Cloning and expression of a gamma-interferon-inducible gene in monocytes: a new member of a cytokine gene family

To define activation-specific sequences in human peripheral blood lymphocytes (PBL), a cDNA library was constructed by subtractive hybridization using resting and stimulated PBL pairs. Stimulation of PBL was achieved by triggering with mitogenic anti-CD2 (T11) monoclonal antibodies. Differential library screening with cDNA probes derived from stimulated versus resting PBL led to identification of two novel sequences, termed HC11 and HC14. The predicted primary and secondary structure of HC11 deduced from the translated nucleotide sequence suggests that the gene encodes a secreted protein of 99 amino acids (aa), including a 23 aa residue leader sequence. Surprisingly, Northern blot analysis demonstrated that HC11 mRNA is induced predominantly in peripheral blood non-T cells. Subsequently, we observed that the HC11 mRNA is induced in macrophages and the monocytic line U-937 by gamma-IFN, raising the possibility that T cell-derived gamma-IFN induced upon anti-CD2 stimulation activated monocytes to express HC11 RNA. In support of this notion, neutralizing anti-gamma-IFN monoclonal antibody inhibits the induction of HC11 mRNA in PBL activated through anti-CD2 antibodies. These findings suggest that there is a molecular cascade involving T cell-produced lymphokines and monokines which serve as a means for intercellular communication. Transient expression of HC11 cDNA results in a readily detectable specific set of protein bands in SDS-PAGE analysis of supernatants from radio-labeled COS cells, consistent with HC11 encoding a secreted product(s). Protein sequence comparison reveals homology with other members of a recently described inducible cytokine family whose functions are yet to be defined.     

Differential responsiveness to CD3-Ti vs. CD2-dependent activation of human intestinal T lymphocytes

Human lamina propria lymphocytes were recovered from surgical specimens of unaffected large bowel and compared with peripheral blood lymphocytes (PBL) from the same individual. We investigated their capacities to respond to triggering stimuli delivered by mitogenic monoclonal antibodies directed at the T cell receptor/CD3 complex or, alternatively, the CD2 glycoprotein. In marked contrast to PBL, expression of the interleukin 2/interleukin 2 receptor system was markedly reduced in LPL following activation through the CD3/T cell receptor. Perhaps, more important, responses to CD2 triggering were similar or even stronger in LPL as opposed to PBL. This indicates that the CD2-dependent alternative pathway of T cell activation might serve a functional role in local immunity in the gut.     

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.     

Expression of the costimulator molecules, CD80, CD86, CD28, and CD152 on lymphocytes from neonates and young children

The expression of CD80, CD86, CD28, and CD152 were examined on peripheral blood lymphocytes from adults, neonates (cord blood lymphocytes) and young children (2-20 months of age). There was no difference in the expression of CD80 or CD86 between adult and neonatal B cells, either resting or activated. A higher percentage of resting T cells expressed CD28 in neonates and young children compared to adults. CD28 expression was similar on adult and neonatal T cells activated with PMA and ionomycin. However, CD28 was expressed at greater intensity on a higher percentage of neonatal T cells than adult T cells stimulated with CD3. CD152 expression was lower on neonatal T cells than adult T cells stimulated with PMA and ionomycin and undetectable on neonatal T cells stimulated with CD3. In contrast, intracellular CD152 was equivalent in adult and neonatal T cells stimulated with PMA and ionomycin, suggesting trafficking of CD152 to the cell surface may be differentially regulated in neonatal T cells. Since the T cell response is determined by the balance of signals received from CD28 and CD152, high levels of CD28 expression and lower surface expression of CD152 on neonatal T cells may represent specialisation to promote activation of neonatal T cells.     

生长激素分泌过多患者外周血淋巴细胞CD2mRNA的表达

应用ＲＮＡ斑点杂交法，测定了１２例生长激素（ＧＨ）分泌过多患者及１０例正常人外周血淋巴细胞ＣＤ２ｍＲＮＡ的表达，发现患者ＣＤ２ｍＲＮＡ的表达较之正常人有显著下降，提示ＧＨ分泌过多对Ｔ淋巴细胞ＣＤ２ｍＲＮＡ的表达有显著影响。此结果为研究内分泌系统产生的肽类激素对免疫系统的影响提供了新资料。     

T lymphocytes from the normal human peritoneum contain high frequencies of Th2-type CD8+ T cells

The majority of peritoneal T lymphocytes have been shown to be CD8⁺ and to co-express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)-4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8⁺ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)-γ and exhibited high alloantigen-specific cytolytic activity, some clones secreted IL-4 and IL-5 but no detectable IFN-γ. These Th2-type CD8⁺ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8⁺ T cell may occur in different immune compartments.     

Comparison of signals delivered through CD3 and CD2 for T-cell activation: the role of calcium influx and interleukin 1

In the absence of monocytes, resting T lymphocytes extensively purified from human peripheral blood failed to proliferate when stimulated with a mixture of calcium ionophore, which elevates intracellular calcium levels, and TPA, which activated protein kinase C. A third signal, i.e., the triggering via CD3 or CD2 molecules, was necessary in order to observe proliferation. These highly purified T cells required the presence of monocytes in both CD3 and CD2 systems for their proliferation. Exogenous interleukin 1 clearly substituted for monocytes in CD2- but not in CD3- triggered T-cell proliferation. In contrast, the effect of CD2 and CD3 antibodies on Ca++ influx was apparently not dependent on the presence of monocytes. In the presence or absence of the monocytes, CD3, as well as certain combinations of CD2 monoclonal antibodies including the D66 monoclonal antibody, were able to increase the intracellular calcium concentration as measured by Quin 2 fluorescence. EGTA, a Ca++ chelator, completely inhibited CD2- and CD3- mediated T-cell proliferation, indicating that calcium uptake is necessary during the T-cell proliferation. The addition of TPA abrogated the inhibitory effect of EGTA and completely restored the response of the T cells stimulated by CD3, but not by CD2, monoclonal antibodies. In the CD2 pathway, EGTA-inhibited proliferation of T cells could be completely restored by addition of exogenous interleukin 2 as well as exogenous recombinant interleukin 1. Our results indicate that EGTA inhibits the production of interleukin 1 but has no direct effect on either interleukin 2 production or on Tac antigen expression. In this system, recombinant interleukin 1 alpha demonstrated a more potent ability for restoring the T-cell response than did recombinant interleukin 1 beta. These results suggest that interleukin 1 could act as a potent costimulatory factor in the non-antigen-specific T-cell activation.     

Activation of resting T lymphocytes by cross-linked anti-CD3 (T3)

In this study we have examined the role of small numbers of adherent cells in the stimulation of highly purified resting T lymphocytes by cross-linked monoclonal anti-CD3 antibodies (T3). T cells were obtained by 3 cycles of purification, using adherence to plastic surface, nylon wool column separation and rosetting with 2-aminoethyl isothiouronium bromide-treated sheep red blood cells. They were stimulated either with T3 antibodies or with solid-phase rabbit anti-mouse IgG-T3 complexes. In standard high density cultures (1 X 10(5)-2 X 10(5) cells), soluble T3, interleukin (IL) 1 and IL 2, used separately, did not induce proliferation of T cells. Soluble T3 together with IL 2 slightly activated T cells to proliferate. Rabbit anti-mouse IgG-T3 complexes attached to the plastic wells induced stimulation in some cultures. The response was markedly increased after addition of IL 2, but not IL 1. The irregular response of these cultures suggested the presence of another variable signal. When cell numbers were reduced (12 X 10(3) cells), neither cross-linking of the CD3-Ti complex nor addition of exogenous IL 1 or IL 2, alone or in combination, stimulated the T cells to increase DNA synthesis. A positive response, comparable to complete peripheral blood mononuclear cells, was restored with 0.5% supplemented adherent cells, provided that IL 2 was present.     

Relative increase of inflammatory CD4+ T cells in the cerebrospinal fluid of multiple sclerosis patients and control individuals.

Of three patients with multiple sclerosis (MS) and two non-MS individuals a large number of CD4+ T cell clones was obtained from the cerebrospinal fluid and peripheral blood by direct limiting dilution. The CD4+ T cell clones from cerebrospinal fluid and peripheral blood lymphocytes were compared for their cytotoxic activity and lymphokine production. Cytotoxic capacity of cloned T cells was analysed with the use of anti-CD3 antibodies and target cells bearing Fc receptors for murine IgG. Recently, we demonstrated the existence of two different subsets of human CD4+ T cell clones by phenotypic and functional criteria. One type of CD4+ T cell with anti-CD3 mediated cytotoxic activity, in analogy with murine studies, is the inflammatory or TH1 subtype, the main producer of interleukin (IL-2), interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF)-alpha, -beta, whereas the other type of CD4+ T cell clone lacked anti-CD3 mediated cytotoxicity and produced minimal amounts of IL-2 concomitant with reduced levels of IFN-gamma and TNF-alpha, -beta. The present study demonstrates that among three MS patients, relatively more inflammatory CD4+ T cell clones with cytotoxic activity and IFN-gamma and TNF-alpha, -beta production were derived from the cerebrospinal fluid as compared with peripheral blood lymphocytes. Also among control individuals more inflammatory CD4+ T cell clones could be obtained from the cerebrospinal fluid as from the peripheral blood. The enrichment of inflammatory CD4+ T cells, therefore, appears to be physiological rather than associated with the disease.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.     

Identification of a bovine surface antigen uniquely expressed on CD4-CD8- T cell receptor gamma/delta+ T lymphocytes

In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.     

CD3+ WT31- peripheral T lymphocytes lack T44 (CD28), a surface molecule involved in activation of T cells bearing the alpha/beta heterodimer

We have applied two-color fluorescence cytofluorometric techniques to the analysis of the distribution of T44 and CD3 antigens in peripheral blood human lymphocytes. While most CD3+ cells co-expressed T44 antigen, a small distinct subset was CD3+ T44- (2-10% of CD3+ cells). This cell subset also did not react with the WT31 monoclonal antibody (mAb), specific for an alpha/beta framework determinant of the T cell receptor (TCR). Lack of T44 antigen expression was also observed in purified CD3+ WT31- polyclonal populations that had been cultured in medium containing interleukin 2 (IL2) and as well as greater than 30 clones expressing the CD3+4-8-WT31- surface phenotype. Immunoprecipitation experiments confirmed that expression of T44 molecules was confined to CD3+ WT31+ peripheral blood T cells. While conventional CD3+ WT31+ cells produced IL2 in response to mAb directed to CD2, CD3 or T44 surface molecules, CD3+ WT31- cells did not respond to anti-T44 mAb but released IL2 following stimulation with anti-CD2 or anti-CD3 mAb. Therefore, assuming that anti-T44 mimicks the effect of a still undefined natural ligand our data suggest that T cells expressing the gamma-gene surface product may be signalled by stimuli which differ, at least in part, from those acting on CD3+ WT31+ T lymphocytes.     

CK226: a novel surface molecule involved in human T cell activation

In this report we describe a novel surface molecule, termed CK226, which mediates activation of human T lymphocytes. This molecule was identified by the monoclonal antibody (mAb) CK226 that had been raised against the human leukemic T cell line CEM/K. The CK226 mAb reacted with 10%-35% of human peripheral blood lymphocytes, virtually all monocytes, polymorphonuclear leukocytes and large granular lymphocytes. As demonstrated by two-color cytofluorimetric analysis, the majority of CK226+ lymphocytes were CD3+, whereas a minority coexpressed surface immunoglobulins. Biochemical analysis showed that the surface molecules immunoprecipitated by CK226 mAb from lysates of 125I surface-labeled cloned T lymphocytes were represented by two distinct polypeptides. Their apparent molecular mass was 160 kDa and 80 kDa under nonreducing conditions and 75 kDa and 80 kDa under reducing conditions. CK226 mAb induced proliferation of peripheral blood lymphocytes and interleukin 2 release. Cell proliferation was optimal in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Finally all T cell clones analyzed for their responsiveness to CK226 mAb released interleukin 2 in the presence of the antibody, regardless of their CD4+ or CD8+ phenotype. Antibody-induced modulation of CD3/T cell receptor molecules inhibited further responses of cloned cells to CK226 mAb. Thus, we conclude that CK226 mAb defines a novel surface molecule which initiates an alternative pathway of T cell activation in humans.     

Profiles of lymphokine activities and helper function for IgE in human T cell clones

A large panel of phytohemagglutinin (PHA)-induced T cell clones (690 in total), established from four different human lymphoid tissues (peripheral blood, tonsils, lymph nodes and spleens) by a high-efficiency cloning technique, was characterized according to their pattern of lymphokine production. The majority of both CD4+ and CD8+ clones from all lymphoid tissues produced interleukin (IL) 2 and/or interferon (IFN)-gamma in response to 24-h stimulation with PHA. In contrast, higher proportions of IL 4-producing clones were found among CD4+ clones from tonsils and spleens than from peripheral blood and lymph nodes, whereas only a minority of CD8+ clones from all lymphoid tissues were found to produce IL 4. It was not possible to divide the CD4+ (helper/inducer) clones on the basis of their pattern of lymphokine activity into two clear-cut groups analogous to Th1 and Th2 helper clones described in mice. Although 21 out of 503 (4%) CD4+ T cell clones produced IL 4, but not IFN-gamma or IL 2, and 208 (41%) produced IL 2 and/or IFN-gamma, but not IL 4, a total number of 185 (37%) CD4+ clones showed the ability to produce IL 4 plus IL 2 and/or IFN-gamma. All types of CD4+ T cells (as classified according to their pattern of lymphokine activity) provided help for IgG production in allogeneic B cells. In contrast, helper function for IgE was detectable only among the IL 4-producing clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Human CD4+ T-Cell Clones that Secrete Helper Factor(s) for B-Cell Proliferation and Maturation

Human peripheral blood lymphocytes (PBL) were activated with K46M, a m mitogenic monoclonal antibody against La-reactive T lymphocyte surface structures. The cultures were expanded in the presence of interleukin 2 (IL-2). After 1 month of culture, the activated T cells were cloned by limiting dilution at 0.5 cells/well. Five clones with the CD3+CD4+ phenotype and one clone with the CD3+CD8+ phenotype were obtained. The CD3+CD8+ clone (K99) displayed a strong major histocompatibility complex (MHC)-unrestricted cytolytic activity against MOLT-4 and a weaker reactivity against the bladder tumour cell lines T24 and RT4. The natural killer (NK)-susceptible K562 cells were not lysed. Two of the CD3+CD4+ clones (K91 and K914) showed a helper activity in pokeweed mitogen (PWM)-induced IgG production by B cells. These cells differed in the expression of CD45R and CDw29 antigens, as defined by the monoclonal antibodies 2H4 or D10D11 and 4B4. When stimulated with PWM for 48 or 72 h, clone K91 and an additional CD4-positive clone (K913) secreted a factor into the supernatants which helped B cells to produce IgG. The K913 supernatant also induced some IgM production. The supernatant obtained after similar stimulation of K914 cells was inactive. None of these supernatants induced B cells to proliferate when tested together with phorbol myristate acetate (PMA). However, when K91 and K914 cells were activated with phytohaemagglutinin (PHA) for 48 or 72 h, the supernatant from K91 was strongly helpful in B-cell proliferation, whereas the supernatant from K914 cultures was only moderately active. In conclusion, we have established human T helper clones that release different factors supporting either B-cell proliferation or maturation when stimulated with PWM or PHA.     

Immunodeficiency with low expression of the T cell receptor/CD3 complex. Effect on T lymphocyte activation.

We report the consequences of low expression of the T cell receptor (TcR)/CD3 complex by T lymphocytes from a 4-year-old boy with a mild immunodeficiency. TcR/CD3 expression was found to be deficient on both resting and activated T cells, using both anti-CD3 and anti-TcR alpha/beta monoclonal antibodies. As shown by immunofluorescence and immunoprecipitation studies, residual expression (corresponding to about 10% of normal) was detectable on resting and activated TcR alpha/beta+ T cells. Other T cell membrane receptors were normally expressed. The functional consequences of this TcR/CD3 expression deficiency included an absence of T cell proliferation, interleukin 2 receptor expression and calcium flux following anti-CD3 and anti-CD2 antibody-triggered T cell activation. Antigen (tetanus toxoid, Candida and allogeneic cell)-induced proliferation was detectable. In contrast, cytotoxic T cell activity towards allogeneic cells was deficient. These findings shed light on the function of the TcR/CD3 complex and indicate that the expression of a limited number of TcR/CD3 receptors may be sufficient to trigger antigen-specific T cell activation (and, possibly, differentiation) and that anti-CD3 antibody-induced T cell activation differs somewhat from antigen/major histocompatibility complex molecule-induced activation. These results also confirm that the CD2 pathway of T cell activation is CD3 dependent.