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-.     

Co-transfer of B cells converts resistance into susceptibility in T cell-reconstituted, Leishmania major-resistant C.B-17 scid mice by a non-congnate mechanism

Resistance to infection of mice with Leishmania major parasitesis dependent on the production of IFN- by CD4⁺ T helper cells.C.B-17 scid mice, lacking both T and B cells, succumb very quicklyto the infection, but develop resistance if reconstituted withappropriate numbers of T cells from BALB/c mice. In this model,we studied the role of B cells with regard to their abilityto influence disease outcome and to function as antigen-presentingcells for T cells. For this purpose, we reconstituted scid mice(H-2^d) with either T cells or with T and B cells obtained from(BALB/c x BALB.B)F₁ mice (H-2^d x ^b), and infected them withL. major parasites 1 day after reconstitution. Mice reconstitutedwith T cells alone cured the disease, whereas additional B cellreconstitution led to susceptibility. Healing was associatedwith a predominant T_h1-type response. In all mice, L. mayor-specificT cell proliferation was restricted to the MHC phenotype ofthe recipient (H-2^d) but not to that of the donor (H-2^d x ^b),indicating that there was no detectable contribution of donorB cells in the priming of a T cell response. Furthermore, Bcells, when purified from infected BALB/c mice, were unableto stimulate a L. mayor-specific CD4⁺ T cell clone (L1/1) withoutaddition of exogenous antigen, in contrast to macrophages fromthe same animal. These data suggest that B cells, in vivo, donot carry L. major antigen in a form capable of activating specificCD4⁺ T cells. Therefore, B cells promote disease by means otherthan cognate interaction with CD4⁺ T 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.     

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.     

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.     

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.     

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.     

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

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

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.     

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.     

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.     

Induction of Th2 responses to soluble proteins is independent of B cell tolerance status

Injection of high doses of monomeric human gamma globulins (dHGG)In naive, adult mice causes antigen-specific tolerance of Bcell and T_h1 lymphocytes, while inducing the selective expansionof antigen-specific T^h2 cells. Several parameters of toleranceinduction were analyzed in this work, In order to establishwhether B cell tolerance and T_h1 unresponsiveness were functionallyrelated in this in vivo model. By varying the antigen form andsite of injection, we demonstrate in this work that T_h1 unresponsivenessto HGG is not a consequence of peripheral B cell tolerance.In particular, mice pretreated with heat-aggregated antigen(HAHGG) or F(ab')² HGG were found to develop a strong humoralresponse while displaying a defective T_h1 response. In fact,these animals developed a strong T^h2 response in vivo, demonstratingthat selective expansion of antigen-specific T_h2 cells in thismodel is not a consequence of B cell tolerance or antigen captureby Fc receptor-expressing cells. We conclude that while B celltolerance in this model is only observed in response to deaggregatedantigen, Injection of all forms of adjuvant-free, protein antigensinduces T helper precursor cells to differentiate into T_h2-typehelper cells in vivo Irrespectively of the B cell tolerancestatus     

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     

Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection

Very little is known about the conformational properties ofthe MHC molecules that are able to signal positive selectionof a given TCR. To try to understand these parameters and todetermine whether these requirements are shared with interactionsduring negative selection and antigen recognition, we have studiedselection and antigen recognition of a transgenic TCR (specificfor lymphocytic choriomeningitls virus glycoprotein and H-2D^b)in the context of two D^b mutants, H-2^bm13 and H-2^bm14. The datashowed that the transgenic TCR was not positively selected bythe H-2^bm14 haplotype but, interestingly, enhanced positiveselection was seen in H-2^bm13 mice. The transgenic TCR couldnot be negatively selected In H-2^bm13animals persistently infectedwith the virus (neonatal virus carrier mice), nor could thetransgenic TCR be activated by H-2^bm13 infected cells in vivoor in vitro. These experiments show that although a TCR maybe selected by a mutant MHC molecule, the corresponding viralantigen cannot be recognized in the context of the mutant MHCmolecule, as Judged by both negative selection and T cell reactivityin vivo and in vitro. The ‘enhanced’ positive selectionoccurring in the context of D^bm13 suggests that a differentconformation of the MHC molecule is able to select the sameTCR and also that various TCR-ligand avidities may permit positiveselection.     

Effects of different antigenic microenvironments on the course of CD8+ T cell responses in vivo

The influence of microenvironment on the course of CD8⁺ T cellresponses in vivo was investigated by injecting H-2K^b-specificT cells from donor TCR transgenlc (TCR-Tg) mice into H-2K^b-tagmice. H-2K^b expression in recipients was either ubiquitous (CBKmice) or restricted to myeloid and erythroid cells (Kßmice). Donor T cells proliferated as extensively and acquiredsimilar surface phenotypes in spleen of both recipient types.Thus, neither the restricted pattern of H-2K^b expression northe significantly reduced level of H-2K^b expression by myeloldcells in Kß recipients affects the ability of thesplenic microenvironment to prime T cell proliferation in vivo.However, an unsustained burst of cytolytic activity was generatedrapidly in spleen of CBK recipients, whereas relatively littlecytolytic activity was generated in Kß spleen. Thisindicates that effector T cells were not generated efficientlyin spleen of Kß recipients even though extensive Tcell proliferation was taking place in this microenvironment.Furthermore, activated donor T cells dispersed rapidly throughoutprimary and secondary lymphoid organs of Kß recipients,whereas few T cells migrated from spleen in CBK recipients.Consequently, the course of CD8⁺ T cell responses and the anatomicaldistribution of activated T cells are profoundly influencedby the nature of the antigenlc microenvironment encounteredin vivo. We conclude that T cells rapidly proliferate and acquirenew tissue-homing characteristics but do not differentiate intocytolytic effector cells at the site of priming when they encountermyelold cells expressing low levels of antigen in vivo.     

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression

BALB/c mice are susceptible to cutaneous lelshmanlasls uponinfection with Leishmania major while C57BL/6 are not. Thereis a major promastigote surface protease (PSP or gp63) whichis available in both native and recomblnant forms, and for whichthe primary amlno acid sequence is known. Immunization withPSP has been shown to offer some protection against challengewith the live organism. Therefore, we attempted to develop apeptide vaccine with PSP peptldes. In the first experiments,recall prollferatlve responses to PSP were measured using aset of 15mer peptldes spanning the entire PSP molecule whichallowed designation of major determinant regions in BALB/c,C57BL/6, and CBA mice. Several of these determinants were promiscuousand shared almost the identical core amlno acid residues inthe different strains. Immunization with major determinant peptldeswas recalled vigorously with L. major soluble antigen as wellas with PSP. The response to peptide was almost entirely T_h1as measured by a localized ELISA assay for single-cell productionof IFN-. A similar assay for IL-5, which overcomes problemsof sensitivity and inhibition by lymphoklnes produced by T_h1cells, Indicates very little production of T_h1 cells even byBALB/c. It was found that if a major responsive peak was examinedby recall with overlapping peptldes, the highest, central peptidegave a mainly Th1 response while the boundary, less efficientpeptldes gave more of a T_h2 response. Possible reasons for thiswere discussed. These results point to the importance of selectingthe exactly appropriate peptide in considering a vacclnogenthat might protect susceptible individuals. Even the choiceof a somewhat immunogenlc peptide within the determinant envelopemight actually exacerbate infection by steering the responsein a T_h2 direction.     

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.