Adiponectin modulates NK‐cell function

Adiponectin (APN) has been shown to exert antiinflammatory effects in various disease models but little is known concerning its regulation of NK‐cell function. Here, we show that the majority of human CD56^dim NK cells express surface Adiponectin receptor (AdipoR) 1 and 2 while most CD56^high NK cells are AdipoR‐negative. Toll‐like receptor (TLR) ligand‐induced IFN‐γ production was diminished by APN while it had no influence on NK‐cell cytotoxicity. In contrast only a small subpopulation of murine NK cells expresses surface AdipoRs, but about 90% store them intracellularly. APN‐deficient knockout (KO) mice had elevated frequencies of NK cells. However, cytotoxic degranulation of NK cells was decreased in APN knockout (APN‐KO) animals. Accordingly, frequencies of CD11b^highCD27^high and CD94^high effector NK cells and expression of NKG2D were lower in APN‐KO mice. Upon CVB3 infection NK‐cell function was restored in APN‐KO mice. Our data suggest that in addition to its antiinflammatory effects APN also influences the numerical and differentiation status of NK cells, which may further impact the outcome of immune‐mediated diseases in APN‐KO mice.     

MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells

MEK kinase 1 (MEKK1) is a potent JNK-activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 activity (Mekk1(DeltaKD)) exclusively in their hematopoietic system, we show that MEKK1 has a negative regulatory role in the generation of a virus-specific immune response. Mekk1(DeltaKD) mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8(+) T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8(+) T cell expansion in Mekk1(DeltaKD) mice following VSV infection is virus-specific and the frequency of virus-specific T cells is significantly higher (more than threefold) in Mekk1(DeltaKD) as compared to WT animals. Moreover, the hyper-expansion of T cells seen in Mekk1(DeltaKD) mice after VSV infection is a result of increased proliferation, since a significantly higher percentage of virus-specific Mekk1(DeltaKD) CD8(+) T cells incorporated BrdU as compared to virus-specific WT CD8(+) T cells. In contrast, similar levels of apoptosis were detected in Mekk1(DeltaKD) and WT T cells following VSV infection. These results strongly suggest that MEKK1 plays a negative regulatory role in the expansion of virus-specific CD8(+) T cells in vivo.     

Activation of p38 mitogen-activated protein kinase is critical step for acquisition of effector function in cytokine-activated T cells, but acts as a negative regulator in T cells activated through the T-cell receptor

Peripheral blood CD4⁺ CD45RO⁺ T cells activated in vitro are able to induce expression of tumour necrosis factor‐α (TNF‐α) in monocytes via a contact‐dependent mechanism. Activation is achieved either with interleukin‐2 (IL‐2)/IL‐6/TNF‐α over an 8‐day period or cross‐linking CD3 using anti‐CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen‐activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti‐CD3 activated T cells, p38 MAPK is a negative regulator for anti‐CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte‐derived TNF‐α) or production of T‐cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine‐induced effector function and promotes cell proliferation and cytokine production.     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Prostaglandin E2 is a negative regulator on human plasmacytoid dendritic cells

Prostaglandin E2 (PGE2), a major lipid derived from the metabolism of arachidonic acid, is an environmentally bioactive substance produced by inflammatory processes and acts as a cAMP up-regulator that plays an important role in immune responses. It has been reported that PGE2 has the ability to inhibit the production of interleukin-12 by myeloid dendritic cells (MDCs) and macrophages, and then induce preferential T helper type 2 (Th2) cell responses. However, little is known of the function of PGE2 for plasmacytoid dendritic cells (PDCs), which may contribute to the innate and adaptive immune response to viral infection, allergy and autoimmune diseases. In the present study, we compared the biological effect of PGE2 on human PDCs and MDCs. PGE2 caused the death of PDCs but MDCs survived. Furthermore, we found that, whereas PGE2 inhibited interferon-alpha production by PDCs in response to virus or cytosine-phosphate-guanosine, it inhibited interleukin-12 production by MDCs in response to lipopolysaccharide (LPS) or poly(I:C). Although both virus-stimulated PDCs and LPS-stimulated MDCs preferentially induced the development of interferon-gamma-producing Th1 cells, pretreatment with PGE2 led both DC subsets to attenuate their Th1-inducing capacity. These findings suggest that PGE2 represents a negative regulator on not only MDCs but also PDCs.     

Basic difference between receptor repertoire of effector cytotoxic T cells and their secondary precursors (memory cells)

Laboratory of Immunochemistry, All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 6, pp. 694–697, June, 1988.     

Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells

Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1-/- background relieved the functional defects of vav1-/- T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor-triggered Ca2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.     

免疫磁珠负性分离纯化小鼠脾脏CD8+ T细胞

目的 探讨小鼠脾脏CD8 T细胞的免疫磁珠负性分选方法,并对分选后所得细胞进行纯度、活力及功能检测.方法 以免疫磁珠负性分选法从小鼠脾脏细胞中分离CD8 T细胞,流式细胞术检测所得细胞的纯度,台盼蓝检测细胞活力并用ConA刺激检测增殖能力. 结果 经过流式细胞仪测定免疫磁珠负性分选后的小鼠脾脏CD8 T细胞纯度达到(91.6±3.6)%,台盼兰染色细胞活力为(94.9±3.2)%,ConA刺激72 h后有(56.3±1.7)%的细胞增殖.结论 免疫磁珠负性分选法能够分选出高纯度的CD8 T细胞,并且不影响分选靶细胞的细胞活力和功能.     

CD8+ T cells and immunoregulatory networks in asthma

It is well established that infection with respiratory viruses can cause acute local inflammation in humans and is a leading cause in the hospitalization of asthmatics. Less well recognized is the potential for viral infections to actually protect against the development of asthma, as are the cellular mechanisms which might underlie such protection. This review outlines the basic immunological pathways involved in atopic asthma and details the currently recognized cellular mechanisms induced by respiratory viral infections which can protect against the development of asthma. Specifically, it appears that virus infection induced memory T cells that remain in tissues, e.g. the lung and airways, can under certain circumstances create a local cytokine milieu which inhibits the development of ensuing allergic immune responses at that site. One key aspect of this immune modulation is the cytokine-dependent communication which occurs between the innate and the adaptive immune systems. The mechanistic principles underlying this form of immunomodulation should be taken into consideration when developing future forms of therapy and rational vaccine design.     

T cells and their eons-old obsession with MHC

T cells bearing receptors made up of α and β chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self-peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse Vβ8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC α-helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto-recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells.     

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.     

Gamma/delta T cells and the diagnosis of coeliac disease.

Gamma/delta T cells are increased in the gut epithelium of patients with coeliac disease compared with normal controls. The aim of this study was to determine whether the increase in gamma delta intraepithelial lymphocytes (IEL) is specific for coeliac disease, in which case it could be of diagnostic importance. Biopsies were obtained from children with no intestinal disease, coeliac disease, cow-milk-sensitive enteropathy/post-enteritis syndrome (CMSE PES) and miscellaneous other enteropathies (n = 67). Intraepithelial CD3+ and gamma delta T cells were identified in frozen sections using peroxidase immunohistochemistry. In normal biopsies there were 0-7 gamma delta IEL/100 cells in the epithelium. In untreated coeliac patients this increased to 9-22 gamma delta IEL/100 cells in the epithelium (P = 0.000004). Of 27 patients with morphologic intestinal damage which was not due to coeliac disease, four with CMSE/PES had gamma delta IEL/100 cells in the epithelium in the same range as the patients with coeliac disease. Of these, two had high densities of CD3+ IEL in the epithelium and were indistinguishable from patients with untreated coeliac disease. The other two could be excluded as possible coeliacs because their CD3+ IEL/100 epithelial cells were in the normal range. Thus an increase in gamma delta IEL is not specific for coeliac disease. However, enumeration of both of gamma delta IEL and CD3+ IEL densities will be useful in the exclusion of coeliac disease as a diagnosis in some children.     

脂联素抗人脐静脉内皮细胞氧化损伤的作用研究

目的:研究脂联素（APN）对过氧化氢(H2O2)诱导人脐静脉内皮细胞 (HUVECs) 脂质过氧化的影响。方法：采用Annexin V-FITC标记后流式细胞检测方法观测H2O2引起的细胞凋亡情况。用硫代巴比妥酸微量法测定丙二醛(MDA),黄嘌呤氧化酶法测总超氧化物歧化酶(SOD),可见光法测过氧化氢酶(CAT）。体外自由基捕捉实验检测APN对超氧阴离子和羟自由基的清除率。结果：APN能明显抑制H2O2引起的细胞凋亡。APN预处理HUVECs后,H2O2（200 μmol/L,6 h）所致的细胞膜脂质过氧化反应明显减弱,MDA产生减少,CAT和SOD活性增加,但仅以APN孵育HUVECs对SOD和CAT活性没有明显的影响。结论：结果表明APN对超氧阴离子和羟自由基有明显的清除效果。APN通过抗脂质过氧化发挥抑制细胞凋亡,保护血管内皮细胞抵抗损伤的作用。     

Ssu72 is a T-cell receptor-responsive modifier that is indispensable for regulatory T cells

The homeostatic balance between effector T cells and regulatory T cells (Tregs) is crucial for adaptive immunity; however, epigenetic programs that inhibit phosphorylation to regulate Treg development, peripheral expression, and suppressive activity are elusive. Here, we found that the Ssu72 phosphatase is activated by various T-cell receptor signaling pathways, including the T-cell receptor and IL-2R pathways, and localizes at the cell membrane. Deletion of Ssu72 in T cells disrupts CD4⁺ T-cell differentiation into Tregs in the periphery via the production of high levels of the effector cytokines IL-2 and IFNγ, which induce CD4⁺ T-cell activation and differentiation into effector cell lineages. We also found a close correlation between downregulation of Ssu72 and severe defects in mucosal tolerance in patients. Interestingly, Ssu72 forms a complex with PLCγ1, which is an essential effector molecule for T-cell receptor signaling as well as Treg development and function. Ssu72 deficiency impairs PLCγ1 downstream signaling and results in failure of Foxp3 induction. Thus, our studies show that the Ssu72-mediated cytokine response coordinates the differentiation and function of Treg cells in the periphery.     

Memory precursor phenotype of CD8+ T cells reflects early antigenic experience rather than memory numbers in a model of localized acute influenza infection

The mechanistic basis of memory T‐cell development is poorly defined. Phenotypic markers that define precursors at effector stages have been characterized for acute systemic infections with high antigen load. We asked whether such markers can identify memory precursors from early effectors (d6) to late memory (>d500) for two immunodominant CD8⁺ responses during the course of a localized low‐load influenza infection in mice. CD8⁺ T cells stained with the D^bNP₃₆₆ and D^bPA₂₂₄ tetramers were characterized as IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi or IL‐7Rα^hiKLRG1^lo. While the D^bNP₃₆₆‐ and D^bPA₂₂₄‐specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi and IL‐7Rα^hiKLRG1^lo phenotypes on effector, but not naïve, CD8⁺ populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP₃₆₆ and PA₂₂₄ peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL‐7Rα^hiKLRG1^lo phenotype had a poor predictive value in identifying memory precursors in the spleen and at the site of infection. Greater consistency in influenza‐specific IL‐7Rα^hiKLRG1^loCD8⁺ T‐cell numbers was found in draining lymph nodes, suggesting that this may be the preferential site for memory establishment and maintenance following localized virus infections.     

Regulation of regulatory T cells in cancer

The inflammatory response to transformed cells forms the cornerstone of natural or therapeutically induced protective immunity to cancer. Regulatory T (Treg) cells are known for their critical role in suppressing inflammation, and therefore can antagonize effective anti-cancer immune responses. As such, Treg cells can play detrimental roles in tumour progression and in the response to both conventional and immune-based cancer therapies. Recent advances in our understanding of Treg cells reveal complex niche-specific regulatory programmes and functions, which are likely to extrapolate to cancer. The regulation of Treg cells is reliant on upstream cues from haematopoietic and non-immune cells, which dictates their genetic, epigenetic and downstream functional programmes. In this review we will discuss how Treg cells are themselves regulated in normal and transformed tissues, and the implications of this cross talk on tumour growth.     

Regulation of antigen-expressing dendritic cells by double negative regulatory T cells

TCRαβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) double negative (DN) Tregs comprise 1-3% of peripheral T lymphocytes in mice and humans. It has been demonstrated that DN Tregs can suppress allo-, xeno- and auto-immune responses in an Ag-specific fashion. However, the mechanisms by which DN Tregs regulate immune responses remain elusive. Whether DN Tregs can regulate DCs has not been investigated previously. In this study, we demonstrate that DN Tregs express a high level of CTLA4 and are able to down-regulate costimulatory molecules CD80 and CD86 expressed on Ag-expressing mature DCs (mDCs). DN Tregs from CTLA4 KO mice were not able to downregulate CD80 and CD86 expression, indicating that CTLA4 is critical for DN Treg-mediated downregulation of costimulatory molecule expression on Ag-expressing mature DCs. Furthermore, DN Tregs could kill both immature and mature allogeneic DCs, as well as Ag-loaded syngeneic DCs, in an Ag-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. These data demonstrate, for the first time, that DN Tregs are potent regulators of DCs and may have the potential to be developed as a novel immune suppression treatment.