Immune evasion in cancer:
role of myeloid-derived suppressor cells

Field of Interest

Tumor-induced T cell tolerance is a major mechanism that facilitates tumor progression and limits the efficacy of immune-based therapeutic interventions. My group is focused on the characterization of myeloid-derived suppressor cells (MDSCs), one of the cell type recruited by tumors to impair T cell function. Within the past few years, my group contributed to demonstrate the role of MDSCs as potent suppressor of the antitumor immune responses, both in the tumor microenvironment and lymphoid organs. We found that it was possible to rouse lymphocytes and induce cancer cell killing by interfering with the activity of two enzymes, arginase and nitric oxide synthase, highly expressed in MDSCs.
Based on our previous findings, we are currently trying to address different issues related to MDSC biology:

Novel drugs affecting L-arginine metabolism in tumor environment as potential adjuvants for adoptive cell therapy

It has been proposed that alterations of the L-arginine metabolism in the tumor environment impair T cell functions in mice. Induction of nitric oxide synthase (NOS2), which generates nitric oxide (NO), and arginase 1 (ARG1), which depletes the L-arginine milieu, in MDSCs and in tumor cells themselves, block mouse tumor-infiltrating lymphocyte (TIL) activation and proliferation. Under conditions of low L-arginine concentrations caused by enhanced ARG1 activity, NOS2 generates superoxide (O^2-). In the tumor environment, NO reacts with O^2- to generate peroxynitrite (ONOO^-), a highly reactive oxidizing agent that nitrates tyrosine on proteins, thus inhibiting activation-induced protein tyrosine phosphorylation. We have confirmed the existence of enhanced peroxynitrite production in human cancers, such as prostate, colon, and liver carcinomas. Moreover, we generated and patented new drugs able to inhibit intratumoral peroxynitrite generation. Preliminary in vitro studies indicated that these drugs are able to restore proliferation and cytotoxic activity of T cells from tolerant, tumor-bearing mice. Moreover, among the several compounds we identified one specific drug (AT38) that inhibits intratumoral generation of RNS in vivo. We want to explore whether AT38 can increase the recruitment of tumor-specific CD8⁺ T cells inside the tumor and reduce tumor growth when given in combination with adoptive immunotherapy. Preliminary data suggest that AT38 drugs can relieve the nitration of chemokines present in tumor-microenvironment, a novel tumor-induced mechanism of post-translational modification that negatively affect the chemokine function.

Role of microRNAs in c ontrollin MDSC generation and function

MicroRNAs (miRs), whose expression varies during the differentiation and maturation of human and mouse haematopoietic cells, can regulate myelopoiesis by interaction with specific target mRNAs. We performed a miR expression analysis of MDSCs by microarrays and, to extend further our analysis to poorly expressed and potentially new miRs, by creating and sequencing MDSC-specific miR libraries. These approaches allowed us to identify miRs differentially expressed in MDSCs in comparison to myeloid cells present in a normal context, which do not possess suppressive activity. We can use cytokines to mimic maturation and differentiation of MDSCs form bone marrow cells. This guarantees an optimal model for in vitro analysis of miR activity. Preliminary experiments, in which we forced the expression of mature miRs found to be down-regulated in tumor-infiltrating MDSCs, showed a significantly decrease in the percentage of the immunosuppressive CD11b⁺ Gr-1^low/- cells. Moreover, these cultures had lost completely the suppressive activity against antigen-activated T lymphocytes. To study how these miRs modulate haematopoiesis and the immune response in vivo, we are planning to infect haematopoietic progenitor cells with lentivirus encoding the miRs before reconstituting the bone marrow in sublethally irradiated mice. Bone-marrow chimeras will be transplanted with different tumors able to recruit MDSCs. We will evaluate tumor growth and mice survival. We will also assess whether miR expression is able to change primary and secondary lymphoid organ structure through histological and flow cytometrical analysis. In particular, we will focus on lymphoid and myeloid lineages. At the same time we will study, both in vitro and in vivo, the immunocompetence of these mice.

Adjuvant role of chemotherapy depends on the effect on a early myeloid-committed, proliferating population

Myeloablation before adoptive transfer of TILs has given promising results in the therapy of some tumors such as human metastatic melanoma. Despite these encouraging results, complete regression of established tumors following adoptive therapy occurred rarely, even in immunodeficient mice that do not require myeloablation prior to ACT. In both immunocompetent and immunodeficient mice, administration of chemotherapeutic drugs provided a significant adjuvant activity on ACT, at doses that had either no adverse effect nor any impact on tumor progression. This adjuvant property was long lasting and associated with the disappearance of a population of proliferating myeloid cells endowed with immunosuppressive function on antigen-activated CD8⁺ T lymphocytes. These cells represented a discrete fraction of MDSCs characterized by the ability to proliferate. These cells appear to possess "stemness" properties and share an hematologic niche with a subset of CD11b^-/Gr-1⁺ lymphoid cells that we are currently characterizing. These studies are conducted in collaboration with the group coordinated by Dr. Paolo De Coppi.

Lymphonanocarriers for the treatment of metastatic cancer

We are part of an european multidisciplinary consortium that is attempting to combine nanotechnology and biomedicine for the development of anticancer treatments targeted specifically to the lymphatic nodes. Taking advantage of the ability of selected nanoparticles to localize to the lymph nodes, we plan to deliver drugs and immunostimulating complexes to this compartment, preventing the process of metastatic spreading through lymphatic vessels. We aim at reaching an advanced preclinical evaluation stage of these novel nanocarriers systems.

Arginase 1 in cancer progression and tumor immune escape

The isoform 1 of arginase (Arg1) in tumor-bearing mice is mainly expressed in tumor-infiltrating myelomonocytic cells. The activity of Arg1, is induced by various factors present in the tumor microenvironment, such us cytokines, hypoxia, cAMP, and prostaglandins. The major aim of this project is to investigate how Arg1 can influence tumor development and progression. Although Arg inhibitors have been used, both in vitro and in vivo, their non-specific effects prevent to draw definitive conclusions on the role of Arg1 as tumor promoter and landscaper gene. We plan to investigate the Arg1 role in vivo by means of different approaches: evaluate the biological effects of Arg1 expression in a transgenic mouse model where macrophages possess an enzyme form that is constitutively active rather than inducible; evaluate the biological effects of the Arg1 inactivation in a conditional knock-out (KO) mouse where the enzyme inactivation is accomplished only in myelomonocytic cells (conditional KO mouse is necessary because the Arg1 systemic deletion is lethal, being the liver enzyme critical to assure the nitrogenous waste); evaluate the kinetics of Arg1 expression during tumor progression in a novel Arg1 reporter mouse model.

Synoptic CV

Honours

Selected Publications (VIMM)

Additional Publications

Selected Seminars

Contact

Vincenzo Bronte Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 228 Fax:  (+39) 049 7923 260

Last updated: March 2007, VB ·

T cell activation in health and diseases

Field of Interest

Figure 1.The actin-binding protein FLNa is required for CD28-induced raft organization and recruitment into the immunological synapse. Jurkat T cells expressing the raft marker MyrPalm-mCFP (in red in the figure) were transfected (B) or not (A) with Myc-tagged FLNaD10-12, a FLNa fragment that functions as a dominant-negative mutant which prevents CD28 interaction with endogenous FLNa. In this figure, two confocal images of fixed conjugates (between Jurkat cells and SEE-pulsed 5-3.1/B7 antigen presenting cells) with raft recruitment into the immunological synapse in the presence of wt FLNa (A) and loss of the recruitment when the FLNaD10-12 mutant is expressed (B). Scale bars, 10 µm.

Figure 1.The actin-binding protein FLNa is required for CD28-induced raft organization and recruitment into the immunological synapse. Jurkat T cells expressing the raft marker MyrPalm-mCFP (in red in the figure) were transfected (B) or not (A) with Myc-tagged FLNaD10-12, a FLNa fragment that functions as a dominant-negative mutant which prevents CD28 interaction with endogenous FLNa. In this figure, two confocal images of fixed conjugates (between Jurkat cells and SEE-pulsed 5-3.1/B7 antigen presenting cells) with raft recruitment into the immunological synapse in the presence of wt FLNa (A) and loss of the recruitment when the FLNaD10-12 mutant is expressed (B). Scale bars, 10 µm.
[click image to enlarge]

My group studies signals modulating the immune response in physiopathological conditions. We try understanding how to help lymphocytes to fight cancer or viruses on the one side, and, on the other, how to block improper lymphocyte activation and thus autoimmune diseases.

Throughout its life, a naïve T lymphocyte continually circulates between blood and lymph nodes searching for the right partner, an antigen-presenting cell (APC) carrying peptide-MHC complexes specific for its antigen-recognition receptors. After this encounter occurring in a specialized area of the lymph nodes, T cells migrate into the inflamed tissue and exert their effector functions. We can therefore identify three distinct phases in T lymphocyte physiology: 1) T cell migration; 2) T cell priming; 3) T cell migration and effector functions.

Figure 2. Mitochondria fission is required for recruitment of the organelles to the cell uropod, phenomenon that constrains lymphocyte polarization and migration. Jurkat T cells were co-transfected with mtRFP and empty vector (A-C), mtRFP and the pro-fission protein DRP1 (D-F), or mtRFP and the pro-fusion protein OPA1 (G-I). Confocal images of mitochondrial shape in resting (A, B, D, E, G, H) or polarized cells (C, F, I). (A, D, G) Volume-rendered 3D reconstructions of mitochondrial network. When transfected with DRP1 the cells showed fragmented mitochondria (E) and, after chemokine treatment, a polarized phenotype (F). In contrast, when OPA1 was overexpressed, the cells showed fused mitochondria (H), and the impaired polarization in response to chemokine, as shown in (I). Scale bar, 5 µm.

Figure 2. Mitochondria fission is required for recruitment of the organelles to the cell uropod, phenomenon that constrains lymphocyte polarization and migration. Jurkat T cells were co-transfected with mtRFP and empty vector (A-C), mtRFP and the pro-fission protein DRP1 (D-F), or mtRFP and the pro-fusion protein OPA1 (G-I). Confocal images of mitochondrial shape in resting (A, B, D, E, G, H) or polarized cells (C, F, I). (A, D, G) Volume-rendered 3D reconstructions of mitochondrial network. When transfected with DRP1 the cells showed fragmented mitochondria (E) and, after chemokine treatment, a polarized phenotype (F). In contrast, when OPA1 was overexpressed, the cells showed fused mitochondria (H), and the impaired polarization in response to chemokine, as shown in (I). Scale bar, 5 µm.
[click image to enlarge]

1) Cell migration

We have recently demonstrated that leukocyte migration is controlled by mitochondrial shape and position inside the cells. We found that during leukocyte migration mitochondria specifically concentrate at the uropod by a process involving rearrangements of their shape and suggested that mitochondria redistribution is required to fuel the motor of migrating cells.

2) T cell priming

We study the interaction between T cells and APCs. In particular, we analyze the role of plasma membrane lipid microdomain — known as "lipid rafts" — and chemokine receptors at the immunological synapse.

3) T cell effector functions

Prostate cancer is the second leading cause of malignancy-related mortality in males in the Western world and available treatments for its metastatic form have demonstrated weak curative efficacy. It is therefore necessary to find alternative therapeutic approaches prostate cancer and immunotherapy may represent an interesting possibility. To analyze the modulation of T cell responses by the prostate tumor environment, we performed a study based on the use of collagen gel-matrix supported organ cultures of human prostate carcinomas. Our results identified a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

Synoptic CV

Honours

Selected Publications (VIMM)

Additional Publications

Selected Seminars

Contact

Antonella Viola Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel. lab.:  (+39) 049 7923 230 Fax:  (+39) 049 7923 260

Last updated: March 2007, AV ·