In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.     

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.     

T cell anergy is programmed early after exposure to bacterial superantigen in vivo

We have investigated the effects of the protein synthesis inhibitor,cycloheximide (CHX), on the induction of post-thymic T celltolerance in mice primed with the bacterial superantigen, Staphylococcusaureus enterotoxin B (SEB). A single injection of 1 mg CHX preventedprotein synthesis in splenic cells for <6 h in vivo. Theconcomitant administration of SEB and CHX prevented inductionof SEB-specific anergy, but did not interfere with the deletionof SEB-speclfic V_ß8⁺ T cells by activation-induced,programmed cell death. When CHX was given 24 h after SEB administrationthe expression of anergy was not affected. These findings suggestthat anergy and deletion represent independent processes. Furthermore,these observations, together with the fact that SEB retainsthe potential to induce anergy in specific T cells 8 h afterpriming in vivo, imply that the determination of alternate fates(anergy or death) occurs at early time points after SEB injection     

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.     

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V_ß6⁺ T cell eliminationin the lymph nodes and thymus during Mls-1^a mouse ontogeny.Our results suggest that induction of tolerance to Mls-1^a antigensinvolves mechanisms other than clonal deletion of immature Tcells in the thymus. Mature CD4⁺CD8^– (CD4SP) T cells wereaffected by Mls-1^a antigens earlier than immature thymocytepopulations. Up to 2 weeks after birth, reduced frequenciesof V_ß6⁺ T cells were detected only in CD4SP cellsfrom the thymus and lymph nodes, and generation of CD4SP cellsin the thymus was blocked at least 1 week earlier than thatof their CD4⁺CD8^loTCR^hl immature precursors. The number of V_ß6⁺CD4SPT cells increased during the first 2 weeks of life and remainedconstant thereafter. We thus found no evidence of deletion ofmature V_ß6⁺CD4SP T cells, as the reduced frequenciesin adult mice can be attributed to the dilution of previouslygenerated cells in lymphoid organs of growing mice, which increasein cellulartty after birth. V_ß6⁺CD4⁺ T cells wereactivated in vivo shortly after birth, as shown by a selectiveincrease in IL-2 receptor a chain expression in the thymus andlymph nodes from day 0 to day 2 after birth. It is thereforelikely that endogenous expression of Mls-1^a antigen shortlyafter birth activates V_ß6⁺CD4SP T cells and rendersthem anergic. This process of tolerance induction may precedethe clonal deletion of immature T cells in the thymus, describedin the adult mouse.     

Age-dependent changes in the response to staphylococcal enterotoxin B

In the present study we investigated the response of old miceto staphylococcal enterotoxin B (SEB) immunization. Old micewere susceptible to lethal toxic shock, probably mediated bytumor necrosis factor-, although lethal toxic shock was notobserved in young mice. Old mice were able to produce more IL-2and IL-4 than young mice in response to in vivo immunizationwith SEB. V_ß8⁺CD4⁺ T cells of old mice expanded lessin vivo and were not deleted in response to SEB. However, inspite of the absence of clonal deletion, SEB was found to induceenergy of SEB reactive cells in old mice, as demonstrated byreduced in vitro T cell proliferation to SEB and reduced invitro IL-2 and IL-4 production.     

Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production

Particular subsets of T cells expressing the NK1.1 antigen havebeen proposed to play an immune regulatory role by their fastand strong production of cytokines, in particular IL-4. We soughtto determine factors driving the functional differentiationof NK1.1⁺ T cells. Since NK1.1⁺ T cells are exquisitely sensitiveto IL-7 stimulation, we analyzed the development, selectionand IL-4 production of NK1.1⁺ T cells in IL-7 deficient mice(IL-7–/–mice). Besides a sharp reduction of allT cell subsets, NK1.1⁺ T cells develop at normal relative frequenciesin IL-7–/–;mice. They also undergo a normal selectionprocess, as revealed by the biased V_ß TCR repertoireidentical to the one in IL-7+/+ mice. However, NK1.1₊ T cellsfrom IL-7+/+ mice were found to be impaired in IL-4 and IFN-production in in vitro and in vivo models. In addition, IL-7was able to restore IL-4 production by NK1.1⁺ thymocytes fromIL-7–/– mice. Finally, IL-7 but not IL-4 was ableto maintain and increase IL-4 production by NK1.1⁺ thymocytesfrom normal mice. These data suggest that the functional maturationof NK1.1⁺ T cells requires a cytokine-driven differentiationprocess, in which IL-7 plays a major role.     

Impaired CD28-mediated co-stimulation in anergic T cells

We have investigated a CD28 co-stimulation in anergic T cellsin staphylococcal enterotoxin Binoculated mice by stimulatingthe cells with a plate-coated anti-TCR antibody in the presenceor absence of an anti-CD28 antibody. CD28 co-stimulation increasedthe levels of IL-2 and IL-4 mRNAs in nalve CD4⁺V_ß8⁺T cells. However, it did not increase the levels of IL-4 mRNAat all and only partially increased those of IL-2 mRNA in anergicT cells. It was demonstrated that CD28 co-stimulation was impairedso that it no longer stabilized cytoklne mRNAs in anergic cells.The levels of IL-4 mRNA in response to TCR stimulation werehigher in anergic T cells than those in nalve T cells in spiteof the defective CD28 co-stimulation in the former cells. Anergyinduction and generation of a T_h2-type immune response in vivoare discussed     

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.     

Activation and re-activation potential of T cells responding to staphylococcal enterotoxin B

To elucidate the parameters that lead to superantigen inducednon-responsiveness, an in vitro model for studying primary andsecondary responses to the bacterial superantlgen staphylococcalenterotoxin B (SEB) was established. Upon re-activation withSEB, in vitro SEB primed T cells show an early proliferativeresponse that ‘quenches’ in time and is severelyimpaired 3 days after re-stimulatlon. Despite their overallimpaired proliferative capacity and IL-2 production, these Tcells are able to produce IFN-_ß and to up-regulateactivation markers CD69 and IL-2R upon re-stimulation with SEB,demonstrating that SEB non-responsiveness is not absolute. Rather,it reflects the inability to mount an ongoing proliferativeresponse upon re-stimulation with SEB. Our results also demonstratethat SEB-induced non-responsiveness is not simply the resultof presentation in the absence of co-stimulation, since presentationof SEB on highly purified dendritic cells during the primaryresponse did not prevent the induction of non-responsiveness.Aspreviously shown, SEB induces a T_h1 phenotype in respondingCD4⁺ T cells. Skewing towards a T_h2 phenotype by adding IL-4and antibodies to IFN-_ß did not prevent the inductionof non-responsiveness by SEB. Interestingly, T cells pretreatedwith plate-bound anti-CD3 and anti-V_ß8 were also non-responsiveto SEB re-stimulation. Thus, non-responsiveness to SEB (definedhere as inability to produce IL-2 and proliferate) seems toreflect an intrinsic inability of previously activated T cellsto respond to SEB, probably reflecting differences in signaltransduction pathways used by naive versus previously activatedT cells.     

Activation mediated CD4+ T cell unresponsiveness during acute Toxoplasma gondii infection in mice

infection of mice with Toxoplasma gondii has been shown to inducea transient state of immune down-regulation. Earlier reportshave demonstrated the role of cytokines, in particular IL-10,in this host response. Here evidence is presented that T.gondll,a major opportunistic pathogen of the newborn and those withAIDS, is able to induce CD4⁺ T cell population Increased involume by day 7 post-infection and expressed T cell maturationmarkers (CD44^hl, Il-2r^hl,Mel-14^fo). Further noted was a clonalactivation of several CD4+ T cells subsets expressing the V_ßchain of the TCR. At day 7 post-infection, partial reductionof all CD4⁺ T cells to mltogen or parasite antigen stimulationwas observed, In particular V_ß5 T cells. Additionof rlL-2 partially restored the CD4⁺ T cell proliferative responsein Vitro. The T cell activation marker CTLA-4 could not be detectedand te co-stimulatory molecule, CD28, was down-regulated. Elctrophoneticand morphologic analysis of these cells post0culture demostrateda DNA fragmentation pattern and cell death consistent with apoptosis.These studies demonstrate for the first time in a protozoanparasite that activation-induced CD4⁺ T cell unresponslvenessoccurs during actue T.gondll infection in mice, and may be importantin immune down-regulation and parasite persistence in the infectedhost.0     

Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

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.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.     

IL-4 producing CD4+ TCR{alpha} {beta}int liver lymphocytes: influence of thymus, {beta}2-microglobulin and NK1.1 expression

The present report describes developmental, phenotypic and functionalfeatures of unconventional CD4⁺ TCRß lymphocytes.In C57BL/6 mice, the majority of liver lymphocytes expressingintermediate intensity of TCRß (TCRß^int)are CD4⁺NK1.1⁺ and express a highly restricted TCR V_ßrepertoire, dominated by V_ß8 with some contributionby V^ß7 and V^ß2. Although these cells expressthe CD4 co-receptor, they are present in H2-l Aß (Aß)^+/–gene disruption mutants but are markedly reduced in ß₂-microglobulin(ß₂m)^–/– mutant mice and hence are ß₂mdependent. Thymocytes expressing the CD4⁺NK1.1⁺ TCR ßphenotype are also (ß₂m) contingent, suggesting thatthese two T lymphocyte populations are related. The CD4⁺NK1.1⁺TCRßlymphocytes in liver and thymus share several markers such asLFA-1⁺, CD44⁺, CD5⁺, LECAM-1^– and IL-2Rßa. TheCD4⁺NK1.1 ⁺ TCRß^int liver lymphocytes were not detectedin athymic nuinu mice. We conclude that ß₂m expressionis crucial for development of the CD4⁺NK1.1⁺ TCRß^intliver lymphocytes and that thymus plays a major role. CD4⁺ TCRß^intliver lymphocytes were also identified in NK1.1⁺ mouse strains,there lacking the NK1.1 marker. We assume that the NK1.1 moleculeis a characteristic marker of the CD4⁺TCR"^int liver lymphocytesin NK1.1⁺ mouse strains, although its expression is not obligatoryfor their development. The liver lymphocytes from +₂m^+/–,but not from +₂m^–/–mice are potent IL-4 producersin response to CD3 or TCRß engagement and the IL-4production by liver lymphocytes was markedly reduced by treatmentwith anti-NK1.1 mAb. We conclude that the CD4+NK1.1⁺ TCRß^intliver lymphocytes are capable of producing IL-4 in responseto TCR stimulation.     

Identification of two binding sites in staphylococcal enterotoxin B that confer specificity for TCR V{beta} gene products

The enterotoxlns produced by Staphytococcua af1reus are potentmitogens. They stimulate T cells in an oligocional fashion thatIs dependent on the expression of particular variable regiongene elements in the ß-chaln of the TCR (V_ß).The fourth hypervarlable loop of the TCR ß-chaln Isgenerally regarded as the site of contact for both viral andmlcroblal superantigens. Recently, residues 60 and 61 of staphylococcalenterotoxin B (SEB) have been highlighted as central to theinteraction of this toxin with the TCR. We have, therefore,analysed a series of toxins with mutations at these positionsto investigate how amlno add substitutions affect the abilityof mutant toxins to stimulate both human and mouse T cells.Each of the variant toxins induced proliferation in a murineV_ß8.3 T cell clone, whereas a V_ß8.1 T cellclone only responded to native toxin. A panel of nine humanT cell clones expressing six different V_ß elements,all of which responded to native SEB, was tested for reactivityto the variant toxins. Only one V_ß19.1⁺ T cell clonewas found to be sensitive to substitution at positions 60 and61 In a manner analogous to the murine V_ß8.1 T celldone. Seml-quantitatlve analysis of the TCR V_ß expressionof human T cell lines expanded with native and mutant SEB revealedthat none of the variant toxins could stimulate T cells thatexpressed V_ß19.1. Taken together, these results suggestthat the interaction of mouse V_ß8.1 and human V_ß19.1TCRs with SEB differs from other TCRs. Sequence comparisonsof the different TCR V_ß chains indicated that residuesin the second complementarity determining region (CDR2) interactwith the 60–61 loop of SEB. Therefore, a minimum of twodistinct binding modules confer specificity to the interactionof the TCR with SEB.     

Altered response of CD4+ T cell subsets to Plasmodium chabaudi chabaudi in B cell-deficient mice

Mice depleted of B cells from birth by treatment with anti-_µantibodies can control but not clear an infection with the malariaparasite Plasmodium chabaudi chabaudi (AS). Splenic CD4⁺ T cellsfrom these mice were unable to mount a significant T_h2 responseto the parasite in vitro as shown by much lower precursor frequenciesof T_h cells for antibody production and of IL-4-producing cellscompared with the response of control-treated mice. CD4⁺ T cellsof the anti-_µ-treated mice which respond to antigens ofP. chabaudi chabaudi maintained a T_h1 phenotype throughout primaryinfection, in contrast to control mice in which a sequentialappearance of T_h1 and T_h2 responses was observed. These datashow that T_h1 responses in anti-_µ-treated mice are sufficientto control parasitemia but not to eliminate an infection. Thedata further suggest that depletion of B cells by treatmentwith anti-_µ; antibodies reduces the generation of theT_h2 subset during a primary response to P. chabaudi chabaudi.     

In vitro effects of HgCl2 on murine lymphocytes. II. Selective activation of T cells expressing certain v{beta}TCR

In vivo administration of HgCI₂ causes autoimmune manifestationsin susceptible rats and mice. We have, previously shown thatmercury is a unique molecule that can primarily activate murineT lymphocytoes to transformation and proliferation in vitro.To test whether a specific TCR repertoire predisposes the autoimmunedevelopment induced by HgCI₂ and our hypothesis that mercurymay, function as a superantigen, we examined the TCR V_ßrepertoire in HgCI₂-stimulated T cells from the responder BALB/cor SJL mice and the non-responder DBA/2 mice. We found a selectiveactivation of T cells bearing a certain set of TCR V_ßchains in response to HgCI₂, e.g. V_ß6, V_ß8,V_ß10, and V_ß14 in the BALB/c strain. Moreover,depletion of V_ß8⁺ T cells, a family predoininantlyexpanded in the BALB/c strain upon HgCI₂ stimulation, profoundlyinhibited the response to HgCI₂ in this strain. An alternativeselection of V_ß segments, involving V_ß6,V_ß7 and V_ß14, was observed in the SJL strainin which the V_ß8 family is genetically deleted. Mechanism(s)whereby mercury modulates the immune system under a stringentgenetic control and a possible therapeutic regime against mercury-inducedautoimmune disease by administration of antibody specific tothe TCR V_ß region are discussed.     

Skewed T cell receptor V{alpha} repertoire among superantigen reactive murine T cells

Reactivity of murine T cells with viral or bacterial superantigensis clearly correlated with the expression of TCR V_ßdomains. Thus, T cells responding to the minor lymphocyte stimulatorylocus (Mls-1^a) or staphylococcal enterotoxin B (SEB) expresspredominantly TCR V_ß6 or V_ß8.2 respectively.We have investigated the involvement of the other major variableelement of the TCR, the V domain, in these superantigen responses.Using a panel of anti-TCR V mAbs, It is demonstrated that theTCR V repertoire among superantigen stimulated V_ß6⁺or V_ß8.2⁺ blasts (responding to Mls-1^a or SEB respectivelyin vitro) is altered in comparison with anti-CD3 stimulatedcells expressing the same V domains. Furthermore, the TCR Vrepertoire is strongly skewed in TCR V_ß8.2 transgenicmice that have undergone extensive peripheral clonal deletionafter SEB injection. These data imply that the V domain influencessuperantigen recognition by sthe TCR.