Supplementary Table 1, Figures 1-4 from Nicotinamide Blocks Proliferation and Induces Apoptosis of Chronic Lymphocytic Leukemia Cells through Activation of the p53/miR-34a/SIRT1 Tumor Suppressor Network

Supplementary materials and methods

Supplementary Tables 1 and 2. Supplementary Table 1. List of the genes differentially expressed in CD11b+Gr1+ splenocytes from tumor-bearing, compared to control mice. Supplementary Table 2. List of the genes differentially expressed in CD38high, compared to CD38low, CD11b+Gr1+ splenocytes from tumor-bearing mice.

Supplementary Figures 1-8. Supplementary Fig. 1. CD11b+Gr1+ populations were isolated by FACS from spleens of tumor-bearing p120-/- and control mice. Supplementary Fig. 2. CD38 is expressed by the CD11b+Gr1+ population in a murine model of Barrett's Esophagus. Supplementary Fig. 3. The HNM007 tumors induce CD38 expression on MDSC population similarly to the autochthonous p120-/- tumors. Supplementary Fig. 4. Immunohistochemical analysis of HNM007 tumors co-injected with CD38High or CD38Low MDSCs. Supplementary Fig. 5. CD38high CD11b+Gr1+ cells produce higher levels of iNOS and pNFκB. Supplementary Fig.6. Network analysis of differentially regulated genes in CD38high and CD38low MDSCs shows upregulation of RB1 pathway. Supplementary Fig.7. Distribution of G-MDSC, M-MDSC and mature monocytes is perturbed in tumor-bearing Cd38-/- mice. Supplementary Fig. 8. CD38 is expressed on human MDSC-like cell population that is expanded in the peripheral blood of advanced-stage cancer patients.

Human amnion epithelial cells (hAEC) can be efficiently isolated from full-term amnion membrane and have been gaining recognition as advanced medical products. Such cells originate directly from the embryo during the early phase of development and exert a crucial function in the establishment of a tolerogenic environment, to avoid maternal immune rejection. Amnion cell immuno-modulation may be exploited, but additional efforts are required to establish the mechanisms underlying such capacity. The way to fully clarify such an issue is so far long. Here we overview current knowledge on the effects on innate or adaptive immune cells offered by intact hAEC or secreted mediators, pinpointing the mechanisms to date elucidated by our group and others. We move from the description of hAEC general features to molecular intermediaries generating effects directly or indirectly on immune cells. We focus on the role of non-canonical HLA class I molecules, with emphasis on HLA-G, but expand such analysis on adenosinergic mediators, cytokines, and hAEC-derived microvesicles. Finally, we report the ongoing clinical trials exploiting hAEC multipotency and immune modulation.

ObjectiveCD38 is a type II glycoprotein highly expressed on plasmablasts and on short- and long-lived plasma cells, but weakly expressed by lymphoid, myeloid, and non-hematopoietic cells. CD38 is a target for therapies aimed at depleting antibody-producing plasma cells. Systemic sclerosis (SSc) is an immune-mediated disease with a well-documented pathogenic role of B cells. We therefore analyzed CD38 expression in different subsets of peripheral blood mononuclear cells (PBMCs) from a cohort of SSc patients.MethodsCell surface expression of CD38 was evaluated on PBMCs from SSc patients using eight-color flow cytometry analysis performed with a FacsCanto II (BD). Healthy individuals were used as controls (HC).ResultsForty-six SSc patients (mean age 56, range 23-79 years; 38 females and 8 males), and thirty-two age- and sex-matched HC were studied. Twenty-eight patients had the limited cutaneous form and eighteen the diffuse cutaneous form of SSc. The mean disease duration was 7 years. Fourteen patients were on immunosuppressive therapy (14 MMF, 5 RTX). The total percentages of T, B and NK cells were not different between SSc and HC. Compared to HC, SSc patients had higher levels of CD3+CD38+ T cells (p<0.05), higher percentage (p<0.001) of CD3+CD4+CD25+FOXP3+ regulatory T cells, lower percentage (p<0.05) of CD3+CD56+ NK T cells. Moreover, SSc patients had higher levels of CD24highCD19+CD38high regulatory B cells than HC (p<0.01), while the amount of CD24+CD19+CD38+CD27+ memory B cells was lower (p<0.001). Finally, the percentages of circulating CD38highCD27+ plasmablasts and CD138+CD38high plasma cells were both higher in the SSc group than in HC (p<0.001). We did not observe any correlations between these immunophenotypes and disease subsets or duration, and ongoing immunosuppressive treatment.ConclusionsThe increased expression of CD38 in peripheral blood plasmablasts and plasma cells of SSc patients may suggest this ectoenzyme as a candidate therapeutic target, under the hypothesis that depletion of these cells may beneficially downregulate the chronic immune response in SSc patients. Validation of this data in multicenter cohorts shall be obtained prior to clinical trials with existing anti-CD38 drugs.

Background & Aim Placenta is a non-controversial and readily available source of stem cells for regenerative medicine. We previously reported that human amnion epithelial cells (hAEC) from term placenta are not tumorigenic, have immunomodulatory and anti-inflammatory properties and once transplanted differentiate into functional hepatocyte-like cells. In preclinical studies with immune-competent mice, hAEC engrafted and survived without administration of immunosuppressive drugs, resulting in correction of metabolic liver diseases or the reversal of acute liver failure. In clinical settings allogenic hAEC have been transplanted without immunosuppression. However, little or null is known about immunomodulatory cells, as hAEC. During the past years we studied and identified molecular pathways this novel stem cells has, driving immunoregulatory capacity. Methods, Results & Conclusion We performed a complete surface screening of hAEC, profiling all the molecules commonly described on other stem as well as somatic cells. Amnion characteristically lacks HLA class 2 expression and expresses both class 1a and non-polymorphic class 1b (responsible for maternal immune-toleration of the fetus). We quantified the level of expression of HLA-G and HLA-E molecules both as membrane-bound and soluble forms. Recently, purinergic mediators, hydrolyzed by plasma membrane nucleotidases, have also been identified to regulate immune cell response, thus we quantified the level of expression of all ecto-enzymes in hAEC preparations. Surprisingly, hAEC constitutively express all known ecto-enzymes. In addition, we proved constitutive expression of of CD47 (‘don't-eat-me’ signal) and complement system (CD55/CD59). Immunogenicity of the hAEC was confirmed on purified immune effector cells (T-, B- and NK-cells). Conclusions High level expression of ecto-enzymatic axis and non-canonical HLA molecules likely play a key role in immunological tolerance and long-term acceptance of the human xeno-cell graft in immunocompetent mice. The ability to treat the most common (liver) diseases with one stem cell therapy without the administration of immunosuppressive drugs could be a “game changer” and will greatly expand the number of patients who could receive cellular therapy. Based on their safety and the successful preclinical studies, approval was granted to begin banking of hAEC under cGMP condition at Karolinska Institutet, and to perform hAEC transplants on 10 patients with liver disease without immunosuppression. Placenta is a non-controversial and readily available source of stem cells for regenerative medicine. We previously reported that human amnion epithelial cells (hAEC) from term placenta are not tumorigenic, have immunomodulatory and anti-inflammatory properties and once transplanted differentiate into functional hepatocyte-like cells. In preclinical studies with immune-competent mice, hAEC engrafted and survived without administration of immunosuppressive drugs, resulting in correction of metabolic liver diseases or the reversal of acute liver failure. In clinical settings allogenic hAEC have been transplanted without immunosuppression. However, little or null is known about immunomodulatory cells, as hAEC. During the past years we studied and identified molecular pathways this novel stem cells has, driving immunoregulatory capacity. We performed a complete surface screening of hAEC, profiling all the molecules commonly described on other stem as well as somatic cells. Amnion characteristically lacks HLA class 2 expression and expresses both class 1a and non-polymorphic class 1b (responsible for maternal immune-toleration of the fetus). We quantified the level of expression of HLA-G and HLA-E molecules both as membrane-bound and soluble forms. Recently, purinergic mediators, hydrolyzed by plasma membrane nucleotidases, have also been identified to regulate immune cell response, thus we quantified the level of expression of all ecto-enzymes in hAEC preparations. Surprisingly, hAEC constitutively express all known ecto-enzymes. In addition, we proved constitutive expression of of CD47 (‘don't-eat-me’ signal) and complement system (CD55/CD59). Immunogenicity of the hAEC was confirmed on purified immune effector cells (T-, B- and NK-cells).

Adenosine (ADO) is an immunosuppressive molecule, which suppresses the immune responses by interacting with specific receptors expressed by immune effector cells. ADO is produced from ATP through the enzymatic activities of CD39 and CD73. Alternatively, ADO can be generated starting from NAD+, which is metabolized by the concerted action of CD38, CD203a/PC-1, and CD73. The role of ADO in immunity has been characterized in the last years in physiology and in pathological settings. This review examines a panel of reports focused on the functions of ADO in the context of human autoimmune/inflammatory diseases and the selected animal models. The final aim is to consider the role of adenosinergic ectoenzymes and ADO receptors as novel therapeutic targets for selected diseases.

Human myeloma cells grow in a hypoxic acidic niche in the bone marrow. Cross talk among cellular components of this closed niche generates extracellular adenosine, which promotes tumor cell survival. This is achieved through the binding of adenosine to purinergic receptors into complexes that function as an autocrine/paracrine signal factor with immune regulatory activities that i) down-regulate the functions of most immune effector cells and ii) enhance the activity of cells that suppress anti-tumor immune responses, thus facilitating the escape of malignant myeloma cells from immune surveillance. Here we review recent findings confirming that the dominant phenotype for survival of tumor cells is that where the malignant cells have been metabolically reprogrammed for the generation of lactic acidosis in the bone marrow niche. Adenosine triphosphate and nicotinamide-adenine dinucleotide extruded from tumor cells, along with cyclic adenosine monophosphate, are the main intracellular energetic/messenger molecules that serve as leading substrates in the extracellular space for membrane-bound ectonucleotidases metabolizing purine nucleotides to signaling adenosine. Within this mechanistic framework, the adenosinergic substrate conversion can vary significantly according to the metabolic environment. Indeed, the neoplastic expansion of plasma cells exploits both enzymatic networks and hypoxic acidic conditions for migrating and homing to a protected niche and for evading the immune response. The expression of multiple specific adenosine receptors in the niche completes the profile of a complex regulatory framework whose signals modify multiple myeloma and host immune responses.

Multiple myeloma (MM) derives from malignant transformation of plasma cells (PC), which accumulate in the bone marrow (BM), where microenvironment supports tumor growth and inhibits anti-tumor immune responses. Adenosine (ADO), an immunosuppressive molecule, is produced within MM patients' BM by adenosinergic ectoenzymes, starting from ATP (CD39/CD73) or NAD(+) [CD38/CD203a(PC-1)/CD73]. These ectoenzymes form a discontinuous network expressed by different BM cells. We investigated the expression and function of ectoenzymes on microvesicles (MVs) isolated from BM plasma samples of patients with MM, using asymptomatic forms of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as controls. The percentage of MVs expressing ectoenzymes at high levels was higher when derived from MM patients than controls. BM CD138(+) PC from MM patients expressed high levels of all ectoenzymes. Paired MVs samples confirmed a higher percentage of MVs with high ectoenzymes expression in MM patients than controls. Pooled MVs from MM patients or controls were tested for ADO production. The catabolism of ATP, NAD(+), ADPR and AMP to ADO was higher in MVs from MM patients than in those from controls. In conclusion, our results confirmed the hypothesis that MVs in MM niche are main contributor of ADO production. The ability of MVs to reach biological fluids strongly support the view that MVs may assume diagnostic and pathogenetic roles.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eμ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Immunotherapy is a growing area in the fight against cancer. Monoclonal antibodies (mAb) present an attractive concept due to the selective targeting of tumor cells, providing the promise of greater efficacy and better tolerability. mAbs exert their effects by engaging cells of the immune system, or by acting independently of these through the direct induction of apoptosis. So far, the search for effective mAbs in myeloma had been somewhat disappointing, however, recent progress suggests that mAb therapy may become a reality in the near future.

The interactions taking place between mother and embryo have been the focus of detailed studies in recent years, where pregnancy is considered as an in vivo transplant. The immune systems of the mother and the embryo together establish a condition of tolerance, which lasts throughout the pregnancy. Alongside immunogenetic components, a contribution is provided by the ectoenzyme network, a chain of surface molecules mainly operating in closed environments and potentially providing inhibitory or activator signals. One of the soluble products of the ectoenzyme network with immunosuppressory potential is adenosine, a purine nucleoside that plays multiple roles in almost all tissues and organs. The hypothesis behind the work was studied in patients with recurrent pregnancy loss (RPL), an event which remains unexplained in over 50 percent of cases. To this aim, we analyzed the expression of CD39 (ectonucleoside triphosphate diphosphohydrolase 1, ENTPD1) and CD73 (ecto-5’-nucleotidase, NT5E), the main pathway for adenosine generation, in samples obtained from women with RPL. The study included the evaluation of the expression of TNF-alpha (a pro-inflammatory cytokine) and of an alternative pathway of adenosine generation run by CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) and PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1). The results of this study highlight the existence of a network of surface enzymes expressed at the maternal/fetal interface and addressed to the production of adenosine. Perturbation of this network may induce a rescue pathway driven by CD38 and ENPP1. Ectoenzyme and inflammation may be considered now key elements in orchestrating the events leading to the interruption of pregnancy in the RPL sample analyzed and at the same potentially becoming therapeutic targets.