Michael Papamichail

Interactions Between Human Mesenchymal Stem Cells and Natural Killer Cells

Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T‐cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK‐to‐MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto‐toxicity against HLA‐class I– expressing targets. Some of these effects require cell‐to‐cell contact, whereas others are mediated by soluble factors, including transforming growth factor–β1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC‐mediated NK‐cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large‐scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fi‐broblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA‐class I and induces low HLA‐DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF‐cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.

Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium.

The aim of the study was to investigate whether a combination of mesenchymal stem cells (MSCs) capable of differentiating into cardiac myocytes and endothelial progenitors (EPCs) that mainly promote neoangiogenesis might be able to facilitate tissue repair in myocardial scars. Previous studies have shown that intracoronary transplantation of autologous bone marrow stem cells results in improvement of contractility in infracted areas of human myocardium. Eleven patients with an anteroseptal myocardial infarction (MI) underwent transcoronary transplantation of bone marrow‐derived MSCs and EPCs to the infarcted area through the left anterior descending artery. Eleven age‐ and sex‐matched patients served as controls. Wall motion score index was significantly lower at follow‐up in the transplantation (P = 0.04) but not in the control group. On stress echocardiography, there was improvement of myocardial contractility in one or more previously nonviable myocardial segments in 5 out of 11 patients (all with recent infarctions) and in none of the controls (P = 0.01). Restoration of uptake of Tc99m sestamibi in one or more previously nonviable myocardial scars was seen in 6 out of 11 patients subjected to transplantation and in none of the controls (P = 0.02). Cell transplantation was an independent predictor of improvement of nonviable tissue. Intracoronary transplantation of MSCs and EPCs is feasible, safe, and may contribute to regional regeneration of myocardial tissue early or late following MI.

An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry

The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initially used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodimer-1, a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

Tumor-Specific CD4+ T Lymphocytes from Cancer Patients Are Required for Optimal Induction of Cytotoxic T Cells Against the Autologous Tumor

This study focuses on the specific CD4+ T cell requirement for optimal induction of cytotoxicity against MHC class II negative autologous tumors (AuTu) collected from patients with various types of cancer at advanced stages. CD4+ T cells were induced in cultures of cancer patients’ malignant effusion-associated mononuclear cells with irradiated AuTu (mixed lymphocyte tumor cultures (MLTC)) in the presence of recombinant IL-2 and recombinant IL-7. Tumor-specific CD4+ T cells did not directly recognize the AuTu cells, but there was an MHC class II-restricted cross-priming by autologous dendritic cells (DCs), used as APC. CD8+ CTL, also induced during the MLTC, lysed specifically AuTu cells or DCs pulsed with AuTu peptide extracts (acid wash extracts (AWE)) in an MHC class I-restricted manner. Removal of CD4+ T cells or DCs from the MLTC drastically reduced the CD8+ CTL-mediated cytotoxic response against the AuTu. AWE-pulsed DCs preincubated with autologous CD4+ T cells were able, in the absence of CD4+ T cells, to stimulate CD8+ T cells to lyse autologous tumor targets. Such activated CD8+ T cells produced IL-2, IFN-γ, TNF-α, and GM-CSF. The process of the activation of AWE-pulsed DCs by CD4+ T cells could be inhibited with anti-CD40 ligand mAb. Moreover, the role of CD4+ T cells in activating AWE-pulsed DCs was undertaken by anti-CD40 mAb. Our data demonstrate for the first time in patients with metastatic cancer the essential role of CD4+ Th cell-activated DCs for optimal CD8+ T cell-mediated killing of autologous tumors and provide the basis for the design of novel protocols in cellular adoptive immunotherapy of cancer, utilizing synthetic peptides capable of inducing T cell help in vivo.

Cell Culture Medium Composition and Translational Adult Bone Marrow‐Derived Stem Cell Research

The use of fetal calf serum (FCS) for the culture of cells to be used in clinical trials raises potential hazards that cannot be neglected, but this is a regulatory issue. However, as specifically regards the isolation and expansion of human mesenchymal stem cells (MSCs), unfortunately serum‐free media have not yet been defined. The alternative of using autologous serum is feasible only for the minority of clinical protocols involving low numbers of MSCs, because a minimum concentration of 10% in the culture medium is required. Besides, because allogeneic serum results in MSC growth arrest and death, use of pooled human serum does not represent an alternative. Finally, vast numbers of MSCs cultured in FCS‐containing media have already been used in many clinical trials targeting a variety of disorders, without any significant side effects, including ventricular arrhythmia.

Natural killer lymphocytes: biology, development, and function

Natural killer (NK) lymphocytes represent the first line of defense against virally infected cells and tumor cells. The role of NK cells in immune responses has been markedly explored, mainly due to the identification of NK cell receptors and their ligands, but also through the analysis of mechanisms underlying the effects of various cytokines on NK cell development and function. A population of lymphocytes that shares function and receptors with NK cells is represented by natural killer T (NKT) cells. NKT lymphocytes are regulators of both innate and adaptive immune responses, but have also been reported to function as effector antitumor cells. The marked progress in our understanding of the biology, development, and function of NK/NKT cells has provided the basis for their potential application in tumor clinical trials.

Combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies for cancer therapy

Accumulating evidence suggests that despite the potency of cytotoxic anticancer agents, and the great specificity that can be achieved with immunotherapy, neither of these two types of treatment by itself has been sufficient to eradicate the disease. Still, the combination of these two different modalities holds enormous potential for eliciting therapeutic results. Indeed, certain chemotherapeutic agents have shown immunomodulatory activities, and several combined approaches have already been attempted. For instance, chemotherapy has been proven to enhance the efficacy of tumor cell vaccines, and to favor the activity of adoptively transferred tumor-specific T cells. A number of mechanisms have been proposed for the chemotherapy-triggered enhancement of immunotherapy response. Thus, chemotherapy may favor tumor cell death, and by that enhance tumor-antigen cross-presentation in vivo. Drug-induced myelosuppression may induce the production of cytokines favoring homeostatic proliferation, and/or ablate immunosuppression mechanisms. Furthermore, the recently reported synergy between monoclonal antibodies and chemotherapy or peptide vaccination is based upon the induction of endogenous humoral and cellular immune responses. This would suggest that monoclonal antibodies may not only provide passive immunotherapy but can also promote tumor-specific active immunity. This article will review several strategies in which immunotherapy can be exploited in preclinical and clinical studies in combination with other agents and therapeutic modalities that are quite unique when compared with “conventional” combination therapies (ie, treatments with chemotherapeutic drugs or chemotherapy and radiotherapy based protocols). The results from these studies may have significant implications for the development of new protocols based on combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies, suggesting an exciting potential for therapeutic synergy with general applicability to various cancer types. Given the complicity of immune-based therapies and cancer pharmacology, it will be necessary to bring together cancer immunologists and clinicians, so as to provide a robust stimulus for realizing the successful management of cancer in the near future.

A New Era in Anticancer Peptide Vaccines

The use of synthetic peptides as vaccines aimed at the induction of therapeutic CD8‐positive T‐cell responses against tumor cells initially experienced great enthusiasm, mostly because of advances in vaccine technology, including design, synthesis, and delivery. However, despite impressive results in animal models, the application of such vaccines in humans has met with only limited success. The therapeutic activity of vaccine‐stimulated, tumor‐specific, CD8‐positive T cells can be hampered through the physical burden of the tumor, tolerance mechanisms, and local factors within the tumor microenvironment. Recently, accumulating evidence has suggested that combining a peptide‐based therapeutic vaccination with conventional chemotherapy can uncover the full potential of the antitumor immune response, increasing the success of immunotherapy. In addition, therapeutic vaccination in the preventive setting has been extremely effective in eliciting antitumor responses in preclinical tumor models and has demonstrated good promise clinically in patients with minimal residual disease. The rationale behind preventive vaccination is that patients with minimal tumor burden still have a fully competent immune system capable of developing robust antitumor responses. Finally, therapeutic CD8‐positive T‐cell peptide vaccines have been improved by coimmunization with T‐helper epitopes expressed on long peptides. Cancer 2010.

TH1 AND TH2 SERUM CYTOKINE PROFILES CHARACTERIZE PATIENTS WITH HASHIMOTO'S THYROIDITIS (TH1) AND GRAVES' DISEASE (TH2)

Objectives: The aim of this study was to document the pattern of immune response, assessed by the measurement of both Th1 and Th2 serum cytokines, in patients suffering from autoimmune thyroid disease and toxic nodular goiter. Methods: Both Th1 and Th2 serum cytokine levels were assayed in patients suffering from Graves’ disease (GD, n = 25), Hashimoto’s thyroiditis (HT, n = 21), and toxic nodular goiter (TNG, n = 7) and compared with corresponding levels of 25 healthy controls. Serum concentrations of IL-2, IL-1β, INF-γ, TNF-α, IL-12, IL-15, IL-10, IL-18, IL-4 and IL-5 were assayed in fasting serum samples. Results: It was found that patients with HT had higher IL-2 serum levels (12.16 ± 0.66 pg/ml) compared to patients with TNG (9.25 ± 0.84 pg/ml), GD (7.86 ± 0.30 pg/ml) and controls (7.36 ± 0.45 pg/ml; p = 0.0001), higher INF-γ levels (7.60 ± 0.33 pg/ml) compared to patients with TNG (5.77 ± 0.55 pg/ml), GD (5.74 ± 0.24 pg/ml) and controls (5.09 ± 0.27 pg/ml; p = 0.0009), higher IL-12 levels (3.57 ± 0.19 pg/ml) compared to patients with TNG (2.57 ± 0.21 pg/ml), GD (2.48 ± 0.13 pg/ml) and controls (2.59 ± 0.23 pg/ml; p = 0.004), and higher IL-18 levels (27.52 ± 1.75 pg/ml) compared to patients with TNG (18.71 ± 2.24 pg/ml), GD (15.44 ± 1.39 pg/ml) and controls (15.16 ± 1.62 pg/ml; p = 0.0002). In contrast, patients with GD had higher serum levels of IL-4 (4.11 ± 0.33 pg/ml) compared to patients with HT (3.0 ± 0.16; p = 0.02) and higher IL-5 levels (4.22 ± 0.30 pg/ml) compared to patients with TNG (3.21 ± 0.58 pg/ml), HT (2.75 ± 0.16 pg/ml) and controls (2.0 ± 0.19 pg/ml; p = 0.0001). Patients had lower IL-1β serum levels (TNG 2.45 ± 0.20, HT 2.52 ± 0.14, GD 2.68 ± 0.12 pg/ml) compared to controls (3.6 ± 0.20 pg/ml; p = 0.008). Conclusions: The above findings suggest that a Th1 pattern of immune response characteristic of cellular immunity is dominant in HT, whereas the predominance of Th2 cytokines in GD indicates a humoral pattern of immune reaction.