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 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.     

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

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

Effects of non-MHC background genes on the induction of CD4+ T cells that prevent rejection of a highly immunogenic tumor, FBL-3

This study showed that non-MHC genes common to (DBA/2 H-2^d)and (DBA/1 H-2^q) gave rise to suppressor T (T_a) cells in thehybrid F₁ mice between C57BL/6 (B6) strain in the antl-FBL-3tumor responses. FBL-3, a Friend virus-induced tumor cell lineof B6 mouse origin, is highly immunogenic as shown by findingsthat syngenelc and hybrid F₁ mice with several other inbredstrains rejected up to 3 x 10⁷ tumor cells inoculated s.c. andgenerated potent CTL responses after mixed lymphocyte tumorcell culture. In contrast to these mice, (B6 x DBA/2) and (B6x DBA/1)F1 mice did not reject the tumor as the tumor dosesincreased. Progressive tumor growth in these F₁ mice was blockedby an I.p. Injection of cyclophosphamlde (250 mg/kg) on day10, but not on day 5, after tumor cell inoculation. Antl-CD4(GK1.5) mAb exerted similar therapeutic effects against tumorwhen given twice, between day 0 and 10, whereas the additionalinjection of antl-CD8 mAb enhanced the tumor growth in micethat otherwise rejected the tumor. Thus, In the response of(B6 x DBA/2)F, mice to FBL-3 tumor cells, CD4⁺ T₈ seemed todown-regulate the immunologically mediated regression of thetumor produced by CD8⁺ CTL. This was evidenced by limiting dilutionculture analyses, which showed that the frequency of an FBL-3-speclflcCTL precursor in the (B6 x DBA/2)F1 mice that rejected the tumorwith antl-CD4 mAb was 7- to 9-fold higher than that in micein which the tumor regressed spontaneously. That more than onegene was involved in suppressor T cell induction was shown bythe tumor growth pattern in (B6 x DBA/2)F₁ x B6 backcross andB6D2F2 mice.     

Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance

It is well established that BALB/c mice can be protected fromfatal infection with Leishmania major by prophylactic intravenous(i.v.) immunization with irradiated parasites. Protection iscritically dependent on the route of injection with i.v. injectionbeing protective and subcutaneous injection not protective.We used this BALB/c-L major model system to investigate thisphenomenon. We analyzed quantitatively the parasite-specific,CD4⁺ T cell mediated immune responses by limiting dilution.Subcutaneous vaccination resulted in priming of CD4⁺ precursorT cells, whereas i.v. vaccination was ineffectual. Moreover,i.v. injection prevented the increase in the number of specificprecursor cells induced by infection of normal mice during thefirst weeks post-challenge with virulent parasites. We showhere that this was not due to the elimination of the virulentchallenge parasites as a result of immunity nor to inefficientantigen presentation of the irradiated organisms after i.v.injection, The data presented here suggest that i.v. injectionresults in tolerizatlon rather than immunization. Tolertzationas a mechanism of host protection is consistent with earlierobservations that transient immunosuppresslon results in cureof L. major infection in BALB/c mice. Transfer of antigen presentingcells (APC) isolated from spleens of mice injected previouslywith irradiated parasites mimicked to some extent the effectof i.v. immunization with irradiated parasites. The possibleinvolvement of these APC in decreasing the parasite-specificT cell response is discussed.     

Over coming the crypticity of a viral T cell Determinant by insertion into a chimeric bacterial protein

Among the potential T cell determinants contained In a proteinantigen, the T cell response only focuses on a few immunodomlnantT cell determinants, whereas cryptic epitopes remain hiddento the Immune system. In the present work, we have studied theantigen processing and presentation of the C3: 93-115 sequenceof Mahoney pollovlrus VP1 protein, which Is Immunodomlnant InH-2^d but cryptic In H-2_s and H-2_q mouse MHC haplotypes. Forthis purpose, we genetically Inserted the C3 determinant Intofive internal sttes of a bacterial protein, the maltose bindingprotein of Escherichla coll (MalE). In four out of five Insertionsites of MalE, the C3 determinant retained Its Immunodominancewhen the purified hybrid proteins were Injected to BALB/c (H-2_d)mice. Moreover, In SJL/J (H-2^d) mice, in three out of five MalE-C3constructs, the new structural environment of the cryptic C3epltope rescued Its processing and its in vivo presentationto T cells. In contrast, In DBA/¹ (H-2^d) mice, although MalE-C3chimeric proteins were correctly processed in vitro, the C3epitope remained cryptic in vivo. In this case, the impairmentto stimulate a T cell response in vivo was correlated with ashort time persistence of C3 peptides bound to Aq moleculesat the surface of live antigen-presenting cells. These resultsemphasize the role of flanking residues on the lack of processingof cryptic determinants and the Importance of the life spanof peptlde-MHC complexes to stimulate T cell responses.     

Modulation of specific T cell responses by concurrent infection with Leishmania major and LP-BM5 murine leukemia viruses

C57BL/6 mice infected with a murine leukemia virus (MuLV) mixturedesignated LP-BM5 develop an immunodeficiency syndrome termedMAIDS, characterized by a variety of T and B cell abnormalities,including elevated levels of IgE, suggesting that IL-4 expressionis increased in these animals. It has been suggested that theimmunodeficiency associated with MAIDS is caused by a conversionof immune responses normally characterized by T_h1 developmenttowards a T_h2- dominated response. Mice of the same strain,infected with Leishmania major, mount a protective T_h1 responsewith the induction of high levels of IFN- and undetectable IL-4.We therefore infected mice with L. major at differing time pointsbefore and after virus infection and assessed the effects onT cell responsiveness, cytokine production and survival to L.major, as well as the effect on MAIDS-associated pathology.We have also immunized C57BL/6 mice with trinitrophenol-keyholelimpet haemocyanln (TNP-KLH), which leads to a predominantlyT_h2 response, and compared the effects of MAIDS on the responseto TNP-KLH with the effect of MAIDS on L. major infection. Ourresults show that significant immunodeficiency with regard toinfection by L. major is only apparent after 8 weeks of LP-BM5MuLV infection, by which time T and B cell defects are welladvanced. Further, we have found that the strongly polarizedT_h1 response stimulated by L. major infection can modulate theeffect of MAIDS on T cells, leading to the survival of antigen-specificT cells. Our results suggest that the impairment of immune responsesto either TNP–KLH or L. major is due not to an alterationof the balance of T_h1/T_h2 subsets but to a general loss of reactivityin antigen-specific CD4⁺ cells. However, prior activation ofT_h1 but not T_h2 cells can inhibit the development of lymphoproliferationand immunodeficiency caused by MAIDS.     

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.     

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.     

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.     

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge

DNA vaccination offers the advantages of viral gene expressionwithin host cells without the risks of infectious virus. Likeviral vaccines, DNA vaccines encoding internal influenza virusproteins can induce immunity to conserved epitopes and so maydefend the host against a broad range of viral variants. CD8⁺cytotoxic T lymphocytes (CTL) have been described as essentialeffectors in protection by influenza nucleoprotein (NP), althougha lesser role of CD4⁺ cells has been reported. We immunizedmice with plasmids encoding influenza virus NP and matrix (M).NP + M DNA allowed B6 mice to survive otherwise lethal challengeinfection, but did not protect B6-ß₂m(–/–)mice defective in CD8⁺ CTL. However, this does not prove CTLare required, because ß₂m(–/–) mice have multipleimmune abnormalities. We used acute T cell depletion in vivoto identify effectors critical for defense against challengeinfection. Since lung lymphocytes are relevant to virus clearance,surface phenotypes and cytolytic activity of lung lymphocyteswere analyzed in depleted animals, along with lethal challengestudies. Depletion of either CD4⁺ or CD8⁺ T cells in NP + MDNA-immunized BALB/c mice during the challenge period did notsignificantly decrease survival, while simultaneous depletionof CD4⁺ and CD8⁺ cells or depletion of all CD90⁺ cells completelyabrogated survival. We conclude that T cell immunity inducedby NP + M DNA vaccination is responsible for immune defense,but CD8⁺ T cells are not essential in the active response tothis vaccination. Either CD4⁺ or CD8⁺ T cells can promote survivaland recovery in the absence of the other subset.     

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-.     

Regulation of chronic colitis in athymic nu/nu (nude) mice

The objective of this study was to assess the roles of NK cells,B cells and/or intraepithelial lymphocytes (IEL) in suppressingthe development of colitis in nude mice reconstituted with CD4⁺CD45RB^highT cells. BALB/c nude mice were lethally irradiated and reconstitutedwith bone marrow from different immunodeficient mice to generateathymic chimeras devoid of one or more lymphocyte populations.Transfer of CD4⁺C45RB^high T cells into chimeric recipients devoidof B cells, T cells and IEL produced severe colitis within 6–8weeks, whereas transfer of these same T cells into B cell- andT cell-deficient or T cell-deficient chimeras produced littleto no gut inflammation. In addition, we found that nude micedepleted of NK cells or RAG-1^–/– mice reconstitutedwith IEL failed to develop colitis following transfer of CD45RB^highT cells. Severe colitis could, however, be induced in nude miceby transfer of activated/T_h1 CD4⁺CD45RB^low T cells. Taken together,our data suggest that IEL, but not B cells or NK cells, playan important role in suppressing the development of chroniccolitis in this model. In addition, our data demonstrate thatsuppression of disease may be due to polarization of naive CD4⁺cells toward a non-pathogenic and/or regulatory phenotype.     

V{beta}6-bearing T cells are involved in resistance to Trypanosoma cruzi infection in XID mice

BALB.xid mice, carrying an X-linked mutation leading to theabsence of CD5⁺ B cells, are highly resistant to Trypanosomacruzi Infection. These mice clear blood parasites In the acutephase of infection and do not develop the inflammatory Infiltrationcharacteristically observed in the chronic phase of susceptiblestrains of mice. We have shown that the resistance of BALB.xldIs dependent on the production of high levels of IFN-y. Natural(adoptive foster) or artificial (In vivo Injection of blockingantibodies) treatments of BALB.xld induced deletion of CD4⁺and CD8⁺ cells bearing V_ß6 TCR. The absence of V_ß6lymphocytes considerably reduced resistance to infection. Furthermore,in BALB.xld lacking this minor fraction of the T cell repertoire,almost 50% of the IFN-y production is lost. This indicates thatV_ß6-bearing T cells are either directly or Indirectlyinvolved in the production of IFN-y and, thus, important foran effective immune response during the acute phase of experimentalChagas' disease.     

Ly-49 G2+ NK cells are responsible for mediating the rejection of H-2b bone marrow allog rafts in mice

NK cells can mediate the specific rejection of bone marrow butnot solid tissue allografts in lethally irradiated mice. NKcells are also responsible for the phenomenon of ‘hybridresistance’ in which F₁ hybrid H-2 heterozygous mice canreject parental H-2 homozygous bone marrow grafts. Ly-49C andLy-49 G2 are markers identified on subsets of NK cells. WhileLy-49C⁺ NK cells have been demonstrated to mediate the specificrejection of H-2^d bone marrow allografts, the role of the Ly-49G2⁺ NK subset is unclear because depletion of this subset invivo did not affect splenic NK activity against tumor targets.Through bone marrow transplantation typing studies, we demonstratethat Ly-49 G2⁺ NK cells complement Ly-49C⁺ NK cells in thatthey specifically mediate the rejection of H-2^b bone marrowallografts in lethally irradiated mice. In support of this,depletion of the Ly-49C⁺ NK subset in vivo also enhanced theability of the mice to reject H-2^b bone marrow cells suggestingthat the depletion was augmenting the ability of the Ly-49 G2⁺NK cells to reject the marrow allografts. Depletion of Ly-49G2⁺ NK cells in F₁ hybrid mice abrogated their ability to rejectparental H-2^b but not H-2^d bone marrow grafts. Therefore, Ly-49G2 denotes, a subset of NK cells that appears to play a criticalrole in the recognition of H-2^b bone marrow cells in allogeneicand F₁ hybrid mice.     

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.     

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.     

Tolerance induction by elimination of subsets of self-reactive thymocytes

Immature thymocytes expressing TCRs which confer reactivityto self-MHC molecules are subject to efficient elimination asa result of negative selection. Previously, we have identifieda lineage of H-2K^b Tg mice, CD2K^b-3, which fails to reject skingrafts from mice expressing H-2K^b even though H-2K^b-specrficcytotoxlc T cells can be generated in vitro. We now show thatbone marrow derived cells are responsible for tolerance inductionand that tolerance is acquired, at toast in part, by negativeselection in CD2K^b-3 mice. Thymocytes expressing two differenttransgenic TCR (TCR-T_g) clonotypes conferring reactivity toH-2K^b are eliminated prior to the CD8⁺CD4⁺ stage of differentiationin double Tg (CD2K^b-3xTCR-T_g)F₁ mice. As in other cases wherethymocytes from TCR-T_g mice develop in the presence of deletingligands, large numbers of TCR⁺ CD8^–CD4^– T cellsaccumulate in double Tg mice. However, these T cells fail torespond to H-2K^b in vitro but can be activated with immobilizedanti-clonotyplc antibody. Consequently, thymocytes expressingthese types of TCR molecules represent a fraction of H-2K^b-reactivethymocytes which are unable to mature into T cells capable ofmounting H-2K^b-specific cytotoxic responses. Presumably, precursorsof H-2K^b-spicific cytotoxlc T cells found in the periphery ofCD2K^b-3 mice express a distinct repertoire of TCR moleculesconferring reactivity to H-2K^b. We consider potential explanationsto account for this discrepancy and their wider implications,including the possibility that the repertoire of thymocyteaable to recognize setf-H-2Kb molecules In CD2K ^b-3 mice is dividedinto distinct subsets; those which are, and those which arenot, subject to negative selection.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.