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.     

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.     

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.     

Dysregulated expression of the IL-2 receptor {beta}-chain abrogates development of NK cells and Thy-1+ dendritic epidermal cells in transgenic mice

The IL-2 receptor ß-chain (IL-2Rß), a specificity-determiningsubunlt In the IL-2R complex with a restricted tissue distributionpattern, Is essential for signal transductlon. Our previousstudies demonstrate that the continuous treatment of mice withanti-IL-2Rß) resulted in the complete disappearanceof NK cells and Thy-1⁺ dendritic epidermal cells (Thy-1⁺ dEC),suggesting that signals through IL-2Rß are criticallyinvolved in development of these lymphocyte subsets. However,these lymphocyte subsets are reported to be apparently unaffectedIn the IL-2-deficient mice. To further examine the biologicalroles of the IL-2Rß, transgenic mice carrying theIL-2Rß transgene were generated. In these mice, highlevels of the cell surface expression of the IL-2Rßwere observed in essentially all hematopoietic lineage cells,and CD4⁺ T cells as well as CD8⁺ T cells showed vigorous cellproliferation upon IL-2 stimulation. Surprisingly, NK cellsmarked with a high expression of NK1.1 in the spleen and Thy-1⁺dEC in the skin were completely absent in transgenic mice. However,the development of other lymphocyte subsets Including conventionalßTCR ⁺ cells, TCR⁺ cells and B cells remained apparentlyintact. From these observations together with previous dataon IL-2-deficlent mice, we speculate that factors, other thanIL-2 that utilizes the IL-2Rß as its functional receptorsubunlt, may have a vital role in the development of NK cellsand Thy-1⁺ dEC. Implications for possible In vivo functionsof over-expressed IL-2Rß are discussed.     

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.     

Reactions of anti-immunoglobulin sera with synthetic T cell receptor peptides: implications for the three-dimensional structure and function of the TCR {beta} chain

The derived amino acid sequence of the human TCR ßchain shows considerable homology to lg light chains in itsvariable (V) and constant (C) domains, and in its joining segment(J). We assessed the cross-reactivity between TCR ßchains and lg light chains by synthesizing a set of nested,overlapping 16-mer peptides that duplicated the sequence thatcorresponds to the continuous VDJC sequence of TCR ßchain and determining the capacity of rabbit antisera to humanor murine lgs to react with these peptides. The reactivitieswe observed were consistent with homologies to and x lightchains. The strongest reactivity in ELISA binding and competitiveinhibition was with a peptide that corresponds to the ‘swtchpeptide’ of light chains. The sequence is encoded by theC-terminal region of the J segment (Fr4) and the N-termlnusof the C region. Other regions reactive with anti-light chainsera corresponded respectively to CDR1 and Fr3 segments of theV region, and a segment of the constant region predicted toloop out of the tight globular structure. The peptide immunochemicalresults, coupled with the identification of specific regionsof sequence correspondence between TCR ß and the characterized light chain Mcg, allowed us to develop a three-dimensionalmodel of the ß chain consistent with its role in antigenrecognition and response to superantigens.     

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.     

Early expression of Ig {micro} chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage

During B cell development, V-J rearrangements at the Ig heavyµ chain (IgH µ chain) locus occur in early cyclingprecursors (pro-B stage). Subsequently, rearrangements at theIg light (IgL) chain locus occur in late resting precursors(small pre-B stage). To study the effects of µ chain expressionon the rate of B cell development, purified hematopoletic stemcells (HSC) bearing a µ chain transgene or wild-type HSCwere transferred Into Immunodeficlent RAG-2^-/-mice and B celldevelopment was followed over time. In addition, cycling B cellprecursors were pulse-labeled by the Injection of BrdU intotransgenlc and wild-type mice, and the production of BrdU-labeledk⁺ and ⁺ B cells was followed over time. These experiments suggestedthat early expression of the µ chain from the transgenesignificantly shortened the duration of the pro-B stage andImmediately drove the precursors to differentiate into smallpre-B cells. By contrast, the presence of the transgene didnot affect the small pre-B stage, where IgL rearrangements occur.Thus, k and rearrangements occurred only after the arrest ofcell cycling as previously shown in wild-type mice, even whenthe µ chain is artificially expressed earlier in B celldevelopment.     

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.     

Transforming growth factor-{beta} inhibits IL-4 and IFN-{gamma} production by stimulated human T cells

The present study investigates the effect of transforming growthfactor (TGF-ß on the production of IL-4 and IFN- bythe leukemia T_h0 type cell line HUT78, by freshly Isolated humanT cells, and by antigen specific human T cell clones. We foundthat IL-4 and IFN-ß, but not IL-2, production by stimulatedHUT78 cells was inhibited by TGF-ß1. TGF-ß1also reduced the accumulation of IL-4 and IFN- specific mRNAin stimulated HUT78 cells. However, IL-2 and IL-7 co-stimulatedIL-4 and IFN- production, whereas IL-1, IL-3, IL-5, IL-6, IL-8,tumor necrosis factor- or granulocyte macrophage colony stimulatingfactor had no effect. Because IL-2 is an Important helper cytokinefor the production of IL-4 and IFN-, we investigated whethersignal transductlon through the IL-2 receptor is Impaired byTGF-ß1. We found that tyrosine phosphorylatlon inresponse to IL-2 In HUT78 cells was strongly inhibited by ashort prelncubatlon with TGF-ß1. Evidence for an antagonisticrole for TGF-ß1 and IL-2 comes from the finding thathigh doses of IL-2 could partially overcome TGF-ß1mediated inhibition of IL-4 and IFN- production. Similar toIts effect on HUT78 cells, TGF-ß1 also inhibited IL-4and IFN- production by freshly Isolated T cells as well as byhuman T cell clones. Taken together, our experiments show thatthe IL-2 dependent cytokines IL-4 and IFN- are both negativelycontrolled by TGF-ß under conditions where IL-2 productionIs unaffected by a mechanism which partially involves an inhibitionof IL-2/1L-2R signal transductlon. These data Identify TGF-ßand IL-2 as mutual antagonists in the regulation of IL-4 andIFN- production.     

A novel form of self tolerance dictated in the thymus of transgenic mice with autoreactive TCR {alpha} and {beta} chain genes

Transgenic (TG) mice with TCR and ß chain genes froma CD4-dependent auto-l-A^k reactive T cell clone were generated.H-2^k TG mice had a large number of thymic and splenic CD4 Tcells expressing the autoreactive TCR without manifestationof autolmmunlty. The cells were not anergic, as they could respondto autologous antigen presenting cells and antl-TCR antibodiesin vitro to proliferate and to produce interleuklns. Variousdegrees of down-regulation of CD2 and CD44 was observed in TGmice, Indicating the presence of a defective co-stlmulatoryprocess in TG T cells. These features indicate that the selftolerance in autoreactive TCR TG mice is due not to clonal deletionand anergy but to a novel mechanism where T cells cannot sufficientlyrespond to normally existing self ligand in vivo. That suchan in vivo unresponsiveness of autoreactive T cells is dictatedin the thymus during CD4 T cell differentiation as an atypicalform of positive selection of autoreactive T cells was suggestedby the abnormal surface expression of CD69 and HSA.     

Intra- and inter-allelic ordering of T cell receptor beta chain gene assembly

Allelic exclusion at the TCRbeta locus mandates that gene assembly be regulated in a manner that permits feedback inhibition of further complete TCRbeta rearrangements upon pre-TCR expression. Here we show that assembly of TCRbeta chain genes from Vbeta, Dbeta and Jbeta gene segments is intra-allelically ordered, proceeding primarily through DJbeta, and not VDbeta, intermediates. This ensures that Vbeta to DJbeta rearrangement, which can be feedback inhibited, is the final step in the assembly process. A newly assembled VDJbeta rearrangement must be tested to determine if it is in-frame before Vbeta to DJbeta rearrangement is permitted on the alternate allele. This inter-allelic ordering may occur through a general inefficiency of Vbeta to DJbeta rearrangement and/or through static differences in accessibility of the two TCRbeta alleles. However, we find that within the regulatory context of allelic exclusion, Vbeta to DJbeta rearrangement proceeds to completion on both alleles. Furthermore, all possible VDJbeta rearrangements are not completed on one allele before Vbeta to DJbeta rearrangement is initiated on the alternate allele. Together, these data support a dynamic model of inter-allelic accessibility that permits the ordered and efficient assembly of complete variable region genes on both TCRbeta alleles during T cell development.     

Ligand-induced autoregulation of IL-2 receptor {alpha} chain expression in murine T cell lines

The IL-2 receptor (IL-2R) is composed of three chains a, ßand . In mice, contrary to the human system, we have previouslydemonstrated that the IL-2Rß complex does not bindIL-2. Therefore, mouse IL-2 response is completely dependenton the expression of the IL-2R gene product. T cell clones expressingmouse IL-2Rß and the human IL-2R transgene have beenstudied. When cells are grown in IL-4, mouse IL-2R is not expressed.However, exposure to IL-2 leads to the expression of the endogenousmurine IL-2R subunit. The T cell line expressing mouse IL-2Rand human IL-2Rß can grow in IL-2 but does not expressendogenous murine IL-2 R. Transfection of these cells with thehuman IL-2R gene restores the capacity to induce murine IL-2R.This result demonstrates that IL-2-IL-2R interactions are requiredfor induction of IL-2R. The kinetics of induction and deinductionof murine IL-2R have been studied using clone 18.III. From negativecells, expression of murine IL-2R is a very slow phenomenon.From cells fully expressing IL-2R, deinduction is a two-stepprocess: after a rapid decrease of IL-2R the cells continueto express, for a long period of time, basal levels of murineIL-2R. When cells expressing basal levels of IL-2R are exposedto IL-2, induction of IL-2R is a very rapid phenomenon. Theautoregulatory loop formed by IL-2-IL-2R therefore displaysdifferent levels of functioning.     

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.     

Role of oxidative damage and IL-1{beta}-converting enzyme-like proteases in Fas-based cytotoxicity exerted by effector T cells

The Implication of oxidative damage and/or intact mitochondrialfunction in physiological Fas-based cytotoxicity has been testedusing the cytolytic hybridoma d11S and the CD8⁺ CTL clone KB5.C20,previously stimulated to express Fas ligand (FasL) on theirsurface, as effectors and U937 or U937-p^° cells (depletedof mitochondrial DNA) as targets. Immobilized anti-Fas mAb,which induced death of U937 cells, Inhibited the growth of U937-p°cells but without inducing cell death. By contrast, FasL-expressingeffectors readily killed both targets, with induction of DNAfragmentation, in 20 h assays. These results demonstrate thelack of involvement of mitochondrial-derived free radicals and/orIntact mitochondrial function in physiological Fas-based cytotoxicity.Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas)with a polyunsaturated fatty acid, which induces cell deaththrough the generation of lipid free radicals, resulted in thepotentiation of Fas-based cytotoxicity. This potentiating effect,but not Fas-based cytotoxicity Itself, was eliminated by thephysiological antioxidant vitamin E. On the other hand, theIL-1ß-converting enzyme (ICE)-like protease tetrapeptideinhibitor Ac-YVAD-cmk partially Inhibited Fas-based cytotoxicity,while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was amuch more effective inhibitor of Fas-Induced apoptosis. It wasconcluded that Fas-Induced cytotoxicity was clearly dependenton ICE-LIke protease activation, and especially on that of CPP32in Fas-sensitive cells, including mitochondrial DNA-depletedones.     

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.     

Differential effect of transforming growth factor-{beta}1 on the activation of human naive and memory CD4+ T lymphocytes

Transforming growth factor-ß1 (TGF-ß1) canhave stimulatory or inhibitory effects on cell growth. For severalcell types, the effect of TGF-ß1 was found to correlatewith the differentiation stage of the cells and the presenceof other cytoklnes. We have studied here the influence of TGF-ß1on CD4⁺ T cell activation in relation to the differentiationstage of the cells by evaluating the effect of TGF-ß1on the prollferatlve responses of purified CD4⁺CD45RA⁺ (unprfmed)and CD4⁺CD45RO⁺ (primed) lymphocytes. Under certain conditions,TGF-ß1 exerted a co-stlmulatory effect on peripheralblood CD4⁺CD45RA⁺ T cells whereas the outgrowth of CD4⁺CD45RO⁺T cells was suppressed in any activation system tested. Theenhancement of prollferatlve responses by TGF-ß1 inTCR/CD3 or CD2 stimulated cultures of CD45RA⁺ cells involvedup-regulatlon of CD25 expression and was dependent on the presenceof exogenous IL-2 or CD28 mAbs; IL-7 driven proliferatlve responseswere suppressed by TGF-ß1. These observations wereconfirmed in experiments with purified cord blood (CB) CD4⁺T cells inasmuch as addition of TGF-ß1 caused a 2-to 7-fold increase in IL-2 driven proliferatlve responses ofthese cells. Finally we show that, in contrast to the effectof TGF-ß1 during primary stimulation of CB CD4⁺ Tcells, TGF-ß1 suppressed T cell proliferation for40% in secondary cultures of these cell. Our findings indicatethat TGF-ß1 Is a blfunctlonal regulator of CD4⁺ Tcell growth in vitro, with co-stimulatory capacities duringCD45RA⁺ T cell mediated primary responses and growth suppresslveeffects during secondary responses of CD45RO⁺ T cells.