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.     

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-.     

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.     

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.     

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.     

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.     

Altered response of CD4+ T cell subsets to Plasmodium chabaudi chabaudi in B cell-deficient mice

Mice depleted of B cells from birth by treatment with anti-_µantibodies can control but not clear an infection with the malariaparasite Plasmodium chabaudi chabaudi (AS). Splenic CD4⁺ T cellsfrom these mice were unable to mount a significant T_h2 responseto the parasite in vitro as shown by much lower precursor frequenciesof T_h cells for antibody production and of IL-4-producing cellscompared with the response of control-treated mice. CD4⁺ T cellsof the anti-_µ-treated mice which respond to antigens ofP. chabaudi chabaudi maintained a T_h1 phenotype throughout primaryinfection, in contrast to control mice in which a sequentialappearance of T_h1 and T_h2 responses was observed. These datashow that T_h1 responses in anti-_µ-treated mice are sufficientto control parasitemia but not to eliminate an infection. Thedata further suggest that depletion of B cells by treatmentwith anti-_µ; antibodies reduces the generation of theT_h2 subset during a primary response to P. chabaudi chabaudi.     

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.     

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.     

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.     

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     

Natural killer cell activity in lymphocytic choriomeningitis virus-infected {beta}2-microglobulin-deficient mice

We have investigated the induction and role of natural killer(NK) activity in lymphocytic choriomeningitis virus (LCMV)-infectedß₂-microglobulin-deficient (ß₂m^–)mice. We demonstrate that LCMV infection is more effective thanpolyinosinic:poiycytidylic acid (poly I:C) at stimulating NKactivity in ß₂m^– .In addition, ß₂m^–NK cells respond poorly to in vitro treatment with IL-12. Thetarget specificity of the virally induced NK cells is similarto that previously reported for chemically induced ß₂m^–NK cells. In both cases they can lyse YAC-1 tumor cells butare unable to kill ß₂m^– orß₂m⁺ T cellblasts. We have also found that the time course of inductionof NK and cytotoxic T lymphocyte (CTL) activity by LCMV in ß₂m^–mice is delayed compared with normal mice. Maximal NK and CTLactivity is attained at day 8 and 10 post-infection respectivelyin ß₂m^– compared with day 4 and 6—8 inB6 mice. Whereas normal mice die {small tilde}7 days followingintracranial infection with LCMV, the course of disease in ß₂^–mmice is protracted and characterized by a marked loss of bodyweight. We show that although the CD4⁺ CTL response in thesemice is intimately involved in mediating weight loss, the virus-inducedNK cells do not appear to play a role in the disease.     

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

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

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.     

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.     

Resistance of BALB/c mice to Leishmania major infection is associated with a decrease in the precursor frequency of antigen-specific CD4+ cells secreting interleukin-4

BALB/c mice are highly susceptible to infection with the protozoanparasite Leishmania major and develop a chronic fatal disease.They can, however, be manipulated to resist disease and thishas been shown to correlate with increased expression of IFN_–mRNA and the absence of IL-4 mRNA in the draining lymph nodesand spleens of these animals. Here we show that anti-IL-4 oranti-CD4 treatment of BALB/c mice resulted in a reduction inthe size of the lesion and in the number of parasites in thedraining lymph nodes compared with untreated mice. The precursorfrequency of CD4⁺ T cells proliferating in response to Leishmaniaantigens in vitro in the treated animals was not significantlydifferent from untreated animals. Analysis of the lymphokinessecreted by the clonal progeny of these cells showed that theprecursor frequency of IL-4 secreting clones was at least 10-foldlower in animals treated with either mAb. However, there wasno reciprocal increase in the precursor frequencies of IFN_–secreting clones. Comparisons of the total number of precursorsof specific CD4⁺ cells secreting IFN_– showed that anti-CD4-treatedanimals, which are resistant to disease, had considerably fewerfor the first 6 weeks than untreated mice with chronic disease.Protection of BALB/c mice was therefore associated with a reductionin the numbers of precursors of cells secreting IL-4 withouta concomitant increase in the number of precursors of IFN_–secreting cells.     

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via {alpha}4 integrin-VCAM-1 interaction

The present investigation examines the localization and migrationof purified T cell subsets in comparison with B cells, CD8 Tcells and CD4⁺CD8^– single-positive thymocytes. CD4 T cellsubsets in the rat are defined by mAb MRC OX22 ( anti-CD45RC),which distinguishes resting CD4 T cells (CD45RC⁺) from those(CD45RC^–) which have encountered antigen in the recentpast– subpopulations often referred to as ‘naive’and ‘memory’. Purified, ⁵¹Cr-labelled CD45RC⁺ CD4T cells broadly reflected the migration pattern of CD8 T cellsand B cells. Early localization to the spleen was followed bya redistribution to mesenteric lymph nodes (MLN) and cervicallymph nodes ( CLN) , B cells migrating at a slightly slowertempo. There was almost no localization of these subpopulationsto the small or large intestine [Peyer's patches (PP) excluded].In contrast, CD45RC^– CD4 T cells (indistinguishable insize from the CD45RC⁺ subset) localized in large numbers tothe intestine; they were present here at the earliest time point(0.5 h) , persisted for at least 48 h but did not accumulate,indicating a rapid exit. Numerically, localization of CD45RC^–CD4 T cells in the MLN could be accounted for entirely by afferentdrainage from the intestine. Unexpectedly, CD45RC^– CD4T cells (but not other subsets) localized and accumulated inthe thymus. In vivo treatment with mAb HP2/1 against the integrin₄ subunit inhibited almost entirely CD45RCT^– CD4 T cellmigration into the PP (98.1%), intestine (87.1%) , MLN (89.1%)and thymus (93.5%) migration into the CLN was only reduced byhalf. To distinguish between recognition of MAdCAM-1 and VCAM-1by ^4–containing integrins, recipients were treated withmAb 5F10 against rat VCAM-1. Except for the thymus and a smallreduction in CLN, localization of CD45RC^– CD4 T cellswas unaffected; entry to the thymus was almost completely blocked(92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC^–CD4 T cells alone showed enhanced localization to the gut andPP, probably via ₄ß₇-MAdCAM-1 interaction; ( II) thatmany CD45RC^– cells entered nonmucosal LN independentlyof ₄ integrin or VCAM-1; and (III) that entry of mature recirculatlngCD45RC^– CD4 T cells into the thymus across thymic endothellumwas apparently regulated by ₄ integrln-VCAM-1 interaction.     

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.     

Mel14+CD4+ T cells from aged mice display functional and phenotypic characteristics of memory cells

Aging is accompanied by an increased fraction of memory CD4⁺T cells. Despite the fact that human memory cells have beenreported to produce high levels of IL-2, studies in mice andman indicate an age-related decline in IL-2 production. In thepresent study, we examined whether these conflicting resultsdepend on the activation pathway employed in a comparison ofphenotypically distinct CD4⁺ T cells from young and aged mice.Our data indicate an age-related decline in IL-2 productionby CD4⁺ T cells when the cells were stimulated with concanavalinA in the presence of accessory cells or the combination of immobilizedanti-CD3 and soluble anti-CD28. However, when CD4⁺ T cells wereonly stimulated with Immobilized anti-CD3, an age-related increasein IL-2 production was observed. This age-related increase inIL-2 could be attributed to the ability of CD4⁺ T cells fromaged mice to produce IL-4 on this stimulation, since anti-IL-4inhibited the IL-2 production In these cultures to levels foundwith cells from young mice. The addition of exogenous IL-4 greatlyenhanced the IL-2 production of CD4⁺ T cells from young miceto levels far beyond that of the aged counterparts, emphasizingthe dominant role of IL-4 In the induction of IL-2 stimulatedwith immobilized anti-CD3. No differences were observed in theactivation requirements of Mel14^– CD4⁺ T cells from youngand aged mice. However, Mel14⁺ CD4⁺T cells from aged mice werefunctionally and phenotypically more mature than their youngcounterparts, since they were capable of IL-2 and IL-4 productionin response to antl-CD3 without the need of CD28 triggeringand expressed Pgp-1 and ICAM-1 in a higher density. Our dataindicate therefore that Mel14 is not a stable marker for naiveCD4⁺ T cells and might not be appropriate to distinguish thesecells from memory cells.     

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.