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.     

Interallelic complementation of {beta}-subunit defects in fibroblasts of patients with propionyl-CoA carboxylase deficiency microinjected with mutant cDNA constructs

Propionic acidemia results from deficiency of propionyl-CoAcarboxylase (PCC) activity. PCC is a biotin-dependent, mitochondrialenzyme composed of - and ß-subunits (structure, ₄ß₄),with the -subunit containing the biotin ligand. About two-thirdsof fibroblast lines from patients with mutations in the PCCB(ß-subunit) gene show interallelic complementationin cell fusion experiments (the pccB and pccC subgroups of thepccBC major group defining ß-subunit mutations, wherepccBxpccC fusions show complementation). We previously identifiedthe mutations in several pccB or pccC cell lines and suggestedthat point mutations or small, in-frame insertions or deletionswere likely responsible for the complementation obtained betweenß-subunit defects. To test this hypothesis, we haveintroduced five different mutations (three pccB and two pccC)that fit these criteria into a PCC ß-subunit cDNAplasmid expressed from a cytomegalovirus promoter. The cDNAplasmids were microinjected into mutant fibroblasts and thecells were assayed by radioautographic detection of ¹⁴C-propionateincorporation into cellular macromolecules. Four different mutations(Pro228Leu or dupKICK140 from pccB or lle408 or Arg410Trp frompccC) complemented cells from complementation subgroups in apattern congruent with the results obtained in cell fusion experiments.The fifth mutation, Arg536Asn, which was found both in a complementingpccB and a non-complementing pccBC cell line, failed to complementany of the mutant cell lines. The high proportion of mutantcell lines that show interallelic complementation, the diversityof mutations responsible for it, and the extent of heteroallelismbecoming apparent in affected patients suggests that partialcomplementation between allelic mutations may contribute tothe clinical heterogeneity observed in propionic acidemia duetos ß-subunit defects.     

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.     

Differential regulation of V(D)J recombination during development of avian B and T cells

The lymphold immune system is comprised of two major cell types,B cells and T cells, originally identified in avian species.Although both lineages arise from hematopoletic stem cells,avian B cells require a period of development in the bursa ofFabricius while T cells undergo development in the thymus. Eachcell type expresses a lineage-specific antigen receptor encodedby genes created by the rearrangement of Individual membersof variable (V), diversity (D), and joining (J) gene segmentfamilies during embryonic development. In this report, we demonstratethat productive rearrangement of the TCR ß gene occursexciusively in the thymus during normal development. TCR ßrearrangements involving gene segments from the V_ß1gene family can be detected beginning on day 12 of development,while rearrangements involving the other family of V_ßgene segments, V_ß2, were first detected on day 14of embryogenesis. In contrast, productive rearrangements ofIg light (IgL) and heavy (IgH) chain genes were not restrictedto the bursa of Fabricius. Instead, V_H-DJ_H heavy chain rearrangementsand V_L-J_L light chain rearrangements were detected primarilyin the embryonic spleen, beginning as early as embryonic day10, even in birds bursectomized at 60 h of development. Withinthe spleen, Ig rearrangementwas confined to the subset of cellsthat express the chB6 surface protein. Unlike bursal lymphocytes,which express the recomblnase activating gene (RAG)-2 but notRAG-1, splenic B cell precursors also express RAG-1. The dataindicate that, while B cell precursors initiate recombinationprior to migration of the bursa of Fabricius, T cell precursorsundergo V(D)J recombination following migration to the thymus.Thus, distinct developmental mechanisms appear to regulate theprocess of receptor rearrangement during avian B and T celldevelopment.     

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.     

Violation of allelic exclusion of the T cell receptor {beta} genes in a helper T cell clone

The 4-hydroxy-3-nltrophenylacetyl coupled to chicken gamma globulinspecific and I-A^b restricted helper T cell clone IH4 carriesa V_{ß8 1}–D_ß2.1–J_ß2.3-rearrangementon one and a V_ß16–D_ß2.1–J_ß2.5-rearrangementon the other chromosome. Both rearranged ß genes aretranscribed and the products of both genes are expressed onthe cell surface. This result implicates the absence of allelicexclusion of the T cell receptor ß genes in this Tcell clone. The finding of a pseudoleader 5' to the functionalleader exon of the V_ß16 gene suggests that this geneis not efficiently translated and that the amount of V_ß16chains synthesized is not sufficient to activate the inhibitorymechanism preventing a second V_ß to D_ßJ_ßrearrangement.     

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.     

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.     

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.     

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.     

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.     

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.     

Rearrangement and expression of {chi} light chain genes can occur without {micro} heavy chain expression during differentiation of pre-B cells

The kinetics of light (L) chain gene rearrangement and expressionon mRNA and protein level has been studied with four stromalcell/IL-7 reactive, long-term in vitro proliferating pre-B celllines and clones, two from fetal liver of normal mice and twofrom fetal liver of E_µH-bcl-2 transgenic (bcl-2-tg) mice.These pre-B cell lines and clones are DJ_H-rearranged on bothH chain alleles. Two of the clones harbor H chain rearrangementswhich do not allow the expression of V_HDJ_H rearranged H chaingenes as _µH chain proteins. Upon removal of IL-7 fromthe pre-B cell cultures all four cell lines rearrange V_H-DJ_Hand V_L-J_L gene segments, loose the surface expression of c-kit,CD43, and surrogate light chain, as well as the capacity tobe clonable on stromal cells in the presence of IL-7. Pre-Bcells from normal mice die by apoptosis during differentiation,while those from bcl-2-tg mice do not. All four lines and clonesexpress comparable levels of mRNA for _µH and _µLchains with the same time kinetics during 3 days of differentiation.However, only two of the four pre-B cell lines and clones express_µH chain protein, whereas all four pre-B cell lines andclones express _µL chain protein at comparable levels between2x10⁵ and 1.40x10⁶ _µL chain molecules per cell. Theseresults suggest that _µH chain expression is not mandatoryfor rearrangement and normal expression of _µL chain geneswhen pre-B cells differentiate to B cells.     

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.     

T cell unresponsiveness correlates with quantitative TCR levels in a transgenic model

Two lines of transgenic mice were developed which differ intheir level of expression of V_ß11. To determine therole of TCR density in tolerance induction, these mice werebred with I-E expressing mice and were investigated for toleranceinduction. T cells expressing V_ß11 at high densityare deleted by negative selection. T cells expressing <10%of the normal TCR receptor density are not subject to negativeselection and were not activated in vitro. There is a correlationin receptor density in both strains of mice with in vitro activation.These findings support the notion that there is a definablequantitative signal threshold which is critical for toleranceinduction.     

Neonatal impaired response to viral superantigen encoded by MMTV(SW) and Mtv-7

MMTV(SW) is an exogenous mouse mammary turnor virus that codesfor a superantigen sharing the same V_ß specificityas Mtv-7 (Mis-1^a). Neonatal mice infected by suckling-infectedmilk show a deletion of the CD4⁺ V_ß6⁺ T cell subsetwithin 8 weeks. In contrast, adult mice infected by injectionof the virus in the footpad have a much faster deletion, whichoccurs within 2 weeks. In the present work, we investigatedpossible mechanisms for the different kinetics of deletion inthe adult and newborn mice. To find out if the route of infectioncould be responsible for this discrepancy, we infected 5-day-oldand adult mice by injection in the footpad. Our results demonstratethat the route of infection is not responsible for the delayedkinetics of reactive T cell deletion since newborn mice injectedwith the virus show similar kinetics to neonates infected bymaternal milk. To exclude differences in viral spreading betweenthe two models, we used a PCR assay to detect proviral DNA.Spreading of the virus was shown to occur at a similar rateor even more rapidly in neonates than in adults. We also comparedthe activation induced by MMTV(SW) or Mis-1^a spleen cells inthe draining lymph node in neonatal and adult mice and showedthat a poor local activation is induced in neonates comparedwith adults. In vitro, neonatal T cell reactivity to anti-V_ß6antibody was also impaired. Thus, the delay in clonal deletioncould be linked to impaired expression, presentation and/orresponse to the viral superantigen. Our results suggest thatthe initial response to MMTV(SW) could be of importance forthe kinetics of reactive T cell deletion.     

Interaction between T cells and murine acquired immunodeficiency virus superantigen: effect of second signal on T cell reactivity to the MAIDS virus superantigen

A murine acquired Immunodeficiency (MAIDS) virus transformedB cell line, B6-1710, was shown to express superantigen activityand stimulate T cell hybridomas to produce IL-2. However, Tcell clones and lines from which B6-1710 reactive hybridomaswere established failed to proliferate upon stimulation withB6-1710, while B6-1710 cells were capable of presenting bacterialsuperantigen to stimulate both T cell hybridomas and clones.Proliferative response of T cells to MAIDS virus superantigencould be detected by the addition of the pharmacological agentphorbol myristate acetate (PMA). Thus, B6-1710 seems to lacka stimulatory activity necessary for the prollferative responseof T cells to the MAIDS viral superantigen. In the analysisof the response of naive splenic T cells to the MAIDS superantigen,T cells recovered from a culture stimulated with B6-1710 cellsin the presence of PMA showed no dominant TCR V_ß chainusage, while cells recovered from a culture stimulated withB6-1710 alone were dominated by T cells expressing V_ß5,11, and 12 TCR chains. These results suggest that T cells bearinga majority of TCR V_ß chains are capable of respondingto B6-1710 superantigen. The avidity of interaction betweenT cells and viral superantigen may differ significantly amongT cells and those T cells exhibiting weak interaction with MAIDSvirus superantigen may require additional signals, such as PMA,for an in vitro proliferative response to B6-1710 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.     

Characterization of a new member of the human /-adaptin gene family from chromosome 22q12, a candidate meningioma gene

A 140 kb homozygous deletion from 22q12 In one menlngioma directedus towards the cloning and characterization of a new memberof the human ß-adaptln gene family (named BAM22).Adaptins are essential for the formation of clathrin coatedvesicles in the course of intracellular transport of receptor-ligandcomplexes. The BAM22 gene is totally inactivated In the tumorwith homozygous deletion. Northern blot analysis of 70 sporadicmeningiomas showed specific loss of expression in 8 tumors,suggesting inactivation of BAM22. Based on this, we proposeBAM22 as a second chromosome 22 locus Important in meningiomadevelopment, after the neuroflbromatosis type 2 gene.