Interaction of suplatast tosilate (IPD) with chloride channels in human blood eosinophils: a potential mechanism underlying its anti-allergic and anti-asthmatic effects

Introduction Alterations in chloride ion channels have been implicated in the induction of changes in cell shape and volume. Because blood and tissue eosinophilia are hallmarks of bronchial asthma, in this study we examined the role of chloride channels in the underlying effects of suplatast tosilate (IPD), an anti‐allergic drug, in human blood eosinophils. Methods Eosinophils were isolated and purified from the blood of allergic asthmatic donors. Chloride ion currents were recorded using the whole‐cell patch‐clamp technique in freshly isolated eosinophils. The current–voltage relationship of whole‐cell currents in human blood eosinophils was calculated and recorded. The effect of chloride channel blockers was examined on superoxide release, eosinophil chemotaxis as measured by the Boyden chamber, and eosinophil adhesion to endothelial cells. Radioligand binding studies with [³H]IPD and competition curves with chloride channel blockers were performed. Results IPD increased both inward and outward chloride currents in human blood eosinophils. IPD in 1 ng/mL did not have significant effect on chloride current. However, at 5 ng/mL IPD activated both outward and inward currents in human blood eosinophils. Chloride channel blockers inhibited IPD‐induced respiratory burst in eosinophils, eosinophil chemotaxis, and eosinophil adhesion to endothelial cells. All these effects of IPD on chloride current and the resultant functional responses in human blood eosinophils were not due to its basic salt, p‐toluenesulphonic acid monohydrate. Human blood eosinophils contained specific binding sites for [³H]IPD with K_D and B_max values of 187.7±105.8 nm and 58.7±18.7 fmol/10⁶ cells, respectively. Both NPPB and DIDS competed, in a dose‐dependent manner, for the specific binding of [³H]IPD in human blood eosinophils. Conclusion These data suggest that the anti‐allergic and anti‐asthmatic effects of IPD could be due to its interaction with chloride channels in human blood eosinophils.     

Impaired interleukin-2 synthesis and T cell proliferation following antibody-mediated CD3 and CD2 or CD28 cross-linking in trans: evidence that T cell activation requires the engagement of costimulatory molecules within the immunological synapse

Although the T cell costimulatory molecules CD2 and CD28 are enriched within the immunological synapse (IS), it has been suggested that costimulatory molecules need not be localized to the contact site between a T cell and an antigen-presenting cell (APC) in order to costimulate T cell activation. To determine whether CD2 or CD28 engagement outside of the IS is sufficient to costimulate T cell activation, we compared mouse T cell responses to anti-CD3 and anti-CD2 monoclonal antibodies (mAbs) or anti-CD3 and anti-CD28 mAbs immobilized on the same, i.e., in cis, or on different, i.e., in trans, 10 micron polystyrene microspheres. In comparison to T cells that were stimulated with co-immobilized anti-CD3 and anti-CD2 or anti-CD28 mAbs, DNA synthesis, interleukin (IL)-2 production, and cellular proliferation were all severely impaired following T cell stimulation with anti-CD3 and anti-CD2 mAbs or anti-CD3 and anti-CD28 mAbs on different microspheres. Deficient cellular proliferation and IL-2 synthesis by T cells that experienced CD3 and CD2 or CD28 cross-linking in trans provides evidence that costimulatory molecules must function in the context of the IS for optimal T cell activation.     

协同刺激分子CD28在结核杆菌抗原体外激活人外周血γδT细胞中的作用

目的 :为了探讨CD2 8协同刺激分子在结核杆菌 (Mtb)低分子多肽抗原体外激活人外周血γδ T细胞中的作用。方法 :采用激发型抗CD2 8单抗模拟第二信号 ,Mtb低分子多肽抗原作为刺激原 ,对纯化的人外周血T细胞进行体外刺激和培养 ;用流式细胞仪检测γδ T细胞上CD2 8分子的表达、γδ T细胞的增殖效应及活化的γδ T细胞上CD6 9分子的表达。结果 :人外周血γδ T细胞中有 5 0 %左右表达CD2 8分子 ;抗CD2 8单抗协同Mtb抗原可刺激γδ T细胞的活化和增殖 ;但抗CD2 8单抗或Mtb抗原单独刺激则无作用。活化的γδ T细胞表面表达CD6 9分子。结论 :Mtb抗原在选择性活化人外周血γδ T细胞时需要第二信号的参与 ;CD2 8在Mtb抗原激活γδ T细胞时可提供协同刺激信号 ;CD6 9可作为γδ T细胞的早期活化标志。     

The enigma of CD57+CD28– T cell expansion—anergy or activation?

Expansion of a CD57⁺CD8^– T lymphocyte subset has been reported in HIV and human cytomegalovirus (HCMV) infection. Almost all of these T cells lack CD28 expression. While CD28^– cells are often associated with anergy, some authors believe their expansion in HIV infection precipitates immunodeficiency. We studied 15 randomly chosen patients with immune activation and observed that CD57⁺CD28^– T cell expansion may occur in various conditions and to the same degree as in HIV infection without resulting in immunodeficiency. Triple colour flow cytometry also revealed that the CD57 and CD28 antigens are coexpressed in only 3% of CD8⁺ T cells, irrespective of the underlying condition, so that almost all CD57⁺CD8⁺ cells are always CD28^–. Analysis of Fas (CD95) expression with respect to CD28 expression on CD4⁺ and CD8⁺ T cells from 10 additional patients indicated no increased commitment to apoptosis in CD28^– T cells. Semiquantitative polymerase chain reaction (PCR) comparing CD28⁺ and CD28^–CD8⁺ T cells with respect to cytokine gene expression (tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-1β) in five renal transplant patients with expansion of the CD57⁺ subset detected no cytokine gene expression deficit in CD28^– T cells. A direct association of increased proportions of CD57⁺CD28^–CD8⁺ T cells with immunodeficiency/anergy is disputed.     

A recombinant bispecific single-chain antibody induces targeted,supra-agonistic CD28-stimulation and tumor cell killing

Endowing tumor cells with costimulatory signals for T cell activation has emerged as a promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor-associated antigens, to achieve targeted T cell costimulation in vivo. Here we report the production and purification of rM28, a recombinant bispecific single-chain antibody directed to a melanoma-associated proteoglycan and to the costimulatory CD28 molecule on human T cells. We found that a dimer of the recombinant molecule, bound to tumor target cells, induced pronounced T cell activation in peripheral blood mononuclear cell preparations without additional TCR/CD3 stimulation being required. The lytic activity generated after 3 days of stimulation effectively prevented tumor cell growth. However, it was unspecific and predominantly mediated by non T cells. Our findings demonstrate that presentation of a CD28 antibody within a suitable recombinant, bispecific format may result in a "targeted supra-agonistic stimulation" of the CD28 molecule, which leads to effective tumor cell killing after induction of unspecifically lytic cells.     

肺癌患者外周血T淋巴细胞及亚群CD28表达和活化的改变

目的检测共刺激分子CD28在肺癌患者外周血T淋巴细胞及亚群中的表达密度和细胞活化程度的变化,并探讨该变化与肺癌临床分期的关系.方法用三色荧光直标流式细胞法分别检测38例正常人与42例肺癌患者中血T淋巴细胞亚群中CD28的阳性率以及CD28+T淋巴细胞亚群活化率.结果与正常人相比,肺癌患者外周血中的CD3+CD28+,CD4+CD28+,CD8+CD28+淋巴细胞百分比均有下降(P＜0.05).淋巴细胞及其T亚群中的CD25+CD28+,CD38+CD28+淋巴细胞百分比亦有所下降(P＜0.05),但在不同临床分期的患者之间无显著性差异(P＞0.05).结论肺癌患者外周血T淋巴细胞的CD28表达密度及CD28+T细胞的活性均有降低,但下降幅度与临床分期无关.     

Preferential elimination of CD28+ T cells in systemic lupus erythematosus (SLE) and the relation with activation-induced apoptosis

CD28 on T cells provides a potent costimulatory signal for T cell activation. Down-regulation of CD28 on peripheral T cells has been reported in certain clinical conditions, but full studies on the mechanism and biological significance have not been performed. Our extensive phenotype analysis of peripheral blood lymphocytes (PBL) from SLE patients revealed that the absolute number of CD28⁺ T cells of both CD4 and CD8 phenotypes was selectively decreased, while that of CD28⁻ T cells was maintained. CD28⁺ T cells from SLE patients exhibited mostly normal proliferative responses to both CD28-dependent and -independent stimulations. In contrast, CD28⁻ T cells were hyporesponsive to anti-CD3 stimulation in both SLE and normal controls. These results implied that the selective decrease of CD28⁺ T cells in SLE does not result from a hyporesponsiveness of CD28⁺ T cells. To investigate the reason for the selective loss of CD28⁺ T cells, we determined the appearance of apoptotic cells in culture with or without anti-CD3 stimulation. Apoptotic cells defined by merocyanine (MC)540 were gradually increased from 12 h to 24 h. Anti-CD3-induced apoptosis of CD28⁺ T cells was significantly accelerated in SLE, whereas apoptosis of CD28⁻ T cells was hardly detected in both SLE and normal controls. Comparative analysis between CD28⁺ and CD28⁻ T cells on CD95 (Fas) and Bcl-2 expression, which are related to activation-induced cell death (AICD), did not show a major difference, although CTLA4, which has been demonstrated to transmit an apoptosis-inducing signal, was expressed only on CD28⁺ T cells. Our results suggest that CD28-mediated costimulation influences T cell susceptibility to AICD and may be involved in T cell lymphopenia in SLE.     

Frontline: Optimal T cell activation requires the engagement of CD6 and CD166

The T cell surface glycoprotein, CD6 binds CD166 in the first example of an interaction between a scavenger receptor cysteine-rich domain and an immunoglobulin-like domain. We report that in human these proteins interact with a K(D) =0.4-1.0 microM and K(off) > or =0.4-0.63 s(-1), typical of many leukocyte membrane protein interactions. CD166 also interacts in a homophilic manner but with around 100-fold lower affinity (K(D) =29-48 microM and K(off) > or = 5.3 s(-1)). At concentrations, that will block the CD6/CD166 interaction, soluble monomeric CD6 and CD166 inhibit antigen-specific human T cell responses. This is consistent with extracellular engagement between CD6 and CD166 being required for an optimal immune response.     

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation

Activation of naive CD4⁺ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4⁺ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4⁺ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4⁺ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4⁺ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.     

Co-stimulation via CD28 induces activation of a refractory subset of MRL-Ipr/Ipr T lymphocytes

Peripheral lymphoid tissues of Ipr mice contain a large proportionof TCRß/CD3⁺CD4^–CD8^– T cells that lacksurface CD2 and express the B cell isoform of CD45, B220. Thissubset of T cells does not proliferate or produce IL-2 in responseto mitogenic signals or TCR–CD3 ligation. At the sametime, these abnormal T cells display several characteristicsof an activated phenotype. Collectively, these properties ofIpr CD4^–CD8^– T cells have functional parallels withanergic T cells. A critical co-stimulatory molecule implicatedin the prevention of or recovery from anergy is CD28, whichbinds the ligand BB1/B7 on certain accessory cells. Ipr CD4^–CD8^–T cells express normal levels of CD28 which is capable of transducinga strong proliferative signal to these cells in co-stimulationwith mitogens. However, proliferation of Ipr CD4^–CD8^–T cells in response to CD28 co-stimulation does not reach thelevels observed in normal T cells stimulated under similar conditions.Stimulation with anti-CD28 mAb in conjunction with phorbol myristateacetate and lonomycin promotes cell cycling in the CD2^–subset of CD4^–CD8^– T cells, and results in a slightinduction of CD2 levels during the course of the culture period.However, the majority of cells obtained at the end of the cultureperiod remain TCRß+ CD4^–CD8^–, CD2^low/–and B220^high, similar to freshly isolated CD4^–CD8^–Ipr T cells. In contrast, if IL-2 is included in the cultures,a strong shift toward a CD2⁺ phenotype is observed by a majorityof the Ipr T cells. Upon repeat stimulation, these Ipr CD4^–CD8^–T cells can now proliferate in an IL-2-dependent manner whenstimulated with only anti-CD3 mAb or mitogens, in the absenceof exogenous IL-2 or anti-CD28 mAb. These data show that thehyporesponsiveness of Ipr CD4^–CD8^– T cells doesnot result from a lack of CD28 expression, that it is not afixed state, and that it can be reversed by the induction ofcell cycling in the presence of IL-2. These observations extendthe parallels between Ipr CD4^–CD8^– T cells and anergicT cells.     

Differential proliferative responses in subsets of human CD28+cells delineated by BB27 mAb

We report the Identification of a novel 140 kDa disulflde-llnkeddimer expressed by a subset of peripheral blood T lymphocytes.This molecule, which Is recognized by mAb BB27, is also detectedon cells of the myelomonocytlc lineage. In the T cell lineage,Its expression Is positively modulated after lymphocyte activation.A series of double-labeling experiments revealed that BB27 mAbIdentifies new CD4 and CDS cell subsets different from thosedefined by CD45RA, CD45RO, CD26, CD29, CD31, and CD38. Finally,BB27 mAb also subdivides the CD28 subset. Of the utmost interestIs the finding that a proliferative response to CD28 mAb andphorbol myrlstate acetate stimulation is exclusively obtainedin the CD28⁺BB27⁺ cell subset, whereas the CD28⁺BB27^–subset falls to proliferate.     

Phenotypic and functional characteristics of CD28+ and CD28- cells from chagasic patients: distinct repertoire and cytokine expression

Chronic human Chagas' disease ranges from an asymptomatic to a severe cardiac clinical form. The involvement of the host's immune response in the development and maintenance of chagasic pathology has been demonstrated by several groups. We have shown that activated T-cells lacking CD28 expression are increased in the peripheral blood of chagasic patients (CP), suggesting a relationship between these cells and disease. In order to better characterize this cell population, determining their possible role in immunoregulation of human Chagas' disease, we evaluated the expression of TCR-Vbeta regions 2, 3.1, 5, 8 and 17, as well as the expression of IFN-gamma, TNF-alpha, IL-4 and IL-10 by CD28+ and CD28- cells from polarized indeterminate and cardiac CP. Flow cytometric analysis demonstrated equivalent TCR-Vbeta usage between CD4+CD28+ and CD4+CD28- cells from all groups (chagasic and healthy controls). However, there was a predominance of Vbeta5 expression in the CD28+ and CD28- populations in the CP groups (indeterminate and cardiac). Interestingly, CD8+CD28- cells from CP, but not from nonchagasic individuals, displayed a reduced frequency of most analysed Vbetas when compared with the CD8+CD28+ subpopulation. Comparison of V-beta expression in CD28+ or CD28- cell populations among individuals from different groups also showed several interesting differences. Functionally, cardiac CP displayed a higher frequency of IFN-gamma, TNF-alpha and IL-4 producing lymphocytes than indeterminate CP. Correlation analysis between the frequency of cytokine expressing cells, and the frequency of CD4+ T-cells with differential expression of CD28 demonstrated that CD4+CD28- T-cells were positively correlated with TNF-alpha in cardiac and with IL-10 in indeterminate CP, suggesting that these cells might have an important regulatory role in human Chagas' disease.     

Dual function of recombinant human CD58: inhibition of T cell adhesion and activation via the CD2 pathway.

To produce large quantities of recombinant CD58 (rCD58) glycoproteins for biochemical and functional studies, a cDNA clone containing the phosphatidylinositol-linked form of human CD58 was expressed in insect cells using the baculovirus system. Gel filtration showed rCD58 to form soluble oligomeric aggregates which were functionally, antigenically, and biochemically similar to their natural counterpart. Sequence analysis of the amino- and carboxy-terminal ends of released rCD58 protein revealed that the 28 amino acid signal peptide was accurately removed. In contrast, the hydrophobic C-terminal peptide was not removed. rCD58 binds to its natural ligand CD2 with a dissociation constant Kd = 5 x 10(-8) M, which is equivalent to the affinity of physiological T cell adhesion mediated by the membrane bound CD2-CD58 receptor-ligand pair. Rosette formation of human T lymphocytes with sheep and human erythrocytes was completely abrogated. In addition, the mixed lymphocyte reaction was significantly inhibited by rCD58. Moreover, cytotoxicity of human NK clones (CD2+CD3-) was inhibited by rCD58 similar to inhibition by CD58 mAbs. In contrast, rCD58 synergized with mitogenic CD2R mAbs in T cell triggering. These data demonstrate that rCD58 might serve as a biological immunomodulator which influences T cell adhesion and activation.     

CD28 signals the differential control of regulatory T cells and effector T cells

One possible means of driving antigen‐specific immune suppression is to expand or induce antigen‐specific FoxP3‐expressing Treg cells. One way of activating and expanding these specialized cells, both in vitro and in vivo, is by strong costimulation via CD28 with an agonistic anti‐CD28 monoclonal antibody, called anti‐CD28 superagonist (CD28SA). However, CD28SA also strongly activates conventional T (Tconv) cells to secrete proinflammatory cytokines and, under certain conditions, causes serious cytokine release syndrome. In this issue of European Journal of Immunology, Tabares et al. [Eur. J. Immunol. 2014. 44: 1225–1236] address how CD28SA can be used for the differential control of human Treg and Tconv cells to suppress immune responses without serious adverse effects. They show that, depending on the dose of the antibody or by comedication of cortico‐steroid, the selective expansion of Treg cells can be achieved without significantly activating Tconv cells to produce inflammatory cytokines. This difference in CD28 signal sensitivity between the two populations can be exploited for better control of immune responses.     

凋亡细胞体外抑制T细胞活化

目的：研究凋亡细胞对Ｔ细胞活化的影响。方法：以ＣｏｎＡ刺激小鼠脾细胞增殖体系为研究对象,观察凋亡细胞预处理后对ＣｏｎＡ刺激的脾细胞ＣＤ６９表达的影响。结果：凋亡细胞可以抑制Ｔ细胞活化,而活细胞或坏死细胞却不能。同时抑制效果与凋亡种类诱导方式无关,而与凋亡细胞数量密切相关。结论：研究证明了凋亡细胞可以主动抑制Ｔ细胞活化,这一发现拓宽了目前对凋亡的认识,表明凋亡细胞主动调节免疫反应在一些生命的基本现象     

T lymphocyte co-signaling pathways of the B7-CD28 family

The past years have witnessed significant advance in our understanding of critical roles of T cell co-signals inB7-CD28 family molecules in regulating T cell activation and tolerance.New co-signaling molecules have beenidentified and their functions have been delineated.These co-signaling pathways play overlapping and distinctregulatory roles at various stages of a T cell response.By expressing in appropriate time and location,thesepathways have different regulatory functions through independent receptors or on different subsets oflymphocytes.Precise understanding of these pathways will allow the development of novel approaches totreatment of human diseases such as cancer,viral infection,autoimmune diseases and transplantation rejection.Cellular & Molecular Immunology.2004;1(1):37-42.     

CD28-mediated costimulation impacts on the differentiation of DC vaccination-induced T cell responses

Costimulatory signals such as the ones elicited by CD28/B7 receptor ligation are essential for efficient T cell activation but their role in anti-tumour immune responses remains controversial. In the present study we compared the efficacy of DC vaccination-induced melanoma specific T cell responses to control the development of subcutaneous tumours and pulmonary metastases in CD28-deficient mice. Lack of CD28-mediated costimulatory signals accelerated tumour development in both model systems and also the load of pulmonary metastases was strongly increased by the end of the observation period. To scrutinize whether lack of CD28 signalling influences priming, homing or effector function of Trp-2(180-188)/K(b)-reactive T cells we investigated the characteristics of circulating and tumour infiltrating T cells. No difference in the frequency of Trp-2(180-188)/K(b)-reactive CD8+ T cells could be demonstrated among the cellular infiltrate of subcutaneous tumours after DC vaccination between both genotypes. However, the number of IFN-gamma-producing Trp-2-reactive cells was substantially lower in CD28-deficient mice and also their cytotoxicity was reduced. This suggests that CD28-mediated costimulatory signals are essential for differentiation of functional tumour-specific CD8+ T-effector cells despite having no impact on the homing of primed CD8+ T cells.