Dietz, Allan B.

Abstract Imatinib mesylate (imatinib, Gleevec®, Novartis, Basel, Switzerland) inhibits T cells in vitro and in vivo (Dietz et al., Blood104: 1094–1099, 2004; Cwynarski et al., Leukemia18: 1332–1339, 2004). The drug blocks T cell cycle progression rather uniquely as it neither inhibits expression of CD69, an early marker of T cell activation, nor induces apoptosis. To characterize the molecular effects of imatinib leading to this mode of T-cell inhibition, we measured the changes in transcriptome (by Affymetrix U133 chips), proteome and phosphoproteome (by Western blotting, differential phosphoprotein expression and mass spectrometry). We found that phytohemagglutinin activated T cells pre-treated with imatinib had reduced expression of 983 transcripts and increased expression of 271 transcripts when compared to untreated PHA activated T cells by the factor of 1.5 or more (p<0.05). Among the prominently down-regulated transcripts were granzyme B, CTLA-4 and IL-2-receptor α-chain (CD25), all characteristic of activated T cells, as well as cyclins D2 and D3 and cyclin-dependent kinases 3, 4 and 7, the molecules regulating cell cycle progression. Among the up-regulated transcripts were Kruppel-like transcription factors 2 and 7, and p27, a finding compatible with the observed cell cycle inhibition. Furthermore, we selected and identified 30 proteins from 2-D gels that were up-regulated and/or hyperphosphorylated in imatinib treated activated T cells. Among these were four heterogeneous ribonucleoproteins, three lamins and γ-actin, all components of the nucleoskeleton at the interface of chromatin and inner nuclear membrane and involved in replication and transcription (Herrmann and Foisner, Cell. Mol. Life Sci.60: 1607–1612, 2003; Shumaker et al. Curr. Opinion Cell Biol.15: 358–366, 2003). Thus, imatinib-borne interference with T cell signal transduction affects the nuclear structure indicating for the first time that nucleoskeleton structural changes are associated with T cell activation status.

Abstract Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

AbstractCD34+ cells are haematopoietic stem cells used therapeutically in patients undergoing radiation or chemotherapy due to their regenerative potential and ability to restore the haematopoietic system. In animal models, CD34+ cells have been associated with therapeutic angiogenesis in response to ischaemia. Several trials have shown the potential safety and efficacy of CD34+ cell delivery in various cardiovascular diseases. Moreover, Phase III trials have now begun to explore the potential role of CD34+ cells in treatment of both myocardial and peripheral ischaemia. CD34+ cells have been shown to be safe and well-tolerated in the acute myocardial infarction (AMI), heart failure, and angina models. Several studies have suggested potential benefit of CD34+ cell therapy in patients with coronary microvascular disease as well. In this review, we will discuss the therapeutic potential of CD34+ cells, and describe the pertinent trials that have used autologous CD34+ cells in no-options refractory angina, AMI, and heart failure. Lastly, we will review the potential utility of autologous CD34+ cells in coronary endothelial and microvascular dysfunction.

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L‐MSCs) are superior immunomodulators and contribute to liver‐mediated tolerance. L‐MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs). Trilineage differentiation of L‐MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L‐MSCs are different than the A‐MSCs and BM‐MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L‐MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L‐MSCs (versus A‐MSCs/BM‐MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ–producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L‐MSCs was not dependent on cell‐to‐cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3‐dioxygenase for complete reversal. In conclusion, L‐MSCs appear to be uniquely well‐equipped immunomodulatory cells, and they are more potent than A‐MSCs and BM‐MSCs in that capacity, which suggests that they may contribute to liver‐induced systemic tolerance.

AbstractPurpose:Patients with HER2+ breast cancer benefit from trastuzumab-containing regimens with improved survival. Adaptive immunity, including cytotoxic T-cell and antibody immunity, is critical to clinical efficacy of trastuzumab. Because Th cells are central to the activation of these antitumor effectors, we reason that HER2 patients treated with trastuzumab may benefit by administering vaccines that are designed to stimulate Th-cell immunity.Patients and Methods:We developed a degenerate HER2 epitope–based vaccine consisting of four HLA class II–restricted epitopes mixed with GM-CSF that should immunize most (≥84%) patients. The vaccine was tested in a phase I trial. Eligible women had resectable HER2+ breast cancer and had completed standard treatment prior to enrollment and were disease free. Patients were vaccinated monthly for six doses and monitored for safety and immunogenicity.Results:Twenty-two subjects were enrolled and 20 completed all six vaccines. The vaccine was well tolerated. All patients were alive at analysis with a median follow-up of 2.3 years and only two experienced disease recurrence. The percent of patients that responded with augmented T-cell immunity was high for each peptide ranging from 68% to 88%, which led to 90% of the patients generating T cells that recognized naturally processed HER2 antigen. The vaccine also augmented HER2-specific antibody. Immunity was sustained in patients with little sign of diminishing at 2 years following the vaccination.Conclusions:Degenerate HLA-DR–based HER2 vaccines induce sustainable HER2-specific T cells and antibodies. Future studies, could evaluate whether vaccination during adjuvant treatment with trastuzumab-containing regimens improves patient outcomes.

Biliary complications (strictures and leaks) represent major limitations in living donor liver transplantation. Mesenchymal stem cells (MSCs) are a promising modality to prevent biliary complications because of immunosuppressive and angiogenic properties. Our goal was to evaluate the safety of adipose‐derived MSC delivery to biliary anastomoses in a porcine model. Secondary objectives were defining the optimal method of delivery (intraluminal versus extraluminal) and to investigate MSC engraftment, angiogenesis, and fibrosis. Pigs were divided into 3 groups. Animals underwent adipose collection, MSC isolation, and expansion. Two weeks later, animals underwent bile duct transection, reanastomosis, and stent insertion. Group 1 received plastic stents wrapped in unseeded Vicryl mesh. Group 2 received stents wrapped in MSC‐seeded mesh. Group 3 received unwrapped stents with the anastomosis immersed in an MSC suspension. Animals were killed 1 month after stent insertion when cholangiograms and biliary tissue were obtained. Serum was collected for liver biochemistries. Tissue was used for hematoxylin‐eosin and trichrome staining and immunohistochemistry for MSC markers (CD44 and CD34) and for a marker of neoangiogenesis (CD31). There were no intraoperative complications. One pig died on postoperative day 3 due to acute cholangitis. All others recovered without complications. Cholangiography demonstrated no biliary leaks and minimal luminal narrowing. Surviving animals exhibited no symptoms, abnormal liver biochemistries, or clinically significant biliary stricturing. Group 3 showed significantly greater CD44 and CD34 staining, indicating MSC engraftment. Fibrosis was reduced at the anastomotic site in group 3 based on trichrome stain. CD31 staining of group 3 was more pronounced, supporting enhanced neoangiogenesis. In conclusion, adipose‐derived MSCs were safely applied to biliary anastomoses. MSCs were locally engrafted within the bile duct and may have beneficial effects in terms of fibrosis and angiogenesis.

Abstract Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients. Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry. Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays. Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor, and novel therapeutic strategies that make a substantial impact on outcomes are sorely needed. Immunotherapies have shown great promise in the treatment of a number of cancers in recent year, and a concerted effort is being made to apply these treatment paradigms to GBM. However, many GBM patients exhibit profound immunosuppression, likely limiting the efficacy of such approaches. The mechanisms of this immunosuppression are poorly understood, but tumor-derived extracellular vesicles (EVs), may play a role. We demonstrate that GBM-derived EVs induce the development of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). We further demonstrate that these EV-induced monocytic cells are immunosuppressive, resulting in impaired T cell proliferation upon co-culture. We found that the immunosuppressive effects of tumor-derived EVs appear to be driven by the induction of these immunosuppressive monocytes, as EV treatment of T cells did not significantly impact T cell proliferation. Further, we sought to characterize the important of programmed death ligand 1 (PD-L1) in the induction of these immunosuppressive monocyte populations. We found PD-L1 to be expressed in the EVs from GBM cell lines, and that modulation in PD-L1 expression via either constitutive overexpression or shRNA-mediated knockdown resulted in concordant changes in expression in tumor-derived EVs. We demonstrate that PD-L1 is important for the induction of NCM but not for MDSCs. Taken together, these findings point to a significant role for tumor-derived EVs in the induction of immunosuppressive monocytes in GBM, and that these cells may be a driving force of systemic immunosuppression. PD-L1 is one factor expressed in EVs that has immunomodulatory properties, but additional EV cargo likely plays a major role in the induction of immunosuppressive cells.

Abstract Recent successes in cancer immunotherapy have provided new avenues for the treatment of glioblastoma multiforme (GBM). Several immunotherapeutic approaches are now being utilized to treat patients with GBM including checkpoint inhibition, autologous/CAR-T cell therapy, and cancer vaccines. Preliminary data suggests that the majority of patients have sub-optimal clinical responses to these therapies. The impaired anti-tumor immune responses observed in these patients are likely a consequence of immune system dysfunction contributed to by a variety of factors that include diminished antigen presentation/detection, leukopenia, alterations in cytokine levels and cellular mediators. We have previously demonstrated that patients with GBM exhibit profound immune suppression resulting from both tumor and treatment related effects via a comprehensive quantitation of peripheral blood leukocytes by flow cytometry. We hypothesize that the depth and breadth of immunosuppression will significantly affect responses to immunotherapy. Prominent phenotypic abnormalities observed in GBM patients include elevated levels of CD14+HLA-DRlo/neg monocytes and reduced absolute CD4 T cell counts. We will present data how these and other phenotypes may potentially influence responses to patients treated with dendritic cell vaccines. Our goal is to understand the role of these cells in the context of immunosuppression not only to facilitate the development of targeted immunotherapies to circumvent their effects, but also to potentially utilize them as biomarkers for understanding diverse responses to immunotherapies.

AbstractThe interest in the use of human dendritic cells in cancer immunotherapy calls for efficient ex vivo methods of dendritic cell education. To extend the range of methods available, we generated phenotypically characteristic dendritic cells from peripheral blood monocytes incubated with granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected them with an adenovirus containing a humanized version of green fluorescent protein as a marker of gene expression. The levels of expressed protein were high, but they were further increased in combination with cationic liposomes. In comparison to transfection efficiency of the homologous expression plasmid, adenovirus-mediated gene transfer was substantially more efficient. With the aid of liposome-mediated infection, gene transfer into CD83+ dendritic cells was highly effective, resulting in more than 90% of the cells expressing the transgene.