Origin of CD8^＋ Effector and Memory T Cell Subsets

It is well accepted that CD8＋ T cells play a pivotal role in providing protection against infection with intracellular pathogens and some tumors. In many cases protective immunity is maintained for long periods of time （immunological memory）. Over the past years, it has become evident that in order to fulfill these multiple tasks, distinct subsets of effector and memory T cells have to be generated. Until today, however, little is known about the underlying mechanisms of subset differentiation and the timing of lineage fate decisions. In this context, it is of special importance to determine at which level of clonal expansion functional and phenotypical heterogeneity is achieved. Different models for T cell subset diversification have been proposed; these differ mainly in the time point during priming and clonal expansion （prior, during, or beyond the first cell division） when differentiation programs are induced. Recently developed single-cell adoptive transfer technology has allowed us to demonstrate that individual precursor cell still bears the full plasticity to develop into a plethora different T cell subsets. This observation targets the shaping of T cell subset differentiation towards factors that are still operative beyond the first cell division. These findings have important implications for vaccine development, as the modulation of differentiation patterns towards distinct subsets could become a powerful strategy to enhance the efficacy and quality of vaccines. Cellular ＆ Molecular Immunology.     

Immune Response Induced in Vitro by CD16− and CD16+ Monocyte-Derived Dendritic Cells in Patients with Metastatic Renal Cell Carcinoma Treated with Dendritic Cell Vaccines

Monocyte-derived dendritic cells (mDC) are increasingly used as cancer vaccines. However, human monocytes are a heterogeneous cell population. We showed previously that DC derived from a monocyte subset expressing CD16 (16+mDC) stimulated allogeneic naïve T lymphocytes to secrete higher levels of IL-4 than DC derived from regular CD14^highCD16^? monocytes (16?mDC). Th1-type responses have been associated with effective antitumor responses, thus the use of mDC containing 16+mDC as cancer vaccines might be disadvantageous. Here, we evaluate the primary and memory immune response elicited in vitro by 16+mDC and 16?mDC in five patients with metastatic renal cell carcinoma vaccinated with autologous mDC pulsed with tumor lysates (TuLy) and keyhole limpet hemocyanin (KLH). After therapy, three of the five patients had stable disease. Surprisingly, patients with longer survival showed the highest amount of peripheral blood CD16+ monocytes. Analysis of KLH-specific antibodies revealed high titers of IgG2 in patients with longer survival. CD4+ T lymphocyte proliferation against KLH and TuLy increased after treatment, and some patients showed an augmented rate of CD4+ T lymphocyte proliferation against KLH (3/5) and TuLy (2/3) when 16+mDC were used as antigen presenting cells (APC). Before treatment, the IFN-γ/IL-4 ratio against TuLy and KLH was higher when using 16?mDC as APC, but after vaccination four of five patients had an increased ratio for TuLy with 16+mDC. These results suggest that the immune response elicited by 16?mDC and 16+mDC is modified when memory or naïve T cells are stimulated, and 16+mDC could favor a stronger and more beneficial antitumoral Th1 memory response in vivo.     

The Qa-1 Dependent CD8^＋ T Cell Mediated Regulatory Pathway

The immune system has evolved a variety of regulatory mechanisms to ensure the peripheral self-tolerance as well as the optimal capacity to elicit effective anti-infection immunity. At present, there is no satisfactory conceptual framework to explain how the peripheral immunity is regulated at a biological system level, which enables the immune system to perform its essential functions to mount effective immunity to virtually any foreign antigens but avoid harmful immune responses to self. In this regard, during the past few years, an “affinity/avidity model of peripheral T cell regulation” has been proposed and tested, which opens up a new paradigm to understand how the peripheral immunity, to both self and foreign antigens, is regulated. The paradigm is based on the discovery of a subset CD8^＋ T cells with TCRs which specifically recognize a unique set of self-peptides presented by the MHC class Ib molecule Qa-I differentially expressed on T cells as a function of the affinity/avidity of T cell activation. These Qa-1 restricted CD8^＋ T cells represent an example of how the immune system utilizes a unified mechanism to regulate adaptive immunity to both self and foreign antigens. Thus, by selectively down-regulating T cells of intermediate affinity/avidity, to any antigens, the immune system controls the adaptive immunity without the necessity to distinguish self from non-self in the periphery at the level of T cell regulation. Cellular ＆ Molecular Immunology. 2005;2（3）：161-167.     

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8^＋ T Cells Ex Vivo

Artificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of co-stimulating receptors of CD28 and 4-1BB, respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32/CD86/4-1BBL cell with OKT3 monoclonal antibody against CD3, named K32/CD86/4-1BBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8^＋ T cell activation, promoting CD8^＋ T cell proliferation, division, and long-term growth, inhibiting CD8^＋ T cell apoptosis, and enhancing CD8^＋ T cell secretion of IFN-T and perforin. Furthermore, antigen-specific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 days. This approach was robust, simple, reproducible and economical for expansion and activation of CD8^＋ T cells and may have important therapeutic implications for adoptive immunotherapy. Cellular ＆ Molecular Immunology.     

Cell–Cell Junctional Proteins in Cardiovascular Mechanotransduction

Cell–cell junctional proteins play important structural and functional roles in several physiological systems. Recent studies have illuminated key aspects in the relationship of junctional proteins with normal cell and tissue function as well as various pathologies. In this review article, the roles of cell–cell junctional proteins will be presented in four classes: adherens junctions, desmosomes, gap junctions, and tight junctions, and discussed primarily in the context of cardiovascular cell and tissue physiology and pathophysiology. The functions of the proteins are described from the perspective of mechanotransductive regulation of physiological and disease processes, with focus being laid on more biomechanical aspects, such as cell adhesion, migration, and mechanosignaling.     

CD21-independent Infection of Epstein-Barr Virus in Human Signet Ring Gastric Carcinoma Cell Line

Epstein Barr virus ( EBV), a ubiquitous human her pesvirus, is the etiologic agent of infectious mononucleo sis, and is closely associated with several human malig nancies including Burkitt′s lymphoma (BL), undifferenti ated nasopharyngeal carcinoma (NPC) and opportunisticlymphoma in immunocompromised hosts. In recent years,there have been increasing evidences of association of EBVwith additional malignancies, such as gastric carcinoma.EBV has been found in tumor cells of m…     

B Cell Deficiencies

Genetics of IgA deficiency and CVID;X-linked agammaglobulinemia;     

Microfluidic Platforms for Studies of Angiogenesis,Cell Migration,and Cell–Cell Interactions

Recent advances in microfluidic technologies have opened the door for creating more realistic in vitro cell culture methods that replicate many aspects of the true in vivo microenvironment. These new designs (i) provide enormous flexibility in controlling the critical biochemical and biomechanical factors that influence cell behavior, (ii) allow for the introduction of multiple cell types in a single system, (iii) provide for the establishment of biochemical gradients in two- or three-dimensional geometries, and (iv) allow for high quality, time-lapse imaging. Here, some of the recent developments are reviewed, with a focus on studies from our own laboratory in three separate areas: angiogenesis, cell migration in the context of tumor cell-endothelial interactions, and liver tissue engineering.     

Stem and Progenitor Cell Expansionin Co-culture of Mobilized CD34~ Cells and Osteopetrotic Mouse Stroma

1 IntroductionCulture systems capable of expanding and/or maintaining hematopoietic stem cells will not only facilitate our understanding of stem cell biology, but also broaden clinical applications. Among various in vitro hematopoietic culture systems, co-cultures of marrow or CD34~ cells with an adherent stromal layer that can produce cytokines and extracellular matrix components most effectively supports long-term hematopoiesis (LTC), mimicking the bone marrow micro-environment.The OP-9…     

Interferon-γ Prevents Death of Bystander Neurons during CD8 T Cell Responses in the Brain

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-γ (IFN-γ). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-γ-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-γ-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-γ-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-γ-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-γ-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-γ protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.Controlling viral infections in the central nervous system is a very demanding task for the immune system as there is a substantial risk of collateral damage.¹ It is often difficult to assess the extent of damage caused by the antiviral immune response to nonrenewable cells in the brain. The reason for this difficulty is that the cytopathic activities of viruses and immune cells do not necessarily result in distinct pathologies. In experimental settings, the complexity of the system can be reduced if model viruses are used that have adopted noncytolytic replication strategies. A suitable pathogen for studying these issues is Borna disease virus (BDV).BDV can replicate in neurons and other cell types of the central and peripheral nervous system of mice and other mammals without damaging the host cells.^2,3 The noncytotoxic nature of BDV was most convincingly demonstrated in MRL/MpJ (MRL) mice lacking functional alleles of the β2-microglobulin gene, which, as a consequence, lack mature CD8 T cells. Unlike wild-type MRL mice that frequently develop fatal neurological disease after infection with BDV, mutant MRL mice lacking mature CD8 T cells are completely resistant to BDV-induced disease although the virus replicates to very high levels in the brain of such mice.⁴ Neurological disease in this model system thus seems to result exclusively from the activity of antiviral CD8 T cells, which recognize virus-infected cells in the brain. This view was recently confirmed by a study in which some features of the BDV-induced neurological disease were successfully recapitulated by adoptive transfer of virus-specific CD8 T cells into recipient mice that express viral antigen derived from a transgene that is active in either astrocytes or neurons.⁵Immune cell infiltrates are abundantly present in the brain of diseased wild-type MRL mice, but the infected organ usually shows no striking pathological alterations. However, in infected MRL mice lacking functional alleles of the IFN-γ gene, a selective and characteristic destruction of neurons was noted in the CA1 region of the hippocampus in about 50% of the mice.⁴ This destruction of neurons was paralleled by an influx of eosinophils, a cell type that is usually not found in the brain of BDV-infected wild-type mice.⁴ Interestingly, the CA1 neurons in these mice usually remain uninfected in mice.⁴The cause of the selective death of hippocampal neurons during BDV-induced brain inflammation in the absence of IFN-γ is not known. Several possible causes have been discussed, including excitotoxicity resulting from glutamate receptor overstimulation, as described for CA1 damage after measles virus,⁶ Sindbis virus^7,8 and HIV infection,⁹ and the possibility that the influx of eosinophils caused the neuronal damage possibly by secretion of toxic granule proteins, cytokines, or lipid mediators.¹⁰ Here we report that excitotoxicity and eosinophil influx are most likely not the cause of the observed CA1 neuron damage. Rather, our data suggest that the disease-inducing CD8 T cells play a decisive role. Interestingly, CA1 neuron damage was observed if CD8 T cells from IFN-γ-deficient but not from wild-type mice were adoptively transferred into uninfected mice that express transgene-encoded viral antigen selectively in astrocytes. These results suggest that the observed CA1 neuron death is a bystander phenomenon of T cell-mediated recognition of antigen-expressing astrocytes that only becomes evident if IFN-γ is absent.     

Therapeutic Potential of CD1d-Restricted Invariant Natural Killer T Cell–based Treatment for Autoimmune Diseases

CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of T cells that recognize glycolipid antigens presented by the CD1d molecule. iNKT cells participate in various kinds of immunoregulation due to a potent ability to produce a variety of cytokines. Recent advances in studies of novel synthetic glycolipid ligands has led to new strategies to manipulate the pleiotropic functions of iNKT cells. The molecular mechanism of selective cytokine production by glycolipid ligands will be discussed. We will also focus on the possible therapeutic application of such ligands for the clinical treatment of various autoimmune diseases.     

Apoptosis: A Programme of Cell Death or Cell Disposal?

Based primarily on morphological features, apoptosis was described as the cell death that occurs during physiological situations, whereas necrosis was observed during acute harmful conditions. Apoptosis was, therefore, associated with a programme of cell death, as opposed to necrosis, considered an accidental, uncontrolled, pathological cell death. The apoptotic machinery was first unravelled in the nematode Caenorhabditis elegans, where a protease called CED-3 was central to the execution of cells destined to die. Inactivation of ced-3 gene prevents developmental cell death in the worm, an observation that reinforces the notion that apoptosis holds the switch between life and death. In mammals, proteins homologous to CED-3, members of the family collectively called caspases, are considered the executioner proteases responsible for generating fundamentally all aspects of apoptosis. However, inhibition of the so-called executioner caspases (i.e. inhibition of apoptosis) does not prevent cell death to occur. Consequently, in mammals, the decision switch between life and death resides upstream of the activation of caspases and the ensuing apoptotic cell death. Therefore, apoptosis is not a programme of cell death but purely a termination step of a cell death programme, responsible for proper disposal of the already-committed, dying cells.     

The CD100 Receptor Interacts with Its Plexin B2 Ligand to Regulate Epidermal γδ T Cell Function

γδ T?cells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. In?vitro blocking of plexin B2 or CD100 inhibited γδ T?cell activation. Furthermore, CD100 deficiency in?vivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ T?cell response to keratinocyte damage. Ligation of CD100 in γδ T?cells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providing?a mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ T?cells and the neighboring cells they support.     

Acquired pMHC I Complexes Greatly Enhance CD4＋ Th Cell＇s Stimulatory Effect on CD8＋T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

CD4＋ helper T （Th） cells play pivotal roles in induction of CD8＋ CTL immunity. However, the mechanism of CD4＋ T cell help delivery to CD8＋ T cells in vivo is still elusive. In this study, we used ovalbumin （OVA）-pulsed dendritic cells （DCovA） to activate OT-II mouse CD4＋ T cells, and then studied the help effect of these CD4＋ T cells on CD8＋ cytotoxic T lymphocyte （CTL） responses＋ We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter （RIP）-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex （pMHC） I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination ＋ ＋ ＋ with TCR transgenlc OT-I mouse CD8 T ceils, the tolerogenic dosage of CD4＋ Th cells with acquired pMHC I, but not CD4＋ （Kb-/-） Th cells without acquired pMHC I were able to cause diabetes in 8/10 （80%） RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4＋ T cell-mediated help in CD8＋ CTL induction. Cellular ＆ Molecular Immunology. 2008;5（6）：407-415.     

Increased CD8+ T Cell Apoptosis in Scleroderma Is Associated with Low Levels of NF-κB

Our objectives were (1) to compare lymphocyte subpopulation apoptosis rates in SSc patients versus healthy controls and (2) to compare Bcl-2 and NF-kappa B expression in cultured CD8 lymphocytes of SSc patients versus controls. Peripheral blood samples were obtained from 27 SSc patients meeting the American College of Rheumatology criteria for SSc and 28 healthy individuals. Mononuclear cells were isolated by Ficoll-Hypaque density gradient separation and cultured for 48 hr. For determination of apoptosis within specific cell populations, samples were labeled with PE-conjugated monoclonal antibody to CD8, CD4, and a FITC-conjugated monoclonal antibody to Annexin V. Flow cytometry was carried out with a FACS operating with Cellquest software. CD8+ lymphocytes were positively selected with magnetic microbeads conjugated to antihuman CD8. CD8 T cells were separated, then incubated with activation for 48 hr, and NF-kappa B and Bcl-2 analysis was carried out using Western immunoblotting. The CD4:CD8 ratio was increased in SSc compared to controls (2.6 +/- 1.13 vs.1.87 +/- 0.76; P = 0.018). The spontaneous apoptosis rate of SSc CD8 lymphocytes was increased compared to that of controls of (21.9 +/- 13.7 vs. 13.3 +/- 9.9; P = 0.019). No difference was found in the rate of CD4 apoptosis of SSc patients versus controls (9.8 +/- 5.2 vs. 7.18 +/- 4.89%; P = ns). The expression of NF-kappa B in SSc CD8 lymphocytes was decreased compared with that of CD8 lymphocytes from healthy controls (144 +/- 13 vs. 188 +/- 11; P = 0.018). Whereas expression of Bcl-2 was similar in activated CD8+ T cells of SSc patients and healthy controls, CD8+ T cell apoptosis rate was found to be in reverse correlation with expression of NF-kappa B in these cells ( r = - 0.53, P = 0.029). The increased CD4:CD8 ratio in SSC may result from increased CD8+ T cell apoptosis. Increased SSc CD8 T cell apoptosis is associated with low levels of NF-kappa B.     

Endothelial Cell–Smooth Muscle Cell Co-Culture in a Perfusion Bioreactor System

Vascular endothelial cells (EC) are exposed to a complex biomechanical environment in vivo and are responsible for relaying important messages to the underlying tissue. EC and smooth muscle cells (SMC) communicate to regulate vascular development and function. In this work, a vascular perfusion bioreactor is used to grow tubular constructs seeded with EC and SMC under pulsatile shear stress in long-term co-culture to study the effects of EC on SMC function. SMC seeded into porous poly(glycolic acid) tubular scaffolds are cultured in the bioreactor for 25 days. Constructs are seeded with EC on day 10 or day 23 creating 2-day (short-term) or 15-day (long-term) EC and SMC co-cultures. Long-term EC–SMC co-culture significantly increases cell proliferation and downregulates collagen and proteoglycan deposition compared to short-term co-culture. After 25 days of culture, 15-day co-culture constructs have a more uniform cell distribution across the construct thickness and SMC express a more contractile phenotype compared to 2-day co-culture constructs. These data demonstrate strong interactions between SMC and EC in the bioreactor under physiologically relevant conditions. Thus, the vascular construct perfusion bioreactor is an important tool to investigate cell–cell and cell–extracellular matrix interactions in vascular cell biology and tissue engineering.     

Post-Transplant CD34+ Selected Stem Cell “Boost” for Mixed Chimerism after Reduced-Intensity Conditioning Hematopoietic Stem Cell Transplantation in Children and Young Adults with Primary Immune Deficiencies

Mixed chimerism and eventual graft loss occurs in a proportion of children with primary immune deficiencies receiving alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens before allogeneic hematopoietic stem cell transplantation (HSCT). We investigated the usefulness of a CD34⁺ selected stem cell “boost” without conditioning to treat mixed chimerism in children and young adults who received predominantly an alemtuzumab, fludarabine, and melphalan RIC regimen for primary immune deficiencies and reported the outcomes. Patients with a primary immune deficiency disorder who were either enrolled on a prospective CD34⁺ boost study for treatment of mixed chimerism from 2011 to 2014 (n = 9) or treated with a CD34⁺ boost on a clinical basis from 2014 to 2016 (n = 3) were included in this analysis. Response to a CD34⁺ boost was defined as a rise in donor chimerism by ≥15% with donor chimerism of at least 20%, stabilization was defined as a rise in chimerism by <15% with donor chimerism ≥ 20%, and no response was defined as any decline in donor chimerism or need for a second HSCT after a CD34⁺ boost. Twelve patients received alemtuzumab, fludarabine, and melphalan. Median age was 4.5 years (range, .9 to 20.6), and median whole blood donor chimerism before the boost was 25% (range, 3% to 61%). Three patients (25%) met criteria for response, 1 patient (8%) was considered to have stabilization, and 8 patients (67%) had no response 12 months after the boost. None of the patients developed any complications from a CD34⁺ boost, including no acute graft-versus-host disease (GVHD). All patients are alive with a median follow-up of 32 months (range, 8 to 79). We conclude that a CD34⁺ selected stem cell boost can be considered for treatment of mixed chimerism after alemtuzumab, fludarabine, and melphalan RIC HSCT in children and young adults with primary immune deficiencies. Approximately one-third of patients can be expected to benefit from a CD34⁺ selected stem cell boost and may avoid the need for a second HSCT. Lack of any GVHD or toxicity makes a stem cell boost an attractive option compared with donor lymphocyte infusions for treatment of mixed chimerism.     

Crucial Role of CD4+CD 25+ FOXP3+ T Regulatory Cell,Interferon-γ and Interleukin-16 in Malignant and Tuberculous Pleural Effusions

The differential diagnosis between malignant and tuberculous exudative pleural effusions is an important clinical problem. The aim of current study is to evaluate the frequencies of T-regulatory cells (Treg) on the basis of distinct phenotypes in the differential diagnosis between malignant and tuberculous pleural effusion. In addition, to evaluate Interferon-gamma (IFN- γ) and interleukin-16 (IL-16) levels and their correlation to Treg cells in malignant and tuberculous pleural effusions. Sixty patients with pleural effusion (26 tuberculous and 34 malignant) and 20 healthy controls were included in the study. Pleural fluid and peripheral blood were assessed for frequencies of T regs, IL-16, and IFN-γ. Pleural effusions from both tuberculous and malignant groups represented significantly higher levels (more in TB) for the following cell populations than peripheral blood: total lymphocytes, CD3+lymphocyte, CD4+CD25+lymphocyte and Treg (CD4+ CD25+FoxP3+). Levels of IL-16 and IFN-γ in tuberculous group were significantly higher than that in malignant group. Regulatory T cells, INF-γ and IL-16 are new important tools for differentiation between tuberculous and malignant pleural effusion.     

Towards Developing a Malaria Vaccine Based on CD4 T Cell Mediated Immunity in Blood Stage of Malaria Infection

Twenty-one years after malaria antigens were first cloned avaccine still appears to be a long way off. There havebeen periods of great excitement and in model systemssubunit vaccine homologues can induce robust protection.However, significant challenges e…