Dimitrios Mougiakakos

Multipotent mesenchymal stromal cells and the innate immune system

Multipotent mesenchymal stromal cells (MSCs) have unique immunoregulatory and regenerative properties that make them an attractive tool for the cellular treatment of autoimmunity and inflammation. Their underlying molecular mechanisms of action together with their clinical benefit — for example, in autoimmunity — are being revealed by an increasing number of clinical trials and preclinical studies of MSCs. However, autoimmunity and therapy-related alloimmunity are not only triggered and sustained by responses of the adaptive immune system; there is growing evidence that components of the innate immune system also have a key role. It is therefore important to study the crosstalk between MSCs and innate immunity, which ranges from the bone marrow niche to injured tissue.

Immature Immunosuppressive CD14+HLA-DR−/low Cells in Melanoma Patients Are Stat3hi and Overexpress CD80, CD83, and DC-Sign

Myeloid-derived suppressor cells (MDSC) have emerged as key immune modulators in various tumor models and human malignancies, but their characteristics in humans remain to be unequivocally defined. In this study, we have examined circulating CD14(+)HLA-DR(-/low) MDSC in 34 advanced malignant melanoma (MM) patients. Their frequency is significantly increased and associated with disease activity. Contrary to the common notion that MDSC are a heterogeneous population of exclusively immature cells, we find the coexpression of markers associated with mature phenotype. We show for the first time the overexpression of CD80, CD83, and DC-Sign in human MDSC. Further, increased levels of signal transducer and activator of transcription 3 (Stat3), an important regulator in MDSC development and function, were noted in MM-MDSC. Stat3 was altered toward an active, phosphorylated state in the HLA-DR(-) population of CD14(+) cells and was more reactive to activating stimuli in patients. Importantly, inhibition of Stat3 abolished their suppressive activity almost completely. The described MM-MDSC use arginase in conjunction with other yet undefined mechanisms to suppress CD4(+) and CD8(+) T cells. Several observations suggest a redox imbalance in MDSC and indicate an important role of Stat3-dependent oxidative stress in MDSC-mediated T-cell suppression. These results emphasize the diversity of MDSC in human cancer and provide potential targets for therapeutic interventions.

CLL-cells induce IDOhi CD14+HLA-DRlo myeloid-derived suppressor cells that inhibit T-cell responses and promote TRegs

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population that shares certain characteristics including an aberrant myeloid phenotype and the ability to suppress T cells. MDSCs have been predominantly studied in malignant diseases and findings suggest involvement in tumor-associated immune suppression. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults. Immune defects occur already at early disease stages and impact the clinical course. We assessed presence, frequency, association to other immune parameters, and functional properties of circulating CD14(+) cells lacking HLA-DR expression (HLA-DR(lo)) in patients with untreated CLL. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14(+)HLA-DR(lo) cells was significantly increased in CLL patients. Furthermore, MDSCs suppressed in vitro T-cell activation and induced suppressive regulatory T cells (TRegs). The MDSC-mediated modulation of T cells could be attributed to their increased indoleamine 2,3-dioxygenase (IDO) activity. CLL cells induced IDO(hi) MDSCs from healthy donor monocytes suggesting bidirectional crosstalk between CLL-cells, MDSCs, and TRegs. Overall, we identified a MDSC population that expands in CLL. The exact mechanisms responsible for such accumulation remain to be elucidated and it will be of interest to test whether antagonizing suppressive functions of CLL MDSCs could represent a mean for enhancing immune responses.

Naturally occurring regulatory T cells show reduced sensitivity toward oxidative stress-induced cell death.

Although the authors of several studies report elevated numbers of immunosuppressive regulatory T cells (Tregs) in hematologic and solid malignancies, the underlying mechanism is not fully clarified. Cancer is associated with oxidative stress mediated through reactive oxygen species produced by malignant cells, granulocytes, tumor-associated macrophages, and myeloid-derived suppressor cells. Oxidative stress is known to have detrimental effects on natural killer (NK) and T cells during chronic inflammatory conditions and cancer. Paradoxically, greater numbers of Tregs can be detected at tumor sites, indicating that Tregs can persist in this environment of increased oxidative stress. We demonstrate that Tregs, especially naive CD45RA(+), exhibit reduced sensitivity to oxidative stress-induced cell death and maintain their suppressive function, a phenomenon that may be attributed to their observed high antioxidative capacity. This newly described characteristic could explain their enrichment in malignancies associated with increased levels of oxidative stress.

The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

Increased thioredoxin-1 production in human naturally occurring regulatory T cells confers enhanced tolerance to oxidative stress

Levels of regulatory T cells (Tregs) are increased in different cancer types as well as in inflammatory diseases, such as rheumatoid arthritis. Treg accumulation may result from aberrant proliferation and trafficking as well as greater resilience to oxidative stress compared with conventional T cells. This enhanced antioxidative capacity of Tregs possibly serves as feedback inhibition during inflammation and prevents uncontrolled immune reactions by favoring survival of suppressor rather than effector cells. In this study, we demonstrate that human Tregs express and secrete higher levels of thioredoxin-1, a major antioxidative molecule. Thioredoxin-1 has an essential role in maintaining their surface thiol density as the first line of antioxidative defense mechanisms and is sensitive to proinflammatory stimuli, mainly tumor necrosis factor-α, in a nuclear factor-κB-dependent fashion. The antiapoptotic and oncogenic potential of (secreted) Trx-1 suggests that it may exert effects in Tregs beyond redox regulation.

Immunosuppressive CD14+HLA-DRlow/neg IDO+ myeloid cells in patients following allogeneic hematopoietic stem cell transplantation.

Myeloid-derived suppressor cells (MDSCs) have emerged as a heterogeneic immunoregulatory population that can expand in response to inflammatory signals. Predominantly studied in cancer, MDSCs suppress T cells utilizing various mechanisms. In allogeneic hematopoietic stem cell transplantation (allo-HSCT) therapy-related toxicity and alloreactivity increase inflammatory cytokines that might favor an MDSC accumulation. To address this question, circulating CD14+HLA-DRlow/neg cells were studied retrospectively in 51 allo-HSCT patients. These cells represent one of the few well-described human MDSC subsets under physiological and pathological conditions. The frequency of CD14+HLA-DRlow/neg cells was significantly increased after allo-HSCT, especially in patients with acute graft-versus-host disease. Compared to healthy donor cells they were pSTAT1low (phosphorylated signal transducer and activator of transcription) and indoleamine 2,3-dioxygenase (IDO)high. Serum levels of granulocyte colony-stimulating factor and interleukin-6, which both have been linked to MDSC induction, correlated positively with the frequency of CD14+HLA-DRlow/neg cells. In vitro dysfunction of patient T cells, such as reduced proliferative capacity or CD3ζ-chain expression, was rescued by blocking the IDO activity of CD14+HLA-DRlow/neg cells. Overall, we identified a T-cell-suppressive monocytic population that expands after allo-HSCT. The mechanisms responsible for such accumulation remain to be elucidated. It will be of great interest to prospectively investigate the influence of these cells on the graft-versus-tumor and -host reaction.

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses.

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the bodys innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

Mitochondrial metabolism contributes to oxidative stress and reveals therapeutic targets in chronic lymphocytic leukemia

Alterations of cellular metabolism represent a hallmark of cancer. Numerous metabolic changes are required for malignant transformation, and they render malignant cells more prone to disturbances in the metabolic framework. Despite the high incidence of chronic lymphocytic leukemia (CLL), metabolism of CLL cells remains a relatively unexplored area. The examined untreated CLL patients displayed a metabolic condition known as oxidative stress, which was linked to alterations in their lymphoid compartment. Our studies identified mitochondrial metabolism as the key source for abundant reactive oxygen species (ROS). Unlike in other malignant cells, we found increased oxidative phosphorylation in CLL cells but not increased aerobic glycolysis. Furthermore, CLL cells adapted to intrinsic oxidative stress by upregulating the stress-responsive heme-oxygenase-1 (HO-1). Our data implicate that HO-1 was, beyond its function as an antioxidant, involved in promoting mitochondrial biogenesis. Thus ROS, adaptation to ROS, and mitochondrial biogenesis appear to form a self-amplifying feedback loop in CLL cells. Taking advantage of the altered metabolic profile, we were able to selectively target CLL cells by PK11195. This benzodiazepine derivate blocks the mitochondrial F1F0-ATPase, leads to a surplus production of mitochondrial superoxide, and thereby induces cell death in CLL cells. Taken together, our findings depict how bioenergetics and redox characteristics could be therapeutically exploited in CLL.

Intratumoral forkhead box P3-positive regulatory T cells predict poor survival in cyclooxygenase-2–positive uveal melanoma

Forkhead box P3 (FOXP3)‐positive regulatory T cells (Tregs) are key mediators of peripheral tolerance and suppress efficient antitumor responses. Prostaglandin E2 (PGE2) produced by inducible cyclooxygenase‐2 (COX‐2) can lead to Treg induction. COX‐2 expression has been linked to tumorigenesis and growth in various malignancies. The objective of the current study was to investigate whether Tregs infiltrate uveal melanomas (UMs) and whether their prevalence is linked to COX‐2 expression and the prediction of overall survival (OS).