V{beta}6-bearing T cells are involved in resistance to Trypanosoma cruzi infection in XID mice

BALB.xid mice, carrying an X-linked mutation leading to theabsence of CD5⁺ B cells, are highly resistant to Trypanosomacruzi Infection. These mice clear blood parasites In the acutephase of infection and do not develop the inflammatory Infiltrationcharacteristically observed in the chronic phase of susceptiblestrains of mice. We have shown that the resistance of BALB.xldIs dependent on the production of high levels of IFN-y. Natural(adoptive foster) or artificial (In vivo Injection of blockingantibodies) treatments of BALB.xld induced deletion of CD4⁺and CD8⁺ cells bearing V_ß6 TCR. The absence of V_ß6lymphocytes considerably reduced resistance to infection. Furthermore,in BALB.xld lacking this minor fraction of the T cell repertoire,almost 50% of the IFN-y production is lost. This indicates thatV_ß6-bearing T cells are either directly or Indirectlyinvolved in the production of IFN-y and, thus, important foran effective immune response during the acute phase of experimentalChagas' disease.     

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice

Autoimmunity is known to increase in aging. A possible factorcould be an alteration in the T cell repertoire wIth advancingage. Antibodies to the variable region of the ß chainof the TCR activate T cells and can serve as probes for analysisof the T cell repertoire. We have used V_ß3 and V_ß17aantibodies to determine the presence and functionality of normallydeleted T cells bearing potentially self-reactive TCR in peripherallymphoid tissue and blood from aged (SJL/J x BALB/c) F₁ LAF₁and BALB/c mice. Although an occasional 20- to 24-month-oldmouse exhibited V_ß3⁺ or V_ß17a⁺ T cells intheir lymph nodes or peripheral blood lymphocytes (PBL) slightlyabove the range for normal young mice of these I-E⁺ strains,there was no striking ‘escape’ from the normal thymicdeletion process. However, responsiveness to anti-V_ß3and anti-V_ß17a was slightly higher In aged, and particularlyIn aged thymectomlzed (TX), than in young mice. This was incontrast to proliferative responses to stimulation with antibodyto the normally expressed V_ß8 which were lower inthe lymph nodes from aged than from young mice. The PBL of some30- to 36-month-old mice were also examined. Enhanced numbersof ‘forbidden’ V_ß bearing T cells wereseen more frequently at this age. In spite of the age-relateddecrease in overall CD4/CD8 T cell ratios in all organs, themice with relatively high V_ß17a⁺ T cells exhibitedproportionally more CD4⁺ cells in that V_ß population.We conclude that the ‘forbidden’ T cells that respondto anti-V_ß stimulation in the 20- to 24-month-oldmice are most likely of extra-thymic origin, since they weremore readily detectable in aged TX mice. Potentially self-reactiveCD4 (and CD8) single-positive T cells were detectable in PBLonly in very aged (30–36 months old) euthymic mice.     

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.     

HLA-DR and H-2E transgenes differentially mediate TCR-specific positive selection

The use of HLA transgenic mice in models of immunity and diseaseassumes that human MHC molecules are able to contribute towardthe positive selection of the mouse TCR repertoire. As an initialstep towards analysis of this we have compared the relativeability of DR/Eß or E/Eß complexes to induceT cell receptor (TCR) positive selection in H-2Ea and HLA-DRAtransgenic mice lacking endogenous E. The results show that,like E/Eß, the hybrid DR/ß complexes arecapable of mediating positive selection of V_ß2⁺;,V_ß6⁺, and V_ß10⁺ cells. However, differenceswere found between the effects of the two transgenes. Thus,while V_ß6⁺ cells were efficiently selected in bothH-2Ea and DRA transgenic mice, positive selection of V_ß10⁺cells was less apparent in the DRA transgenic mice. Variationbetween Ea and DRA transgenic mice is consistent with the notionthat this process is dependent on differential binding of endogenouspeptides to the E/Eß and DR/Eß complexes.Furthermore, contrary to expectations, in neither set of micewas positive selection limited solely to the CD4⁺ subset. Thus,examples were found in which V_ß-specific positiveselection was confined to either the CD4⁺ or CD8⁺ subsets, andothers in which both subpopulations were concomltantly increased.In the case of V_ß2 positive selection, H-2Ea transgenicmice showed expansion of these cells in both the CD4⁺ and CD8⁺subpopulations whlle in DRA transgenic mice this occurred predominantlyin the CD8⁺ subpopulatlon.     

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.     

Expression of an ovalbumin-specific V{beta}8.2 TCR transgene inhibits collagen arthritis in B10.Q mice

Previous studies have illustrated the importance of T cellsbearing ß TCRs in the induction and development ofcollagen induced arthritis (CIA) in mice. However, the scopeof TCR usage in CIA has yet to be clearly defined. Given theinherent diversity of the TCR repertoire, the relative flexibilityof the arthritogenic TCR repertoire specific for type II collagen(CII) is not clear. Therefore, we chose to examine the influenceof a highly skewed TCR repertoire on CIA. Arthritis susceptibleB10.Q (H-2^q) mice were mated with C57L (H-2^b) animals expressingan ovalbuminspecific V_ß8.2 TCR transgene (Tg) andTg⁺ offspring were further backcrossed to B10.Q. HomozygousH-2^a/q, V_ß8.2 Tg⁺ mice displayed a high level of V_ß8.2⁺T cells in peripheral blood. However, expression of some endogenousV_ß TCR, such as V_ß14, was still detected.Upon immunization with bovine CII in adjuvant, V_ß8.2Tg⁺ mice were highly resistant to CIA when compared with Tg^–littermates. Analysis of sera demonstrated a marked reductionin antibody specific for homologous mouse CII as well as heterologousbovine CII in Tg⁺ animals. Interestingly, V_ß8.2 Tg⁺mice still mounted good antibody responses following immunizationwith human thyroglobulin, indicating that the skewed TCR repertoireaffected anti-CII but not antithyroglobulin responses. Thus,our findings show that constraints placed on the TCR repertoireInhibit pathogenic responses against CII and suggest that inH-2^q mice the arthritogenlc TCR repertoire bears only limitedflexibility.     

Analysis of T cell repertoire and function in mice transgenic for the human V{beta}3 TCR

We have constructed mice containing the human V_ß3TCR gene from the influenza virus haemagglutinin specific humanCD4⁺ T cell clone HA1.7. Similar cell yields were obtained fromtransgenic and non-transgenic lymphoid tissue, with normal levelsof T cells and with no unusual bias of the CD4 or CD8 subpopulations.Immunostaining and FACS analysis of transgenic thymocytes, spleen,and mesenteric lymph nodes revealed that the majority of T cellsexpressed the human V_ß3 TCR on the cell surface. Smallnumbers of cells expressing murine TCR_ßchain werealso detected. Polymerase chain reaction analysis revealed thatan extensive V TCR repertoire was used in the human V_ß3transgenic mice. Lymphocytes from the spleen and bmesentericlymph nodes of transgenic mice were assessed for functionalactivity in vitro. Isolated cells were stimulated with mitogenor superantigen, as well as directly through the TCR-CD3 complex,and their ability to proliferate and secrete lymphokines analysed.Cells from transgenic mice responded well after stimulationwith phytohaemagglutinin, concanavalin A, anti-CD3 antibody,anti-CD3 antibody with phorbol ester, and Staphylococcus aureusenterotoxin B, and also showed alloreactivity in a mixed lymphocytereaction. Minimal levels of response were detected after stimulationwith murine TCRß antibody. Together, these data suggestthat a human TCRß chain is able to associate witha murine TCR chain, to form a fully functional surface TCR-CD3complex.     

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.     

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.     

Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

The onset of T_h1 immunity is in part regulated by genetic background.To elucidate the cell type carrying critical factors determiningthe T_h1 response, we employed Rag-2^–/– mice on Leishmaniamajor-susceptible BALB/c and -resistant B10.D2 backgrounds.By using bone marrow (BM) chimeras generated by the transplantationof B10.D2 BM cells into BALB/c-Rag-2^–/– mice, andvice versa, it was shown that hematopoietic cells carry factorsdetermining the disease outcome and T_h1 response against L.major infection. B10.D2-Rag-2^–/– mice reconstitutedwith BALB/c CD4⁺ T cells exhibited a T_h1 response and controlledL. major infection. Wild-type BALB/c mice inoculated with L.major-parasitized B10.D2-Rag-2^–/– splenocytes alsoexhibited a T_h1 response and a mild disease outcome, whereassuch a T_h1 response was not induced when CD11c⁺ dendritic cells(DCs) were depleted from parasitized B10.D2-Rag-2^–/–splenocytes. T_h1 response was reconstituted by the additionof L. major-parasitized B10.D2 DCs but not L. major-parasitizedBALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/–splenocytes. These results indicate that DCs determine the outcomeof the disease upon L. major infection.     

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.     

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.     

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.     

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.     

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites

The site of negative selection in the thymus has been inferredfrom a range of different experiments. Analysis of thymic deletionof Vß5⁺, V_ß11⁺ or Vß17a⁺ cellsH-2E transgenic mice led to the theory that negative selectionoccurs predominantly in the medulla (specifically, through presentationby medullary dendritic cells). Other experiments investigatedwhether transgenic TCR are deleted at the double-positive (DP)or single-positive stage following encounter with peptide ligand:by flow cytometric analysis deletion is generally found to occurat the DP thymocyte stage and as these cells are found predominantlyin the cortex, it has been inferred that this is the key siteof negative selection. The visualization of apoptotic thymocytesin situ has recently been reported for specific examples ofnegative selection. Using a panel of TCR transgenic lines inwhich negative selection occurs at different stages of thymocytedevelopment, we have used TUNEL staining to analyse the anatomicalsites of thymocyte apoptosis. For the first time we have beenable to compare directly the sites of deletion induced by theendogenous cognate peptides or by endogenous superantigen. Weshow that generalization from the medullary deletion of V_ß5⁺,V_ß11⁺ or V_ß17a⁺ cells by the endogenoussuperantigens Mtv 8 and 9 and from limited examples of corticaldeletion by exogenous peptide administered to TCR transgenicmice is over-simplified. Apoptotic thymocytes in mice lackingMtv superantigens are indeed localized in the cortex. However,when deletion is induced by cognate self peptide, apoptosiscan occur in the cortex, the medulla or at the junction betweenthe two.     

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.     

TCR repertoire to proteolipid protein (PLP) in multiple sclerosis (MS): homologies between PLP-specific T cells and MS-associated T cells in TCR junctional sequences

In the pathogenesis of multiple sclerosis (MS), autoimmune Tcells reactive with proteolipid protein (PLP) may play a crucialrole. We determined 23 TCR (ß-chain sequences of limitingdilution T cell lines (TCL) selected against a synthetic peptide,PLP 95–116, 105–124 or 139–155, from the peripheralblood of three Japanese MS patients with the DR2, w15 haplotype(Tl, SK and OK). Fourteen sequences were originated from Tl,seven from SK and two from OK. The PLP-reactive TCL utilizedvarious V_ß and J^ß; gene segments, but therewas significant bias in the V_ß and J_ß usage.Overutilization of the V_ß2 family and dominant usageof the J_ß2.5 subfamily was seen in PLP 105–124-reactiveand 95–116-reactive TCL respectively. More remarkably,a majority of the TCL were found to express ß-chainCDR3 motifs that appear to be unique to MS brain infiltrates.In contrast, these motifs were only rarely seen in control TCRsequences from peripheral blood or from a TCL selected againsttetanus toxoid. In several cases, the _ßCDR3 homologiesbetween the PLP-reactive T cells and MS brain T cells were extensive,owing to the shared motifs in combination with the surroundingamino acid identities. These results indicate that PLP-specificT cells may be involved in the immunopathology of MS.     

Analysis of T cell antigen receptors of myelin basic protein specific T cells in SJL/J mice demonstrates an {alpha} chain CDR3 motif associated with encephalitogenic T cells

Experimental autoimmune encephalomyelitis (EAE) is an animalautoimmune disease mediated by CD4⁺ T cells. Analysis of TCRexpression revealed that limited TCR elements (V^ß8.2,V2 or 4) were utilized by myelin basic protein (MBP) specificT cells In mice with H-2^U haplotype and Lewis rats. The usageof a particular ß chain complementarity determiningregion 3 (CDR3) motif has also been shown. However, It remainsunclear to what extent these observations can be extrapolated.Here we studied the TCR sequences of MBP 89–101/1-A^s specificT cell clones derived from SJL/J mice, using the polymerasechain reaction on reverse transcribed mRNA. Although the V_ßusage was less restricted than In H-2^U mice, they predominantlyutilized v_ß17a and expressed LGG or related motifsin the V_ß - D_ß - J_ß junctions.Furthermore, a single chain rearrangement between V1.1 andJBBM142 with no N region diversity was preferentially used.Concordantty, Immunization with a peptlde corresponding to the chain CDR3 was found to significantly alter the clinical courseof EAE. Comparison of the published TCR junctional regions demonstratesthat the CDR3 motifs (LGG in ß chain, CA*R*NY motifIn chains) are expressed by other encephalitogenic clones.Notably, the CA*R*NY was conserved in PL/J mice clones thatrecognize a distinct MBP–MHC determinant. It suggeststhat an antigen-independent mechanism may contribute to conservingthe a chain motif. The Implications of these observations arediscussed.