumab (atezo) is a humanized programd death-ligand 1 (PD-L1) monoclonal antibody that blocks the interaction between PD-L1 and the programd death-1 and B7.1 receptors, reinvigorating anticancer immune responses.

Abstract 3684: Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models

Abstract AZD9150, a gen2.5 antisense oligonucleotide (ASO) targeting human STAT3, has improved drug-like properties compared to previous generation ASO therapeutics, including increased stability and resistance to nucleases, reduced proinflammatory effects, and enhanced potency. We have previously reported that in tumors, STAT3 ASOs are taken up preferentially in stromal and immune cells of the tumor microenvironment (TME). Since AZD9150 is selective for human STAT3, we used a surrogate ASO (muSTAT3 ASO) to explore the pharmacodynamics of ASO-mediated STAT3 inhibition in syngeneic and genetically engineered mouse (GEM) tumor models, focusing on effects in the TME. In mice bearing subcutaneous CT-26 tumors, treatment with muSTAT3 ASO at 50 mg/kg, s.c., on a qdx5/wk schedule decreased STAT3 levels in immune cell subsets in the tumor and in circulating leukocytes by 40 - 60%, similar to the decrease in STAT3 achievable in circulating leukocytes in human patients after AZD9150 treatment. In a Nanostring analysis (nCounter mouse immunology panel) of CT-26 tumors from muSTAT3 ASO treated mice, CD163 (M2 immune suppressive macrophage marker) was the gene most consistently and significantly downregulated, by an average of 84% in three independent experiments, and was confirmed by immunohistochemistry (IHC). Flow cytometry analysis of myeloid subpopulations - tumor associated macrophages (F4/80+ TAMs), monocytic myeloid derived suppressor cells, and granulocytic cells - showed a decrease in TAMs averaging 69% across three independent experiments. The analysis was extended to include IHC for arginase (Arg, a marker of functional immune suppression activity). Subpopulations of cells identified included Arg+, CD163+, and Arg+CD163+. Treatment with muSTAT3 ASO decreased these populations by 79%, 88% and 97% respectively, compared to control treatment. These populations were also analyzed in two GEM tumor models - the KPC pancreatic cancer model, and a PTEN -/- prostate cancer model - which have a TME more representative of that found in tumors in the clinic. While the specific changes varied across the models, likely reflecting differences in TME makeup, a reduction in immune suppressive cell populations was present in both GEM models, including a decrease in CD163+ cells of 79% (along with modest antitumor activity) in the PTEN -/- prostate model after muSTAT3 ASO treatment. These results indicate that selective STAT3 inhibition can reduce immune suppressive cell populations in the TME, and suggest that STAT3 inhibition has the potential to enhance the antitumor activity of T-cell targeted therapies, such as those targeting the PD1-PDL1 axis. In support of this hypothesis, we observed that addition of muSTAT3 ASO to anti-PD-L1 Ab treatment significantly enhanced the antitumor activity of PD-L1 Ab treatment in two subcutaneous syngeneic tumor models, CT-26 and A20. Citation Format: Rich Woessner, Vasu Sah, Patricia McCoon, Shaun Grosskurth, Nanhua Deng, Rachel DuPont, Deborah Lawson, Lourdes Pablo, Corinne Reimer, Marco A. De Velasco, Hirotsugu Uemura, Juliana Candido, Paul Lyne. Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor 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 3684. doi:10.1158/1538-7445.AM2017-3684

Randomized, open-label phase Ib/II study of atezolizumab with or without daratumumab in previously treated advanced or metastatic non-small cell lung cancer (NSCLC).

TPS9102 Background: Daratumumab (DARA), a human CD38 monoclonal antibody, is approved for treatment of relapsed/refractory multiple myeloma (RRMM). DARA produces deep clinical responses in RRMM and induces T-cell expansion through reduction of immune suppressive cell populations (CD38+ myeloid-derived suppressor cells and regulatory T and B cells). Atezolizumab (atezo) blocks programmed death-ligand 1 (PD-L1) and was recently approved for metastatic NSCLC that progressed on or during platinum therapy based on data showing improved overall survival (OS) in the atezo vs docetaxel treatment arm. A combination of DARA and atezo may improve clinical responses in NSCLC by enhancing anti-tumor T-cell responses facilitated by checkpoint inhibition. This study will assess the anti-tumor activity and safety profile of DARA plus atezo vs atezo alone in patients (pts) with previously treated advanced or metastatic NSCLC. Methods: This is an ongoing phase 1b/2 randomized, open-label, multicenter study of atezo (1200 mg intravenous [IV]; Day 2 of Cycle 1 and Day 1 of each 21-day cycle thereafter) alone or in combination with DARA (16 mg/kg IV weekly for 3 cycles [Days 1, 8, and 15] and then Day 1 of each 21-day cycle thereafter). Eligible pts (≥18 years) must have advanced or metastatic NSCLC and received standard platinum-based therapy with disease progression or intolerance to therapy. ECOG performance status of ≤1 and known PD-L1 tumor status are required. Pts previously treated with anti-CD38 therapy, including DARA, CD137 agonists, or immune checkpoint inhibitors are excluded. The primary endpoint is overall response rate. Secondary outcomes include safety, duration of response, clinical benefit rate (≥16 weeks duration), progression-free survival, OS, and pharmacokinetics and immunogenicity of DARA and atezo when given in combination. Approximately 96 pts will be enrolled; 6 pts will receive combination therapy in a safety run-in cohort for evaluation of dose-limiting toxicity followed by 90 pts randomly (1:1) assigned to the 2 treatments. Clinical trial information: NCT03023423.

Phase 1 study to evaluate the safety and efficacy of immunotherapy with tremelimumab and durvalumab in multiple myeloma patients receiving high dose chemotherapy and autologous stem cell transplant (HDT/ASCT) + peripheral blood lymphocyte (PBL) reinfusion.

TPS8051 Background: Multiple myeloma (MM) remains an incurable hematologic malignancy despite the advent of new classes of drugs, including immunomodulatory agents, proteasome inhibitors, and monoclonal antibodies. The success and synergistic activity of immunotherapy (IMT) in solid tumors and hematologic malignancies has fueled their investigation in MM. HDT/ASCT as consolidation or as treatment for relapse remains a cornerstone for improving overall survival. HDT/ASCT transiently eliminates immune-suppressive cell populations and provides a viable IMT platform. Reinfusion of PBLs harvested pre-HDT induces immune responses, supporting its inclusion in IMT combinations. This study evaluates the effect of IMT, using tremelimumab (T), an anti-CTLA-4 monoclonal antibody, and durvalumab (D), an anti-PD-L1 monoclonal antibody, together with autologous PBL reinfusion and starting T ± D at Day 100 and earlier (Day 30) post-ASCT. Methods: This ongoing Phase 1, open-label, multicenter study (NCT02716805) evaluates the safety and preliminary efficacy of T and D administered on 2 schedules in MM patients at high risk for relapse as outlined below. Cohort initiation requires dose-limiting toxicity in &lt; 2/6 patients in the previous cohort. The primary endpoint is safety. Secondary endpoints are objective response rate per IMWG, minimal residual disease, progression free and overall survival, and 100-day ASCT-related mortality. Exploratory endpoints include immunological effects and immune response. Enrollment opened 18 Nov 2016. As of 31 Dec 2016, 1 patient is enrolled in Cohort 1; enrollment is ongoing. Clinical trial information: NCT02716805. [Table: see text]

Improved Antitumor Activity of a Therapeutic Melanoma Vaccine through the Use of the Dual COX-2/5-LO Inhibitor Licofelone

Immune-suppressive cell populations impair antitumor immunity and can contribute to the failure of immune therapeutic approaches. We hypothesized that the non-steroidal anti-inflammatory drug licofelone, a dual cyclooxygenase-2/5-LO inhibitor, would improve therapeutic melanoma vaccination by reducing immune-suppressive cell populations. Therefore, licofelone was administered after tumor implantation, either alone or in combination with a peptide vaccine containing a long tyrosinase-related protein 2-peptide and the adjuvant α-galactosylceramide, all formulated into cationic liposomes. Mice immunized with the long-peptide vaccine and licofelone showed delayed tumor growth compared to mice given the vaccine alone. This protection was associated with a lower frequency of immature myeloid cells (IMCs) in the bone marrow (BM) and spleen of tumor-inoculated mice. When investigating the effect of licofelone on IMCs in vitro, we found that the prostaglandin E2-induced generation of IMCs was decreased in the presence of licofelone. Furthermore, pre-incubation of BM cells differentiated under IMC-inducing conditions with licofelone reduced the secretion of cytokines interleukin (IL)-10 and -6 upon lipopolysaccharides (LPS) stimulation as compared to untreated cells. Interestingly, licofelone increased IL-6 and IL-10 secretion when administered after the LPS stimulus, demonstrating an environment-dependent effect of licofelone. Our findings support the use of licofelone to reduce tumor-promoting cell populations.

High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

Osteocyte Apoptosis Attracts Myeloma Cells to Bone and Supports Progression through Regulation of the Bone Marrow Microenvironment

Multiple Myeloma (MM) pathogenesis and progression is intricately tied to the bone marrow (BM) microenvironment, and understanding this microenvironment is of upmost importance. Osteocytes are the most abundant cell type in bone, comprising over 90% of the cellular population. These cells were long thought to be primarily inert mechanical sensors, however osteocyte regulation of the BM is increasingly appreciated. Still, very little is known about the role of osteocytes in the context of cancer. We initially evaluated MM patient BM biopsies and determined that osteocyte apoptosis is increased compared to MGUS and healthy BM. We further validated this finding in the SCID-hu mouse model - increased osteocyte death was seen in the primary human bone grafts that were injected with aggressive MM cells compared to those injected with less aggressive MM cells. Surprisingly, and more interestingly, increased osteocyte apoptosis/death was also found in the contralateral, un-injected human bone grafts from mice with aggressive MM. Importantly, these changes in the secondary bone grafts occurred prior to detection of MM cells. In the present study, we investigated if and how osteocyte apoptosis at distant bone sites, before MM metastasis, feeds back to MM cells to support dissemination and progression.We developed a unique, syngeneic mouse model of MM in which osteocytes can be specifically ablated in vivo. Mice expressing the Diphtheria toxin receptor (DTR) in mature osteocytes (RIKEN BioResource Center, Japan) were crossed with C57Bl/KaLwRij mice, a strain that when injected with syngeneic 5TGM1 MM cells develops many characteristics of human MM. The resulting 8-week old progeny expressing the DTR gene and on the C57Bl/KaLwRij background were intraperitoneally (i.p.) injected with Diphtheria toxin (DT,12.5 μg/kg) or PBS as control (n=7 per group). After 7 days, mice were injected intravenously (i.v.) with 2x106 5TGM1 MM cells expressing firefly Luciferase. Weekly bioluminescent imaging and ELISA for IgG2bκ (secreted by 5TGM1 cells) tracked tumor location and growth over time. Remarkably, compared to control mice (DTR+PBS), osteocyte ablated (DTR+DT) mice showed significantly enhanced tumor homing to bone by bioluminescent imaging and significantly increased total tumor burden by both bioluminescent imaging and sera IgG2bκ ELISA.To understand mechanistically how osteocyte apoptosis enhances MM-bone homing and tumor progression, we evaluated BM microenvironmental changes after osteocyte apoptosis was induced. Syngeneic DTR mice were injected at 8 weeks with DT or PBS (n=3-4 per group). Mouse legs were collected after 7 days: one leg for sectioning and one leg for isolating BM cells and BM extracellular fluid. Flow cytometry was performed on BM cells and BM extracellular fluid was evaluated using a cytokine/chemokine array. Leg sections were stained via immunohistochemistry (IHC). Cytokine arrays of BM extracellular fluid revealed substantial increases of multiple cytokines/chemokines important for MM dissemination and progression (i.e. IL-6, HGF) in DTR+DT mice. Additionally, bone destruction-associated molecules DKK1, RANKL, and M-CSF were also increased in DTR + DT mice compared to DTR+PBS control mice. IHC showed increased numbers of osteocytes positive for sclerostin, HIF-1α and RANKL. Finally, flow cytometric analysis suggested heightened immune suppression in the BM as multiple immune-suppressive cell populations were enhanced in DTR+DT mice compared to DTR+PBS mice. These include myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and a regulatory B cell subset (Breg).In conclusion, we have successfully generated a syngeneic model of MM in which osteocytes are selectively ablated in vivo after a single injection of DT. Using this model, our data demonstrate that osteocyte apoptosis supports MM homing and growth in bone, likely as a result of an altered BM microenvironment. Interestingly, these changes include enhanced MM supportive/attractive soluble molecules, support of bone destruction, and enhanced immune suppression. Combined, our data suggests that osteocyte apoptosis is important for developing a pre-metastatic niche and contributes to MM progression in bone. Therefore, preventing osteocyte apoptosis may help stall and prevent dissemination of MM, and possibly other cancers, to bone. DisclosuresNo relevant conflicts of interest to declare.

Anti-Human T-Lymphocyte Immunoglobulin (ATG)-Induced T Regulatory Cells and Their Soluble Factors Suppress T Cell Proliferation: Potential Role in Allogeneic Stem Cell Transplantation

Polyclonal anti-human T-lymphocyte immunoglobulin(ATG) have been recently shown, in two randomized studies, to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells is suggested as one of the possible mechanisms involved. The aim of our current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance in patients receiving intravenous ATG (ATG-Fresenius ® S, Neovii Biotech) as part of their pre HSCT conditioning.Phenotypic characterization of regulatory cells markers revealedthattwo days culture of peripheral blood mononuclear cells (PBMCs) with ATG-F (30-120 µg/ml) resulted in significant increase in CD25 expression on CD4+ T cells. The percentages of cells expressing CTLA4, GITR, CD95 and FoxP3 was also significantly elevated on CD4+ cells compared to rabbit IgG-treated PBMCs. In addition, expression of CD127 and VLA-4 molecules was significantly decreased on CD4+CD25+ cells upon ATG-F treatment (p<0.01).Next, tolerance ability of ATG-F-induced cells was examined. Addition of ATG-F-treated cells to autologous PBMCs stimulated with antiCD3/antiCD28 antibodies resulted in significant (50-75%) inhibition of cell proliferation (p<0.001), measured by CFSE and Ki67 staining. Moreover, CD69 cell expression and interferon-γ (IFNγ) proinflammatory cytokine secretion were reduced by 50-60% and 65-90%, respectively, in the presence of ATG-F-activated Treg cells (p<0.01).Importantly, addition of cyclosporine A to the induction culture with ATG-F interfered with the ATG-induced regulatory phenotype acquisition, suggesting the involvement of interleukin-2 in ATG-mediated activity.In order to purify the tolerizing population, sorting of CD4+CD25+CD127-low cells (considered as viable regulatory T cells) from ATG-F-treated culture was performed. Sorted cells demonstrated greater suppressive potency than bulked pre-sorted cell population when added to autologous stimulated PBMCs. Of note, Treg-depleted fraction was still able to suppress the proliferation, albeit less efficiently then sorted Treg cells, suggesting that ATG-F is capable to induce multiple immune suppressive cell populations that should be further defined.To explore the possible involvement of soluble factor(s)-mediated mechanisms, in addition to the involvement of cell to cell contact mechanism, conditioned medium (CM) produced by ATG-F-primed cells was applied on stimulated autologous PBMCs. Addition of CM produced by ATG-F-treated cells, but not IgG-treated cells, resulted in significant suppression (30-65%, p<0.01) of T cell proliferation and activation, indicating the presence of soluble factors secreted by ATG-F-primed suppressive cells. Indeed, significant dose- and time-dependent induction of TGFβ secretion was observed in ATG-treated cells. To this end, addition of TGFβ receptor kinase inhibitor SB-431542 interfered with suppressive activity of ATG-F-primed cells, enabling partial rescue of proliferation and IFNγ secretion in response to antiCD3/antiCD28 activation. Similar results were obtained with anti-TGFβ neutralizing antibodies.Finally, characterization of phenotype and frequencies of regulatory immune populations in peripheral blood of 26 patients undergoing allogeneic transplantation with conditioning regimen including ATG-F (15mg/kg) was performed. Consistent with our ex vivo results, transient increase in percent of circulating CD4+CD25+CD127-low cells was detected in the ATG-F treated patients on days 14, 21 and 28 after HSCT. Furthermore, elevated levels of TGFβ were detected in the patients' plasma at day 28 and remaining high at day 60 post HSCT.Our results demonstratethat in vitro treatment with ATG-F is capableto induce functional Treg cells. Suppressive ability of ATG-F-induced cells was both contact and soluble-factors dependent and was partially promoted by TGFβ. Patients undergoing allogeneic HSCT with ATG-F-including conditioning demonstrated increased frequencies of circulating Treg cells and elevated plasma levels of TGFβ. Altogether, our data further support the use of ATG-F, a potent inducer of Treg cells, for prevention of GVHD post HSCT and potentially other therapeutic applications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transplant tolerance: a new role for IL-34.

Immune-suppressive cell populations, including Tregs and suppressor monocytes, have been implicated in long-term survival of allografts in both human transplant recipients and animal models. The factors that drive differentiation and function of these cell populations are not completely understood. In this issue, Bézie and colleagues identify IL-34 as an important mediator of allograft tolerance in a rat model of heart transplantation. Their data support a model in which IL-34 production by Tregs promotes a population of suppressive macrophages that in turn promote Treg differentiation. The results of this study support further exploration of the immunosuppressive properties of IL-34.

Myeloid-Derived Suppressor Cells: Critical Cells Driving Immune Suppression in the Tumor Microenvironment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress innate and adaptive immunity. MDSCs are present in many disease settings; however, in cancer, they are a major obstacle for both natural antitumor immunity and immunotherapy. Tumor and host cells in the tumor microenvironment (TME) produce a myriad of pro-inflammatory mediators that activate MDSCs and drive their accumulation and suppressive activity. MDSCs utilize a variety of mechanisms to suppress T cell activation, induce other immune-suppressive cell populations, regulate inflammation in the TME, and promote the switching of the immune system to one that tolerates and enhances tumor growth. Because MDSCs are present in most cancer patients and are potent immune-suppressive cells, MDSCs have been the focus of intense research in recent years. This review describes the history and identification of MDSCs, the role of inflammation and intracellular signaling events governing MDSC accumulation and suppressive activity, immune-suppressive mechanisms utilized by MDSCs, and recent therapeutics that target MDSCs to enhance antitumor immunity.

Abstract 3537: Manipulation of local and systemic immune suppression by GPI-anchored immune stimulatory proteins

Abstract Each day in the United States alone, approximately 3400 people are diagnosed while another 1500 people die from cancer. Breast cancer is the most common form of cancer among females and accounts for the second leading cause of death among female cancer patients. In order to be effective in a therapeutic setting, current immunotherapies must be able to overcome the multiple layers of tumor-induced immune suppression such as modulation of MHC expression and costimulatory molecules (CD80/CD86), expression of inhibitory molecules PDL-1 and GITRL and the secretion of immunosuppressive factors such as VEGF and TGF-β into the tumor microenvironment. These immunosuppressive factors promote the recruitment, differentiation and expansion of regulatory immune cells such as myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the tumor microenvironment. Using the 4T07 murine breast cancer model we investigated the effects of expressing GPI-anchored immune stimulatory molecules (GPI-ISMs), namely cytokines (IL-2, IL-12) and the costimulatory protein B7-1, on the surface of the tumor cells. BALB/c mice were challenged subcutaneously (s.c.) with either wild-type 4T07 cells (4T07-WT) or 4T07 cells expressing GPI-ISMs. We observed significant splenomegaly in the mice challenged with 4T07-WT cells relative to the mice challenged with 4T07 cells expressing GPI-ISMs. This observed splenomegaly correlated with tumor size and a 4-5 fold increase in the percentage of splenic CD11b+Gr1+ MDSCs indicating the role of active immune suppression in the tumorigenicity of 4T07 breast cancer cells. We then conducted studies to analyze the effect of GPI-ISMs on infiltrating cells into the tumor microenvironment as well as in the spleen and draining lymph nodes (dLNs). Three groups of mice were challenged (s.c.) with the following cells mixed in a 1:1 ratio with BD Matrigel™ (a solubilized basement membrane preparation derived from a mouse sarcoma): 4T07-WT, 4T07-B7/IL-12 or PBS (control). Seven days post challenge, the Matrigel/tumor, spleen and dLNs were harvested from the mice, digested and analyzed for cellular infiltrates by flow cytometry. We observed that the expression of GPI-ISMs on the surface of tumor cells led to reduced angiogenesis as evidenced by a reduced level of blood vessels and decreased presence of CD4+CD25+FOXP3+ regulatory T cells and CD11b+Gr1+ MDSCs locally at the tumor site and dLNs as well as systemically in the spleen. Additionally, there was a decrease in CD8+PD1+ exhausted T cells at the tumor site. Along with the inhibition of immune suppressive cell populations, the GPI-ISMs increased the presence of CD4+ and CD8+ T cells as well as dendritic cells and B cells. These observations suggest that components of the active immune suppression evident in this model can be inhibited by expressing GPI-ISMs on the surface of the 4T07 tumor cells and could be effective in a therapeutic setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3537. doi:1538-7445.AM2012-3537