The ex vivo Microenviroments in MLTC of Poorly Immunogenic Tumor Cells Facilitate Polarization of CD4^＋ CD25^＋ Regulatory T Cells

CD4^＋CD25^＋ regulatory T （TR） cells play an important role in maintaining a balanced peripheral immune system. Recent studies have shown that TR cells may also play a key role in suppressing anti-tumor immune response. In order to investigate the tumor immune microenvironment and its influence on TR polarization, poorly immunogenic tumor cell line Ds （C57BL/6, H-2^b）, immunogenic tumor cell lines FBL3 （C57BL/6, H-2^b） and H22 BALB/c, H-2^d） were used to establish the syngeneic/allogeneic, poorly immunogenic/immunogenic mixed lymphocytes-tumor cell culture （MLTC）. Our results revealed that the proportion of CD4^＋CD25^＋ T cells in MLTC of syngeneic primed splenocytes stimulated with D5 tumor cells was higher than that with H22 cells （0.43% vs 0.044%, and the similar results appeared in allogeneic splenocytes stimulated with D5 tumor cells （0.39% vs 0.04%）. The splenocytes stimulated with supernatant from syngeneic MLTC of D5 tumor cells demonstrated higher proportion of CD4^＋CD25^＋ cells than that from allogeneic MLTC of D5 tumor cells, and the splenocytes stimulated with supernatant from syngeneic or allogeneic MLTC of H22 tumor cells generated lower proportion of CD4^＋CD25^＋ T cells than that of D5 tumor cells. The TGF-β1 and Th2-oriented cytokines （IL-4 and IL-10） were dominated in supernatants of syngeneic MLTC of poorly immunogenic tumor cells. Our results provided useful information for studying the mechanisms underlying tumor immune surveillance as well as for the tumor immunotherapy.     

Thymic Nurse Cells Support CD4^-CD8^＋ Thymocytes to Differentiate into CD4^＋CD8^＋ Cells

Thymic nurse cells （TNCs） represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4^-CD8^＋ cells differentiated into CD4^＋CD8^＋ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4^-CD8^＋ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4^＋CD8^＋ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4^-CD8^＋ thymocytes to differentiate into CD4^＋CD8^＋ T cells by direct interaction.     

Deficiency of Mouse CD4^＋CD25^＋Foxp3^＋ Regulatory T Cells in Xenogeneic Pig Thymus-Grafted Nude Mice Suffering from Autoimmune Diseases

Xenogeneic thymus transplantation can efficiently induce specific immune tolerance to donor antigens in athymic recipients. However, many nude mice suffer from autoimmune diseases （AID） for over 10 weeks after xenogeneic thymus transplantation. CD4^＋CD25^＋Foxp3^＋ regulatory T （Treg） cells were recently determined to play a pivotal role in keeping immune tolerance in humans and mice. Thus, we investigated this subpopulation of Treg cells in the periphery of pig thymus-grafted nude mice suffering from AID. Our results showed that the expression of Foxp3, CTLA-4 and GITR on mouse CD4^＋CD25^＋ T cells and the ratio of CD4^＋CD25^＋Foxp3^＋ Treg cells to CD4^＋ T cells were significantly decreased in the periphery of pig thymus-grafted nude mice suffering from AID, compared with healthy pig or mouse thymus-grafted nude mice. Furthermore, mouse CD4^＋CD25^＋ T cells in pig thymus-grafted nude mice suffering from AID showed more severe deficiency in immunosuppressive function compared with the counterpart in xenogeneic pig or syngeneic thymus-grafted nude mice without AID. Thus, the decreased frequency, altered phenotype and functional deficiency of mouse CD4^＋CD25^＋ Treg cells in pig thymus-grafted nude mice may contribute to the development of AID in this model.     

Naturally Occurring Self-Reactive CD4^＋CD25^＋ Regulatory T Cells： Universal Immune Code

Naturally occurring thymus-arisen CD4^＋CD25^＋ regulatory T （Treg） cells are considered to play a central role in self-tolerance. Precise signals that promote the development of Treg cells remain elusive, but considerable evidence suggests that costimulatory molecules, cytokines, the nature of the TCR and the niche or the context in which the T cell encounters antigen in the thymus play important roles. Analysis of TCR from Treg cells has demonstrated that a large proportion of this population has a higher avidity to self-antigen in comparison with TCR from CD4^＋CD25^＋ cells and that peripheral antigen is required for their development, maintenance, or expansion. Treg cells have been shown to undergo expansion in the periphery, likely regulated by the presence of self-antigen. Many studies have shown that the involvement of Treg cells in the tolerance induction is antigen-specific, even with MHC-mismatched, in transplantation/graft versus host disease （GVHD）, autoimmunity, cancer, and pregnancy. Theses studies concluded a vital role for self-reactive Treg cells in maintenance of the body integrity. Based on those studies, we hypothesize that self-reactive Treg cells are shared among all healthy individuals and recognize same self-antigens and their TCR encodes for few dominant antigens of each organ which defines the healthy self. These dominant self antigens can be regarded as ＂universal immune code＂. Cellular ＆ Molecular Immunology.     

A naturally occurring CD8^＋CD122^＋ T-cell subset as a memory-like Treg family

Despite extensive studies on CD4^＋CD25^＋ regulatory T cells （Tregs） during the past decade, the progress on their clinical translation remains stagnant. Mounting evidence suggests that naturally occurring CD8^＋CD122^＋ T cells are also Tregs with the capacity to inhibit T-cell responses and suppress autoimmunity as well as alloimmunity. In fact, they are memory-like Tregs that resemble a central memory T cell （TcM） phenotype. The mechanisms underlying their suppression are still not well understood, although they may include IL-IO production. We have recently demonstrated that programmed death-1 （PD-1） expression distinguishes between regulatory and memory CD8^＋CD122^＋ T cells and that CD8^＋CD122^＋ Tregs undergo faster homeostatic proliferation and are more potent in the suppression of allograft rejection than conventional CD4^＋CD25^＋ Tregs. These findings may open a new line of investigation for accelerating effective Treg therapies in the clinic. In this review, we summarize the significant progress in this promising field of CD8^＋CD122^＋ Treg research and discuss their phenotypes, suppressive roles in autoimmunity and alloimmunity, functional requirements, mechanisms of action and potential applications in the clinic.     

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.     

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.     

Type 1 CD8^＋ T Cells are Superior to Type 2 CD8^＋ T Cells in Tumor Immunotherapy due to Their Efficient Cytotoxicity, Prolonged Survival and Type 1 Immune Modulation

CD8^＋ cytotoxic T （Tc） cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8^＋ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8^＋ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin （OVA）-pulsed dendritic cell （DCovA）-activated CD8^＋ type 1 Tc （Tcl） cells secreting IFN-T, and CD8^＋ type 2 Tc （Tc2） cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor （TCR） transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tcl and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8^＋ Tc2, Tcl cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8^＋ Tcl and Tc2 cells skewed the phenotype of CD4^＋ T cells toward Thl and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8^＋ Tcl and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8^＋ T cells for adoptive T cell therapy of cancer.     

Potent Dendritic Cell Vaccine Loaded with Latent Membrane Protein 2A （LMP2A）

Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.     

Defect of CD8^＋ Memory T Cells Developed in Absence of IL-12 Priming for Secondary Expansion

IL-12 priming plays an important role in stimulation of CD8^＋ effector T cells and development of CD8^＋ memory T （Tm） cells. However, the functional alteration of CD8^＋ Tm cells developed in the absence of IL-12 priming is elusive. In this study, we investigated the capacity of secondary expansion of CD8~ Tm cells developed from transgenic OT I CD8^＋ T cells. The latter cells were in vitro and in vivo stimulated by ovalbumin （OVA）-pulsed dendritic cells [DCovA and （IL-12^-/-）DCovA] derived from wild-type C57BL/6 and IL-12 gene knockout mice, respectively. We demonstrated that IL-12 priming is important not only in CD8^＋ T cell clonal expansion, but also in generation of CD8^＋ Tm cells with the capacity of secondary expansion upon antigen re-encounter. However, IL-12 signaling is not involved in CD8^＋ Tm cell survival and recall responses. Therefore, this study provides useful information for vaccine design and development. Cellular ＆ Molecular Immunology.     

I L- 15 trans-presentation regulates homeostasis of CD4^＋ T lymphocytes

Interleukin-15 （IL-15） is essential for the survival of memory CD8^＋ and CD4^＋ T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4^＋ T cells. Adoptive transfer of splenocytes from non-obese diabetic （NOD） mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.II15^-/- mice when compared to NOD.scid recipients. The increased accumulation of CD4^＋ T cells is also observed in NOD.II15^-/- mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the I L- 15Ra chain, but not those lacking the common gamma chain, also show increased accumulation of CD4^＋ T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4^＋ T cells and requires trans-presentation of IL-15. CD4^＋ T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4^＋ T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-7 production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4^＋ T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.     

The current immune function of hepatic dendritic cells

While only a small percentage of the liver as dendritic cells, they play a major role in the regulation of liver immunity. Four major types of dendritic cell subsets include myeloid CD8α^-B220^-, lymphoid CD8α^＋B220^-, plasmacytoid CD8α^-B220^＋, and natural killer dendritic cell with CD8α^-B220^-NK1.1^＋ phenotype. Although these subsets have slightly different characteristics, they are all poor naive T cell stimulators. In exchange for their reduced capacity for allostimulation, hepatic DCs are equipped with an enhanced ability to secrete cytokines in response to TLR stimulation. In addition, they have increased level of phagocytosis. Both of these traits suggest hepatic DC as part of the innate immune system. With such a high rate of exposure to the dietary and commensal antigens, it is important for the hepatic DCs to have an enhanced innate response while maintaining a tolerogenic state to avoid chronic inflammation. Only upon secondary infectivity does the hepatic DC activate memory T cells for rapid eradication of recurring pathogen. On the other hand, overly tolerogenic characteristics of hepatic DC may be responsible for the increase prevalence of autoimmunity or liver malignancies.     

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.     

Cell-mediated immunity imbalance in patients with intrahepatic cholestasis of pregnancy

Decidual lymphocytes may mediate fetal trophoblast recognition and regulate maternal immune reaction and play an essential role in the maintenance of normal pregnancy. The aim of this study was to compare the percentage of T cells, natural killer （NK） cells and natural killer T （NKT） cells within decidual parietalis of normal pregnant controls （NP） and patients with intraheptic cholestasis of pregnancy （ICP）, and to investigate the production of interleukin-4 （IL-4）, interferon-γ （IFN-γ） in the culture supernatant of decidual parietalis mononuclear cells （DPMCs）. Compared with controls, the decidua parietalis from ICP were characterized with significant increased percentages of CD3^＋CD56^＋ cells, CD3^＋CD56^＋ cells, CD56^＋CD16＋ cells, CD56^＋CD16^＋ cells, CD56^＋NKG2D＋ ceils, and the significant decreased percentages of CD3^＋ cells, CD3^＋CD4^＋ cells. There were no differences found for the percentage of CD3^＋CD8^＋ cells, CD56^＋NKG2A^＋ cells between control and study group. In addition, the enhanced concentration of IFN-γ was presented in culture supernatant of DPMCs from ICP. It was suggested that the increased NK cells, NKT cells and the decreased T cells in the decidual parietalis and over-secretion of IFN-γ could be correlated with the pathophysiology of ICP patients. Cellular ＆ Molecular Immunology. 2007;4（1）：71-75.     

Anti-Idiotypic Regulatory Responses Induced by Vaccination with DNA Encoding Murine TCR Vα5 and Vβ2

There is evidence suggesting that anti-idiotypic regulation against T cells plays a role in maintaining homeostasis in the immune system, although its mechanism is not fully understood. By using DNA constructs encoding the TCR Vα5.2 and Vβ2.1 chains derived from ：in ovalbumin （OVA）-specific T cdl clone （OVA-T）, we herein demonstrated that vaccination with TCR-DNA effectively induced anti-idiotypic cellular as well as humoral responses. Serum samples from the TCR-DNA-vaccinated BALB/c mice were able to stain T cells in an idiotype-specific manner. CD4^＋ T cells from the TCR-DNA-vaccinated mice proliferated in response to stimulation with irradiated syngeneic OVA-T calls and secreted interferon-y but very little IL-4. Splenocytes from the TCR-DNA-vaccinated mice showed strong idiotype-specific CTL activity against the OVA-T cdls. Furthermore, adoptive transfer of the CD4^＋ or CD8^＋ T cells from the TCR-DNA-vaccinated mice resulted in hyporesponsiveness of syngeneic recipients. These results demonstrated that vaccination with DNA encoding TCR can effectively activate anti-idiotypic regulatory responses in vivo and thus providing a useful way for immunological intervention.     

Generation and Regulation of CD8＋ Regulatory T Cells

Research into the suppressive activity of CD4＋FoxP3＋ T regulatory cells （Treg） has defined a sublineage of CD4＋ cells that contribute to self-tolerance and resistance to autoimmune disease. Much less attention has been given to the potential contribution of regulatory sublineages of CD8＋ cells. Analysis of a small fraction of CD8＋ cells that target autoreactive CD4＋ cells through recognition of the MHC class Ib molecule Qa-1 in mouse and HLA-E in human has revitalized interest in CD8＋ Treg. Here we summarize recent progress and future directions of research into the role of this CD8＋ sublineage in resistance to autoimmune disease. Cellular ＆ Molecular Immunology. 2008; 5（6）：401-406.     

Blockade of Tim-3 Pathway Ameliorates Interferon-γ Production from Hepatic CD8^＋ T Cells in a Mouse Model of Hepatitis B Virus Infection

T cell immunoglobulin- and mucin-domain-containing molecule-3 （Tim-3） has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus （HBV） infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8^＋ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8^＋ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8^＋ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection. Cellular ＆ Molecular Immunology.     

Preparation and Characterization of HLA-A＊0201 Tetramer Loaded with IE-1316.324 Antigenic Peptide of Human Cytomegalovirus

Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8^＋ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A＊0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp6549s.50a has been successfully applied to investigate HCMV-specific CD8^＋ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A＊0201 tetramer loaded with another dominant epitope derived from immediate early （IE）-1 316.324 （VLEETSVML, VLE） of HCMV （A2-VLE） and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8^＋ T cells of one HLA-A2^＋ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8^＋ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8^＋ T cells. Cellular ＆ Molecular Immunology.