{alpha}{beta} and {gamma}{delta} T cells in the immune response to the erythrocytic stages of malaria in mice

Mice lacking T cells with ß TCR (TCR ß–/–)or TCR (TCR –/–) were infected with the erythrocyticstages of the malaria parasite, Plasmodium chabaudi chabaudi(AS). Mice without T cells could control and reduce a primaryinfection of P. chabaudi with a slight delay in the time ofclearance of the acute phase of infection and significantlyhigher recrudescent parasitaemias compared with control intactmice. TCR –/– mice had higher levels of both serumIg and malaria-specific antibodies of the isotypes IgG3 andIgG1 compared with control mice. TCRß–/–mice, despite a striking increase in NK1.1⁺ cells and the presenceof T cells, were unable to clear their infection. Althoughthe plasma of TCR ß–/– mice containedall Ig isotypes before and during a primary infection, theywere unable to produce significant levels of malaria-specificIgG antibodies, suggesting that in the absence of ßT cells T cells are not able to provide efficient help forantibody production.     

Phenotypic and functional assessment of intraepithelial lymphocytes bearing a 'forbidden' {alpha}{beta} TCR

Differences in the surface antigen phenotype, such as the expressionCD8 as an homodimer or the lack of Thy-1, on Intestinal Intraepitheliallymphocytes (IEL) are related, In part, to alternative differentiationpathways. The relationship of IEL lacking the pan-T cell markerCD5 to these IEL, their TCR repertoire and function has notbeen examined directly. We explored the TCR repertoire and functionof the CD5^– IEL subset In relation to the expression ofthe ‘autospecific’ V_ß6 TCR in Mls-1^a miceand to TCR. The results indicate that CD5 expression was absenton the majority of TCR IEL (96.9%) and on a significant proportionof TCR ß IEL (25.0%). Virtually all IEL In DBA/2 (Mls-1^a)mice that expressed the ‘autospecific’ V_ß6TCR were CD5^–, and this correlated with the expressionof CD8 . To assess the functional capacity of this subset ofIEL, we examined proliferation and IL-2 production in responseto TCR activation. Although CD5^– IEL proliferated in responseto anti-CD3, IEL bearing TCR V_ß6, In Mls-1^a mice,were not responsive to TCR-mediated activation. Similarly, TCR IEL were not responsive to stimulation by anti-TCR antibodies.The addition of exogenous IL-2, however, reconstituted the prollferativeresponse of both TCR IEL and the TCR V_ß6 expressingIEL. We conclude that the lack of CD5 defines a unique subsetof intraepithelial T cells expressing either TCR or ßthat Include potentially autoreactive cells that remain anergicin the absence of IL-2.     

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.     

Peripheral lymphoid development and function in TCR mutant mice

We describe the development and function of the peripheral lymphoidsystem of mutant mice rendered deficient in either ßor T cells via targeting of TCR genes In embryonic stem cells.In the spleen of ß T cell-deficient mice, T cellsdo not compensate in numbers for the lack of ß Tcells, but B cells do. ß T cell-deficient mice areunable to mount an antibody response to ovalbumln and do notreject skin allografts. Natural killer cell function is notimpaired in any of the mutant mice. TCR mutant mice will proveuseful in dissecting differential functions of ßand T cells in vivo.     

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activateB-1 cells in vivo in normal mice and anti-red blood cell autoantibodytransgenic mice (HL mice). To assess the exact role of IL-5in proliferation and activation of peritoneal B-1 cells, weanalyzed IL-5 receptor chain-deficient HL (IL-5R^–/–x HL) mice generated by the cross between IL-5R^–/–and HL mice. In IL-5R^–/– x HL mice, Ig-producingB-1 cells in the peritoneal cavity were negligible, althoughthe total number of B-1 cells in the peritoneal cavity wereas many as 30% of that in HL mice. Moreover, LPS- or IL-10-induceddifferentiation of B-1 cells into antibody-producing cells wasseverely impaired in IL-5R^–/– x HL mice. We alsoused in vivo 5-bromo-2'-deoxyuridine labeling to estimate theproliferation of B-1 cells in IL-5R^–/– mice. Theabsence of IL-5R did not affect spontaneous proliferation ofperitoneal B-1 cells. However, induced proliferation of peritorealB-1 cells by oral administration of LPS was markedly impairedin IL-5R^–/– mice. These results suggest that IL-5is required for activation-associated proliferation of B-1 cellsbut not for their spontaneous proliferation and support theidea that IL-5 plays an important role on the induction of autoantibodyproduction from B-1 cells.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

CD2 expression on murine intestinal intraepithelial lymphocytes is bimodal and defines proliferative capacity

The CD2 molecule is normally expressed on nearly all murinelymphocytes, and is co-stimulatory in T cell activation viathe antigen receptor (TCR). A naturally occurring T lymphocytepopulation that is bimodal for CD2 expression was found in theintestinal intraepithelial lymphocytes (IEL). TCRß⁺IEL contain CD2^– and CD2⁺ cells of approximately equalproportion, while TCR⁺ IEL are predominantly CD2^–. Theproliferative response of IEL to stimulation with an anti-CD3mAb or with PMA plus ionomycin co-segregated with CD2 expression;the CD2⁺ subset proliferated vigorously under these conditionswhile the CD2^– subset was much less responsive. The respondingCD2⁺ IEL contained both TCRß⁺ and TCR⁺ cells. However,activation of the CD2^– IEL with anti-CD3 mAb resultedin only the expansion of TCR⁺ IEL, while activation with PMAplus ionomycin did not promote expansion of either the TCRß⁺or the TCR⁺ IEL. These findings parallel observations in theautoimmune lpr mouse, where massive numbers of peripheral TCRß⁺CD4^–CD8^–T cells that lack CD2 expression are also hyporesponsive tomltogenic stimulation. The apparent energy of CD2^–TCRß⁺IEL, as well as CD2^– T cells from lpr mice, demonstratesthat the absence of CD2 on TCRß⁺ T lymphocytes co-segregateswith nonresponsiveness.     

Maturation of medullary thymic epithelium requires thymocytes expressing fully assembled CD3-TCR complexes

Unlike meduilary thymic epithelial cells (TEC) of normal mice,meduilary TEC of TCR^– SCID mice are immature and disorganized.In order to assess directly the role of TCR⁺ cells in the developmentof medullary TEC, we bred mice which co-expressed the SCID geneticdefect and transgenes encoding clonotypic TCR chains. Immunohistologicexamination revealed that meduilary thymic epithelial cellsfrom TCRß transgenic SCID mice, whose thymocytes onlyexpress TCRß chains that inefficiently associate withCD3 and , components, remained immature and disorganized. Incontrast, meduilary TEC from TCRß transgenic SCIDmice, whose thymocytes express fully assembled CD3--TCRßcomplexes were mature and organized. Interestingly, the abilityof TCRß⁺-⁺-CD3³ thymocytes to induce maturation ofmeduilary TEC appeared not to be related to the antigen specificityof the TCR as thyml from positively selecting, negatively selectingand non-selecting TCRß transgenic SCID mice all possessedinduced meduilary thymic epithelial cells. In addition, we foundthat induction of meduilary TEC cells was associated with thepresence of meduilary thymocytes, including those of the CD4-CD8-TCRß⁺phenotype. The present findings demonstrate that fully assembledCD3--TCR complexes are required to induce maturation of meduilarythymic epithelial cells and indicate that thymocyte inductionof meduilary thymic epithelial cells may result from signalingindependently of their clonotyplc chains.     

Predominant expression of invariant V{alpha}14+ TCR {alpha} chain in NK1.1+ T cell populations

A novel T cell subset characterized by cell surface NK1.1⁺ TCRß⁺expression was investigated for its TCR usage, particularlythat of invariant V14 TCR, which was found to be preferentiallyused in peripheral CD4^–CD8^–T cells developed atextrathymic sites. We found that NK₊ ß T cell subsetsaccount for 0.4% in thymocytes, 5% in the splenic T cells and40.5% in the bone marrow T cells. Among these NK⁺ ßT cells, two distinct subsets were detected; cell surface TCRV14⁺and V14^– subpopulations. Almost all of NK⁺ ßthymocytes express V14 mRNA; however, only<20% were positive,while >80% were negative or undetectable for V14 TCR expressionon the cell surface in the thymus. Similarly,50% of NK⁺ ßT cells in spleen and bone marrow are V14⁺; as revealed by FACS.TCR repertoire analysis by nucleotide sequences on inverse PCRproducts demonstrated that most NK⁺ ß T cells expressan invariant TCR encoded by the V14J281 gene with a 1 base N-regionin all tissues. Thus, invariant V14 TCR is uniquely expressedon NK T cells, and can be a marker to distinguish NK, NK T andT cells.     

Defective IL-5-receptor-mediated signaling in B cells of X-linked immunodeficient mice

Murine (m) IL-5 induces proliferation and differentiation ofboth Ly-1⁺; B cells and activated conventional B cells. X-linkedimmunodeficient (XID) mice do not respond to thymus-independenttype II antigens, and have an abnormal response to a varietyof activation signals through Ig receptors, CD40 and cytokinereceptors. Furthermore, XID mice show a B cell specific defect,reflected in decreased numbers of IL-5R⁺ B cells and reducedresponsiveness of IL-5R⁺ B cells to mIL-5. We generated IL-5Rtransgenic (5R-Tg) mice in which B cells expressed recombinantIL-5R. We crossed male 5R-Tg mice with female XID mice and usedtheir offspring to determine the IL-5 responsiveness of theseB cells. All B cells of F₁ male mice carrying the xid gene togetherwith the transgene expressed the recombinant IL-5R. However,those mice lacked Ly-1 B cells and their B cells acquired responsivenessto mIL-5. Interestingly, XID-5R-Tg B cells, but not XID B cells,acquired mIL-5 proliferatlve and Ig-secretory responsivenessonly in the presence of suboptimal doses of Ilpopolysaccharide.Stimulation of these B cells with mIL-5 plus phorbol myristateacetate induced proliferation, but not Ig secretion. These resultsindicate that the impaired mIL-5 responsiveness of B cells inXID mice is due to an abnormality of IL-5R-mediated signalingwhich may correlate with the xid gene mutation, alteration ofa single amino acid of Bruton's tyroslne kinase.     

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.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Alteration of a polyclonal to an oligoclonal immune response to cecal aerobic bacterial antigens in TCR{alpha} mutant mice with inflammatory bowel disease

Since lumenal bacteria have been postulated to play an Importantrole in the pathogenesis of inflammatory bowel disease (IBD),we investigated the humoral response to cecal aerobic bacterialantigens by Western blot analysis in TCR⁺ mice which spontaneouslydevelop IBD. The sera from TCR⁺ mice revealed an alterationof the recognition pattern against aerobic bacterial antigensfrom polyclonal to oligoclonal with age. This alteration wasnot observed in TCR⁺ and TCR⁺ mice. The alteration of the recognitionpattern in TCR⁺ mice was associated with production of autoantibodiesagainst tropomyosin and the development of IBD. The unique populationof CD4⁺ TCR^–ß⁺ cells in TCR⁺ mice may be involvedin the recongnition of these bacterial antigens and the absenceof the chain may result in the alteration of immune response.     

T cell receptor-triggered activation of intraepithelial lymphocytes in vitro

Intraepithelial lymphocytes (IEL) of the mouse small intestinewere examined for their potential to respond to TCR signallingin vitro. Purified IEL subsets were activated using mAbs specificfor CD3, TCRßor TCR&. Thy-1⁺IEL, regardless ofTCR type, proliferated equally well in response to anti-TCRmAb with or without exogenous IL-2. In contrast, Thy-1^–TCR, CD8^– IEL required exogenous IL-2 for proliferation.No such requirement was observed for Thy-1^– TCR& IELproliferation. IEL proliferation in the absence of added IL-2was due to an IL-2 secretion/IL-2 receptor (IL-2R) autocrinepathway, since mAbs specific for IL-2 and IL-2R inhibited IELproliferation. Thy-1⁺ CD8ß^– CD4⁺CD8⁺ IEL wereunresponsive to TCR-induced proliferation but exhibited highlevels of cytolytic activity upon TCR-triggerlng. Thy-1^–non-cytolytic IEL were induced to express Thy-1 and cytolytlcactivity following activation in vitro. In addition, the involvementof the co-stimulatory molecule CD28 in IEL activation was tested.CD28 was weakly expressed by fresh IEL and anti-CD28 mAb hadno effect on TCR-triggered proliferation. However, anti-TCRstimulation increased CD28 expression on a subset of TCRßIEL and the addition of anti-CD28 mAb resulted in increasedIL-2 production, but not in increased proliferation. Our resultsindicate that IEL, including the purported extrathymlc CD8ß^–subset, can respond to TCR-driven signals via proliferationand/or cytolytlc activity.     

TCR-mediated hyper-responsiveness of autoimmune Galphai2(-/-) mice is an intrinsic naïve CD4(+) T cell disorder selective for the Galphai2 subunit

Heterotrimeric Gi signaling regulates immune homeostasis, sinceautoimmunity occurs upon disruption of this pathway. However,the role of the lymphocyte-expressed Gi subunits (Gi2 and 3)on T cell activation and cytokine production is poorly understood.To examine this role, we studied T lymphocytes from mice deficientin the Gi2 or Gi3 subunits. Gi2^-/- but not Gi3^-/- splenocyteswere hyper-responsive for IFN- and IL-4 production followingactivation through the TCR. Gi2^-/- T cells had a relaxed costimulatoryrequirement for IL-2 secretion and proliferation compared towild-type cells. Purified naïve Gi2^-/- T cells producedmore IL-2 than naïve wild-type T cells following TCR activation,indicating that the hyper-responsive cytokine profile was notdue to the expanded Gi2^-/- memory T cells, but involved an intrinsicT cell alteration. Cytokine hyper-responsiveness was not seenwhen purified Gi2^-/- T cells were stimulated with phorbol myristicacetate/ionomycin, localizing the alteration to a proximal TCR-specificsignaling pathway. Gi2^-/- CD4⁺ T cells were distinguished fromwild-type or Gi3^-/- T cells by a globally augmented TCR-inducedcalcium response. These findings indicate that Gi2^-/- mice havean intrinsic CD4⁺ T cell abnormality in TCR signaling whichmay be one cause of augmented T cell effector function and Gi2^-/-autoimmune susceptibility.     

CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2 / mice in the absence of TCR {beta} chain expression

Recent studies have shown that TCR ß chain expressioncan effect the differentiation of CD4^–CD8^– double-negative(DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes.The TCR ß chain is expressed on the surface of DPthymocytes in association with CD3, and chains, suggestinga potential role for CD3 components in this signaling process.We now report detection of a very tow level of surface expressionof CD3 on adult DN RAG-2^–/–; thymocytes. This surfaceCD3 was associated with CD3 and chains, as detected by anti-CD3immunoprecipitation analyses. Significantly, injection of anti-CD3mAb into RAG-2^–/– mice led to the accumulation ofan IL-2R^– CD2⁺ DP cell population and a nearly 100-foldincrease in thymic cellularity to essentially normal levels.Together, these data strongly indicate that TCR ßchain-mediated developmental signals are transduced by CD3 componentsand provide potential insights into mechanisms by which TCRß chain expression may effect this process.     

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

The mature TCR is composed of a clonotypic heterodimer (ßor) associated with the invariant CD3 components (, , and ).There is now considerable evidence that more immature formsof the TCR-CD3 complex (consisting of either CD3 alone or CD3associated with a heterodimer of TCR ß and pre-T)can be expressed at the cell surface on early thymocytes. Thesepre-TCR complexes are believed to be necessary for the orderedprogression of early T cell development. We have analyzed indetail the expression of both the pre-TCR and CD3 complex atvarious stages of adult thymus development. Our data indicatethat all CD3 components are already expressed at the mRNA levelby the earliest identifiable (CD4¹⁰) thymic precursor. In contrast,genes encoding the pre-TCR complex (pre-T and fully rearrangedTCR ß) are first expressed at the CD44¹⁰CD25⁺CD4^–CD8^–stage. Detectable surface expression of both CD3 and TCR ßare delayed relative to expression of the corresponding genes,suggesting the existence of other (as yet unidentified) componentsof the pre-TCR complex.     

Intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 are involved in protection mediated by CD3+TCR{alpha}{beta} T cells at the early stage after infection with Listeria monocytogenes in rats

To Investigate the significance of Intercellular adhesion molecule-1(ICAM-1) and leukocyte function-associated antlgen-1 (LFA-1)In host defense against infection with Intracellular parasites,we examined the effects of In vivo pretreatment with mAbs toICAM-1 (1A29) and LFA-1 (WT-1) on the protection against Infectionwith Listeria monocytogenesIn Fisher F344/N rats. Expressionof ICAM-1 and LFA-1 molecules on T cells In spleen, liver andperitoneal cavity of rats was down-regulated after i.p. administrationwith daily doses of 300 µg of either 1A29 or WT-1 for10 days. The survival rate of rats inoculated with viable Listeriawas significantly reduced byIn vivo pretreatment with 1A29 togetherwith WT-1 for 10 days but not by In vivo pretreatment with controlmAb. The numbers of bacteria In the spleen In rats pretreatedwith both 1A29 and WT-1 were significantly increased on day3 and day 6 after Infection with 1 x 10⁷ of viable Listeriacorresponding to 1/30 of LD₅₀ to normal rats. Thus, the resistanceagainst llsterial Infection was severely Impaired by combinationalpretreatment with mAbs In ICAM-1 and LFA-1. As shown In ourprevious report, the early appearance of CD3⁺TCRß^–T cells, presumably TCR cells, was evident In the peritonealcavity and liver of control rats at the early stage after llsterialInfection, while this was suppressed at this stage in rats pretreatedwith both 1A29 and WT1. These results suggest that the ICAM-1and LFA-1 adhesion pathway may be critically involved in protectlveroles of CD3⁺TCR_ß– T cells at the early stageof rat listeriosis.