Establishment of resistance to Leishmania major infection in susceptible BALB/c mice requires parasite-specific CD8+ T cells

Although CD4⁺ T cells are generally accepted to be responsiblefor the determination of resistance to infection in experimentalmurine cutaneous leishmanlasis, a contribution of CD8⁺ lymphocytesto immunity can be demonstrated under certain well-defined conditions.Normally highly susceptible BALB/c mice can be rendered resistantto infection with Leishmania major promastigotes by a singleinjection of monoclonal anti-CD4 antibodies at the beginningof infection. Mice treated in such a way can heal their primarycutaneous lesions and acquire immunity to subsequent challengeinfection. Both the resolution of the primary infection andthe induced state of immunityto reinfection in these mice isshown to be dependent upon the anti-leishmanial effector functionsof CD8⁺ T cells. Furthermore, in contrast to control infectedBALB/c mice, which are unable to mount a delayed-type hypersensitivity(DTH) response to viable parasites, mice cured as a result oftreatment with anti-CD4 antibodies in vivo exhibit a strongDTH response, which can be significantly reduced by injectionof either anti-CD4 or anti-CD8 monoclonal antibodies prior toantigenic challenge with viable promastigotes. Moreover, increasednumbers of specific CD8⁺ T cells, able to transferLeishmania-specificDTH responses, were found in lymphold organs of BALB/c micerendered resistant to infection by immunointervention with anti-CD4monoclonal antibodies at the beginning of infection. Neutralizationin vivo of interleukin 4 during the course of infection in BALB/cmice also enables these otherwise susceptible mice to resolvetheir cutaneous lesions and to decrease the parasite burdenin infected tissues. CD8⁺ T cells are required for both of thesebeneficial effects. Taken together, these results indicate thatin the immune BALB/c mouse, as in the normally resistant CBAmouse, CD8⁺ lymphocytes are involved in the elimination of L.major and in the establishment and maintenance of immunity againstinfection with this parasite.     

O-Acetyl GD3 ganglioside in human peripheral blood T lymphocytes

O-acetyl GD3 ganglioside is a cell surface molecule of someneural, neural crest and renal cells. Here we show, by usingmAbs specific for O-acetyl GD3 (clone 27A) and flow-cytomotric,biochemical or immunological techniques, that it is also expressedat high intensity level on the surface of 49.6% (median) ofthe CD3⁺ cells (T lymphocytes), at medium level in 16.2% ofthe CD16⁺ (natural killer) cells, at very low level in 51.9%of CD14⁺ cells (monocytes) and in 6.9% of CD20⁺ cells (B lymphocytes),but not in other human blood cells. Of the CD4⁺or CD8^* cells,52.6 or 36.5% respectively were 27A⁺. Furthermore, 81.6% ofthe CD45RO⁺ lymphocytes carried the O-acetyl GD3 ganglloslde.It was not detected in the thymus, although its immediate precursor,the GD3 ganglioside, was present in the meduilary thymocytes,suggesting that O-acetyltransferases are regulated by maturationevents taking place in the periphery. The anti-O-acetyl GD3antibodies induced a strong mitogenic response in cultured peripheralblood mononuclear cells, but not in purified T cells. However,in combination with phorbol myristate acetate the antibodiesinduced proliferation also in purified T cells, suggesting thatprotein kinase C priming is needed for this effect. This andthe restricted expression of O-acetyl GD3 suggest a functionalrole for this ganglioside in T cell subpopulations.     

Chimaeric anti-CD4 monoclonal antibody cross-linked by monocyte Fcγ receptor mediates apoptosis of human CD4 lymphocytes

Previous studies have shown that murine anti-CD4 monoclonal antibody, cross-linked by rabbit anti-mouse immunoglobulin, could mediate apoptosis of murine CD4⁺ lymphocytes when they were stimulated by T cell receptor antibody. In this study, we have shown that the murine anti-CD4 monoclonal antibody, OKT4, can induce apoptosis in human CD4⁺ T cells stimulated by the recall antigen tuberculin purified protein derivative (PPD) only when cross-linked by rabbit anti-mouse immunoglobulin. The chimeric anti-CD4 monoclonal antibody, cM-T412 whose Fc fragment is human, was able to cause apoptosis without cross-linking by a second antibody. Similarly, abolition of PPD-induced proliferation of peripheral blood mononuclear cells by cM-T412 did not require cross-linking with rabbit anti-human immunoglobulin. Inhibition of proliferation by cM-T412 could be reduced by pre-treating monocytes with heat-aggregated human IgG. This suggested that monocyte Fcγ receptors might be cross-linking the human Fc of cM-T412. Propidium iodide staining together with immunofluorescence showed that the apoptotic cells were indeed CD4⁺ lymphocytes. It is proposed that during treatment with cM-T412 in autoimmune disease such as rheumatoid arthritis, cM-T412-coated CD4 T cells, when they are subsequently stimulated by the unknown arthritogenic antigen, may undergo apoptotic cell death through cross-linking of cM-T412 on Fey receptor-positive cells within the joint.     

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.     

Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

Novel mAbs reveal potent co-stimulatory activity of murine CD27

Members of the tumor necrosis factor receptor (TNFR) familyare emerging as important molecules implicated in the regulationof proliferation, differentiation and survival of T and B lymphocytes.Among these receptors is CD27, the function of which has thusfar only been studied in the human system, where it amplifiesthe T cell proliferative response induced by TCR triggering.We report here the generation of mAbs to murine CD27, by anefficient method involving the use of transfected Armenian hamsterfibroblasts. Previous analysis had already indicated that murineCD27 mRNA is uniquely expressed in lymphold cells. As determinedwith one of the newly developed antibodies, murine CD27 is expressedon the great majority of both ß and T lymphocytes,on a small population of peripheral B cells, and on a very smallsubset of B220⁺ cells in the bone marrow. This distributionlargely corresponds to that in the human system. However, unlikehuman CD27, which is primarily expressed in mature, medullarythymocytes, murine CD27 is found on all thymocytes, except asubset of CD4^–CD8^– precursors. Upon cross-linking,anti-CD27 mAb amplified the proliferative response of purifiedT lymphocytes to suboptimal stimulation with concanavalin Aat least 4-fold. This indicates that such mAbs can mimick llgandbinding and demonstrates that CD27 also acts as a potent co-stimulatorymolecule in the murine system     

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56ick in CD4 T cells from HIV-1 infected patients

Early in HIV infection, CD4⁺ lymphocytes exhibit the propertiesof an anergic state characterized by unresponslveness to mltogensor to TCR stimulation and by defective IL-2 production. As tyrosinephosphorylation is the earliest of the biochemical events initiatedby stimulation of CD3-TCR, we studied protein tyrosine phosphorylationin purified CD4⁺ lymphocytes from 25 asymptomatic seropositlvepatients (CD4 T cells >350/mm³) previously stimulated invitro by immobilized antl-CD3 mAb or by co-lmmoblllzed antl-CD3and antl-CD28 mAbs. Purified CD4⁺ lymphocytes from HIV-lnfectedpatients exhibited defective early protein tyrosine phosphorylationin response to CD3 activation when compared with normal subjects.This defect was observed mainly in patients in whom prollferatlveresponses to immobilized antl-CD3 ranged from 2 to 50% of controlvalues obtained in healthy donors, and was frequently associatedwith increased cellular levels of p59^fyn and decreased cellularlevels of p56^ick. Interestingly, these defects appeared to correlatewith the degree of impairment in thymidine incorporation. SinceCD28 mAbs have been reported to enhance prollferatlve responsesto the CD3–TCR pathway in cloned murine or human anergicmodels and to induce tyrosine phosphorylation in human T cells,we studied the role of CD28 mAb as a co-signal. Although antl-CD28co-stlmulatlon augmented the prollferatlve responses in bothcontrols and HIV-lnfected patients, It failed to correct thetyrosine phosphorylation pattern in the latter. Our resultssuggest a relationship between defective early protein tyrosinephosphorylation and impairment of proliferatlve responses inCD4 T cells from HIV-lnfected patients, and evidence is providedthat associated altered cellular levels of the fyn and Ick tyrosinekinases might play an important role in the anergic responseobserved early during HIV infection.     

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.     

Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells.

The CAMPATH-1H (CD52) antigen is a 21 000-28 000 MW glycopeptide antigen that is highly expressed on T and B lymphocytes and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The humanized CAMPATH-1H anti-CD52 antibody is extremely effective at mediating depletion of both normal and tumorigenic lymphocytes in vivo and has been used in clinical trials for lymphoid malignancy and rheumatoid arthritis. Cross-linking GPI-anchored molecules, including CD52, on the surface of T lymphocytes in the presence of phorbol 12-myristate 13-acetate or anti-CD3, results in cellular activation. In the present study we have investigated the functional effects of cross-linking CD52 on T and B tumour cell lines. Cross-linking CD52 on either a B-cell line, Wien 133, which expresses high levels of endogenous CD52 or Jurkat T cells transfected and selected to express high levels of CD52 resulted in growth inhibition. This effect showed slower kinetics and occurred in a lower percentage of cells than growth inhibition stimulated via T- or B-cell receptors. Growth inhibition of the Wien 133 line was followed by the induction of apoptosis, which appeared independent of the Fas/Fas L pathway. Wien 133 cells surviving anti-CD52 treatment were selected and cloned and found to have down-regulated CD52 expression, with a characteristic biphasic pattern of 10% CD52-positive, 90% negative by fluorescence-activated cell sorter analysis. Interestingly, surface expression of other GPI-linked molecules, such as CD59 and CD55, was also down-regulated, but other transmembrane molecules such as surface IgM, CD19, CD20, HLA-DR were unaffected. The present study and previous work show that this is due to a defect in the synthesis of mature GPI precursors. Separation of CD52-positive and negative populations in vitro resulted in a rapid redistribution to the mixed population. Injection of CD52-negative cells into nude mice to form a subcutaneous tumour resulted in a substantial increase in expression of CD52. These results suggest that the defect in the Wien 133 cells is reversible, although the molecular mechanism is not clear. These observations have relevance to the clinical situation as a similar GPI-negative phenotype has been reported to occur in lymphocytes following CAMPATH-1H treatment in vivo.     

Alternative complement pathway activation by CD4+ T cells of HIV infected individuals: a possible role in AIDS pathogenesis

The mechanisms by which CD4⁺ T cells are eliminated during HIVinfection are poorly understood. We have previously shown thatHIV infected cell lines activate and fix C3 via the alternativecomplement pathway (ACP). In the present study we examined theability of blood lymphocytes from 40 HIV⁺ individuals to fixC3. A large fraction of the CD4⁺ T cells reacted with anti-gp120antibodies. These cells also carried C3 fragments in vivo andcould further fix C3 if exposed to human serum In vitro. C3activation occurred via the ACP. In some cases exposure of thelymphocytes to human serum under conditions allowing ACP activationresulted in partial elimination of CD4⁺ T cells. The resultssuggest that complement activation and fixation by CD4⁺ T cellsopsonized with HIV particles or gp120 may contribute to theirselective destruction.     

Activation of human T cells through CD6: functional effects of a novel anti-CD6 monoclonal antibody and definition of four epitopes of the CD6 glycoprotein

The CD6 glycoprotein is expressed primarily on lymphocytes andconveys co-activating signals to T cells, but its exact functionand ligand(s) are unknown. A novel mAb, termed UMCD6, was demonstratedto recognize CD6 by immunoprecipitation, Western blotting, andreactivity with COS cells transfected with CD6 cDNA. UMCD6 wasmitogenic for T cells and was strongly synergistic with phorbolester in inducing T cell activation. UMCD6 enhanced the autologousmixed lymphocyte reaction as previously observed with anotheranti-CD6 mAb, anti-T12. The activating effects of UMCD6 weremore striking than those of other anti-CD6 mAbs and encompassedall of the diverse stimulatory properties previously reportedfor other anti-CD6 reagents. However, neither UMCD6 nor otheranti-CD6 antibodies alone or in combination with phorbol esteror IL-2 were able to induce thymocytes to proliferate. Stimulationby UMCD6 is dependent on accessory cell function in a mannernot accounted for simply by antibody crosslinking. UMCD6 didnot induce an increase in cytoplasmic free Ca²⁺, but the CD6activation pathway appears to involve protein kinase C. UMCD6and a panel of seven other anti-CD6 mAbs were used in a seriesof experiments to define four discrete epitopes of CD6 usingthe criteria of antibody cross-blocking, reactivity on reducedWestern blots, and resistance to controlled V8 protease digestion.The functional mAbs UMCD6, 2H1, and antl-T12 each recognizeda different epitope. Taken together, the results of these studiesstrongly reinforce the hypothesis that CD6 plays a significantand distinct role in T cell activation, and suggest that multipleregions of CD6 may be functionally active.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

Cellular basis of the resistance of newborn mice to the pathogenic effects of anti-CD3 treatment

We have analysed the mechanisms underlying the differences Inthe susceptibilities of adult and newborn mice to the pathogeniceffects of antl-CD3 mAbs. Our data show that the thymus cellnumber in adults is reduced by 93% 48 h after one single Injectionof 5 mg/kg of Ab whereas the same dose In newboms induces onlya 30% decrease. In the adult, this effect is associated witha marked depletion of CD4⁺CD8⁺ double positive (DP) cells andwith the appearance of important areas of cell necrosis in thethymic cortex. In newborns, the DP cells are less affected andthe thymic cortex does not present any cell necrosis even afteran injection of 45 mg/kg of mAbs. Pre-treatment of adults withanti-CD4 and antl-CD8 Abs, while completely abolishing the toxicside-effects induced by anti-CD3 mAbs, does not protect thethymus from the depletion of DP cells. In vitro, anti-CD3 mAbsinduce the proliferation of thymocytes and spleen cells fromadults but not from newborns. Tumour necrosis factor-alpha (TNF)Is found in the serum of adults 90 min after the injection ofantl-CD3 but is never detected In the serum of anti-CD3 treatednewborns. Taken together our data support the view that anti-CD3mAbs act by two different mechanisms. The first one resultsfrom the binding of anti-CD3 on the CD3+ thymocytes which Inducesa direct toxicity for only the CD4⁺CD8⁺ cells. The second oneresults from the activation of mature T cells which in turnproduce cytoklnes such as TNFa which could be responsible forthe toxic side-effects observed in adults. Our results thereforeindicate that the inability of the immune system of newbornsand of peripheral T cell-depleted adult mice to respond to anti-CD3mAbs protects them from the toxicity induced by these Abs inimmunocompetent mice.     

CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

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.     

Stimulation and proliferation of CD4+ peripheral blood T lymphocytes induced by an anti-CD4 monoclonal antibody

There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4⁺ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4⁺ cells were induced to proliferate and to synthesize interleukin 2 (IL2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL2-producing clones bearing either CD4 or CD8 on their surface. IL2 production was induced by mAb B66 in CD4+, but not CD8⁺, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4⁺ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4⁺ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.     

Impaired CD28-mediated co-stimulation in anergic T cells

We have investigated a CD28 co-stimulation in anergic T cellsin staphylococcal enterotoxin Binoculated mice by stimulatingthe cells with a plate-coated anti-TCR antibody in the presenceor absence of an anti-CD28 antibody. CD28 co-stimulation increasedthe levels of IL-2 and IL-4 mRNAs in nalve CD4⁺V_ß8⁺T cells. However, it did not increase the levels of IL-4 mRNAat all and only partially increased those of IL-2 mRNA in anergicT cells. It was demonstrated that CD28 co-stimulation was impairedso that it no longer stabilized cytoklne mRNAs in anergic cells.The levels of IL-4 mRNA in response to TCR stimulation werehigher in anergic T cells than those in nalve T cells in spiteof the defective CD28 co-stimulation in the former cells. Anergyinduction and generation of a T_h2-type immune response in vivoare discussed     

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.     

Virgin and memory T cells have different requirements for activation via the CD2 molecule

T cells can be divided into unprimed virgin (T°) and primedmemory (T') subpopulations by their expression of differentisoforms of the leukocyte common antigen. We have separatedthe CD4⁺ T cells into T° and T' subpopulations and examinedtheir capacity to respond to activation signals via the CD2receptor molecule. On stimulation with a mitogenic combinationof anti-CD2 antibodies, the T' population was inducd to expressIL-2 receptor, increased levels of the 4F2 antigen and to proliferate,whereas the response of the T° populations was reflectedsolely by a minimal increase in the 4F2 antigen. The additionof IL-2 or monocytes to T° cells stimulated with anti-CD2antibodies did not enhance their expression of the IL-2 receptoror proliferation. However, T° cells stimulated with thetried of anti-CD2 antibodies, monocytes, and IL-2 respondedwith high levels of IL-2 receptor expression and proliferation.The T° subpopulation could also be induced to respond whencultured with anti-CD2 antibodies and phorbol myristate acetate.The results suggest that in order to respond to stimulationvia the CD2 molecule, virgin T helper cells require additionalsignals that can be jointly provided by monocytes and IL-2.In contrast, memory T helper cells can be activated via CD2signal transduction alone.