Analysis of proteolipid protein (PLP)-specific T cells in multiple sclerosis: identification of PLP 95 116 as an HLA-DR2,w15-associated determinant

Multiple sclerosis (MS) is a putative autoimmune disease thatis linked with HLA-DR2,w15. Proteollpid protein (PLP) is a candidateautoantigen in MS, but the disease-associated epitopes havenot been determined. Using overlapplng and non-overlapping PLPpeptides, we have studied the T cell response to the major hydrophllicdomain PLP 85–159 in the peripheral blood of MS and healthysubjects (HS). Short-term T cell lines (TCL) were selected againsteach peptide using microwell plates and the frequency of peptide-specificTCL was estimated. PLP 95–116-specific TCL were most efficientlygenerated and the frequency was significantly higher in MS comparedwith HS (P <0.05). When compared between DR2,w15⁺ and DR2,w15^–MS, TCL frequency to PLP 95–116 was significantly higherin DR2,w15⁺ MS (P<0.005) and TCL reactive to the overlappingpeptide 105–124 were also increased in DR2,w15⁺ MS (P<0.025). Using DR gene-transfected L cells, we could show thatthe DRB1^*1501 product of the DR2 haplotype presents PLP 95–116to TCL selected against the pepide. These results imply thatPLP 95–116 represents a major epitope for the DR2,w15⁺MS.     

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes

The contributions of individual amino acids in the polymorphic β chain and the conserved chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the β₁ domain and 11 positions in the ₁ domain. The β₁-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR₁ domain were based on the sequences of other human and mouse class II chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the β₁-domain -helix caused substantial reduction in the responses of all the clones. Substitutions at multiple positions in the β₁-domain floor and in the ₁ domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the β₁ domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules. Human Immunology 40, 311–322 (1994)     

Activation of a myelin basic protein-specific human T cell clone by antigen-presenting cells from rhesus monkeys

This study addresses the capacity of peripheral blood mononuclearcells (PBMC) from rhesus monkeys (Macaca mulatta) to presentmyelin basic protein (MBP), a candidate auto-antigen for multiplesclerosis, to MBP-speclfic human CD4⁺ T cell clones. MHC-restrictlonof the human T cell clones was determined with HLA-DR-transfectedL cells, and epitope specificity was established with a panelof overlapping 20-mer peptides. The MHC-DR region of the rhesusmonkeys (Mamu) was characterized serologlcally and by sequenceanalysis. We identified one CD4⁺ HLADRB1*0301-restrlcted T_h1-llkehuman T cell clone (ES-BP8) that was activated to proliferationwith human or rhesus monkey MBP, or peptide MBP 29–48presented by PBMC from six different rhesus monkeys expressingthe Mamu-DRB1*0305 or -DRB1*0306 alleles. After transformationto continuous growth with Herpesvirus salmiri, the T cell clonecould still be stimulated by antigen (Ag) and Ag-presentlngcells (APC) from monkeys. Two other T cell clones with the sameHLA-restriction and the same peptide-specificity did not respondto MBP presented by these rhesus monkeys. The exon 2 sequencesHLA-DRB1*0301, Mamu-DRBV^*0305 and -DRB 1* 0306 differ at positions32, 47, 67, 73 and 86. These amino acid differences are notcritical for the binding of MBP 29–48 and do not abrogate-recognitionby the clone ES-BP8, but interfere with the recognition of thetwo other HLA-DRB1^*0301-restricted T cell clones. In conclusion,studying Ag-presentation from rhesus monkey may provide furtherinsight Into the Interaction of antigenic peptide, TCR and MHC.Furthermore, these results could form a useful basis for theIn vivo transfer of human auto-Ag-specific T cells into rhesusmonkey recipients.     

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.     

Definition of TCR recognition sites on Ld-tum complexes

The P911 variant of the P815 mastocytoma was shown by Lurquinet al. (Cell 58:293,1989) to elicit rapid tumor rejection ina syngeneic host. This rejection was mediated by L^d-restrictedcytotoxic T lymphocytes (CTL) for which targets could be sensitizedby the synthetic peptide designated tum^– (P91A^–.12–24).In a previous study, T cell clones specific for L^d-tum^–complexes displayed very restricted TCR usage and a characteristicTCR motif in the V CDR3 region, predicted to interact with peptide.However, in contrast to the majority of L^d peptide Uganda thatare nonamers, the tum^– peptide is a 13-mer and its sequencedoes not fit the Ld binding motif. Thus, to define shorter versionsof the tum^– 13-mer and residues involved in TCR recognition,nonamer derivatives were synthesized and compared in severaldifferent binding and functional assays. From these comparisons,the peptide TQNHRALDL was found to be the optimal nonamer. CTLrecognition of Ala-substituted analogues of this peptide indicatedthat the Hls and Arg residues at positions 4 and 5 are importantfor TCR contact We propose that these basic residues of thetum^– peptide interact with the previously defined acidicresidues in the CDR3 region of several TCR known to recognizeLd-tum^– complexes.     

Sharing of Four DR-β Sequence Motifs Between HLA-DRB1*1601 and DRB1*1101 Correlates with Frequent Degenerate T-Cell Recognition of HA306–320 Peptide Complexed to these Two Molecules

This paper shows that the seven HA306–320 specific T-cell clones isolated from one individual recognize the peptide complexed to both autologous HLA–DRB1*1101 and allogeneic HLA-DRB1*1601 (or DRB5*0201) molecules. For each T-cell clone, a single T-cell receptor (TCR) is involved in the recognition of these two different peptide-DR complexes as evidenced by cold target competition experiments. Yet, the seven T-cell clones express several different TCRs as judged by Vβ-Jβ usage and fine specificities. Furthermore, one representative clone has the same fine specificity for HA306–320 analogues mutated at epitopic residues irrespective of the use of DR1101 or DR1601 APC. These results suggest that structural differences between DRB1*1101 and DRB1*1601 (or DRB5*0201) do not dramatically influence the orientation of HA306–320 in the grooves such that most residues interacting with TCRs are conserved. In another individual, the same pattern of restriction, i.e. DR1101 + DR1601, was found for several HA306–320 specific clones. Two additional patterns, DR1101 + DR0801 and DR1101 + DR0801 + DR1601, were identified. By comparing DR sequences the authors found that DRB1*1101 and DRB1*1601 share four important motifs, i.e. β85–86, β67–71, β57 and β28–31 supposed to line three distinct HLA-DR pockets. Three of these motifs are also shared with DRB1*0801. All the results further support that the motif similarities allow the peptide to adopt very similar orientations in the cross-reacting DR molecules.     

Characterization of the HLA-restrictive elements of a rubella virus-specific cytotoxic T cell clone: influence of HLA-DR4{beta} chain residue 74 polymorphism on antigenic peptide-T cell interaction

The influence of glutamic acid (E)-alanine (A) dimorphism atposition 74 of the DR4ß chain on cytotoxic T cellrecognition of an antigenic rubella virus peptide, E1(273–284),was studied using a panel of B cell lines and B cell transfectantsexpressing different HLA-DRB1 alleles as antigen-presentingcells and targets in 51Cr-release assays. Only B cell linesexpressing the DRB1^*0403, DRB1^*0406 or DRB 1^*0407 subtypes whichshared a residue, E, at position 74 in the DR4ß chainwhen sensitized with E1(273–284) elicited strong cytotoxicT lymphocyte responses. However, in direct binding and antibodyinhibition assays, it was shown that biotinylated E1(272–285)could bind to DR molecules with residues other than E at position74, including DRB1^*0401, DRB1^*0404 and DRB1^*1101 expressed ontransfectants. E1(272–285) bound with similar affinityto the transfectant with DRB1^*0403, which has E at position74, as well as the transfectant with DRB1^*0404, which does not.When T-B cell engagement rates were compared in cell conjugateassays, the percentage of T-B conjugates was higher when peptide-pulsedtransfectants with DRB1^*0403 were used than with transfectantsexpressing DRB1^*0404. Hence, the HLA DRß1 polymorphismat position 74, while not critical for the binding affinityof E1(272–285) to the HLA molecule, appears to be a primarydeterminant of restricted recognition and subsequent activationof the peptide-specific T cells.     

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.     

Emergence of CD52 , glycosyiphosphatidylinositol-anchor deficient lymphocytes in rheumatoid arthritis patients following Campath-1H treatment

CD52 is a glycosylphosphatidyl-inositol (GPI)-llnked glycoproteinexpressed at high levels on normal T and B lymphocytes and atlower levels on monocytes, while being absent on granulocytesand bone marrow stem cell precursors. The emergence of CD52^– lymphocytes of both T and B cell lineages was observedIn three out of 25 rheumatoid arthritis patients treated withthe humanized antibody Campath-1H in phase II clinical trial.Whereas the majority of CD52^– B cells had disappearedfrom the peripheral blood by 3 months post-treatment, both CD52^–CD4⁺ and CD8⁺ T cells persisted in the circulation for at least20 months. In the two patients that were tested, the GPI-anchoredsurface molecules CD55 and CD59 were also absent on the CD52^–cells, although expression of other cell surface transmembraneproteins (CD3, CD4 and CD2) was unaffected. The CD52^–cells maintained a stable phenotype in vitro despite repeatedre-stimulation in culture. Both CD52^– and CD52⁺ clones,established from one of the patients, responded to a similarextent to several T cell mitogens, as assessed by proliferation,suggesting that a general defect in expression of GPI-llnkedmolecules does not impair T cell activation. These data showthat an immune attack against a GPI-anchored surface moleculecan result in the selection of a GPI-anchor-deficient cell population.Despite the persistence of CD52^– T cells in the peripheralblood, no adverse reactions associated with the presence ofthese cells were noted in any of the patients; in fact theyresponded with longer remission times after Campath-1H treatmentthan the other patients in the trial. Received 16 May 1995, accepted 27 November 1995.     

Development of retrovirally marked human T progenitor cells into mature thymocytes

Retroviral vectors have been used in most human gene therapytrials that have been undertaken. Many of these therapies havefocused on the introduction of genes into hematopoietic stemcells with the goal of obtaining expression in the mature Tlymphocytic progeny. It has proven difficult to achieve expressionin the lymphoid lineage, although several groups have demonstratedlow expression of transduced genes in the myelold lineage. Inthis study we used an in vitro thymic organ culture in whichstem/progenitor cells can develop into T cells and all intermediatestages can be studied and manipulated to investigate the fateof a retrovirally introduced Escherlchla coll LacZ gene in thissystem. Here we show that certain conditions can transduce JurkatT cells, three different antigen-specific T cell clones andCD34⁺CD3^–CD4^–CD8^– thymocytes (progenitor Tcells) with high (>80%) efficiency. Moreover, retroviraltransduction with the LacZ gene does not inhibit T and NK celldifferentiation of progenitor cells in fetal thymic organ cultures(FTOC). The LacZ gene also is functionally expressed at allstages of development, although the expression decreases somewhatduring differentiation. This experimental system, combiningFTOC and retroviral transduction, provides a genetic tool forthe study of human T cell development.     

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ({alpha}1*0501, {beta}1*0201) molecule

Peptide binding to DQ molecules has not previously been described.Here we report a biochemical peptlde-blndlng assay specificfor the BQ2 [I.e. DQ(1^*0501, ß1^*0201)] molecule. Thismolecule was chosen since It shows a strong association to diseasessuch as celiac disease and insulin-dependent diabetes mellitus.Initially we radlolabelled some selected peptides and testedthem for binding to affinity-purified DQ2 molecules. One ofthe peptides, a Mycobacterium bovis (MB) 65 kDa 243–255Ypeptide, displayed a good slgnal-to-noise ratio and was thuschosen as an indicator peptide in the DQ2 binding assay. TheMB 65 kDa 243–255Y peptide bound to DQ2 In a strictlypH-dependent fashion, with optimal binding around pH 5 and onlyweak binding at pH 7.4. The association of the MB 65 kDa 243–255Ypeptide to DQ2 was slow, but once formed, the peptide-HLA complexeswere very stable. The binding of peptides to DQ2 was specific,as shown in Inhibition experiments with a panel of 47 peptides,differing in length, sequence, and origin. The binding of peptidesto DR3 was tested in a similar assay with a Mycobacterium tuberculosis65 kDa 3–13 peptide as the binding indicator. DQ2 andDR3 molecules bound to different sets of peptides. However,the peptide binding to DQ2 and DR3 showed, In general, similarcharacteristics with respect to pH dependence and kinetic parameters,Indicating that the overall rules for peptide binding to DQmolecules are the same as those previously shown for human DRand murlne I-A and I-E molecules.     

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

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

Proliferation and apoptosis of B220+ CD4 CD8 TCR{alpha}{beta}intermediate T cells in the liver of normal adult mice: implication for Ipr pathogenesis

Small numbers of T cells have been isolated from the normalmouse liver and many of these are of the CD4^–CD8^–TCRß⁺phenotype. Larger numbers of such cells are present in the liversof mice homozygous for the Ipr mutation and the liver has beenproposed to be the site of an extrathymlc T cell developmentpathway that is expanded in Ipr/lpr mice. Using a modified separationprocedure that increases the liver T cell yield, we have beenable to characterize a subset of CD4^–CD8^–TCRß^intermediateT cells that express the B220 epltope of the CD45 molecule,and resemble in this and many other ways the accumulating Tcells in Ipr lymph nodes. These cells are an actively dividingpopulation and even in healthy, unmanipulated mice a large proportionof them are undergoing apoptosis. We propose the model thatthe normal liver is a major site for T cell destruction andthat the Ipr defect results in failure of this process withleakage of B220⁺CD4^–CD8^–TCRß⁺ cells fromthe liver to peripheral lymphoid tissues, particularly lymphnodes.     

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.     

Functional expression of a human TCR{beta} gene in transgenic mice

A functionally rearranged TCRß (Tcrb) gene was isolatedfrom a cloned human T helper cell recognizing the CS.T3 epitopeof Plasmodium falciparum with HLA-DR2. Transgenic mice weregenerated by co-injection of the human gene together with themouse Tcrb enhancer. Analysis of transgenic mice shows thatthe functional Tcrb gene of xenogenic, i.e. human, origin exertsallelic exclusion of endogenous Tcrb genes. Cytofluorometricanalysis revealed expression of the human TCRß chainon virtually all thymocytes and peripheral T cells togetherwith endogenous TCRß chains and CD3 components. Nosurface expression of mouse TCRß chain or rearrangementof endogenous Tcr genes was detectable. Expression of the hybridreceptor causes a reduction in the number of thymocytes anda bias for CD4⁺CD8^– T cells in the thymus as comparedwith non-transgenic littermates. Peripheral transgenic T cellsmount a normal prollferative response against allogenelc targetsin mixed lymphocyte reactions. These results show that a hybridmouse/human TCR is able to pass positive and negative selectionin the thymus, and is functional in transgenic mice.     

Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells

Exosomes are small vesicles (60-100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1-hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1-HA(306-318) complexes activated HA/DR1-specific T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.     

Abnormal methylation of imprinted genes in human sperm is associated with oligozoospermia

Genomic imprinting marks in the male germ line are already establishedin the adult germinal stem cell population. We studied the methylationpatterns of H19 and MEST imprinted genes in sperm of controland oligozoospermic patients, by bisulphite genomic sequencing.We here report that 7 out of 15 (46.7%) patients with a spermcount below 10 x 10⁶/ml display defective methylation of H19and/or MEST imprinted genes. In these cases, hypomethylationwas observed in 5.54% (1.2–8.3%) and complete unmethylationin 2.95% (0–5.9%) of H19 clones. Similarly, for the CTCF-bindingsite 6, hypomethylation occurred in 4.8% (1.2–8.9%) andcomplete unmethylation in 3.7% (0–6.9%) of the clones.Conversely, hypermethylation occurred in 8.3% (3.8–12.2%)and complete methylation in 6.1% (3.8–7.6%) of MEST clones.Of the seven patients presenting imprinting errors, two hadboth H19 hypomethylation and MEST hypermethylation, whereasfive displayed only one imprinted gene affected. The frequencyof patients with MEST hypermethylation was highest in the severeoligozoospermia group (2/5 patients), whereas H19 hypomethylationwas more frequent in the moderate oligozoospermia (2/5 patients).In all cases, global sperm genome methylation analysis (LINE1transposon) suggested that defects were specific for imprintedgenes. These findings could contribute to an explanation ofthe cause of Silver–Russell syndrome in children bornwith H19 hypomethylation after assisted reproductive technologies(ART). Additionally, unmethylation of the CTCF-binding sitecould lead to inactivation of the paternal IGF2 gene, and belinked to decreased embryo quality and birth weight, often associatedwith ART.     

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.     

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

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

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.