Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V_β8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V_β8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing “anergy” and apoptotic cell death of V_β8⁺ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V_β8⁺ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)α chains in vivo, although SEB induces IL-2R α in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V_β8⁺ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.     

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.     

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.     

Co-transfer of B cells converts resistance into susceptibility in T cell-reconstituted, Leishmania major-resistant C.B-17 scid mice by a non-congnate mechanism

Resistance to infection of mice with Leishmania major parasitesis dependent on the production of IFN- by CD4⁺ T helper cells.C.B-17 scid mice, lacking both T and B cells, succumb very quicklyto the infection, but develop resistance if reconstituted withappropriate numbers of T cells from BALB/c mice. In this model,we studied the role of B cells with regard to their abilityto influence disease outcome and to function as antigen-presentingcells for T cells. For this purpose, we reconstituted scid mice(H-2^d) with either T cells or with T and B cells obtained from(BALB/c x BALB.B)F₁ mice (H-2^d x ^b), and infected them withL. major parasites 1 day after reconstitution. Mice reconstitutedwith T cells alone cured the disease, whereas additional B cellreconstitution led to susceptibility. Healing was associatedwith a predominant T_h1-type response. In all mice, L. mayor-specificT cell proliferation was restricted to the MHC phenotype ofthe recipient (H-2^d) but not to that of the donor (H-2^d x ^b),indicating that there was no detectable contribution of donorB cells in the priming of a T cell response. Furthermore, Bcells, when purified from infected BALB/c mice, were unableto stimulate a L. mayor-specific CD4⁺ T cell clone (L1/1) withoutaddition of exogenous antigen, in contrast to macrophages fromthe same animal. These data suggest that B cells, in vivo, donot carry L. major antigen in a form capable of activating specificCD4⁺ T cells. Therefore, B cells promote disease by means otherthan cognate interaction with CD4⁺ T cells.     

Frequent deletion of the transgene in T cell receptor {beta} chain transgenic mice

TCR V_ß8.1 transgenic mice were generated using a genomicTCR V_ß gene construct under the control of its promoterand enhancer. Among three lines of transgenic mice, one lineexpressed the transgenic TCR on only 70% of peripheral T cells,while the other two lines expressed it on almost all matureT cells. T cells which lacked expression of the transgenic TCRß chain expressed endogenous TCR ß chains.The molecular basis underlying the lack of transgene expressionin T cells of this line of transgenic mice was investigated.The transgenic TCR^– cells were isolated by two methods.First, Thy-1⁺ V_ß8.1/8.2^– cells were purifiedfrom peripheral T cells using cell sorting. Second, transgenicTCR^– T cell clones were established. In both cases, Southernblotting indicated that V_ß8.1^– T cells had deletedthe transgenic TCR gene. Thus, deletion of the transgenic TCRcan occur in a high proportion of T cells, which allows rearrangementand expression of endogenous TCR ß chains.     

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-{gamma}

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL)activity by T cells of aged mice in vitro, we initially assessedwhether IL-12 could overcome age-related deficits when givento aged mice in vivo. Growth of P815 (H-2^d) was enhanced inaged compared with young BALB/c (H-2^d) mice and tumor growthwas curtailed by IL-12 in both age groups. Unexpectedly, secondaryCTL stimulated ex vivo with P815 were reduced in IL-12-treatedmice compared with controls. Primary CTL generated ex vivo acrossMHC differences in IL-12-treated BALB/c and C57BL/6 young micewere reduced by 90–99%, were dose- and time-dependent,and were associated with reduced allo-stimulated NK-like activityand [³H]thymidine incorporation. IFN- was elevated in sera andin supernatants from allo-stimulated cultures from IL-12-treatedmice, while IL-4 was reduced in such supernatants, suggestingthat, despite reduced CTL, IL-12 was associated with increasedT_h1- and reduced T_h2-type cytokine production. IL-12 also inducedsplenomegaly, primarily due to increased numbers of cells lackingmarkers of mature T, B and NK cells, or macrophages, or polymorphonuclearleukocyte morphology. IFN- mutant mice exhibited reduced splenicenlargement in response to IL-12, suggesting that the splenomegalywas due, in part, to IFN- production. However, reduced CTL generationwas not due entirely to dilution of CTL precursor cells becausespleen cellularity and size increased 3-fold while CTL activitydecreased 10- to 100-fold, and CTL generation normalized toCD8⁺ T effector cells was still significantly reduced in IL-12-treatedmice. Interestingly, purified CD4⁺ and CD8⁺ T cells from IL-12-treatednormal mice exhibited greater proliferative and cytolytic activitiesrespectively compared with controls. Thus, effector T cellsin IL-12-treated mice were not impaired, but exhibited augmentedresponsiveness, suggesting that IL-12 induced complex interactionsamong spleen cell populations and that these effects, in part,are mediated by IFN-.     

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via {alpha}4 integrin-VCAM-1 interaction

The present investigation examines the localization and migrationof purified T cell subsets in comparison with B cells, CD8 Tcells and CD4⁺CD8^– single-positive thymocytes. CD4 T cellsubsets in the rat are defined by mAb MRC OX22 ( anti-CD45RC),which distinguishes resting CD4 T cells (CD45RC⁺) from those(CD45RC^–) which have encountered antigen in the recentpast– subpopulations often referred to as ‘naive’and ‘memory’. Purified, ⁵¹Cr-labelled CD45RC⁺ CD4T cells broadly reflected the migration pattern of CD8 T cellsand B cells. Early localization to the spleen was followed bya redistribution to mesenteric lymph nodes (MLN) and cervicallymph nodes ( CLN) , B cells migrating at a slightly slowertempo. There was almost no localization of these subpopulationsto the small or large intestine [Peyer's patches (PP) excluded].In contrast, CD45RC^– CD4 T cells (indistinguishable insize from the CD45RC⁺ subset) localized in large numbers tothe intestine; they were present here at the earliest time point(0.5 h) , persisted for at least 48 h but did not accumulate,indicating a rapid exit. Numerically, localization of CD45RC^–CD4 T cells in the MLN could be accounted for entirely by afferentdrainage from the intestine. Unexpectedly, CD45RC^– CD4T cells (but not other subsets) localized and accumulated inthe thymus. In vivo treatment with mAb HP2/1 against the integrin₄ subunit inhibited almost entirely CD45RCT^– CD4 T cellmigration into the PP (98.1%), intestine (87.1%) , MLN (89.1%)and thymus (93.5%) migration into the CLN was only reduced byhalf. To distinguish between recognition of MAdCAM-1 and VCAM-1by ^4–containing integrins, recipients were treated withmAb 5F10 against rat VCAM-1. Except for the thymus and a smallreduction in CLN, localization of CD45RC^– CD4 T cellswas unaffected; entry to the thymus was almost completely blocked(92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC^–CD4 T cells alone showed enhanced localization to the gut andPP, probably via ₄ß₇-MAdCAM-1 interaction; ( II) thatmany CD45RC^– cells entered nonmucosal LN independentlyof ₄ integrin or VCAM-1; and (III) that entry of mature recirculatlngCD45RC^– CD4 T cells into the thymus across thymic endothellumwas apparently regulated by ₄ integrln-VCAM-1 interaction.     

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.     

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.