Th1 and Th2 CD4+ T cell clones specific for Plasmodium chabaudi but not for an unrelated antigen protect against blood stage P. chabaudi infection

The host protective immune response to blood stage malaria infection was studied using Plasmodium chabaudi chabaudi (P. chabaudi) in NIH mice. It has been shown previously that CD4⁺ cells are critically required for protection against erythrocytic infection. Mice lacking a functional CD4⁺ cell compartment suffer unremitting patent primary parasitemias for at least 60 days after infection. Here, we report that the adoptive transfer of eight P. chabaudi-specific CD4⁺ T cell clones of either the T_h1 or T_h2 type to mice rendered CD4-depleted by adult thymectomy and anti-CD4 monoclonal antibody therapy fully restored the ability of recipients to control challenge infection. Control T_h1 and T_h2 clones specific for an unrelated antigen, ovalbumin, were unable to confer a comparable level of protection in CD4-depleted mice, even though they received regular doses of the antigen. These data demonstrate that protective immunity to asexual P. chabaudi parasites can be mediated through immune CD4⁺ T cell clones of either the T_h1 or the T_h2 subset.     

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.     

Frequencies of CD4+ T cells reactive with Plasmodium chabaudi chabaudi: distinct response kinetics for cells with Th1 and Th2 characteristics during infection

The functional heterogeneity of the CD4⁺ T cell response toPlasmodium chabaudi has been evaluated. Using a limiting dilutionassay system and a variety of assays to detect -interferon (IFN-),interleukln-2 (IL-2), IL-3, and T helper (T_h) cells for malaria-specificantibody production, the precursor frequencies of P. chabaudl-reactiveT cells have been calculated. The patterns of lymphokines producedby individual microcultures of the limiting dilution assay generallysupported the Idea of two functionally distinct CD4⁺ subsets:one which produces IFN- and IL-2 (T_h1) and one which Is an effectivehelper cell for antibody production (T_h2) However, it couldnot be determined whether the overiapping functions observedin some cultures represented T cells which could produce allfactors or separate clones which were developing In the samewells. During the first 14 days of an erythrocytic Infectionof P. chabaudi the predominant T cell response was of the T_h1-tupe.The frequency of these cells decreased after 14 days. By 3 weeksafter Infection the CD4⁺ T cell response was characterized byT_h2 cells, as defined by their ability to act as helper cellsin the production of malaria-specific antibody. These data supportthe hypothesis that early clearance of P. chabaudi may be antibody-Independentbut that the final clearance mechanism coincides with the appearanceof helper cells and antibody.     

Functional CD4+T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependentepitope of CD45 molecules(JH6.2) and one a natural thymocytotoxicautoantibody (NTA) with an unknown reactive epitope (NTA260),we subdivided splenic CD4⁺ T cells from 2-month-old BALB/c miceinto five phenotypically distinct subsets. CD45RC⁺NTA260^–(SI) cells were phenotypically analogous to CD4⁺ T cells predominatingin newborn mice and produced a significant amount of IL-2, butnot so IL-4, IL-10 or IFN- when stimulated with immobilizedanti-CD3 mAb in vitro. They appeared to consist mainly of naiveT_hP cells. The CD45RC⁺;NTA260⁺ (S II) subset also produced IL-2,but not other cytokines; however, the IL-2 levels produced weremuch higher than seen with the S I subset, thereby suggestingthe predominance of further maturated T_hP cells. The D45RC^–NTA260⁺(S III) subset mainly produced IL-4, IL-10, IFN- and less IL-2,and contained memory cells that helped the secondary antibodyresponse to a recall antigen, and hence contained T_h2 and probablya mixture of T_h0 and T_h1 cells. The CD45RC^–NTA260^–(S IV) subset was a poor responder to the immobilized anti-CD3mAb. The CD45RC^brightNTA260^dull(S V) subset consisted of a smallnumber of cells that were phenotypically analogous to activatedCD4⁺ T cells. While an age-associated decrease in the proportionof S I and less markedly in S II and in turn increase in S IIIsubsets of CD4⁺ T cells occurred in normal BALB/c mice, autoimmunedisease-prone (NZBxNZW)F₁ mice showed a marked age-associateddecrease in the proportion of not only S I, II but also IIIsubsets. As aged (NZBxNZW)F₁ mice carry CD4⁺ T helper cellsfor IgG anti-DNA antibody production, such age-associated polarizationto the S IV subset appears to be critical in the pathogeneslsof autoimmune disease in these mice.     

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-.     

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.     

Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

The onset of T_h1 immunity is in part regulated by genetic background.To elucidate the cell type carrying critical factors determiningthe T_h1 response, we employed Rag-2^–/– mice on Leishmaniamajor-susceptible BALB/c and -resistant B10.D2 backgrounds.By using bone marrow (BM) chimeras generated by the transplantationof B10.D2 BM cells into BALB/c-Rag-2^–/– mice, andvice versa, it was shown that hematopoietic cells carry factorsdetermining the disease outcome and T_h1 response against L.major infection. B10.D2-Rag-2^–/– mice reconstitutedwith BALB/c CD4⁺ T cells exhibited a T_h1 response and controlledL. major infection. Wild-type BALB/c mice inoculated with L.major-parasitized B10.D2-Rag-2^–/– splenocytes alsoexhibited a T_h1 response and a mild disease outcome, whereassuch a T_h1 response was not induced when CD11c⁺ dendritic cells(DCs) were depleted from parasitized B10.D2-Rag-2^–/–splenocytes. T_h1 response was reconstituted by the additionof L. major-parasitized B10.D2 DCs but not L. major-parasitizedBALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/–splenocytes. These results indicate that DCs determine the outcomeof the disease upon L. major infection.     

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.     

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production

Particular subsets of T cells expressing the NK1.1 antigen havebeen proposed to play an immune regulatory role by their fastand strong production of cytokines, in particular IL-4. We soughtto determine factors driving the functional differentiationof NK1.1⁺ T cells. Since NK1.1⁺ T cells are exquisitely sensitiveto IL-7 stimulation, we analyzed the development, selectionand IL-4 production of NK1.1⁺ T cells in IL-7 deficient mice(IL-7–/–mice). Besides a sharp reduction of allT cell subsets, NK1.1⁺ T cells develop at normal relative frequenciesin IL-7–/–;mice. They also undergo a normal selectionprocess, as revealed by the biased V_ß TCR repertoireidentical to the one in IL-7+/+ mice. However, NK1.1₊ T cellsfrom IL-7+/+ mice were found to be impaired in IL-4 and IFN-production in in vitro and in vivo models. In addition, IL-7was able to restore IL-4 production by NK1.1⁺ thymocytes fromIL-7–/– mice. Finally, IL-7 but not IL-4 was ableto maintain and increase IL-4 production by NK1.1⁺ thymocytesfrom normal mice. These data suggest that the functional maturationof NK1.1⁺ T cells requires a cytokine-driven differentiationprocess, in which IL-7 plays a major role.     

T cell subpopulation phenotypes in filarial infections: CD27 negativity defines a population greatly enriched for Th2 cells

Three-color flow cytometric analysis was used to define surfacemarkers which identify the T_h2-type CD4⁺ cells responsible forthe eosinophilia and elevated serum IgE typical of tissue invasivehelminth infections. A group of six mAba to well known cellsurface markers were screened for differential expression onCD4⁺ CD45RO⁺ lymphocytes from normal individuals (NL; n = 6)and filaria-infected patients (PT; n = 10). The majority ofmarkers were expressed equally by both groups, but the CD4⁺CD45RO⁺ cells in the PTs showed significantly higher levelsof expression of HLA-DR than those of NLs (P = 0.014). ThisCD4⁺ HLA-DR⁺ subpopulation was then studied further for itsexpression of an additional 10 activation and adhesion molecules.CD27 showed a trend for lower intensities of expression on PTCD4⁺ HLA-DR⁺ cells than on those of NLs. Analysis of the serumfrom both NLs and PTs revealed that PTs had significantly higherlevels of soluble CD27 and CD25 (IL-2R) In the serum than NLs(P < 0.01 and P = 0.022 respectively) indicating a generalstate of Immune activation and differentiation. Functional analysisof the CD4⁺ HLA-DR⁺ and the CD4⁺ CD27^– subpopulatlonsrevealed that the CD4⁺ HLA-DR⁺ cells produced significantlyhigher levels of IL-5 than the CD4⁺ HLA-DR^– cells (P <0.04), and the CD4⁺ CD27^– cells produced significantlyhigher levels of both IL-4 and IL-5 than the CD4⁺ CD27^–cells (P <0.05 and P <0.001 respectively). Thus, whilethe CD4⁺ CD27^– and CD27⁺ subpopulatlons contain T_h1 andT_h0 cells, only the CD4⁺ CD27^– population contains theT_h2 cells (producing both IL-4 and IL-5).     

Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

Effect of age on the expressed B cell repertoire: role of B cell subsets

Aged humans and experimental animals are impaired in their responsesto most foreign antigens although they produce greater amountsof autoantibodies. We have examined the effect of age on theproduction of antibodies to a prototypic foreign antigen, sheeperythrocytes (SRBC), and to a prototypic autoantigen, bromelain-treatedmouse erythrocytes (BrMRBC), in young and old ice before andafter immunization with SRBC. Old mice express more anti-BrMRBCplaqueforming cell (PFC) antibodies before and an even greaternumber after immunization with SRBC than young mice. Conversely,old mice produce far fewer anti-SRBC PFC than young mice followingimmunization with SRBC. We hypothesized that the differencesin the responses of old mice to BrMRBC and SRBC reflects differencesin the activity of CD5⁺ and CD5^– B cells. To test thishypothesis we immunized young and old mice with foreign antigensreported (and confirmed in our studies) to stimulate CD5⁺ Bcells [TNP-ficoll and phosphorylcholine - keyhole limpet hemocyanln(KLH)] or CD5^– B cells (SRBC and TNP-KLH). We found thatthe PFC response of old mice to antigens mediated by CD5⁺ Bcells was equal to or greater than that of young mice. In contrastthe PFC response of old mice induced by antigens mediated byCD5⁺ B cells was only 10% that of young mice. Thus it appearsthat the immune response of old mice is well maintained forantigens which elicit a CD5⁺ B cell response but not for thosewhich elicit a CD5^– B cell response. This conclusion issupported by our finding that a higher percentage of splenicB cells from old compared to young mice express that V_H11 lghgene family. This V_H gene family is preferentially expressedby CD5⁺ B cells.     

Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-{gamma} in CD4+ T cells

Immunization of BALB/c mice with killed Brucella abortus (BA)has previously been shown to Increase serum lgG2a levels andlong-term T cell clones from these mice secrete T_h1-associatedcytokines: IFN- and IL-2 but not IL-4 or IL-5. We analyzed cytokinegene expression following primary immunization with BA to determinewhen CD4⁺ T cells first express cytokine genes and whether specifichypothesized cytokine patterns (e.g. T_h precursor, T_h0) couldbe identified prior to a T_h1-like pattern. Our results demonstrateda highly consistent and novel pattern of T_h 1/T_h2 cytokine geneexpression characterized by elevated IL-10 and IFN- in CD4⁺T cells which rapidly manifests itself and is sustained forat least 10 days after immunization. No elevation in IL-2 cytokinegene expression was observed and treatment of BA-immunlzed micewith blocking anti- IL-2 antibodies had no effect on the cytokinegene expression pattern, although treatment with anti-IFN antibodiesresulted in increased IL-4, IL-5, and IL-9 cytoklne gene expression,In the absence of any change In IFN- or IL-10 as early as 4days after immunization. These results suggest that a wholepathogen may trigger sufficient costimulatory signals to rapidlyinduce effector T cells in the absence of elevated IL-2 andthat IL-10 Is specifically elevated in certain T_h1-like responses.     

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.     

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.     

B cells play a regulatory role in mice infected with the L3 of Brugia pahangi

Mice infected with the L3 of the filarial nematode Brugia pahangimake a strong T_h2 response characterized by elevated levelsof antigen-specific IL-4, IL-5 and IL-10. Here we show thatB cells from these animals are the major proliferating populationin vitro with depletion of B cells or infection of µMTmice, resulting in reduced levels of antigen-specific proliferation.B cells also act as antigen-presenting cells (APC) to CD4⁺ cellsas demonstrated by the switch in cytokine profiles upon B celldepletion. The efficiency of B cells in antigen presentationis attenuated by IL-10 which down-regulates the expression ofB7-1 and B7-2 on the surface of B cells both in vitro and invivo. Thus, IL-10 may modulate CD4 responses in L3-infectedmice by suppressing the expression of B7 ligands on B cells.In support of this hypothesis, blockade of the IL-10R in vivoresults in increased proliferation of CD4⁺ cells. We proposethat B cells participate in a negative feedback loop: IL-10elicited by infection with L3 and produced by B cells (and CD4⁺cells) down-regulates the expression of B7 molecules on theB cell surface, attenuating their efficiency as APC to CD4⁺T cells and restricting their expansion.     

The Xid defect determines an improved clinical course of murine leishmaniasis in susceptible mice

The course ofLeishmania majorinfection in B cell-defective BALB.Xidmice was investigated. Infected BALB.Xid mice showed a significantiyslower lesion development compared with BALB/c controls accompaniedby a 10– to 30– fold lower parasite burden in lymphaticorgans. The B cell immune response, as quantified by anti-leishmanialantibody production and B cell numbers in lymphatic organs,remained significantly lower in BALB.Xid mice as compared withBALB/c control mice. In accordance with disease development,CD4⁺ T cells from lymph nodes of infected BALB.Xid mice produced6– to 10– fold more IFN-; than the respective Tcells of BALB/c mice, when stimulated with leishmanial antigeninvitro. B cells from lymph nodes and the peritoneal cavitiesof BALB/c mice could be induced to produce 3– to 8–fold more IL-10 than the respective cells from B cell-defectiveBALB.Xid mice. The data thus indicate that the Xid mutationallows for the development of T_h1 cells which confer resistanceto infection withL. major. Moreover, the data suggest that Bcells contribute to susceptibility to L. major infection inBALB/c mice by skewing the T_h cell network towards a T_h2 phenotype.Since the difference in B cell-derived IL–10 productionbetween BALB/c and BALB.Xid mice was more prominent in peritonealB cells, the data support the notion that the skewing of theT cell response may be predominantly mediated by the B1 cellsubset.     

CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo'

The inducible co-stimulator (ICOS, CD278) is essential to theefficient development of normal and pathological immune reactions.Since ICOS-deficient mice have enhanced susceptibility to experimentalallergic encephalomyelitis (EAE), we have functionally analyzeda CD4⁺ICOS⁺ population comprising 6–15% of all CD4⁺ Tcells in secondary lymphoid organs of unmanipulated wild-typemice and checked for their ability to suppress EAE. In C57BL/6mice, CD4⁺ICOS⁺ cells were a major source of cytokines includingIFN-, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cellsshowed preferentially enhanced production of IL-4 or IL-10 butinhibited IFN- production. In contrast, CD4⁺ICOS^– cellsmainly produced IFN-. Interestingly, CD4⁺ICOS⁺ cells partiallysuppressed the proliferation of CD4⁺ICOS^– or CD4⁺CD25^–lymphocytes ‘in vitro’ by an IL-10-dependent mechanism.Furthermore, CD4⁺ICOS⁺ activated and expanded under appropriateconditions yielded a population enriched in cells producingIL-10 and T_h2 cytokines that also suppressed the proliferationof CD4⁺CD25^– lymphocytes. CD4⁺ICOS⁺ cells, before or afterexpansion in vitro, reduced the severity of EAE when transferredto ICOS-deficient mice. In the same EAE model, lymph node cellsfrom ICOS-deficient mice receiving ICOS⁺ cells showed reducedIL-17A production and enhanced IL-10 secretion upon antigenactivation in vitro. Thus, naturally occurring CD4⁺ICOS⁺ cells,expanded or not in vitro, are functionally relevant cells ableof protecting ICOS-deficient mice from severe EAE.     

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