IL-2 regulation of soluble IL-2 receptor levels following thermal injury.

In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2.     

Selective impairment of T lymphocyte activation through the T cell receptor/CD3 complex after cytomegalovirus infection.

Cytomegalovirus (CMV) infection is reported to cause transient immunosuppression in man. In this study we have analysed the effect of CMV on T lymphocyte function in 29 children diagnosed for acute CMV infection. Peripheral blood mononuclear cells (PBMC) obtained from the patients showed a significant specific impairment in their proliferative response to enterotoxins A and C1, to concanavalin A and to the anti-CD3 MoAb OKT3. The impaired responses were corrected with exogenous IL-2. In contrast, stimulation using phytohaemagglutinin, as well as activation signals delivered through the surface molecules CD26 or CD28, elicited normal proliferative responses in CMV PBMC. The results indicate that the T cell anergy associated with CMV infection is restricted to the T cell receptor/CD3 activation pathway.     

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P less than 0.05) and to an anti-CD3 MoAb (P less than 0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA was impaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 or ionophore. Lastly, PHA failed to induce IL-2 alpha receptor (IL-2R alpha) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor (TCR)-specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL-2 and IL-2R alpha) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.     

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.     

Serum markers of T cell activation in relapses of Wegener's granulomatosis.

Levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4) and CD8 (sCD8) were measured by sandwich ELISA as markers for T cell activation in serial serum samples from 16 patients showing 18 histologically proven relapses of Wegener's granulomatosis (WG). Levels of sIL-2R increased from 1065 U/ml (median, range 373-2345 U/ml) 6 months before the relapse to 1684 U/ml (median, range 486-3404 U/ml) at the moment of relapse for the whole group (P = 0.10). The eight major relapses showed a profound rise in sIL-2R levels, from 1008 U/ml (median, range 686-1553 U/ml) 6 months before the relapse, to 1994 U/ml (median, range 1469-3404 U/ml) at the moment of relapse (P < 0.01). The levels of sIL-2R at the moment of relapse were significantly higher at the eight major relapses than at the time of the 10 minor relapses (P < 0.05). Minor relapses were not accompanied by a significant rise in sIL-2R levels. Titres of antineutrophil cytoplasmic antibodies (ANCA) rose by two or more titresteps or from negative to positive in 15/18 patients during the 6 months period before the relapse. In all seven cases with both a rise of the ANCA titre and an at least 25% increase in sIL-2R levels, the rise in ANCA preceded the rise in sIL-2R by at least 1 month. The level of sIL-2R at the moment of relapse correlated with the level of C-reactive protein (r = 0.488, P < 0.05) and with the disease activity score (r = 0.824, P < 0.002). There were no significant changes in levels of sCD4 or sCD8, although the levels of sCD4 tended to be higher at the time of major relapses. We conclude that major relapses of Wegener's granulomatosis are accompanied by systemic T cell activation. T cell activation, however, does not appear to precede the rise in ANCA titre.     

T cell deletion follows chronic antigen specific T cell activation in vivo

Exposure of mice transgenic for a TCR (F5) to cognate peptideantigen results in thymic depletion of CD4⁺ CD8⁺ cells and expansionand activation of peripheral CD8⁺ TCR(tg)⁺ T cells.in the thymusapoptotic DNA ladder is evident as early as 3 h after peptideinjection. Long exposure of intact or thymectomized F5 TCR transgenicmice to peptide antigen leads to depletion of most of the peripheralCD8⁺ T cells bearing the F5 receptor, with the remaining cellshaving lower levels of transgenic TCR compared with non-treatedanimals. In the thymus of intact F5 TCR transgenic mice suchcontinuous exposure to antigen results in the reappearance ofCD4⁺CD8⁺ with lower levels of the transgenic receptor.     

The influence of tetracyclines on T cell activation.

Minocycline has been shown to have an anti-inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell-mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose-dependent inhibition of T cell proliferation and reduction in production of IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). Besides an inhibition of IL-2 production, minocycline exerted its effect on T cell proliferation by induction of a decreased IL-2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3-induced intracellular Ca2+ mobilization. Taken together, the results clearly indicate that minocycline has immunomodulating effects on human T cells.     

Local and systemic effects of co‐stimulatory blockade using cytotoxic T lymphocyte antigen‐4‐immunoglobulin in dinitrofluorobenzene‐ and oxazolone‐induced contact hypersensitivity in mice

Cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4)‐immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA‐4‐Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA‐4‐Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA‐4‐Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose‐dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)‐ and oxazolone‐induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA‐4‐Ig. It was further shown that CTLA‐4‐Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA‐4‐Ig reduces infiltration of activated CD8⁺ T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute‐phase proteins in circulation. Finally, our results suggest that CTLA‐4‐Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA‐4‐Ig induces long‐term immunosuppression of both DNFB‐ and oxazolone‐induced inflammation and our data are the first to compare the effect of this compound in both DNFB‐ and oxazolone‐induced CHS and to show that CTLA‐4‐Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.     

Elevated serum decoy receptor 3 with enhanced T cell activation in systemic lupus erythematosus

Decoy receptor 3 (DcR3/TR6) is a decoy receptor for the Fas ligand (FasL) and can inhibit FasL-induced apoptosis. It has been reported recently that DcR3 can induce T cell activation via co-stimulation of T cells, suggesting that DcR3 may be involved in the pathophysiology of autoimmune diseases. This study aims to analyse the serum DcR3 in patients with systemic lupus erythematosus (SLE) and to investigate the role of DcR3 in the pathogenesis of SLE. Significantly elevated serum DcR3 was observed in SLE patients, and the mean serum DcR3 level was significantly higher for those with active disease [SLE disease activity index (SLEDAI) >/= 10] compared with that in patients with inactive disease (SLEDAI < 10). In addition to reducing activation-induced cell death in activated T cells via neutralization of the FasL, soluble DcR3-Fc enhanced T cell proliferation and increased interleukin-2 and interferon-gamma production via co-stimulation of T cells. Moreover, enhanced T cell reactivity to DcR3-induced co-stimulation was demonstrated in lymphocytes from patients with SLE, suggesting the elevated serum DcR3 may associate with enhanced T cell activation in vivo. These findings are the first to demonstrate that serum DcR3 concentrations are increased in SLE patients, and this may imply a possible role of DcR3 in the pathogenesis of SLE via enhanced T cell hyperreactivity and reduced apoptosis in activated T cells.     

CpG-ODN在T细胞活化和抑制中的作用

CpG-ODN是人工合成的含有CpG基序的ODNs,能够模拟细菌DNA的免疫刺激活性,通过细胞内定位的Toll样受体9活化抗原递呈细胞（APC）,如树突状细胞（DC）和B细胞,分泌大量Th1型细胞因子诱导产生Th1型免疫应答。CpG—ODN不仅增强T淋巴细胞的活化,在特定条件下还可以抑制T淋巴细胞功能。在抗感染、抗肿瘤疾病中,CpG—ODN作为新型的免疫佐剂得到了广泛的应用,但其安全性问题应引起足够的重视。     

Cytolytic effector function is present in resting peripheral T lymphocytes.

Antigen-specific cytotoxic killer lymphocytes (CTLs) represent one of the major effector functions of the immune system. It is well established that, as a consequence of TCR recognition of the antigen-bearing target cell, resting T lymphocytes develop into fully active antigen-specific CTLs. In contrast, natural killer (NK) cells are immediately lytic upon contact with an appropriate target cell. The lytic machinery of CTLs and NK cells is thought to include the contents of their cytoplasmic granules, in particular the pore-forming protein perforin. Here we report direct cytolytic activity by resting peripheral CD3+CD8+ T cells as a result of TCR-CD3 binding to the target cell; the murine OKT3 hybridoma (anti-human CD3) was used as a target. The cytotoxicity was more pronounced in the CD8+CD45RO+ population, which contains 'memory' T cells, than in the reciprocal CD8+CD45RA+ subset; CD8+CD4- mature thymocytes were non-cytotoxic. The cytolytic potential of these populations correlated with the presence or absence of perforin. The results demonstrate that the cytolytic machinery of T cells develops post-thymically and can be immediately triggered by TCR-CD3 stimulation.     

Characterization of H4: a mouse T lymphocyte activation molecule functionally associated with the CD3/T cell receptor

The monoclonal antibody C398.4A was produced by immunizing Armenian hamsters with the mouse T cell clone D10.G4.1. It recognizes a molecule selectively expressed by activated mouse T cells and was named H4. H4 is expressed on the T cell surface about 24 h after activation and peaks at day 7. By contrast, it is not expressed by resting or activated B cells, macrophages, or fibroblasts. It is also expressed by CD4 or CD8 single-positive mature thymocytes. Immunoprecipitation showed that H4 is a disulfide-linked dimer, precipitating as a broad band at about 50–65 kDa under nonreducing conditions and at 25 and 29 kDa under reducing conditions. Deglycosylation of the reduced H4 by N-glycanase gave rise to a single band of about 21 kDa, suggesting that the two chains may be differentially glycosylated forms of the same protein. The H4 expression pattern and biochemical features, together with cross-blocking, co-capping, co-modulation, and immunoprecipitation preclearing experiments showed that H4 is different from other known co-stimulatory molecules such as CD69, CD2, Ly-6, CD25, OX-40, Mac-1 and LFA-1. By in vitro kinase assay, H4 was found to co-precipitate a tyrosine kinase activity that phosphorylated substrates of about 29 and 25 kDa. Co-modulation and co-capping experiments showed that H4 is physically associated with the CD3/T cell receptor. These data suggest that H4 may function as a T cell-specific co-stimulatory molecule and play a role in the T cell response when the activation stimulus is limited either because the antigen is only available in low concentration or has a low agonistic activity.     

GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations

GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2Ralpha) and production of IL-2 and IFN-gamma were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR(-/-) CD4+CD25+ and GITR(+/+) CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25- responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.     

The role of CD2/LFA-3 interaction in antigen- and mitogen-induced activation of human T cells

Binding of LFA-3 to the T cell surface receptor CD2 promotes intercellular adhesion and is costimulatory with anti-CD2 mAbs in 'alternative pathway' activation of T cells. Since all AC-dependent systems of T cell activation are inhibited by anti-LFA-3 mAb, it was asked whether in mitogen- and antigen-induced activation of human T cells, the function of CD2/LFA-3 interaction involves signalling beyond its function in promoting intercellular adhesion. In order to selectively block and reconstitute CD2/LFA-3 interaction while leaving other AC functions available, the response of unseparated PBMC to various T cell mitogens and to allogeneic cells was blocked by a newly developed mAb (G26) to human LFA-3. Addition of purified T11TS, the sheep form of LFA-3 that binds to human CD2 but is not recognized by mAb G26, restored the T cell response to PHA-P but not to ConA, surface aldehydes, anti-CD3 mAb, or allogeneic cells. In addition, purified resting human T cells which were unresponsive to stimulation by lectins or anti-CD3 mAbs were activated by PHA-P in the presence of purified T11TS, demonstrating that provision of LFA-3 is a sufficient accessory cell function in the activation of human T cells by this mitogen. Again, the responses to ConA, cell surface aldehydes, or soluble anti-CD3 mAb were not restored by T11TS. T cell activation by PHA-P, but not by the other polyclonal T-cell activators studied thus seems to be mechanistically similar to 'alternative pathway' activation induced by anti-CD2 mAb in that the costimulatory effect of LFA-3 is independent of its prescence on an accessory cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peripheral blood T lymphocytes in systemic vasculitis: increased T cell receptor Vβ2 gene usage in microscopic polyarteritis

Antigen recognition by T lymphocytes is mediated by cell surface receptors. T cell specificity depends on the variable, diversity and junctional (VDJ) regions of the α and β polypeptide chains of the T cell receptor (TCR). The expression of the variable region genes of the β chain (Vβ) has been analysed to study the involvement of peripheral blood T cells in systemic vasculitis. RNA was extracted from peripheral blood lymphocytes of 12 patients with microscopic polyarteritis, 10 with Wegener's granulomatosis, six with unclassified vasculitis, and 28 healthy age- and sex-matched individuals. Complementary DNA was made from RNA and amplified by the anchored polymerase chain reaction (PCR) using redundant oligonucleotide primers for the TCR Vβ genes. To determine if the dominant usage of a Vβ gene family reflected the presence of particular T cell clones, cDNA was amplified with primers for the specific Vβ gene family. The product was screened for sequence homogeneity by single-stranded conformational polymorphism (SSCP) and cloned to sequence the adjoining TCR (Dβ)Jβ region. A significant increase in the mean percentage expression of the Vβ 2.1 gene was seen in vasculitis patients (11·4+1·0% (mean + s.e.m.)) compared with controls (6·6 + 0·6%; P < 0·003). The most marked increase was seen in microscopic polyarteritis (13·9 + 1·7%; P < 0·0001). There were also increases in the expression of Vβ3, 13 and 14 in peripheral blood of vasculitis patients compared with controls. SSCP analysis of Vβ 2.1 amplified products indicated the presence of oligoclonal bands in a smaller proportion of patients (8/27) than controls (12/28). There was no strong evidence for the conservation of the TCR Vβ 2.1 junctional region sequence data from a sample group of three patients with oligoclonal bands. Thus, a subset of patients with systemic vasculitis, particularly those with microscopic polyarteritis, have increased TCR Vβ 2.1 gene expression in their peripheral blood T cell repertoire. As superantigens binding Vβ 2.1 are postulated to activate T cells with diverse CDR3 sequences, it is proposed that a superantigen is involved in the immunopathogenesis of vasculitis.