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.     

Plasmodium falciparum-specific T cell clones from non-exposed and exposed donors are highly diverse in TCR {beta} chain V segment usage

Humans lacking previous exposure to Plasmodium falciparum typicallyhave a high frequency of malaria-reactive T cells in peripheralblood, which cross-react with antigens from other microorganisms.We studied a large number of malaria-specific human T cell clonesfrom non-exposed and malaria-exposed donors to determine whetherthis response is oligoclonal, and might therefore be generatedby a limited number of cross-reactive epitopes. Most clonesresponded well to schizont antigen from three antigenicallydistinct stocks of P. falciparum. Clones derived from the samedonor tended to show similar patterns of reactivity to a panelof non-malaria antigens from various microorganisms, suggestingthat a limited number of epitopes were recognized by individuals.However, analysis of the usage of V segments of the ßchain of the TCR (TCRBV) revealed no evidence of TCRBV restrictionin the T cell response, either within individual donors or acrossall donors. An apparent skewing towards TCRBV8 in one donorwas shown by two methods to be due to in vitro expansion ofa single clone: (i) Direct sorting of TCRBV8⁺ CD4⁺ T cells fromfresh PBMC did not reveal any enrichment for pRBC-reactive cells;(ii) Sequencing of VDJ regions revealed that the TCRBV8 cloneswere identical. Sequences of non-TCRBV8 clones from this donorshowed major differences in the VDJ junctional region. No differencesin TCRBV repertoire between non-exposed and exposed donors wereobserved. These results exclude the existence of a malarialsuperantigen and suggest that the T cell response to malariaschizont antigen in non-exposed donors is driven by a largenumber of epitopes.     

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.     

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.     

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.     

Functional changes of intestinal intraepithelial lymphocytes during acute graft versus host diease: correlation with phenotype

While the pathological characteristics of acute graft versushost disease (GvHD) have been defined for many target organs,little attention has been paid to the functional changes oflymphocytes in target organs such as the liver and small intestine.We have shown previously, utilizing a nonlethal parent (C57BL/6J)into the F₁ (C57BL/6J x DBA2J F₁) GvHD model, that the intestinalmucosa is infiltrated exclusively by donor T cells with a CD8+phenotype during the first 3 weeks post-GvHD induction. Thepresent study investigated the functional changes associatedwith the phenotypic changes of intestinal intraepithelial lymphocytes(IEL) during the acute phase of GvHD. IEL displayed specificanti-host cytolytic activity during the second and third weekafter GvHD induction. In addition, enhanced cytolytic activity,detected in the presence of anti-CD3 antibody, was apparentduring the second through fourth week, with a peak in the thirdweek after GvHD induction. The IEL also showed enhanced proliferativeresponses to immobilized anti-CD3 during the first 2 weeks ofacute GvHD, while profound inhibition of proliferation was observedin the splenocyte population of the same animals. During weeks2 and 3 post-GvHD induction, the V_ß distribution ofhost IEL remained unchanged while the V_ß distributionof infiltrating donor CD8⁺ cells resembled that of the donorIEL population. In the small intestine, the increased cytolyticand proliferative activity of IEL during the course of the diseasemay provide a rationale for the involvement of this organ inthe pathology associated with GvHD.     

A monoclonal antibody to a determinant of the rat T cell antigen receptor expressed by a minor subset of T cells

A hybridoma producing the monoclonal antibody HIS42 was isolatedfrom a fusion between spleen cells from a BALB/c mouse immunizedwith rat thymocytes and the fusion partner SP2/0. This antibodyrecognizes a minor subset of T cells in every haplotype of rattested so tar. The subpopulation of HIS42 positive T cells containsboth CD4+ and CD8+ cells, in the same ratio as found in theperipheral T cell population. When bound to Sepharose beads,HIS42 induces T cell proliteration in the presence of Interleukin-2.In contrast to lymph node T cells, a number of thymocytes werefound to express HIS42 only in the cytoplasm or together withmembrane expression. Most bright HIS42 surface labelled thymocyteswere also positive for MRC OX-44, a marker predominantly identifyingmature thymocytes. SDS-PAGE analyses of the membrane moleculeslmmunoprecipitated by HIS42 show two bands on unreduced gels.One of these bands (85 kd) runs as two separate bands at 35and 48 kd on reduction. The other much weaker broad band ({smalltilde}100 kd) is hardly affected by reduced conditions. Takentogether these data suggest that HIS42 is directed against adeterminant on the rat T cell receptor for antigen, which iscommon to a small number of T cells.     

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.     

Production of IgG autoantibodies to TCRs in mice infected with the retrovirus LP-BM5

Autoantibodies (AAbs) directed against particular segments ofthe variable region of TCR ß chains occur in normalhumans and in certain autoimmune diseases, but the factors regulatingthe appearance of such antibodies are unknown. We report thatAAbs binding a peptide determinant corresponding to the CDR1of the V_ß domain are elevated in C57BL/6 mice followinginfection with the LP-BM5 murlne leukemia retrovirus mixture,a treatment used to induce murine AIDS. The elevation of thelevel of these AAbs is an early event following retrovlral infectionwhich corresponds in part to the general polyclonal activationof the B cells, but a selectivity for particular V_ßsequences is apparent later. This suggests that the appearanceof these antibodies may play a part in the subsequent developmentof immunodeficiency. Since the antibodies studied are of theIgG isotype, both T cells and B cells are involved in theirelaboration.     

Clonal unresponsiveness results from an interaction between staphylococcal enterotoxin B and T cells expressing unexpected V beta elements.

The ability of staphylococcal toxins to stimulate large numbers of T cells has led to their designation as a superantigen. Previous studies have indicated that activation of T cells bearing particular V beta elements may be responsible for the toxic effects of these bacterial products. However, the widespread expression of functionally similar proteins by unrelated bacterial species suggests the possibility that these products may represent a successful microbial strategy for subversion of the host antibacterial response. We have examined the effects of the staphylococcal enterotoxin B (SEB) on T cell clones that express different V beta elements. We note that SEB stimulates clones bearing previously defined V beta elements to proliferate and to produce cytokines. In addition, we demonstrate that an interaction between SEB and the TCR of clones that express additional V beta elements, including V beta 2 and V beta 6, results in a sterile form of immunological activation. This activation phenotype is characterized by proliferation without detectable cytokine production and is followed by profound immunological unresponsiveness in vitro and in vivo. We propose that reduced levels of antibacterial responses resulting from this form of T cell unresponsiveness may account for the highly conserved expression of superantigens by diverse bacterial species.     

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.     

Neonatal tolerance to MIs-1a determinants: deletion or anergy of V beta 6+ T lymphocytes depending upon MHC compatibility of neonatally injected cells.

Recent investigations in mice revealed that natural immunological tolerance to endogenous minor lymphocyte-stimulating locus 1a (MIs-1a) antigen correlates primarily with deletion of MIs-1a-specific V beta 6+ T lymphocytes in the thymus. Similar mechanisms account for acquired tolerance in some instances since the neonatal injection of MIs-1a-expressing MHC compatible cells in neonatal mice within the first 24 h of life causes clonal deletion of V beta 6+ T cells. Here we demonstrate that V beta 6+ T cells are not deleted in mice neonatally treated with MIs-1a spleen cells expressing allogeneic H-2 molecules. However, when such non-deleted V beta 6+ T cells were tested in vitro, no interleukin 2 (IL-2) secretion or proliferation was observed after MIs-1a stimulation. This non-responsive state could be overcome by addition of exogenous IL-2, consistent with the fact that V beta 6+ cells enlarged and expressed IL-2 receptors upon MIs-1a stimulation. Furthermore, the same neonatally treated mice showed in vitro functional unresponsiveness of cytotoxic T cells but not of IL-2-secreting cells specific for the tolerated allogeneic MHC antigens. Taken together, our data indicate that neonatal tolerance to MIs-1a can be accomplished by either clonal deletion or clonal anergy, and that it does not necessarily correlate with tolerance to MHC determinants.     

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.     

Mel14+CD4+ T cells from aged mice display functional and phenotypic characteristics of memory cells

Aging is accompanied by an increased fraction of memory CD4⁺T cells. Despite the fact that human memory cells have beenreported to produce high levels of IL-2, studies in mice andman indicate an age-related decline in IL-2 production. In thepresent study, we examined whether these conflicting resultsdepend on the activation pathway employed in a comparison ofphenotypically distinct CD4⁺ T cells from young and aged mice.Our data indicate an age-related decline in IL-2 productionby CD4⁺ T cells when the cells were stimulated with concanavalinA in the presence of accessory cells or the combination of immobilizedanti-CD3 and soluble anti-CD28. However, when CD4⁺ T cells wereonly stimulated with Immobilized anti-CD3, an age-related increasein IL-2 production was observed. This age-related increase inIL-2 could be attributed to the ability of CD4⁺ T cells fromaged mice to produce IL-4 on this stimulation, since anti-IL-4inhibited the IL-2 production In these cultures to levels foundwith cells from young mice. The addition of exogenous IL-4 greatlyenhanced the IL-2 production of CD4⁺ T cells from young miceto levels far beyond that of the aged counterparts, emphasizingthe dominant role of IL-4 In the induction of IL-2 stimulatedwith immobilized anti-CD3. No differences were observed in theactivation requirements of Mel14^– CD4⁺ T cells from youngand aged mice. However, Mel14⁺ CD4⁺T cells from aged mice werefunctionally and phenotypically more mature than their youngcounterparts, since they were capable of IL-2 and IL-4 productionin response to antl-CD3 without the need of CD28 triggeringand expressed Pgp-1 and ICAM-1 in a higher density. Our dataindicate therefore that Mel14 is not a stable marker for naiveCD4⁺ T cells and might not be appropriate to distinguish thesecells from memory cells.     

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.     

Non-random employment of V{beta}6 and J{beta} gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR {beta} chain rearrangements

We have studied the usage of V_ß36, D_ß, andJ_ß elements, and the composition of the CDR3 regionsof human fetal TCR ß chain rearrangements In a 17week old fetal thymus and In fetal liver, bone marrow, spleen,and cord blood at 11 and 13 weeks of gestation. These fetalsequences were compared with TCR ß chain rearrangementsobtained from post-partum thymus, adult spleen, and adult peripheralblood mononuclear cells. Both fetal and adult TCR V_ß6rearrangements exhibited a non-random usage of V_ßand J_ß elements. Up to 90% of the sequences obtainedat 11 weeks of gestation used J_ß2 elements, most notablyJ_ß2.1. In the 13 and 17 week old fetal and in theadult tissues, J_ß1 elements were used In 30% of therearrangements while, within the J_ß2 rearrangements,J_ß2.1 and J_ß2.7 were used most frequently.Both fetal and adult TCR ß chain CDR3 regions showednon-random usage of amlno acids. However, the early fetal repertoirewas further limited due to the relative absence of N-reglonsin up to 60% of the 11 and 13 week old TCR ß chainrearrangements, resulting In smaller antigen binding sites.In fetal and adult TCR ß chain rearrangements thedistribution patterns of the length of N-regions and the usageprofiles of J_ß elements were similar in hematopoletlcand peripheral organs, suggesting no apparent preference forparticular TCR ß chain rearrangements. On the whole,the data showed that both the available fetal and adult TCRV_ß6 repertoires are seemingly smaller than expectedon the basis of random usage of V_ß and J_ßelements and amlno acid composition of the CDR3 regions.     

Age-dependent alterations of the T cell repertoire and functional diversity of T cells of the aged

The aging immune system is characterized by the contraction of T cell receptor (TCR) diversity and the de novo expression of NK-related receptors (NKR) on oligoclonal T cells. NKR⁺T cells likely represent a secondary immune diversification as a biological adaptation of aging to ensure host defense despite shrinkage of the TCR repertoire. NKRs are expressed in various combinations even among TCR-identical cells, and are capable of triggering effector pathways in either TCR-independent or TCR-dependent fashion. Understanding the biology of NKR⁺ T cells will be pivotal to the development of strategies to enhance immunity in the elderly.     

Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults

In spite of thymic involution early in life, the numbers of naive CD4(+) T cells only slowly decline in ageing humans implying peripheral post-thymic naive CD4(+) T cell expansion. This proliferation may compensate for continuous activation and death of naive CD4(+) T cells but may also have negative consequences for protective immunity. Here we show that naive CD4(+) T cells that have proliferated in the periphery are characterized by a highly restricted oligoclonal TCR repertoire. Additionally these cells, which constitute the majority of naive CD4(+) T cells in the elderly, display signatures of recent TCR engagement. Our results demonstrate for the first time that peripheral post-thymic proliferation of naive CD4(+) T cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. This age-dependent deterioration of CD4(+) T cell immunity could entail ageing-associated autoimmunity, increased susceptibility to infection or cancer and decreased efficiency of vaccination.     

Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice

We hypothesize that regulatory T-cell (Treg)-deficient strainshave an altered TCR repertoire in part due to the expansionof autoimmune repertoire by self-antigen. We compared the Vβfamily expression profile between B6 and Treg-lacking B6.Cg-Foxp3^sf/Y(B6.sf) mice using fluorescent anti-Vβ mAbs and observedno changes. However, while the spectratypes of 20 Vβ familiesamong B6 mice were highly similar, the Vβ family spectratypesof B6.sf mice were remarkably different from B6 mice and fromeach other. Significant spectratype changes in many Vβfamilies were also observed in Treg-deficient IL-2 knockout(KO) and IL-2R KO mice. Such changes were not observed withanti-CD3 mAb-treated B6 mice or B6 CD4⁺CD25^– T cells.TCR transgenic (OT-II.sf) mice displayed dramatic reductionof clonotypic TCR with concomitant increase in T cells bearingnon-transgenic Vβ and V families, including T cells withdual receptors expressing reduced levels of transgenic V andendogenous V. Collectively, the data demonstrate that Treg deficiencyallows polyclonal expansion of T cells in a stochastic manner,resulting in widespread changes in the TCR repertoire.