Notch signaling in adipose tissue macrophages prevents diet-induced inflammation and metabolic dysregulation.

The importance of macrophages in adipose tissue (AT) homeostasis and inflammation is well established. However, the potential cues that regulate their function remain incompletely understood. To bridge this important gap, we sought to characterize novel pathways involved using a mouse model of diet-induced obesity. By performing transcriptomics analysis of AT macrophages (ATMs), we found that late-stage ATMs from high-fat diet mice presented with perturbed Notch signaling accompanied by robust proinflammatory and metabolic changes. To explore the hypothesis that the deregulated Notch pathway contributes to the development of AT inflammation and diet-induced obesity, we employed a genetic approach to abrogate myeloid Notch1 and Notch2 receptors. Our results revealed that the combined loss of Notch1 and Notch2 worsened obesity-related metabolic dysregulation. Body and AT weight gain was higher, blood glucose levels increased and metabolic parameters were substantially worsened in deficient mice fed high-fat diet. Moreover, serum insulin and leptin were elevated as were triglycerides. Molecular analysis of ATMs showed that deletion of Notch receptors escalated inflammation through the induction of an M1-like pro-inflammatory phenotype. Our findings thus support a protective role of myeloid Notch signaling in adipose tissue inflammation and metabolic dysregulation.

Comprehensive Analysis of 1-Year-Old Female Apolipoprotein E-Deficient Mice Reveals Advanced Atherosclerosis with Vulnerable Plaque Characteristics.

Apolipoprotein E-knockout (Apoe-/-) mice constitute the most widely employed animal model of atherosclerosis. Deletion of Apoe induces profound hypercholesterolemia and promotes the development of atherosclerosis. However, despite its widespread use, the Apoe-/- mouse model remains incompletely characterized, especially at late time points and advanced disease stages. Thus, it is unclear how late atherosclerotic plaques compare to earlier ones in terms of lipid deposition, calcification, macrophage accumulation, smooth muscle cell presence, or plaque necrosis. Additionally, it is unknown how cardiac function and hemodynamic parameters are affected at late disease stages. Here, we used a comprehensive analysis based on histology, fluorescence microscopy, and Doppler ultrasonography to show that in normal chow diet-fed Apoe-/- mice, atherosclerotic lesions at the level of the aortic valve evolve from a more cellular macrophage-rich phenotype at 26 weeks to an acellular, lipid-rich, and more necrotic phenotype at 52 weeks of age, also marked by enhanced lipid deposition and calcification. Coronary artery atherosclerotic lesions are sparse at 26 weeks but ubiquitous and extensive at 52 weeks; yet, left ventricular function was not significantly affected. These findings demonstrate that atherosclerosis in Apoe-/- mice is a highly dynamic process, with atherosclerotic plaques evolving over time. At late disease stages, histopathological characteristics of increased plaque vulnerability predominate in combination with frequent and extensive coronary artery lesions, which nevertheless may not necessarily result in impaired cardiac function.

Rosuvastatin Attenuates Progression of Atherosclerosis and Reduces Serum IL6 and CCL2 Levels in Apolipoprotein-E-deficient Mice.

BACKGROUND/AIM: Apolipoprotein E-deficient (Apoe-/-) mice develop atherosclerotic lesions that closely resemble metabolic syndrome in humans. We sought to investigate how rosuvastatin mitigates the atherosclerotic profile of Apoe-/- mice over time and its effects on certain inflammatory chemokines. MATERIALS AND METHODS: Eighteen Apoe-/- mice were allocated into three groups of six mice each receiving: standard chow diet (SCD; control group); high-fat diet (HFD); and HFD and rosuvastatin at 5 mg/kg/d orally via gavage for 20 weeks. Analysis of aortic plaques and lipid deposition was conducted by means of en face Sudan IV staining and Oil Red O staining. Serum cholesterol, low-density lipoprotein, high-density lipoprotein, plasma glucose and triglyceride levels were determined at baseline and after 20 weeks of treatment. Serum interleukin 6 (IL6), C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor-α (TNFα) levels were measured by enzyme-linked immunosorbent assay at the time of euthanasia. RESULTS: The lipidemic profile of Apoe-/- mice on HFD deteriorated over time. Apoe-/- mice on HFD developed atherosclerotic lesions over time. Sudan IV and Oil Red O-stained sections of the aorta revealed increased plaque formation and plaque lipid deposition in HFD-fed mice compared with SCD-fed mice and reduced plaque development in HFD-fed mice treated with rosuvastatin compared with mice not receiving statin treatment. Serum analysis revealed reduced metabolic parameters in HFD-fed mice on rosuvastatin compared with non-statin, HFD-fed mice. At the time of euthanasia, HFD-fed mice treated with rosuvastatin had significantly lower IL6 as well as CCL2 levels when compared with HFD-fed mice not receiving rosuvastatin. TNFα levels were comparable among all groups of mice, irrespective of treatment. IL6 and CCL2 positively correlated with the extent of atherosclerotic lesions and lipid deposition in atherosclerotic plaques. CONCLUSION: Serum IL6 and CCL2 levels might potentially be used as clinical markers of progression of atherosclerosis during statin treatment for hypercholesterolemia.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs.

Life-threatening 'breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS-CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals (age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, while two neutralized IFN-ω only. No patient neutralized IFN-ß. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population.

Mesenchymal stromal cell delivery as a potential therapeutic strategy against COVID-19: Promising evidence from in vitro results.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic, which was initiated in December 2019. COVID-19 is characterized by a low mortality rate (< 6%); however, this percentage is higher in elderly people and patients with underlying disorders. COVID-19 is characterized by mild to severe outcomes. Currently, several therapeutic strategies are evaluated, such as the use of anti-viral drugs, prophylactic treatment, monoclonal antibodies, and vaccination. Advanced cellular therapies are also investigated, thus representing an additional therapeutic tool for clinicians. Mesenchymal stromal cells (MSCs), which are known for their immunoregulatory properties, may halt the induced cytokine release syndrome mediated by SARS-CoV-2, and can be considered as a potential stem cell therapy. AIM: To evaluate the immunoregulatory properties of MSCs, upon stimulation with COVID-19 patient serum. METHODS: MSCs derived from the human Wharton's Jelly (WJ) tissue and bone marrow (BM) were isolated, cryopreserved, expanded, and defined according to the criteria outlined by the International Society for Cellular Therapies. Then, WJ and BM-MSCs were stimulated with a culture medium containing 15% COVID-19 patient serum, 1% penicillin-streptomycin, and 1% L-glutamine for 48 h. The quantification of interleukin (IL)-1 receptor a (Ra), IL-6, IL-10, IL-13, transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF)-a, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and indoleamine-2,3-dioxygenase (IDO) was performed using commercial ELISA kits. The expression of HLA-G1, G5, and G7 was evaluated in unstimulated and stimulated WJ and BM-MSCs. Finally, the interactions between MSCs and patients' macrophages were established using co-culture experiments. RESULTS: Thawed WJ and BM-MSCs exhibited a spindle-shaped morphology, successfully differentiated to "osteocytes", "adipocytes", and "chondrocytes", and in flow cytometric analysis were characterized by positivity for CD73, CD90, and CD105 (> 95%) and negativity for CD34, CD45, and HLA-DR (< 2%). Moreover, stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation, in comparison to unstimulated cells. The HLA-G isoforms (G1, G5, and G7) were successfully expressed by the unstimulated and stimulated WJ-MSCs. On the other hand, only weak expression of HLA-G1 was identified in BM-MSCs. Stimulated MSCs secreted high levels of IL-1Ra, IL-6, IL-10, IL-13, TGF-ß1, FGF, VEGF, PDGF, and IDO in comparison to unstimulated cells (P < 0.05) after 12 and 24 h. Finally, macrophages derived from COVID-19 patients successfully adapted the M2 phenotype after co-culturing with stimulated WJ and BM-MSCs. CONCLUSION: WJ and BM-MSCs successfully produced high levels of immunoregulatory agents, which may efficiently modulate the over-activated immune responses of critically ill COVID-19 patients.

Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections.

The widespread presence of autoantibodies in acute infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is increasingly recognized, but the prevalence of autoantibodies in infections with organisms other than SARS-CoV-2 has not yet been reported. We used protein arrays to profile IgG autoantibodies from 317 samples from 268 patients across a spectrum of non-SARS-CoV-2 infections, many of whom were critically ill with pneumonia. Anti-cytokine antibodies (ACA) were identified in > 50% of patients infected with non-SARS-CoV-2 viruses and other pathogens, including patients with pneumonia attributed to bacterial causes. In cell-based functional assays, some ACA blocked binding to surface receptors for type I interferons (Type I IFN), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6). Autoantibodies against traditional autoantigens associated with connective tissue diseases (CTDs) were also commonly observed in these cohorts, including newly-detected antibodies that emerged in longitudinal samples from patients infected with influenza. We conclude that autoantibodies, some of which are functionally active, may be much more prevalent than previously appreciated in patients who are symptomatically infected with diverse pathogens.

CD103 integrin identifies a high IL-10-producing FoxP3+ regulatory T-cell population suppressing allergic airway inflammation.

BACKGROUND: Although FoxP3+ regulatory T (Treg) cells constitute a highly heterogeneous population, with different regulatory potential depending on the disease context, distinct subsets or phenotypes remain poorly defined. This hampers the development of immunotherapy for allergic and autoimmune disorders. The present study aimed at characterizing distinct FoxP3+ Treg subpopulations involved in the suppression of Th2-mediated allergic inflammation in the lung. METHODS: We used an established mouse model of allergic airway disease based on ovalbumin sensitization and challenge to analyze FoxP3+ Tregs during the induction and resolution of inflammation, and identify markers that distinguish their most suppressive phenotypes. We also developed a new knock-in mouse model (Foxp3cre Cd103dtr ) enabling the specific ablation of CD103+ FoxP3+ Tregs for functional studies. RESULTS: We found that during resolution of allergic airway inflammation in mice >50% of FoxP3+ Treg cells expressed the integrin CD103 which marks FoxP3+ Treg cells of high IL-10 production, increased expression of immunoregulatory molecules such as KLRG1, ICOS and CD127, and enhanced suppressive capacity for Th2-mediated inflammatory responses. CD103+ FoxP3+ Tregs were essential for keeping allergic inflammation under control as their specific depletion in Foxp3cre Cd103dtr mice lead to severe alveocapillary damage, eosinophilic pneumonia, and markedly reduced lifespan of the animals. Conversely, adoptive transfer of CD103+ FoxP3+ Tregs effectively treated disease, attenuating Th2 responses and allergic inflammation in an IL-10-dependent manner. CONCLUSIONS: Our study identifies a novel regulatory T-cell population, defined by CD103 expression, programmed to prevent exuberant type 2 inflammation and keep homeostasis in the respiratory tract under control. This has important therapeutic implications.

A Multimodal Approach for the Risk Prediction of Intensive Care and Mortality in Patients with COVID-19.

BACKGROUND: Although several studies have been launched towards the prediction of risk factors for mortality and admission in the intensive care unit (ICU) in COVID-19, none of them focuses on the development of explainable AI models to define an ICU scoring index using dynamically associated biological markers. METHODS: We propose a multimodal approach which combines explainable AI models with dynamic modeling methods to shed light into the clinical features of COVID-19. Dynamic Bayesian networks were used to seek associations among cytokines across four time intervals after hospitalization. Explainable gradient boosting trees were trained to predict the risk for ICU admission and mortality towards the development of an ICU scoring index. RESULTS: Our results highlight LDH, IL-6, IL-8, Cr, number of monocytes, lymphocyte count, TNF as risk predictors for ICU admission and survival along with LDH, age, CRP, Cr, WBC, lymphocyte count for mortality in the ICU, with prediction accuracy 0.79 and 0.81, respectively. These risk factors were combined with dynamically associated biological markers to develop an ICU scoring index with accuracy 0.9. CONCLUSIONS: to our knowledge, this is the first multimodal and explainable AI model which quantifies the risk of intensive care with accuracy up to 0.9 across multiple timepoints.

CD8+ T cells in beige adipogenesis and energy homeostasis.

Although accumulation of lymphocytes in the white adipose tissue (WAT) in obesity is linked to insulin resistance, it remains unclear whether lymphocytes also participate in the regulation of energy homeostasis in the WAT. Here, we demonstrate enhanced energy dissipation in Rag1-/- mice, increased catecholaminergic input to subcutaneous WAT, and significant beige adipogenesis. Adoptive transfer experiments demonstrated that CD8+ T cell deficiency accounts for the enhanced beige adipogenesis in Rag1-/- mice. Consistently, we identified that CD8-/- mice also presented with enhanced beige adipogenesis. The inhibitory effect of CD8+ T cells on beige adipogenesis was reversed by blockade of IFN-γ. All together, our findings identify an effect of CD8+ T cells in regulating energy dissipation in lean WAT, mediated by IFN-γ modulation of the abundance of resident immune cells and of local catecholaminergic activity. Our results provide a plausible explanation for the clinical signs of metabolic dysfunction in diseases characterized by altered CD8+ T cell abundance and suggest targeting of CD8+ T cells as a promising therapeutic approach for obesity and other diseases with altered energy homeostasis.

Interferon-λs: Front-Line Guardians of Immunity and Homeostasis in the Respiratory Tract.

Type III interferons (IFNs), also termed lambda IFNs (IFNλs) or interleukins-28/29, constitute a new addition to the IFN family. They are induced upon infection and are particularly abundant at barrier surfaces, such as the respiratory and gastrointestinal tracts. Although they signal through a unique heterodimeric receptor complex comprising IFNLR1 and IL10RB, they activate a downstream signaling pathway remarkably similar to that of type I IFNs and share many functions with them. Yet, they also have important differences which are only now starting to unfold. Here, we review the current literature implicating type III IFNs in the regulation of immunity and homeostasis in the respiratory tract. We survey the common and unique characteristics of type III IFNs in terms of expression patterns, cellular targets, and biological activities and discuss their emerging role in first line defenses against respiratory viral infections. We further explore their immune modulatory functions and their involvement in the regulation of inflammatory responses during chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Type III IFNs are, therefore, arising as front-line guardians of immune defenses in the respiratory tract, fine tuning inflammation, and as potential novel therapeutics for the treatment of diverse respiratory diseases, including influenza virus infection and asthma.

Rationale for the application of RANKL inhibition in the treatment of Langerhans cell histiocytosis.

CONTEXT: Langerhans Cell Histiocytosis (LCH) is a rare disease exhibiting both neoplastic and inflammatory features including abundant cytokine secretion both at the lesional and systemic level. OBJECTIVE: Evaluation of RANKL expression within LCH lesions in various tissues. DESIGN: Cross-sectional study involving paraffin blocks from the diagnostic biopsies of adults with LCH. SETTING: The study was conducted among patients followed in an adult outpatient clinic. SUBJECTS: Eleven patients with active LCH, who were 41.27 ± 3.44 years old, and five patients who were 46.8 ± 7.19 years old with non-LCH diagnosis serving as controls. INTERVENTIONS: RANKL, p65 and CD1a immunostaining of deparaffinized sections from LCH lesions and control tissues. MAIN OUTCOME MEASURE: Comparison of RANKL and p65 expression between LCH lesions, as indicated from concomitant CD1a immunostaining, and control tissue counterparts. RESULTS: A focal positive granular cytoplasmic RANKL staining was found at all lesional sites in a number of cells of the pathological infiltrate, mostly with morphologic features of pathological Langerhans cells (LCs). Compared to control tissues, RANKL positivity in LCH cases showed an excess of staining, both in intensity of staining and the number of stained cells, especially in areas of pathologic infiltration. RANKL staining also coincided with strong p65 NFκB nuclear positivity, especially in lesional infiltrates. CONCLUSIONS: RANKL is highly expressed in active LCH at the lesional level, concomitant to p65 NFκB activation. The use of RANKL inhibition as a rational therapeutic approach of the disease now needs further clinical evaluation.

Toll-like receptor 7 protects from atherosclerosis by constraining "inflammatory" macrophage activation.

BACKGROUND: Toll-like receptors (TLRs) have long been considered to be major culprits in the development of atherosclerosis, contributing both to its progression and clinical complications. However, evidence for most TLRs beyond TLR2 and TLR4 is lacking. METHODS AND RESULTS: We used experimental mouse models, human atheroma cultures, and well-established human biobanks to investigate the role of TLR7 in atherosclerosis. We report the unexpected finding that TLR7, a receptor recognizing self-nucleic acid complexes, is protective in atherosclerosis. In Apoe(-/-) mice, functional inactivation of TLR7 resulted in accelerated lesion development, increased stenosis, and enhanced plaque vulnerability as revealed by Doppler ultrasound and/or histopathology. Mechanistically, TLR7 interfered with macrophage proinflammatory responses to TLR2 and TLR4 ligands, reduced monocyte chemoattractant protein-1 production, and prevented expansion of Ly6C(hi) inflammatory monocytes and accumulation of inflammatory M1 macrophages into developing atherosclerotic lesions. In human carotid endarterectomy specimens TLR7 levels were consistently associated with an M2 anti-inflammatory macrophage signature (interleukin [IL]-10, IL-1RA, CD163, scavenger and C-type lectin receptors) and collagen genes, whereas they were inversely related or unrelated to proinflammatory mediators (IL-12/IL-23, interferon beta, interferon gamma, CD40L) and platelet markers. Moreover, in human atheroma cultures, TLR7 activation selectively suppressed the production of key proatherogenic factors such as monocyte chemoattractant protein-1 and tumor necrosis factor without affecting IL-10. CONCLUSIONS: These findings provide evidence for a beneficial role of TLR7 in atherosclerosis by constraining inflammatory macrophage activation and cytokine production. This challenges the prevailing concept that all TLRs are pathogenic and supports the exploitation of the TLR7 pathway for therapy.

New insights into the role of NK cells in cancer immunotherapy.

Repetitive infusions of ex vivo expanded NK cells induced antitumor T-cell responses in a metastatic lung cancer mouse model. These were further potentiated by Treg depletion. Thus the combination of NK cell-based immunotherapy with other treatment modalities in the direction of adaptive response enhancement might promote long lasting antitumor immunity.

NK cell adoptive transfer combined with Ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses.

Previous work from our laboratory showed that hydrocortisone (HC) combined with IL-15 induces expansion of activated human NK cells. We set up an experimental tumor model to evaluate the use of adoptively transferred, HC plus IL-15 (HC/IL-15)-activated and -expanded murine NK cells in the treatment of syngeneic mice carrying established lung metastases of the CT26 transplantable tumor. We also examined the effect of denileukin diftitox (Ontak) on the depletion of regulatory T cells to enhance the in vivo antitumor immunity induced by the adoptively transferred NK cells. Our results clearly demonstrate that murine DX5(+) NK cells are largely expanded in the presence of IL-15 plus HC while retaining intact their functional status. Moreover, when intravenously infused, they mediated significant antitumor responses against CT26 lung tumors in syngeneic BALB/c animals that were further enhanced upon pretreatment of the tumor-bearing animals with Ontak. Total splenocytes and isolated splenic T cells from NK-treated mice responded in vitro against CT26 tumor cells as evidenced by IFN-γ-based ELISPOT, proliferation, and cytotoxicity assays. Importantly, animals treated with Ontak plus adoptive transfer of HC/IL-15-expanded NK cells significantly retarded CT26 tumor growth after a rechallenge with the same tumor s.c. in their flanks. Taken altogether, our data suggest that NK cell adoptive transfer can trigger adaptive antitumor T cell responses, and regulatory T cell depletion by Ontak is mandatory for enabling HC/IL-15-activated NK cells to promote long-lasting adaptive antitumor immunity.

Induction of potent CD4+ T cell-mediated antitumor responses by a helper HER-2/neu peptide linked to the Ii-Key moiety of the invariant chain.

The Ii-Key fragment from the MHC class II-associated invariant chain (or Ii protein) has been shown to facilitate direct charging of MHC class II epitopes to the peptide binding groove. The purpose of the present study was to test the potential of a series of Ii-Key/HER-2/neu776-790 hybrid peptides to generate increased frequencies of peptide-specific CD4+ T cells over the native peptide in mice transgenic (Tg) for a chimeric human mouse class II molecule (DR4-IE) (H-2b) as well as their antitumor potency. Following in vivo priming, such hybrid peptides induced increased proliferation and frequencies of IFN-gamma producing CD4+ T cells in response to either syngeneic dendritic cells pulsed with native peptide, or HLA-DR4+ human tumor cell lines expressing HER-2/neu. Hybrid peptides were more stable in an off-rate kinetics assay compared to the native peptide. In addition, antigen-specific CD4+ T cells from hybrid peptide immunized DR4-IE Tg mice synergized with HER-2/neu(435-443)-specific CD8+ T cells from HLA-A2.1 Tg HHD (H-2b) mice in producing antitumor immunity into SCID mice xenografted with the HER-2/neu+, HLA-A2.1+ and HLA-DR4+ FM3 human melanoma cell line. High proportions of these adoptively transferred HER-2/neu peptide-specific CD4+ and CD8+ T cells infiltrated FM3-induced tumors (tumor infiltrating lymphocytes; TIL) in SCID mice. CD8+ TIL exhibited long-lasting antitumor activity when cotransferred with CD4+ TIL, inducing regression of FM3 tumors in a group of untreated, tumor-bearing SCID mice, following adoptive transfer. Our data show that Ii-Key modified HER-2/neu776-790 hybrid peptides are sufficiently potent to provide antigen-specific CD4+ TH cells with therapeutic antitumor activity.

CD4+CD25+ regulatory T-cell frequency in HER-2/neu (HER)-positive and HER-negative advanced-stage breast cancer patients.

PURPOSE: CD4(+)CD25(bright) regulatory T cells (Tregs) are increased in patients with several malignancies and correlate with disease stage and prognosis. Breast cancer patients represent a heterogeneous population with unpredictable disease progression even at advanced stages. Circulating Tregs in correlation with HER-2/neu (HER) status and treatment with chemotherapy, either alone or in combination with trastuzumab therapy, were monitored in advanced-stage breast cancer patients. EXPERIMENTAL DESIGN: Circulating Treg frequency and absolute counts of 46 HER(+) and 28 HER(-), stage III and IV, breast cancer patients before therapy and during trastuzumab therapy and/or chemotherapy have been compared with 24 healthy donors and correlated with plasma HER extracellular domain concentration and clinical outcome. RESULTS: Treg frequency in HER(+) patients was significantly increased compared with both HER(-) patients and healthy donors. Trastuzumab therapy, with or without combined chemotherapy, resulted in a progressive decrease of circulating Tregs. Percentage change in Tregs statistically correlated with percentage change in plasma HER extracellular domain. Furthermore, decrease in Tregs correlated with either objective clinical response or stable disease, whereas increased Treg frequency during trastuzumab therapy coincided with disease progression. No statistically significant change in Treg frequency following chemotherapy was observed in HER(-) patients. CONCLUSIONS: Treg cell frequency does not directly correlate with clinical stage in breast cancer, as stage III and IV HER(+) and HER(-) patients exhibit significantly different Treg profiles. Trastuzumab therapy, either alone or combined with chemotherapy, results in decreased Treg frequency in HER(+) advanced patients with an objective clinical response.

An essential role for IL-18 in CD8 T cell-mediated suppression of IgE responses.

The ability of CD8 T cells to suppress IgE responses is well established. Previously, we demonstrated that CD8 T cells inhibit IgE responses via the induction of IL-12, which promotes Th1 and suppresses Th2 responses. In this study, we show that IL-18 also plays an essential role in IgE suppression. In vitro, IL-18 synergized with IL-12 to promote Th1/T cytotoxic 1 and inhibit Th2/T cytotoxic 2 differentiation. OVA-specific TCR transgenic (OT-I) CD8 cells induced both IL-12 and IL-18 when cultured with OVA(257-264) peptide-pulsed dendritic cells. In vivo, IL-18(-/-) mice exhibited higher IgE and IgG1 levels compared with wild-type mice after immunization with OVA/alum. Furthermore, adoptive transfer of CD8 T cells from OVA-primed mice suppressed IgE responses in OVA/alum-immunized mice, but not in IL-18(-/-) mice. IgE suppression in IL-18(-/-) mice was restored if CD8 T cells were coadoptively transferred with IL-18-competent wild-type bone marrow dendritic cell progenitors, demonstrating an essential role of IL-18 in CD8 T cell-mediated suppression of IgE responses. The data suggest that CD8 T cells induce IL-18 production during a cognate interaction with APCs that synergizes with IL-12 to promote immune deviation away from the allergic phenotype. Our data identify IL-18 induction as a potentially useful target in immunotherapy of allergic disease.

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.

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 fibroblast 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.