In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

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.     

CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection

CD69, an ‘activation marker’ that is rapidly inducedon mature T cells after stimulation through the T cell antigenreceptor (TCR) was found to be expressed on 10% of normal thymocytes.All of these CD69⁺ thymocytes express ß TCR, and theyinclude both TCR^lowCD4⁺CD8⁺ and TCR^highCD4⁺CD8^– or CD4^–CD8⁺thymocytes. The CD69⁺ cells can be further segregated into heat-stableantigen (HSA)⁺TCR^*HSA^–TCR^high and HSA⁺TCR^high thymocytepopulations. None of CD69⁺ cells express the mature T cell markerQa-2. Thus CD69⁺ cells present in vivo appear phenotyplcallyto represent transitional cell populations between immatureTCR^lowHSA⁺Qa-2^– double-positive cells and mature TCR^highHSA^–QA-2⁺single-positive cells. In addition, TCR engagement by MHC moleculesis required for CD69 expression in the thymus. Taken together,the CD69 ⁺ thymocytes appear to represent the cells auditioningin positive selection process or they are the cells that havebeen positively selected recently. Analysis of a TCR transgenicmouse model revealed an increased number of CD69⁺ thymocytesin a positively selecting thymus, whereas no CD69⁺ transgenicTCR⁺ thymocytes were observed in the non-selecting thymus. Basedon the results of this study, we suggest that the surface expressionof CD69 serves as a useful marker to identify and trace thosethymocytes that are engaged in the TCR-mediated positive selectionprocess in the thymus.     

3H11, a unique cell surface molecule involved in the function of the CD45RA+subset of CD4+ cells

We have developed a mAb anti-3H11 by immunizing mice with aT cell line derived from the Callithrix Jacchus (common marmoset).anti-3H11 is reactive with 48% of unfractlonated T cells, 62%of CD4⁺ cells and 39% of CD8⁺ cells. Among CD4 cells, anti-3H11preferentially reacts with the CD45RA⁺ T cell subset. The majorityof helper activity for pokeweed mitogen (PWM)-driven B cellIgG synthesis and T cell response to recall antigen such astetanus toxold was found within the 3H11-CD4 cell population,whereas anti-3H11⁺CD4⁺ cells provided poor helper function forPWM-driven B cell IgG synthesis and were more responsive toconcanavalln A and autologous mixed lymphocyte reaction. Biochemicalcharacterization showed that anti-3H11 precipitated a singleprotein band with a relative molecular weight of 32,000 from¹²⁵l-surface labeled cell lysate. Biochemical, phenotyplc andfunctional studies revealed that the 3H11 molecule appearedto be different from previously established molecules on theT cell surface. Interestingly, addition of anti-3H11 to thecombination of CD4 and B cells in the presence of CDS cellsbut not to the combination of CD4 and B cells resulted in enhancementof the suppression of PWM-driven B cell IgG synthesis. Moreover,anti-3H11 had a co-mitogenic effect on T cells via the CD2 andCD3 pathways, and this co-mitogenic activity is restricted tothe CD45RA⁺ T cells. Taken together, our results show that the3H11 molecule is a novel antigen which may play an importantrole in the activation and function of the CD45RA⁺ subset ofT cells.     

Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice

CD4⁺ T cells in the mouse can be subdivided into two fractionsbased on the level of expression of the CD45RB determinant.Previous studies have shown that these subsets are functionallydistinct. We have further characterized the properties of thesesubpopulations in vivo by injecting them into C. B-17 scid mice.The animals restored with the CD45RB^highCD4⁺ T cell populationdeveloped a lethal wasting disease with severe mononuclear cellinfiltrates into the colon and elevated levels of IFN- mRNA.In contrast, animals restored with the reciprocal CD45RB^lowsubset or with unfractionated CD4⁺ T cells did not develop thewasting or colitis. Importantly, the co-transfer of the CD45RB^lowpopulation with the CD45RB^high population prevented the wastingdisease and colitis. These data indicate that important regulatoryinteractions occur between the CD45RB^high and CD45RB^lowCD4⁺T cell subsets and that disruption of this mechanism has fatalconsequences.     

Characterization of virus-primed CD8+ T cells with a type 1 cytokine profile

Infection with lymphocytic chorlomeningitis virus is associatedwith marked polyclonal activation of the CD8⁺ T cell subpopulation.In this report the cytokine production of virus-activated Tcells is analyzed and the producing cells subset is characterizedphenotypically. CoinciB. A. Askonasding with other parametersof cell-mediated immunity, splenic T cells appear which areable to release high amounts of IFN-, but not IL-5, IL-10 ortumor necrosis factor- upon short-term stimulation with anti-CD3in vitro. A similar profile is observed analyzing T cells takenfrom an inflammatory site. Phenotypically, the main cytokine-producingcell subset is found to be CD8⁺ cells targeted for homing toinflammatory sites (VLA-4^hiL-selectin^lo) of which 30–40%were positive by intracellular staining for IFN-. This subsetalso contains all T cells with a cytotoxic potential as measuredby redirected killing. An enhanced cytotoxic potential as wellas an increased capacity to produce IFN- is observed for atleast 2 months after infection and cell sorting analysis revealedthat this could be ascribed to a long-standing increase in thefrequency of CD8⁺Pgp-1^hi cells. Therefore, these results demonstratethat systemic virus infection may exert marked perturbationof the CD8⁺ T cell population resulting in generation of a long-livedsubset of primed cells with important effector potential.     

Activation of CD45-deficient T cell clones by lectin mitogens but not anti-Thy-1

CD45, the leukocyte-common antigen, Is a transmembrane proteintyrosine phosphatase uniquely expressed by cells of hematopoletlcorigin. We have developed CD4⁺ and CD8⁺ T cell clones that aredeficient in the expression of CD45 and have previously shownthat these cells fall to proliferate in response to antigenor cross-linked CD3. These studies have now been extended toshow that stimulation with antl-Thy-1, a mltogenlc signal forthe CD4⁺CD45⁺ and CD8⁺CD45⁺ T cells, falls to induce proliferationin the CD45^– T cells. Examination of the CD8⁺CD45^–T cells correlates antl-Thy-1 unresponslveness with a failureto increase in tyrosine phosphorylatlon. Furthermore, stimulationof CD8⁺CD45⁺ T cells with antl-Thy-1 results in an increasein p56^ick activity but not in CD8⁺CD45^– T cells. In contrastto the results with antl-Thy-1, both the CD4^+– CD45 andCD8⁺CD45^– T cells respond to treatment with lectin mitogens,concanavalln A or phytohemagglutlnln. Lectin-lnduced proliferationwas inhibited by the addition of cyclosporln A. Treatment ofCD45^– T cells with PMA and lonomycln also results in proliferationindicating that activation of protein kinase C in conjunctionwith an increase in intracellular calcium rescues the defectcafsed by CD45 deficiency. The data suggest that CD45 Is requiredfor the activation of tyrosine kinase activity Immediate orprior to transmembrane signaling.     

CD4+Thy1- thymocytes with a Th-type 2 cytokine response

We have identified a small subset of CD3⁺, CD4⁺, CD8^–thymocytes that do not express Thy1 (CD90). This Thy1^–subset represents 1–3.7% of the total number of thymocytesin a naive mouse. CD4⁺Thy1^– thymocytes express high levelsof CD3, intermediate to high levels of heat-stable antigen (HSA),and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They producehigh titers of IL-4 and no IFN- upon stimulation in vitro, aresponse characteristic of T_h2 cells. In the thymi of mice infectedneonatally with a high dose of the retrovirus Cas-Br-E MuLV,the frequency of CD4⁺Thy1^– cells increased ~10-fold. High-dosevirus infection resulted in decreased HSA and increased CD44expression on CD4⁺Thy1^– cells relative to cells from naivemice. CD4⁺Thy1^– cells from high-dose infected mice alsosecreted IL-4 and not IFN- upon in vitro stimulation. We previouslyreported that infection of newborn mice with a high dose ofmurine retrovirus results in the induction of a non-protectiveanti-viral T_h2 T cell response; CD4⁺Thy1^– thymocytes witha T_h2-like cytokine profile may play a role in determining thecytokine bias of this anti-viral response.     

Activation mediated CD4+ T cell unresponsiveness during acute Toxoplasma gondii infection in mice

infection of mice with Toxoplasma gondii has been shown to inducea transient state of immune down-regulation. Earlier reportshave demonstrated the role of cytokines, in particular IL-10,in this host response. Here evidence is presented that T.gondll,a major opportunistic pathogen of the newborn and those withAIDS, is able to induce CD4⁺ T cell population Increased involume by day 7 post-infection and expressed T cell maturationmarkers (CD44^hl, Il-2r^hl,Mel-14^fo). Further noted was a clonalactivation of several CD4+ T cells subsets expressing the V_ßchain of the TCR. At day 7 post-infection, partial reductionof all CD4⁺ T cells to mltogen or parasite antigen stimulationwas observed, In particular V_ß5 T cells. Additionof rlL-2 partially restored the CD4⁺ T cell proliferative responsein Vitro. The T cell activation marker CTLA-4 could not be detectedand te co-stimulatory molecule, CD28, was down-regulated. Elctrophoneticand morphologic analysis of these cells post0culture demostrateda DNA fragmentation pattern and cell death consistent with apoptosis.These studies demonstrate for the first time in a protozoanparasite that activation-induced CD4⁺ T cell unresponslvenessoccurs during actue T.gondll infection in mice, and may be importantin immune down-regulation and parasite persistence in the infectedhost.0     

Changes in the subsets of CD4 + T cells in Trypanosoma musculi infection: delay of immunological cure in young mice and the weak ability of aged mice to control the infection

After a 3 week course (approximately), during which there ismarked lymphoid hyperplasia, Trypanosoma musculi infectionsin young-adult mice are cured by an immune mechanism involvingantibodies of the IgG2a isotype. Both the lymphoid hyperplasiaand IgG2a antibody response are T-cell-dependent events andboth processes appear to be defective in aged mice. The purposeof the studies reported here was to elucidate the effects ofT.musculi infection on subsets of T cells for two reasons: (I)to gain insight into the probable roles of selected cytokines(IL-2, IL-4 and IFN-) in facilitating the production of curative,lgG2a antibodies, and (II) to examine the hypothesis that agingaffects the competence of CD4⁺ T cells to participate in immunologicalcontrol of infections. The major conclusions from these studiesare that: (I) T. musculi infection of mice induces rapid changein the CD4⁺ T cell population toward predominance of the activatedor memory (CD45RB^loCD44^hi) phenotype, cells which produce IFN-,II-3. IL-4 and IL-5, accompanied by profound Inhibition of IL-2production, and (II) in the old mice these changes are superimposedon the natural age-associated changes in the same direction(i.e. toward predominance of CD45RB^loCD44^hi T cells).Thus, inthe old animals, the combined changes of aging and infection,moving in the same direction, are devastating, resulting inthe aged animals being unable, or barely able, to control infection.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-{gamma}

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL)activity by T cells of aged mice in vitro, we initially assessedwhether IL-12 could overcome age-related deficits when givento aged mice in vivo. Growth of P815 (H-2^d) was enhanced inaged compared with young BALB/c (H-2^d) mice and tumor growthwas curtailed by IL-12 in both age groups. Unexpectedly, secondaryCTL stimulated ex vivo with P815 were reduced in IL-12-treatedmice compared with controls. Primary CTL generated ex vivo acrossMHC differences in IL-12-treated BALB/c and C57BL/6 young micewere reduced by 90–99%, were dose- and time-dependent,and were associated with reduced allo-stimulated NK-like activityand [³H]thymidine incorporation. IFN- was elevated in sera andin supernatants from allo-stimulated cultures from IL-12-treatedmice, while IL-4 was reduced in such supernatants, suggestingthat, despite reduced CTL, IL-12 was associated with increasedT_h1- and reduced T_h2-type cytokine production. IL-12 also inducedsplenomegaly, primarily due to increased numbers of cells lackingmarkers of mature T, B and NK cells, or macrophages, or polymorphonuclearleukocyte morphology. IFN- mutant mice exhibited reduced splenicenlargement in response to IL-12, suggesting that the splenomegalywas due, in part, to IFN- production. However, reduced CTL generationwas not due entirely to dilution of CTL precursor cells becausespleen cellularity and size increased 3-fold while CTL activitydecreased 10- to 100-fold, and CTL generation normalized toCD8⁺ T effector cells was still significantly reduced in IL-12-treatedmice. Interestingly, purified CD4⁺ and CD8⁺ T cells from IL-12-treatednormal mice exhibited greater proliferative and cytolytic activitiesrespectively compared with controls. Thus, effector T cellsin IL-12-treated mice were not impaired, but exhibited augmentedresponsiveness, suggesting that IL-12 induced complex interactionsamong spleen cell populations and that these effects, in part,are mediated by IFN-.     

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

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

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V_ß6⁺ T cell eliminationin the lymph nodes and thymus during Mls-1^a mouse ontogeny.Our results suggest that induction of tolerance to Mls-1^a antigensinvolves mechanisms other than clonal deletion of immature Tcells in the thymus. Mature CD4⁺CD8^– (CD4SP) T cells wereaffected by Mls-1^a antigens earlier than immature thymocytepopulations. Up to 2 weeks after birth, reduced frequenciesof V_ß6⁺ T cells were detected only in CD4SP cellsfrom the thymus and lymph nodes, and generation of CD4SP cellsin the thymus was blocked at least 1 week earlier than thatof their CD4⁺CD8^loTCR^hl immature precursors. The number of V_ß6⁺CD4SPT cells increased during the first 2 weeks of life and remainedconstant thereafter. We thus found no evidence of deletion ofmature V_ß6⁺CD4SP T cells, as the reduced frequenciesin adult mice can be attributed to the dilution of previouslygenerated cells in lymphoid organs of growing mice, which increasein cellulartty after birth. V_ß6⁺CD4⁺ T cells wereactivated in vivo shortly after birth, as shown by a selectiveincrease in IL-2 receptor a chain expression in the thymus andlymph nodes from day 0 to day 2 after birth. It is thereforelikely that endogenous expression of Mls-1^a antigen shortlyafter birth activates V_ß6⁺CD4SP T cells and rendersthem anergic. This process of tolerance induction may precedethe clonal deletion of immature T cells in the thymus, describedin the adult mouse.     

Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD+ T lymphocytes

Gangliosides are glycosphingolipids found ubiquitously on thesurface of mammalian cells. They contain a ceramide tail thatis inserted into the membrane and exposed carbohydrate and sialicacid moleties. The non-toxic B subunit oligomer (EtxB) of Escherichiacoli heat-labile enterotoxin (Etx) is a potent immunogen invivo and has profound modulatory effects on EtxB-primed lymphocytesin vitro, properties which are dependent on its ability to bindto GM1 ganglioside receptors. Here, it is shown that cross-linkingGM1 by EtxB causes a differential effect on mature CD4⁺ andCD8⁺ T cells from lymph node cultures proliferating in responseto an unrelated antigen, ovalbumin. Addition of EtxB to suchcultures led to the complete depletion of CD8⁺ T cells comparedwith enhanced activation of CD4⁺ T cells [as measured by expressionof CD25 (IL-2R)]. By contrast, addition of a mutant EtxB, EtxB(G33D),which does not bind to GM1, failed to trigger CD8⁺ T cell depletion.When EtxB was added to isolated non-immune CD8⁺ lymphocytesrapid (12–18 h) alterations in nuclear morphology andthe appearance of sub-G₀/G₁ levels of DNA were induced; propertieswhich are characteristic of cells undergoing apoptosis. EtxB(G33D)failed to trigger apoptosis, indicating that the induction ofthe apoptotic signal was dependent on the binding of GM1. Thesefindings provide an insight into the potent immunogenicity andimmunomodulatory properties of E. coli enterotoxins as wellas heralding a novel method for the selective induction of apoptosisin mature CD8⁺ T lymphocytes.     

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity

The proximal promoter of lck directs gene expression exclusivelyin T cells. To investigate the developmental regulation of thelck proximal promoter activity and its relationship to T celllineage commitment, a green fluorescence protein (GFP) transgenic(Tg) mouse in which the GFP expression is under the controlof the proximal promoter of lck was created. In the adult GFP-Tgmice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^– thymocytes, andthe majority of CD4^–CD8⁺ and CD4^–CD8^– [double-negative(DN)] thymocytes were highly positive for GFP. Slightly lowerbut substantial levels of expression of GFP was also observedin mature splenic T cells. No GFP⁺ cells was detected in non-Tlineage subsets, including mature and immature B cells, CD5⁺B cells, and NK cells, indicating a preserved tissue specificityof the promoter. The earliest GFP⁺ cells detected were foundin the CD44⁺CD25^– DN thymocyte subpopulation. The developmentalpotential of GFP^– and GFP⁺ cells in the CD44⁺CD25^–DN fraction was examined using in vitro culture systems. Thegeneration of substantial numbers of ß and T cells aswell as NK cells was demonstrated from both GFP^– and GFP⁺cells. However, no development of B cells or dendritic cellswas detected from GFP⁺ CD44⁺CD25^– DN thymocytes. Theseresults suggest that the progenitors expressing lck proximalpromoter activity in the CD44⁺CD25^– DN thymocyte subsethave lost most of the progenitor potential for the B and dendriticcell lineage. Thus, progression of T cell lineage restrictionin the earliest thymic population can be visualized by lck proximalpromoter activity, suggesting a potential role of Lck in theT cell lineage commitment.     

Non-obese diabetic (NOD) mice display enhanced immune responses and prolonged survival of lymphoid cells

We report that lymphoid cells originating from the non-obesediabetic (NOD) aftoimmune prone mouse strain are resistant toseveral signals known to induce programmed cell death. In vitroculturlng of lymphoid cells of splenic or lymph node originshowed that B cells and T cells of both CD4⁺ and CD8⁺ phenotypesfrom NOD mice display extended survival in vitro. By cytofluorimetrlcanalysis, Immature CD4⁺CD8⁺ NOD thymocytes were shown to partiallyresist in vivo treatment with corticosterolds. Finally, Immunizationwith protein antigens induced enhanced and prolonged Immuneresponses in NOD mice compared with normal C57BL/6, BALB/c,and C3H/TH control mice. We conclude that the NOD mouse displaysa defect in the mechanl8m(8) mediating programmed cell deathin T and B lymphocytes. These findings provide a novel explanationfor the B cell aberrations observed in the NOD mouse and mayhave Implications for the understanding of the aftoimmune pathogeneslsin this mouse strain.     

The sensitivity of IL-4 production for cAMP inducers is lost in CD4+ T cells from aged mice

CD4⁺ T cell clones have been demonstrated to display a differentialsensitivity for the induction of cAMP. In the present studywe investigated whether the differential sensitivity of CD4⁺T cell clones for cAMP inducers is also applicable to freshlyisolated phenotypically and functionally distinct CD4⁺ T cellsubsets that develop naturally in aging mice. Our results showthat the concanavalin A induced and anti-CD3 induced proliferativeresponse of CD4⁺ T cells from young mice is more sensitive forprostaglandin E₂ (PGE₂) and forskolin than that of their agedcounterparts, although the IL-2 production by these cells wasequally sensitive. In contrast, only a slight or no inhibitoryeffect of these cAMP inducers was found when the cells werestimulated with the combination of phorbol myristate acetateand lonomycln. In contrast to the findings obtained with T_n2clones, IL-4 production by freshly isolated CD4⁺ T cells wasinhibited by the cAMP inducers, whereas exogenous IL-2 had norestorative effect. However, the IL-4 production by CD4⁺ T cellsfrom aged mice was less sensitive than the IL-4 production byCD4⁺ T cells from young mice, although CD4⁺ T cells from agedmice showed significantly higher levels of intracellular cAMPin response to PGE₂. These higher levels of cAMP were relatedto the increased fraction of memory cells in aged mice: theMel-14^– Pgp-1⁺⁺ CD4⁺ T cells responded with at least 2-foldhigher levels of intracellular cAMP than the naive cells inyoung as well as in aged mice. Although memory CD4⁺ T cellsfrom young as well as aged mice responded vigorously to PGE₂by an enhancement of intracellular cAMP, only the IL-4 productionby cells from young mice was significantly inhibited. Therefore,it is not likely that the induction of cAMP is a major eventin the skewing of a primary response towards a T_h2 type of response.