APO-1(CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines

Certain B and T cell lines respond to activation signals, e.g.through the antigen receptor, by undergoing apoptotlc cell death.In T cells it has been recently shown that TCR-mediated apoptosisinvolves APO-1/Fas (CD95) receptor-ligand interaction. To investigatewhether the TCR-CD3 complex can trigger alternative apoptosispathways we generated subclones of the T cell line Jurkat whichwere completely resistant towards APO-1-mediated apoptosis.These Jurkat^R cells differed phenotypically from sensitive parentalJurkat^S cells only by the lack of APO-1 protein expression.Although Jurkat^R cells responded normally to anti-CD3 stimulationby expression of APO-1 ligand they failed to undergo anti-CD3-inducedapoptosis. Thus, in Jurkat cells APO-1 -mediated apoptosis wasthe main, and might be the only, mechanism for anti-CD3-inducedcell death. However, BL-60 B cells, highly sensitive to anti-IgM-inducedapoptosis, did not use the APO-1 receptor-ligand system becausethey failed to express APO-1 ligand mRNA. Taken together, ourresults suggest that malignant T and B cell lines may use APO-1receptor-ligand-dependent and -independent antigen receptor-inducedapoptosis pathways respectively. Similarly, differential pathwaysmay be used by T and B cell subsets.     

Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD+ T lymphocytes

Gangliosides are glycosphingolipids found ubiquitously on thesurface of mammalian cells. They contain a ceramide tail thatis inserted into the membrane and exposed carbohydrate and sialicacid moleties. The non-toxic B subunit oligomer (EtxB) of Escherichiacoli heat-labile enterotoxin (Etx) is a potent immunogen invivo and has profound modulatory effects on EtxB-primed lymphocytesin vitro, properties which are dependent on its ability to bindto GM1 ganglioside receptors. Here, it is shown that cross-linkingGM1 by EtxB causes a differential effect on mature CD4⁺ andCD8⁺ T cells from lymph node cultures proliferating in responseto an unrelated antigen, ovalbumin. Addition of EtxB to suchcultures led to the complete depletion of CD8⁺ T cells comparedwith enhanced activation of CD4⁺ T cells [as measured by expressionof CD25 (IL-2R)]. By contrast, addition of a mutant EtxB, EtxB(G33D),which does not bind to GM1, failed to trigger CD8⁺ T cell depletion.When EtxB was added to isolated non-immune CD8⁺ lymphocytesrapid (12–18 h) alterations in nuclear morphology andthe appearance of sub-G₀/G₁ levels of DNA were induced; propertieswhich are characteristic of cells undergoing apoptosis. EtxB(G33D)failed to trigger apoptosis, indicating that the induction ofthe apoptotic signal was dependent on the binding of GM1. Thesefindings provide an insight into the potent immunogenicity andimmunomodulatory properties of E. coli enterotoxins as wellas heralding a novel method for the selective induction of apoptosisin mature CD8⁺ T lymphocytes.     

Human CD4/CD45RA+ and CD4/CD45RA T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates

T cell activation appears to be regulated by an interplay betweenprotein tyrosine kinases (PTKs) and protein tyrosine phosphatases(PTPases). p56^lck and p59^fyn have been found to associate withCD4 and TCR-CD3 respectively. The CD45 family of transmembranePTPases has been shown to be able to regulate the activitiesof these receptor-associated PTKs in vitro. In man, CD45 containsfive different isoforms whose distribution defines subsets ofT cells having distinct activation requirements and in vitrofunctions.Several groups have reported a physical interaction betweendistinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex.Given the potential regulatory interaction between CD45 andPTKs in CD4⁺ subsets expressing different CD45 isoforms, wehave examined CD4 associated and TCR-CD3^– associated PTKactivities, associated phosphatidyl inositoi (PI) kinases andsubstrates of tyrosine phosphoryiation in CD45RA⁺and CD45RA^–CD4⁺ T cell lines derived from peripheral blood. Both subsetsexpress CD4-assoclated p56^lck and TCR-CD3-associated p59^fynkinases which exhibit identical in vitro phosphoryiation atthe Y-394 and Y-420 autophosphorylation sites respectively.Further, both subsets exhibited PI kinases activity associatedwith CD4-p56^lck. Consistent with these observations, anti-CD3crosslinklng induced the phosphoryiation of a similar spectrumof intracellular substrates in these CD45RA⁺and CD45RA^–CD4⁺ T cell lines. These observations indicate that despitethe possible interaction between CD45 isoforms and CD4 or TCR-CD3,the mere expression of the CD45RA isoform does not in and ofitself alter the presence of receptorassociated kinases or theirintracellular targets.     

Induction of Fas-dependent apoptosis in synovial infiltrating cells in rheumatoid arthritis

Apoptosis is a feature of the synovium of rheumatoid arthritis(RA). We have recently shown that RA synoviocytes were susceptibleto anti-Fas mAb and undergo apoptosis in vitro. To investigatewhether infiltrating mononuclear cells also undergo Fas-dependentapoptosis, double-labeling techniques combined with immunohistochemicalexamination with anti-CD3 mAb and the TdT-mediated dUTP-biotinnick end labeling (TUNEL) method to detect apoptotic cells,or in situ RT assay to detect Fas mRNA, were performed usingfrozen tissue sections. We also examined the in vitro inductionof Fas-dependent apoptosis in freshly isolated synovium infiltratingmononuclear cells (SIM), synovial stromal cells (SSC) and peripheralblood lymphocytes (PBL) using tissues from nine patients withRA and three with osteoarthritis (OA). The results showed expressionof Fas antigen and apoptotic cells in a number of CD3-bearingcells in RA synovial tissues. In vitro treatment with anti-FasmAb produced a significant apoptosis of RA SIM and SSC, whilenone of PBL, and neither SIM nor SSC from OA exhibited apoptosis.Moreover, 50% of CD4⁺, CD3⁺ and CD45RO⁺ cells, and >90% ofFas-expressing cells of RA SIM underwent apoptosis in responseto anti-Fas mAb, as detected by flow cytometry. Our resultssuggest that RA synovial infiltrating lymphocytes acquire highsusceptibility to anti-Fas mAb and undergo apoptosis. Such aphenomenon of infiltrating T cells in RA synovium may play animportant pathophysiological role and suggest a possible therapeuticeffect for anti-Fas mAb in RA.     

Alternative complement pathway activation by CD4+ T cells of HIV infected individuals: a possible role in AIDS pathogenesis

The mechanisms by which CD4⁺ T cells are eliminated during HIVinfection are poorly understood. We have previously shown thatHIV infected cell lines activate and fix C3 via the alternativecomplement pathway (ACP). In the present study we examined theability of blood lymphocytes from 40 HIV⁺ individuals to fixC3. A large fraction of the CD4⁺ T cells reacted with anti-gp120antibodies. These cells also carried C3 fragments in vivo andcould further fix C3 if exposed to human serum In vitro. C3activation occurred via the ACP. In some cases exposure of thelymphocytes to human serum under conditions allowing ACP activationresulted in partial elimination of CD4⁺ T cells. The resultssuggest that complement activation and fixation by CD4⁺ T cellsopsonized with HIV particles or gp120 may contribute to theirselective destruction.     

Induction of unresponsiveness to the superantigen staphylococcal enterotoxin B: cyclosporin A resistant split unresponsiveness unfolds in vivo without preceding clonal expansion

The continuous presence of antigen and powerful immune responses(exhaustive cell proliferation) of llgand reactive T cells arecurrently thought to condition clonal deletion and/or inductionof unresponsiveness to endogenous or exogenous superantigens(SAg). Here we report that in vivo induction of unresponsivenessto the SAg staphylococcal enterotoxin B (SEB) can be an immediateprocess. Within hours a large portion of ligand reactive V_ß8⁺T cells becomes clonally deleted by apoptosis. In parallel,the remaining V_ß8⁺ T cells are unresponsive to SEB,yet at the same time express functional IL-2 receptors (IL-2R)and thus are highly responsive to the growth promoting effectsof IL-2. In a subsequent step refractory IL-2R⁺V_ß8⁺T cells undergo a wave of cell proliferation for 48 h, presumablydriven by IL-2. Thereafter a large proportion of V_ß8⁺T cells succumb to apoptosis, the remaining cells display thehallmarks of split unresponsiveness, i.e.they display a selectivefallure to produce IL-2 upon SEB stimulation in vitrocombinedwith a preserved capability to express functional IL-2R. Earlydeletion and Induction of unresponsiveness to SEB are cyclosporinA (CsA) resistant, while clonal expansion with subsequent celldeletion is blocked by CsA, yet the development of split unresponsivenessis not impaired by CsA. The results suggest that IL-2 drivengrowth of refractory T cells may mimic powerful immune responsesof ligand reactive V_ß8⁺ T cells. Since unresponsivenessto SEB precedes in vivo expansion, the results as such questionthe concept of ‘exhaustive cell proliferation’ asa prerequisite for induction of unresponsiveness. In additionthey suggest that unresponsiveness (tolerance) can be inducedin the presence of CsA.     

Fas (CD95)-independent regulation of immune responses by antigen-specific CD4 CD8+ T cells

Antigen-activated T cells of the CD4⁺CD8^– and the CD4^–CD8⁺phenotype are susceptible to antigen receptor-stimulated celldeath. This form of apoptotic cell death has been shown to bedependent on the expression of the Fas (CD95) antigen and canoccur via an autocrine mechanism involving the concomitant up-regulationof Fas and its ligand on activated T cells. Mutations in genesencoding Fas (lpr) and the Fas ligand (gld) contribute to thedevelopment of an autoimmune syndrome similar to systemic lupuserythematosus in mice. These observations led to the suggestionthat the Fas signaling pathway is an important regulator ofimmune responses in vivo. Here we evaluated the importance ofthe Fas pathway in regulating immune responses by male antigen-specificCD4^–CD8⁺ T cells. We found that the in vivo eliminationof male antigen-activated cells was independent of Fas expressionby these cells. However, the elimination of these activatedcells was inhibited by the transgenic expression of Bcl-2, aprotein that inhibits multiple forms of apoptotic cell death.The transgenic Bcl-2 protein also inhibited the death of maleantigen-activated cells following IL-2 deprivation. Cell deathresulting from IL-2 deprivation occurred efficiently in maleantigen-activated Fas^- cells. We propose that the rapid deletionof male antigen-activated Fas^– cells in vivo is due tolimiting amounts of IL-2 that are available in the microenvironmentof the activated cells at the peak of the response.     

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes

The CAMPATH-1 (CD52) antigen is a 21–28 kDa glycopeptidewhich is highly expressed on lymphocytes and macrophages andis coupled to the membrane by a glycosylphosphatidylinositol(GPI) anchoring structure. The function of this molecule isunknown. However, it is an extremely good target for complement-mediatedattack and antibody-mediated cellular cytotoxicity. The humanizedCAMPATH-1H antibody, which is directed against CD52, is veryefficient at mediating lymphocyte depletion in vivo, and iscurrently being used in clinical trials for lymphoid malignancyand rheumatoid arthritis. It is therefore important to examinethe functional effects of this antibody on different lymphocytesub-populations. Because several other GPI-linked moleculesexpressed on the surface of T lymphocytes are capable of signaltransductlon resulting in cell proliferation, we have investigatedwhether the CAMPATH-1 antigen can also mediate these effects.In the presence of phorbol esters and cross-linking anti-lgantibodies, mAbs specific for CD52 induced proliferation andlymphokine production in highly purified resting CD4⁺ and CD8⁺T lymphocytes. The ret lgG2c YTH 361.10 anti-CD52 antibody,however, was able to activate resting CD4⁺ and CD8⁺ T cellsdirectly without cross-linking or phorbol myristate acetatein the absence of Fc-bearlng cells. Anti-CD52 antibodies alsoaugmented the anti-CD3 mediated proliferatlve response of CD4⁺and CD8⁺ T cells when the two antibodies were co-immobilizedonto the same surface or cross-linked in solution by the samesecond antibody. Both CD4⁺CD45RA and CD4⁺CD45RO T cells werestimulated to proliferate by anti-CD52 antibodies in the presenceof appropriate co-stimulatory factors. Antl-CD52 mAbs did not,however, synerglze with anti-CD2 or CD28 mAb to induce CD4⁺T cell proliferation. The activation of CD4⁺ T cells by antl-CD52antibodies was inhibited by cyclosporin A, suggesting a rolefor the calcineurin-dependent signal transduction pathways.Although CD52 could transduce a signal In T cells, anti-CD52antibodies did not inhibit antigen-specific or polyclonal Tcell responses, suggesting this molecule does not play an essentialco-stimulatory role in normal T cell activation.     

In vitro differentiation and commitment of CD4+ thymocytes to the CD4+ lineage without TCR engagement

Thymocyte positive selection is based on protection of immatureCD4/CD8 double-positive (DP) thymocytes from apoptosis and theirdifferentiation into CD4 or CD8 single-positive (SP) cells.Intracellular signals essential for positive selection appearto be induced through the TCR and some of the accessory moleculesincluding LFA-1, CD4 and CD8 upon Interaction with thymic stromalcells. The signals, however, still remain to be identified.Since physiological levels of glucocorticoids potentially induceor enhance thymocyte apoptosis even in vivo, the signals arelikely to inhibit the apoptotic effect of glucocorticoids. Wehave previously shown that proper cross-linking of TCR-CD3 withLFA-1, CD4 or CD8 inhibited glucocortlcold-lnduced thymocyteapoptosis in vitro, and that a proper combination of the calciumionophore, ionomycin and the protein kinase C (PKC) activator,phorbol 12-myrlstate 13-acetate (PMA), mimicked the inhibitoryeffect. Here we determined whether this combination of ionomycinand PMA induces differentiation of isolated DP thymocytes fromnormal and TCR transgenic mice. We found that pretreatment ofDP thymocytes with ionomycin and PMA followed by 1 day cultureof the cells without the reagents resulted in the differentiationof the cells into CD4 SP and CD4⁺ CD8^lo T cells that have mostlycommitted to the CD4 lineage. The changes in expression of otherdifferentiation markers were also in good accordance with thoseassociated with positive selection, except the final maturation.The results indicate that moderate and transient increases inintracellular Ca²⁺ level and PKC activity induce differentiationand commitment of DP thymocytes to the CD4 lineage, and suggestedthat the biochemical pathway leading to positive selection isbased on a similar mechanism.     

CD45 up-regulation during lymphocyte maturation

CD4⁺CD8⁺ double-positive thymocytes differentiate into CD4⁺and CD8⁺ single-positive T cells during thymic positive selection.This process requires the interaction between the TCR and selfMHC molecules. In this context we have anaiyzed the expressionof CD45, an abundant transmembrane protein tyrosine phosphatase,and describe here its differential surface expression duringT cell maturation. Using four-color FACS analysis of thymocytesfrom normal as well as TCR-transgenic mice we demonstrate thatCD45 is up-regulated only during positive selection concomitantlywith the TCR-CD3 complex and the transient early activationmarker CD69, but that this up-regulation precedes heat stableantigen down-regulation. The tight linkage of the upregulationof the TCR-CD3 complex and CD45 may be required because theCD45 tyrosine phosphatase plays a role in modulating signaltransduction by the TCR-CD3 complex during positive selection.In addition, our findings argue for a regulation mechanism thatadapts the CD45 levels to increasing antigen receptor levelson mature T cells and B cells.     

Inhibition of thymidine synthesis by folate analogues induces a Fas-Fas ligand-independent deletion of superantigen-reactive peripheral T cells

Methotrexate (MTX), a folate antagonist with multiple enzymatictargets, is used in the treatment of malignancies as well asin autoimmune and chronic inflammatory diseases, and ZD1694(tomudex), a water-soluble quinazoline specific inhibitor ofthymidylate synthase (TS), is used in the treatment of adenocarcinomas.In this study, we investigated the effects of these folate analogueson superantigen (SAg)-reactive peripheral T cells in vivo. InBALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokinesecretion, IL-2R (CD25) expression and early deletion of a fractionof SEB-reactive V_ß8⁺ T cells were not impaired by eitherMTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTXand tomudex prevented V_ß8-selective T cell expansion andaccelerated their peripheral elimination. Administration ofthymidine (500 mg/kg/12 h) completely abrogated this effect,indicating that inhibition of TS but not that of other folate-dependentenzymes was the main mechanism involved. Furthermore, a markedincrease of apoptotic cells restricted to the V_ß8⁺ T cellsubset indicated that proliferation inhibition was associatedwith apoptosis. In contrast with peripheral V_ß8⁺ T celldeletion, MTX and tomudex did not prevent the increase of V_ß8⁺thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lprmice further demonstrated that deletion of V_ß8⁺ T cellsinduced by folate analogues was independent of Fas–Fasligand interaction. Our results provide evidence that folateanalogues may selectively delete dividing peripheral T cellsthrough TS inhibition, but do not interfere with other eventstriggered by SAg.     

Cross-linking of the TCR CD3 complex with CD4, CD8 or LFA-1 induces an anti-apoptotic signal in thymocytes: the signal is canceled by FK506

The immunosuppressant FK506 did not block differentiation ofdouble-negative thymocytes into double-positive (DP) cells,but interfered with differentiation of DP cells into maturesingle-positive cells In a fetal thymus organ culture system,suggesting that FK506 inhibits positive selection. The drugalso reduced the number of DP cells recovered after the culture.As positive selection depends on the inhibition of thymocyteapoptosis at Its DP stage by signaling through the TCR-CD3 complexand some of the accessory molecules, including CD4, CDS andLFA-1, we studied the possibility that FK506 enhanced apoptosisby itself or canceled the inhibition of apoptosis. The resultsIndicated that FK506 was hardly toxic or hardly affected antl-CD3-inducedDNA fragmentation in isolated thymocytes In vitro. On the otherhand, upon cross-linking TCR-CD3 together with CD4, CD8 or LFA-1,FK506 markedly enhanced DNA fragmentation and cytolysis. Thedrug, however, hardly or only slightly enhanced these responsesupon cross-linking TCR-CD3 together with CD2, CD28, Thy-1 orH-2K^d. Cross-linking of TCR-CD3 together with CD4, CD8 or LFA-1markedly Inhibited glucocorticoid-induced death and the inhibitionwas canceled by FK506. Furthermore, cross-linking of TCR-CD3together with LFA-1 enhanced Bcl-2 expression In DP cells. Theseresults suggest that cross-linking of TCR-CD3 with CD4, CD8or LFA-1 potentially induces both an apoptosis-lnducing signaland an FK506-sensttive anti-apoptotic signal, and that the lattersignal may be related to an essential signal for positive selection.     

Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes

A mAb J43 has been produced against the product of the mousePD-1 gene, a member of the Ig gene superfamily, which was previouslyisolated from an apoptosis-induced T cell hybridoma (2B4.11)by using subtractive hybridization. Analyses by flow cytometryand immunoprecipitation using the J43 mAb revealed that thePD-1 gene product is a 50–55 kDa membrane protein expressedon the cell surface of several PD-1 cDNA transfectants and 2B4.11cells. Since the molecular weight calculated from the aminoacid sequence is 29,310, the PD-1 protein appears to be heavilyglycosylated. Normal murine lymphoid tissues such as thymus,spleen, lymph node and bone marrow contained very small numbersof PD-1⁺ cells. However, a significant PD-1⁺ population appearedin the thymocytes as well as T cells in spleen and lymph nodesby the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1antigen expression was strongly induced in distinct subsetsof thymocytes and spleen T cells by in vitro stimulation witheither anti-CD3 mAb or concanavalin A (Con A) which could leadT cells to both activation and cell death. Similarly, PD-1 expressionwas induced on spleen B cells by in vitro stimulation with anti-IgMantibody. By contrast, PD-1 was not significantly expressedon lymphocytes by treatment with growth factor deprivation,dexamethasone or lipopolysaccharide. These results suggest thatthe expression of the PD-1 antigen is tightly regulated andinduced by signal transduction through the antigen receptorand do not exclude the possibility that the PD-1 antigen mayplay a role in clonal selection of lymphocytes although PD-1expression is not required for the common pathway of apoptosis.     

Cytokine synthesis and apoptosis by intestinal intraepithelial lymphocytes: Signaling of high density αβ T cell receptor+ and γδ T cell receptor+ T cells via T cell receptor-CD3 complex results in interferon-γ and interleukin-5 production,while low density T cells undergo DNA fragmentation

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3⁺ IEL populations. Stimulation of low- or high-density CD3⁺ IEL via the T cell receptor (TCR)-CD3 complex using monoclonal anti-CD3, anti-αβ TCR or anti-γδ TCR antibodies resulted in opposing effects. In one case, a significant number of the high-density CD3⁺ T cells entered cell cycle from the resting stage (DNA replication was observed) and anti-TCR-CD3 treatment enhanced the numbers of interferon-γ and interleukin-5 spot-forming cells in this cell fraction. In contrast, when the low-density αβ TCR⁺ or γδ TCR⁺ T cells were activated via the TCR-CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR-CD3 complex resulted in their entry into the cell cycle and subsequent interferon-γ and interleukin-5 production in the high-density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low-density fraction of CD3⁺ IEL.     

Reversal of T cell unresponsiveness by augmentation of antigen presenting cell function

We have previously described epltopes of the 18 kDa proteinof Mycobacterium leprae which stimulate T and B cell responsesin different strains of mice. A series of overlapping 20-merpeptides that span the 18 kDa protein were used as immunogensto examine T and B cell recognition of different epltopes. Strain-specificvariation in the epltopes which induce the strongest responseswas affected by genes linked to the H-2 complex and the T cellresponses revealed by re-challenge with antigen were at leastpartially controlled by factors other than T cell specificity.We have examined the responses to one such antigen, peptlde1–20, which contains strongly immunogenlc epltopes forT and B cells. T cells from draining lymph nodes of peptlde1–20 immunized B10.BR, but not BALB/c mice, proliferatedIn vitro In response to rechallenge with peptlde 1–20or whole protein. Immunization with the same peptlde also inducedspecific antibody only in B10.BR mice. However, Immunizationof BALB/c mice results in ‘silent’ priming of Tcells since these can be induced to respond In vitro to thisantigen when cultured with activated macrophages as antigenpresenting cells (APC). The failure of APC from mBALB/c miceprimed with peptlde 1–20 to stimulate CD4⁺ proliferationwhen re-challenged In vitro and the failure to elicit antibodyresponses to peptlde 1–20 are presumably due to the samedefect in antigen-presenting cell function, since presentationof peptlde 1–20 by activated macrophages is sufficientto restore both responses. The failure of APC to stimulate responsesto this antigen in this model may be generally applicable toother cases of apparent non-responsiveness, and may have importantimplications for the understanding of T cell activation requirements.     

T cell activation through Thy-1 is associated with the expression of a surface protein(p100) on a subset of CD4 cells

Thy-1 molecules, which lack a transmembrane domain, can nonethelessinduce T cell activation; it has thus been suggested that aseparate transmembrane molecule associated with Thy-1 is requiredfor signal transduction. We have previously characterized atransmembrane protein with an M_r of 100,000 (p100), which isnon-covalently bound to two glycosyl-phosphatidylinositol (GPI)-linkedmolecules, Thy-1 and ThB. p100 is selectively expressed on theT cell surface and divides peripheral CD4 cells into two subpopulations.This differential expression on CD4 cells allowed us to investigatethe role of p100 in signal transduction through Thy-1 molecules.Here we report that only p100⁺ CD4 cells proliferate and releasecytokines in response to cross-linkage of Thy-1, although bothp100⁺ and p100^– CD4 cells strongly express Thy-1 on theirsurfaces. Control stimulation by anti-CD3 antibodies or concanavallnA induces identical thymidine uptake by the two CD4 cell populations.Interestingly, these two populations of CD4 cells had differentcytokine release profiles after activation through CD3: onlyp100⁺ CD4 cells released high amounts of IL-2 and IFN-, whereasboth populations released IL-4. p100 expression correlates withthe induction of homotypic aggregation of T cells after Thy-1triggering. p100 is associated with kinase activity (fyn andlck), and phosphorylated proteins of 90, 59, 57 and 33 kDa co-precipitatewith Thy-1 only in p100⁺ CD4 cells. Altogether, these data suggestthat p100 is involved in signal transduction through Thy-1.p100 expression by activated CD4 cells in vivo may be relevantto the proposed function of Thy-1 as an accessory signalingmolecule in cell activation.     

On the mechanism of human T cell suppression

Previous evidence from several laboratories suggests that CD8⁺T suppressor cells may be important regulatory elements governingspecific unresponsiveness of lepromatous leprosy patients toM.leprae. To analyse the mechanism of suppression, CD8⁺ Ts cloneswere established from lesions and peripheral blood of lepromatouspatients and tested for ability to suppress antigen-responsiveCD4⁺ Th clones or PBL. Suppression required induction by specificM.leprae antigen, but was effected in an antigen-non-specificfashion. The Ts clones failed to exhibit cytotoxicity of fourantigen-exposed MHC-matched target cells: (i) an ori-SV4O transformedmacrophage line; (ii) EBV transformed B cell lines; (iii) primarymacrophages; and (iv) M.leprae responsive CD4⁺ cells. The possibllitythat Ts clones induce functional inactivation of CD4⁺ clonesin vitro was investigated. M.leprae-responsive CD4⁺ clones werepreincubated with Ts CD8⁺ clones, APC, and antigen for 16 h,after which the CD8⁺ cells were removed. The CD4⁺ clones withM.leprae and APC remained unresponsive to restimulation withAPC and antigen for at least 10 days, although they respondedto IL-2. Addition of IL-2 to the pre- or post- incubation culturesneither prevented the induction of unresponsiveness, nor reversedit. Earlier models of tolerance have suggested that receptoroccupancy in the absence of second signals induces tolerancein B and T cells. Under conditions in which antigen responsesof Th clones were HLA-DR-restricted, the Ts clones were ableto suppress the response of DR mismatched Th clones. Thus, theeffect of the Ts cells, like mechanisms requiring antigen presentationwithout a second signal, appears to be induction of clonal anergyin Th cells, perhaps by a novel mechanism.     

Activation-induced expression of thymic shared antigen-1 on T lymphocytes and its inhibitory role for TCR-mediated IL-2 production

We have produced a hamster mAb, PRST1, which reacts with thymicshared Ag-1 (TSA-1), a product of the Ly6 gene family. By cross-blockingexperiments, we found that TSA-1 is identical to stem cell Ag-2(Sca-2). Using PRST1, the changes of TSA-1/Sca-2 expressionon mature T cells during the activation process were analyzed.Although freshly isolated T cells did not express detectableTSA-1 on their cell surface, in vitro stimulation of T cellswith concanavalln A induced a marked increase of surface TSA-1expression. The increased expression of TSA-1 on T cells wasdetected from 12 h after stimulation and was associated withthe increase of TSA-1 mRNA. In vivo injection of mice with staphylococcalenterotoxin B (SEB) resulted in the enhanced TSA-1 expressionin splenic V_ß8⁺ T cells. This antigen-specific inductionof TSA-1 expression in vivo preceded a detectable increase innumbers of V_ß8⁺ T cells after SEB injection. Functionally,whereas anti-TSA-1 mAb was not mitogenic to T cells, it inhibitedanti-CD3-induced IL-2 production by T cell hybridomas. Theseresults indicate that TSA-1/Sca-2 is a unique marker for T cellactivation and a signal through this molecule may have a negativefeedback role to limit IL-2 production from activated T cellsstimulated through the TCR.