Mesenchymal stem cell therapy for acute respiratory distress syndrome: from basic to clinics

The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has occurred in China and around the world. SARS-CoV-2-infected patients with severe pneumonia rapidly develop acute respiratory distress syndrome (ARDS) and die of multiple organ failure. Despite advances in supportive care approaches, ARDS is still associated with high mortality and morbidity. Mesenchymal stem cell (MSC)-based therapy may be an potential alternative strategy for treating ARDS by targeting the various pathophysiological events of ARDS. By releasing a variety of paracrine factors and extracellular vesicles, MSC can exert anti-inflammatory, anti-apoptotic, anti-microbial, and pro-angiogenic effects, promote bacterial and alveolar fluid clearance, disrupt the pulmonary endothelial and epithelial cell damage, eventually avoiding the lung and distal organ injuries to rescue patients with ARDS. An increasing number of experimental animal studies and early clinical studies verify the safety and efficacy of MSC therapy in ARDS. Since low cell engraftment and survival in lung limit MSC therapeutic potentials, several strategies have been developed to enhance their engraftment in the lung and their intrinsic, therapeutic properties. Here, we provide a comprehensive review of the mechanisms and optimization of MSC therapy in ARDS and highlighted the potentials and possible barriers of MSC therapy for COVID-19 patients with ARDS.

Mesenchymal stem cell therapy for acute respiratory distress syndrome: from basic to clinics

The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has occurred in China and around the world. SARS-CoV-2-infected patients with severe pneumonia rapidly develop acute respiratory distress syndrome (ARDS) and die of multiple organ failure. Despite advances in supportive care approaches, ARDS is still associated with high mortality and morbidity. Mesenchymal stem cell (MSC)-based therapy may be an potential alternative strategy for treating ARDS by targeting the various pathophysiological events of ARDS. By releasing a variety of paracrine factors and extracellular vesicles, MSC can exert anti-inflammatory, anti-apoptotic, anti-microbial, and pro-angiogenic effects, promote bacterial and alveolar fluid clearance, disrupt the pulmonary endothelial and epithelial cell damage, eventually avoiding the lung and distal organ injuries to rescue patients with ARDS. An increasing number of experimental animal studies and early clinical studies verify the safety and efficacy of MSC therapy in ARDS. Since low cell engraftment and survival in lung limit MSC therapeutic potentials, several strategies have been developed to enhance their engraftment in the lung and their intrinsic, therapeutic properties. Here, we provide a comprehensive review of the mechanisms and optimization of MSC therapy in ARDS and highlighted the potentials and possible barriers of MSC therapy for COVID-19 patients with ARDS.

Mesenchymal stem cell therapy for acute respiratory distress syndrome: from basic to clinics

The 2019 novel coronavirus disease (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has occurred in China and around the world. SARS-CoV-2-infected patients with severe pneumonia rapidly develop acute respiratory distress syndrome (ARDS) and die of multiple organ failure. Despite advances in supportive care approaches, ARDS is still associated with high mortality and morbidity. Mesenchymal stem cell (MSC)-based therapy may be an potential alternative strategy for treating ARDS by targeting the various pathophysiological events of ARDS. By releasing a variety of paracrine factors and extracellular vesicles, MSC can exert anti-inflammatory, anti-apoptotic, anti-microbial, and pro-angiogenic effects, promote bacterial and alveolar fluid clearance, disrupt the pulmonary endothelial and epithelial cell damage, eventually avoiding the lung and distal organ injuries to rescue patients with ARDS. An increasing number of experimental animal studies and early clinical studies verify the safety and efficacy of MSC therapy in ARDS. Since low cell engraftment and survival in lung limit MSC therapeutic potentials, several strategies have been developed to enhance their engraftment in the lung and their intrinsic, therapeutic properties. Here, we provide a comprehensive review of the mechanisms and optimization of MSC therapy in ARDS and highlighted the potentials and possible barriers of MSC therapy for COVID-19 patients with ARDS.

An Alternative Dendritic Cell-Induced Murine Model of Asthma Exhibiting a Robust Th2/Th17-Skewed Response

PURPOSE: Simple and reliable animal models of human diseases contribute to the understanding of disease pathogenesis as well as the development of therapeutic interventions. Although several murine models to mimic human asthma have been established, most of them require anesthesia, resulting in variability among test individuals, and do not mimic asthmatic responses accompanied by T-helper (Th) 17 and neutrophils. As dendritic cells (DCs) are known to play an important role in initiating and maintaining asthmatic inflammation, we developed an asthma model via adoptive transfer of allergen-loaded DCs.METHODS: Ovalbumin (OVA)-loaded bone marrow-derived DCs (BMDCs) (OVA-BMDCs) were injected intravenously 3 times into non-anesthetized C57BL/6 mice after intraperitoneal OVA-sensitization.RESULTS: OVA-BMDC-transferred mice developed severe asthmatic immune responses when compared with mice receiving conventional OVA challenge intranasally. Notably, remarkable increases in systemic immunoglobulin (Ig) E and IgG1 responses, Th2/Th17-associated cytokines (interleukin [IL]-5, IL-13 and IL-17), Th2/Th17-skewed T-cell responses, and cellular components, including eosinophils, neutrophils, and goblet cells, were observed in the lungs of OVA-BMDC-transferred mice. Moreover, the asthmatic immune responses and severity of inflammation were correlated with the number of OVA-BMDCs transferred, indicating that the disease severity and asthma type may be adjusted according to the experimental purpose by this method. Furthermore, this model exhibited less variation among the test individuals than the conventional model. In addition, this DCs-based asthma model was partially resistant to steroid treatment.CONCLUSIONS: A reliable murine model of asthma by intravenous (i.v.) transfer of OVA-BMDCs was successfully established without anesthesia. This model more accurately reflects heterogeneous human asthma, exhibiting a robust Th2/Th17-skewed response and eosinophilic/neutrophilic infiltration with good reproducibility and low variation among individuals. This model will be useful for understanding the pathogenesis of asthma and would serve as an alternative tool for immunological studies on the function of DCs, T-cell responses and new drugs.

Adoptive cell transfer therapy for hepatocellular carcinoma / 医学前沿

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. This malignancy is associated with poor prognosis and high mortality. Novel approaches for prolonging the overall survival of patients with advanced HCC are urgently needed. The antitumor activities of adoptive cell transfer therapy (ACT), such as strategies based on tumor-infiltrating lymphocytes and cytokine-induced killer cells, are more effective than those of traditional strategies. Currently, chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved numerous breakthroughs in the treatment of hematological malignancies, including relapsed or refractory lymphoblastic leukemia and refractory large B-cell lymphoma. Nevertheless, this approach only provides a modest benefit in the treatment of solid tumors. The clinical results of CAR-T immunotherapy for HCC that could be obtained at present are limited. Some published studies have demonstrated that CAR-T could inhibit tumor growth and cause severe side effects. In this review, we summarized the current application of ACT, the challenges encountered by CAR-T technology in HCC treatment, and some possible strategies for the future direction of immunotherapeutic research.

Cortical Bone Derived Stem Cells for Cardiac Wound Healing

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Adoptive transfer of multiple stem cell types into failing human hearts has demonstrated safety however the beneficial effects in patients with cardiovascular disorders have been modest. Modest improvement in patients with cardiac complications warrants identification of a novel stem cell population that possesses effective reparative properties and improves cardiac function after injury. Recently we have shown in a mouse model and a porcine pre-clinical animal model, that cortical bone derived stem cells (CBSCs) enhance cardiac function after MI and/or ischemia-reperfusion injury. These beneficial effects of allogeneic cell delivery appear to be mediated by paracrine mechanisms rather than by transdifferentiation of injected cells into vessels and/or immature myocytes. This review will discuss role of CBSCs in cardiac wound healing. After having modest beneficial improvement in most of the clinical trials, a critical need is to understand the interaction of the transplanted stem cells with the ischemic cardiac environment. Transplanted stem cells are exposed to pro-inflammatory factors and activated immune cells and fibroblasts, but their interactions remain unknown. We have shown that CBSCs modulate different processes including modulation of the immune response, angiogenesis, and restriction of infarct sizes after cardiac injury. This review will provide information on unique protective signature of CBSCs in rodent/swine animal models for heart repair that should provide basis for developing novel therapies for treating heart failure patients.

Extrinsic Acquisition of CD80 by Antigen-Specific CD8⁺ T Cells Regulates Their Recall Immune Responses to Acute Viral Infection

CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8⁺ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8⁺ T cells, but not naïve CD8⁺ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8⁺ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8⁺ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8⁺ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8⁺ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8⁺ T cells and APCs. We compared the recall immune responses by memory CD8⁺ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8⁺ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8⁺ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.

Targeting the epitope spreader Pep19 by naïve human CD45RA⁺ regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy

PURPOSE: Beyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance. METHODS: Human polyclonal CD4⁺CD25⁺CD127(lo−) Tregs (127-Tregs) and naïve CD4⁺CD25⁺CD45RA⁺ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ(−/−) mouse model of collagen-induced arthritis. RESULTS: Epitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4⁺CD25⁺ Tregs at the articular joints in a mechanistic and orchestrated way. CONCLUSIONS: We propose human naïve CD4⁺CD25⁺CD45RA⁺ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

Enforced Expression of CXCR5 Drives T Follicular Regulatory-Like Features in Foxp3⁺ T Cells

CXCR5⁺ T follicular helper (Tfh) cells are associated with aberrant autoantibody production in patients with antibody-mediated autoimmune diseases including lupus. Follicular regulatory T (Tfr) cells expressing CXCR5 and Bcl6 have been recently identified as a specialized subset of Foxp3+ regulatory T (Treg) cells that control germinal center reactions. In this study, we show that retroviral transduction of CXCR5 gene in Foxp3⁺ Treg cells induced a stable expression of functional CXCR5 on their surface. The Cxcr5-transduced Treg cells maintained the expression of Treg cell signature genes and the suppressive activity. The expression of CXCR5 as well as Foxp3 in the transduced Treg cells appeared to be stable in vivo in an adoptive transfer experiment. Moreover, Cxcr5-transduced Treg cells preferentially migrated toward the CXCL13 gradient, leading to an effective suppression of antibody production from B cells stimulated with Tfh cells. Therefore, our results demonstrate that enforced expression of CXCR5 onto Treg cells efficiently induces Tfr cell-like properties, which might be a promising cellular therapeutic approach for the treatment of antibody-mediated autoimmune diseases.

Current Perspectives on Emerging CAR-Treg Cell Therapy: Based on Treg Cell Therapy in Clinical Trials and the Recent Approval of CAR-T Cell Therapy / 대한이식학회지

Regulatory T cells (Treg) naturally rein in immune attacks, and they can inhibit rejection of transplanted organs and even reverse the progression of autoimmune diseases in mice. The initial safety trials of Treg against graft-versus-host disease (GVHD) provided evidence that the adoptive transfer of Treg is safe and capable of limiting disease progression. Supported by such evidence, numerous clinical trials have been actively investigating the efficacy of Treg targeting autoimmune diseases, type I diabetes, and organ transplant rejection, including kidney and liver. The limited quantity of Treg cells harvested from peripheral blood and subsequent in vitro culture have posed a great challenge to large-scale clinical application of Treg; nevertheless, the concept of CAR (chimeric antigen receptor)-Treg has emerged as a potential resolution to the problem. Recently, two CAR-T therapies, tisagenlecleucel and axicabtagene ciloleucel, were approved by the US FDA for the treatment of refractory or recurrent acute lymhoblastic leukemia. This approval could serve as a guideline for the production protocols for other genetically engineered T cells for clinical use as well. The phase I and II clinical trials of these agents has demonstrated that genetically engineered and antigen-targeting T cells are safe and efficacious in humans. In conclusion, both the promising results of Treg cell therapy from the clinical studies and the recent FDA approval of CAR-T therapies are paving the way for CAR-Treg therapy in clinical use.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.

Development of Auto Antigen-specific Regulatory T Cells for Diabetes Immunotherapy

CD4⁺ regulatory T cells (Tregs) are essential for normal immune surveillance, and their dysfunction can lead to the development of autoimmune diseases, such as type-1 diabetes (T1D). T1D is a T cell-mediated autoimmune disease characterized by islet β cell destruction, hypoinsulinemia, and severely altered glucose homeostasis. Tregs play a critical role in the development of T1D and participate in peripheral tolerance. Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source of healthy Tregs to treat T1D as they have the ability to produce almost all cell types in the body, including Tregs. However, the right conditions for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) remain undefined, especially molecular mechanisms that direct differentiation of such Tregs. Auto Ag-specific PSC-Tregs can be programmed to be tissue-associated and infiltrate to local inflamed tissue (e.g., islets) to suppress autoimmune responses after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. Developing auto Ag-specific PSC-Tregs can reduce overall immunosuppression after adoptive transfer by accumulating inflamed islets, which drives forward the use of therapeutic PSC-Tregs for cell-based therapies in T1D.

Development of Auto Antigen-specific Regulatory T Cells for Diabetes Immunotherapy

CD4⁺ regulatory T cells (Tregs) are essential for normal immune surveillance, and their dysfunction can lead to the development of autoimmune diseases, such as type-1 diabetes (T1D). T1D is a T cell-mediated autoimmune disease characterized by islet β cell destruction, hypoinsulinemia, and severely altered glucose homeostasis. Tregs play a critical role in the development of T1D and participate in peripheral tolerance. Pluripotent stem cells (PSCs) can be utilized to obtain a renewable source of healthy Tregs to treat T1D as they have the ability to produce almost all cell types in the body, including Tregs. However, the right conditions for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) remain undefined, especially molecular mechanisms that direct differentiation of such Tregs. Auto Ag-specific PSC-Tregs can be programmed to be tissue-associated and infiltrate to local inflamed tissue (e.g., islets) to suppress autoimmune responses after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. Developing auto Ag-specific PSC-Tregs can reduce overall immunosuppression after adoptive transfer by accumulating inflamed islets, which drives forward the use of therapeutic PSC-Tregs for cell-based therapies in T1D.

Application of Cytotoxic T-Lymphocytes for the Treatment of Cytomegalovirus Infection in Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

Human cytomegalovirus (HCMV) infection, a common complication, remains a major risk factor related with patient death after hematopoietic stem cell transplantation (HSCT). Cytotoxic T lymphocytes (CTL) which is crucial to control HCMV infection, can prevent or treat HCMV infection safely and effectively after adoptive infusion. Many studies have been focussed on exploring different methods for preparation of CTL. The method of using antigen presenting cells to stimulate peripheral blood mononuclear cells is simple to operate, easy to conduct large-scale clinical trials. Isolation of CTL from donor-derived PBMC by peptide-tetramer or INF-γ antibody requires a large volume of peripheral blood and high cost for preparation. Third-party CTL can provide an "off-the-shelf" product, but the problem of HLA-mismatch still would be solved. In addition, the clinical efficacy and safety of different methods also vary. This article reviews and compares the current methods to generate CTL and efficacy of the cells after infusions.

The protective role of myeloid-derived suppressor cells in concanavalin A-induced hepatic injury

The mechanism underlying T cell-mediated fulminant hepatitis is not fully understood. In this study, we investigated whether myeloid derived suppressor cells (MDSCs) could prevent the concanavalin A (ConA)-induced hepatitis through suppressing T cell proliferation. We observed an increase in the frequencies of MDSCs in mouse spleen and liver at early stage of ConA treatment, implicating that the MDSCs might be involved in the initial resistance of mice against ConA-mediated inflammation. Subpopulation analysis showed that the MDSCs in liver of ConA-induced mice were mainly granulocytic MDSCs. Adoptive transfer of the bone marrow-derived MDSCs into ConA-treated mice showed that the MDSCs migrated into the liver and spleen where they suppressed T cell proliferation through ROS pathway. In addition, the frequencies of MDSCs in mice were also significantly increased by the treatment with immune suppressor glucocorticoids. Transfer of MDSCs into the regulatory T cell (Treg)-depleted mice showed that the protective effect of MDSCs on ConA-induced hepatitis is Treg-independent. In conclusion, our results demonstrate that MDSCs possess a direct protective role in T cell-mediated hepatitis, and increasing the frequency of MDSCs by either adoptive transfer or glucocorticoid treatment represents a potential cell-based therapeutic strategy for the acute inflammatory disease.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy.</p><p><b>METHODS</b>Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed.</p><p><b>RESULTS</b>Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs.</p><p><b>CONCLUSIONS</b>CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability, long half-life, and low toxicity.</p>

Efficient and durable gene delivery of self complementary adeno-associated virus 6 vector and impact of pre-existing immunity / 生物医学工程学杂志

Recombinant adeno-associated viral (rAAV) vectors are promising vectors for human gene therapy. However, AAV-mediated gene transduction can be hampered because of the pre-existing neutralized natural antibodies (NAbs) in primates. We evaluated transduction efficiency of rAAV6 expressing human alpha-1-anti-trypsin (hAAT) vectors in murine models, and found that these vectors showed stable and high levels of transgene expression. Fluorescence imaging showed that AAV6 expressing enhanced green fluorescent protein (eGFP) by intravenous administration predominantly targeted the liver, but led to self-limited hepatitis. Besides, our study evaluated epidemiology of anti-AAV6 NAb in non-human primates (NHPs) by NAb assay in vitro. The result indicated that 52.17% of NHPs had detectable NAb at 1:5 dilution rate. In vivo passive transfer experiment showed that AAV6 specific neutralizing antibody, even though with low NAb titer, could significantly inhibit rAAV6 transduction.

Generation of CD2+CD8+ NK Cells from c-kit+ Bone Marrow Cells in Porcine

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-kit+ bone marrow cells (c-kit+ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-kit+ BM cells that give rise to CD2+CD8+ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-kit+ BM cells than those of controls. And, we compared the ability of the cytotoxicity of CD2+CD8+ NK cells differentiated by cytokines from c-kit+ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100:1 for 4 h once a week. In results, CD2+CD8+ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-kit+ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-kit+ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

CD43 Expression Regulated by IL-12 Signaling Is Associated with Survival of CD8 T Cells

BACKGROUND: In addition to TCR and costimulatory signals, cytokine signals are required for the differentiation of activated CD8 T cells into memory T cells and their survival. Previously, we have shown that IL-12 priming during initial antigenic stimulation significantly enhanced the survival of activated CD8 T cells and increased the memory cell population. In the present study, we analyzed the mechanisms by which IL-12 priming contributes to activation and survival of CD8 T cells. METHODS: We observed dramatically decreased expression of CD43 in activated CD8 T cells by IL-12 priming. We purified CD43(lo) and CD43(hi) cells after IL-12 priming and analyzed the function and survival of each population both in vivo and in vitro. RESULTS: Compared to CD43(hi) effector cells, CD43(lo) effector CD8 T cells exhibited reduced cytolytic activity and lower granzyme B expression but showed increased survival. CD43(lo) effector CD8 T cells also showed increased in vivo expansion after adoptive transfer and antigen challenge. The enhanced survival of CD43(lo) CD8 T cells was also partly associated with CD62L expression. CONCLUSION: We suggest that CD43 expression regulated by IL-12 priming plays an important role in differentiation and survival of CD8 T cells.

Animal Models of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of unknown etiology that includes two main disease entities-ulcerative colitis and Crohn's disease. Although the pathogenesis of IBD remains unclear, it is widely accepted that genetic, environmental and immunological factors are involved. Animal models of IBD are indispensable for the understanding of the pathogenesis and novel therapeutic applications for IBD. IBD animal models can be divided into several different categories, including models of spontaneous colitis (cotton-top tamarin colitis); inducible forms of colitis (using acetic acid, dextran sulfate sodium and indomethacin); an adoptive transfer model (CD45RB(high) transfer model); genetically engineered models (with IL-10 knockout or TCR-alpha chain knockout mice). However, there is no 'perfect' model for human disease. Investigators must make judicious choices when selecting a model for a particular study. In this review, an overview of the different IBD animal models is provided and the contribution of the models to the current understanding of disease mechanisms is discussed, with the ultimate goal to develop future therapeutic trials.