Interaction of sulfonamide derivatives with the TCR of sulfamethoxazole-specific human alpha beta+ T cell clones

Drugs like sulfamethoxazole (SMX) or lidocaine can be presented to specific human alphabeta+ T cell clones (TCC) by undergoing a noncovalent association with MHC-peptide complexes on HLA-matched APCs. For a better understanding of the molecular basis of the recognition of such drugs by specific TCC, we investigated 1) the fine specificity of the recognizing TCR, 2) the dose-response relationship for the induction of proliferation or cytokine production, and 3) the mechanism of TCR triggering. For that purpose, we tested the reactivity of 11 SMX-specific CD4+ TCC and 2 SMX-specific CD8+ TCC to a panel of 13 different sulfonamide derivatives bearing the same core structure. Five of 13 clones recognized only SMX, while all other clones were responding to as many as 6 different compounds. Some of the compounds needed up to two orders of magnitude higher concentrations than SMX to stimulate TCC, thereby displaying features of weak agonists. Different clones showed clear differences in the minimal drug concentration required for the induction of a proliferative response. Therefore, weaker or stronger agonistic properties were not a characteristic of a given sulfonamide derivative but rather an intrinsic property of the reacting TCR. Finally, the number of down-regulated TCRs was a logarithmic function of the ligand concentration, implicating that specific T cells were activated by serial TCR engagement. Our data demonstrate that, despite the special way of presentation, nonpeptide Ag like drugs appear to interact with the TCR of specific T cells in a similar way as peptide Ags.     

Selective activation of CD8 T cell effector functions by epitope variants of lymphocytic choriomeningitis virus glycoprotein

We provide evidence for selective activation of different effector functions of CD8+ T lymphocytes by altered peptide ligands. A T cell epitope from the glycoprotein of lymphocytic choriomeningitis virus (p33-41) and single amino acid variants thereof were used for primary in vitro induction of CTL clones. When the CTL were analyzed for cytotoxicity, proliferation, IFN-gamma production, and Ca2+ mobilization, we found that some of the clones showed activation of only their cytotoxic effector function when stimulated with variants of their inducing peptides. For one clone, cytotoxic reactivity was readily detected to the inducing peptide and three of four variants, but only the former was also able to trigger proliferation, IFN-gamma production, and Ca2+ mobilization. Another clone also revealed this dichotomy, but in this case some of the altered peptide ligands in addition to the inducing peptide were able to stimulate the full spectrum of effector functions, whereas others only stimulated cytotoxicity. A third clone revealed inefficient triggering of some effector functions by the peptide variants. Our data suggest that, as described for CD4 T cells, altered peptide ligands may lead to partial activation of effector functions of CD8 T cells. In addition, ligands with glycine substitutions in potential TCR contact positions induced CTL, which were able to recognize peptides with a variety of amino acids in the former glycine position.     

Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

T cells express CD28 and CD27 which transduce co-stimulatory signals after interaction with their ligands on antigen-presenting cells (APC). These ligands, CD80, CD86 and CD70, are also expressed to some extent on activated T cells. Here, we show that in human immunodeficiency virus (HIV)-infected individuals, CD28 and CD27 expression is decreased on CD8+ T cells. On the other hand, T cell stimulation in vitro induced high CD80, CD86 and CD70 expression on T cells from HIV-infected individuals. It appeared that an inverted CD4:CD8 T cell ratio could explain this enhanced expression of co-stimulatory ligands. Indeed, high expression levels of CD80, CD86 and CD70 were found on activated CD8+ T cells from HIV- individuals cultured in the absence of CD4+ T cells. Addition of CD4+ T cells prevented this up-regulation. However, in HIV-infected individuals, addition of excess autologous or healthy control CD4+ T cells did not completely counteract up-regulation of co-stimulatory ligand expression on CD8+ T cells. Thus, to some extent, CD8+ T cells in HIV-infected individuals appeared to be refractory to CD4+ T cell-mediated regulation of ligand expression in vitro. Activated T cells from HIV-infected individuals and activated CD8+ T cells from healthy controls were able to act as accessory cells in CD3-induced T cell proliferation, which was dependent on cell-cell contact. Thus, we showed that T cells from HIV-infected individuals express enhanced levels of co-stimulatory ligands upon activation, which provides them with accessory cell properties. Enhanced stimulatory potential of these nonprofessional APC may contribute to persistently high levels of immune activation in HIV infection related to disease progression.     

Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocytic infiltrates of Graves' ophthalmopathy

Lymphocytic infiltration of muscular and connective tissues of the retroorbital (RO) space is a histological hallmark of Graves' ophthalmopathy (GO). We have characterized some phenotypical and functional features of T cells derived from RO infiltrates of four GO patients who were submitted to orbital decompression. Fragments of RO tissue were cultured for 7 days in IL-2-conditioned medium in order to generate T cell lines of in vivo activated T cells. Phenotypical analysis of freshly isolated peripheral blood (PB) lymphocytes both from patients and four healthy controls showed a predominance of CD4+ T cells (CD4/CD8 ratios 1.9:2.5), whereas RO-derived T cell lines displayed almost equal proportions of CD4+ and CD8+ cells (CD4/CD8 ratios 0.9:1.2). RO T cell lines and PB T cells from patients and controls were then cloned using a high-efficiency cloning procedure. The phenotypical and functional features of 153 T cell clones (TCC) derived from RO infiltrates were examined and compared with those of 166 and 236 TCC derived from the PB of patients and controls, respectively. CD4/CD8 ratios ranged from 0.8-1.4 in the series of RO-derived TCC and from 1.9-2.2 in the corresponding series of PB-derived TCC. Assessment of lectin-dependent cytolytic activity showed similar proportions of cytotoxic clones in TCC derived from the PB of patients (37%) and controls (38%); most of the cytolytic TCC was CD8+. In contrast, the proportion of cytolytic RO TCC was markedly higher (106/153 = 69%), including 100% of CD8+ and the majority (59/79 = 75%) of CD4+ clones. When compared to TCC derived from the PB of both patients and controls, RO TCC showed remarkably high proportions of both CD8+ and CD4+ clones with a Th1-like cytokine profile, as documented by their ability to secrete IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4 or IL-5. This study provides evidence that cytolytic T cells with Th1 profile of cytokine production predominate in RO infiltrates of GO, a pattern quite similar to those previously described in thyroid infiltrates of Hashimoto's thyroiditis or Graves' disease. The peculiar cytokine secretion profile of RO T cells may be of importance in the pathogenesis of both the tissue alterations and fibrogenic process observed in GO.     

Ligands of CD4 inhibit the association of phospholipase Cgamma1 with phosphoinositide 3 kinase in T cells: regulation of this association by the phosphoinositide 3 kinase activity

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4+ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i.e. Ca2+ signaling by phospholipase Cgamma1 (PLCgamma1), and ERK-2 activation, suggesting a p21ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCgamma1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCgamma1, both in peripheral CD4+ T lymphocytes and the HUT-78 CD4+ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4+ T cells and HUT-78 CD4+ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCgamma1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCgamma1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes.     

Central T cell tolerance in lupus-prone mice: influence of autoimmune background and the lpr mutation

Lupus-prone mice develop a systemic autoimmune disease that is dependent upon the B cell help provided by autoreactive alphabeta CD4+ T cells. Since autoreactive T cells with high affinity for self peptides are normally deleted in the thymus, their presence in these mice suggests the possibility that intrathymic negative selection may be defective. Here, we directly compared central T cell tolerance in response to a conventional peptide Ag in lupus-prone MRL/MpJ mice with a nonautoimmune strain using an MHC class II-restricted TCR transgene. Our results did not demonstrate any defects after Ag exposure in the induction of intrathymic deletion of immature CD4+ CD8+ thymocytes, in TCR down-regulation, or in the number of apoptotic thymocytes in MRL/MpJ compared with nonautoimmune mice. Furthermore, we found that the lpr mutation had no influence upon the Ag-induced thymic deletion of immature thymocytes. These data support the notion that T cell autoreactivity in MRL/MpJ mice is caused by defects in peripheral control mechanisms.     

TCR-independent pathways mediate the effects of antigen dose and altered peptide ligands on Th cell polarization

We examined the role of the peptide/MHC ligand in CD4+ T cell differentiation into Th1 or Th2 cells using a TCR alphabeta transgenic mouse specific for hemoglobin (Hb)(64-76)/I-Ek. We identified two altered peptide ligands of Hb(64-76) that retain strong agonist activity but, at a given dose, induce cytokine patterns distinct from the Hb(64-76) peptide. The ability of these peptides to produce distinct cytokine patterns at identical doses is not due to an intrinsic qualitative property. Each peptide can induce Th2 cytokines at low concentrations and Th1 cytokines at high concentrations and has a unique range of concentrations at which these distinct effects occur. The pattern of cytokines produced from limiting dilution of naive T cells demonstrated that the potential to develop an individual Th1 or Th2 cell is stochastic, independent of Ag dose. We propose that the basis for the observed effects on the Th1/Th2 balance shown by the altered peptide ligands and the amount of Ag dose involves the modification of soluble factors in bulk cultures that are the driving force that polarize the population to either a Th1 or Th2 phenotype.     

Partial activation of CD8+ T cells by a self-derived peptide

T cells are normally activated when the peptide for which they are specific is presented to them in the context of the appropriate major histocompatibility complex (MHC) (class I and Class II for CD8+ and CD4+ T cells, respectively). An increasing body of evidence indicates that structural homologues of the immunogenic peptide can partially activate or antagonize CD4+ T cells. CD8+ T cells may also be partially antagonized by such peptides, and self-derived peptides of this type may play a role in CD8+ T cell selection in the thymus. Activated CD8+ T cells lyse their targets by perforin-dependent granule exocytosis and by inducing apoptosis mediated by CD95 (also known as Fas or APO1) with its ligand (CD95L). Here we show that a clone of Kd-restricted CD8+ T cells specific for influenza haemagglutinin, which can also be activated in a crossreactive manner by a peptide derived from a myeloma tumour immunoglobulin heavy-chain variable region (IgVH) to kill by both routes, kills only by the CD95-CD95L pathway when stimulated by the corresponding germline IgVH peptide. As this germline IgVH peptide differs from the tumour peptide only at a single position buried in the MHC-binding groove, this indicates that CD95-CD95L-mediated killing can be triggered independently of the perforin-mediated pathway, and can be selectively affected by changes in MHC conformation.     

In the normal repertoire of CD4+ T cells, a single class II MHC/peptide complex positively selects TCRs with various antigen specificities

In the thymus, immature T cells are positively and negatively selected by multiple interactions between their Ag receptors (TCRs) and self MHC/peptide complexes expressed on thymic stromal cells. Here we show that in the milieu of negative selection on physiological self class II MHC/peptide complexes (Abwt), a single class II/peptide complex AbEp52-68 positively selects a number of TCRs with various Ag specificities. This TCR repertoire is semidiverse and not biased toward Ep-like Ags. Our finding implies that the degeneracy of positive selection for peptide ligands exceeds peptide-specific negative selection and is essential to increase the efficiency and diversity of the repertoire so that T cells with the same Ag specificity can be selected by different self MHC/ peptide complexes.     

The cell-mediated response to schistosomal antigens at the clonal level. III. Identification of soluble egg antigens recognized by cloned specific granulomagenic murine CD4+ Th1-type lymphocytes

Granulomatous inflammation in schistosomiasis is a consequence of T cell-mediated hypersensitivity to parasite egg Ag. In the present study we used three consecutive independent chromatographic procedures to fractionate and identify the soluble egg Ag recognized by schistosome-specific, cloned, murine, CD4+ Th1-type lymphocytes, which had been shown previously to be capable of mediating granuloma formation in vivo when adoptively transferred to normal syngeneic hosts challenged with an i.v. injection of eggs. The stimulatory activity resided in two acidic egg molecules, with apparent molecular masses of 64 to 68 kDa and 38 to 42 kDa, each of which ran as a single band on SDS-PAGE after purification. Fast performance liquid chromatography and SDS-PAGE performed under reducing conditions suggested that the two molecules are related and that the 38- to 42-kDa molecule is a subunit of the 64- to 68-kDa molecule. Polyclonal lymphoid cells from schistosome-infected mice were similarly stimulated by the purified 64- to 68-kDa and 38- to 42-kDa molecules, implying that these are sensitizing Ag in the natural disease.     

Phosphatidylinositol 3-kinase participates in p56(lck)/CD4-dependent down-regulation of LFA-1-mediated T cell adhesion

The mechanism inducing cell detachment in Ag-independent adhesion between lymphocytes is poorly understood. Different putative CD4 ligands, anti-CD4 Ab, a DR35-46 peptide mimicking residues 35 to 46 of HLA class II beta1, and a DR134-148 peptide mimicking residues 134 to 148 of HLA class II beta2, were previously found to down-regulate LFA-1-dependent adhesion between CD4+ T cells and HLA class II+ B cells. This down-regulation was shown to be p56(lck) dependent. Here we show that binding of these ligands to CD4 induced the activation of the tyrosine kinase p56(lck) associated with CD4 and also the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) associated with the CD4-p56(lck) complex in the HUT78 cell line. These events were not detected when p56(lck) was dissociated from CD4 in cell lines expressing mutated forms of CD4. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002, and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by CD4 binding. These results strongly suggest that CD4-induced PI3-kinase activation, in the absence of concomitant TCR/CD3 triggering, leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. The mechanism by which PI3-kinase could exert its effect remains unknown. Since PI3-kinase has previously been found to participate in the regulation of cytoskeleton structure, we propose that p56(lck)-associated PI3-kinase activation leads to a cytoskeleton organization unfavorable for LFA-1 function.     

Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.     

Binding of malaria T cell epitopes to DR and DQ molecules in vitro correlates with immunogenicity in vivo: identification of a universal T cell epitope in the Plasmodium falciparum circumsporozoite protein

The efficacy of a malaria peptide vaccine would be enhanced by the inclusion of a parasite-derived universal T cell epitope to ensure that all vaccinees develop parasite-specific cellular and humoral immunity. Two circumsporozoite (CS) protein T cell epitopes, previously identified by CD4+ T cell clones derived from Plasmodium falciparum sporozoite-immunized volunteers, were studied to determine their HLA class II binding potential. One epitope, located in amino acid (aa) 326-345 of the P. falciparum (NF54 strain) CS protein, was "universal" in that it could bind to multiple DR and DQ molecules in vitro. In contrast, the second epitope, T1, which is located in the CS repeat region, was recognized by T cells in the context of DQ6 (DQB1*0603) and did not bind with high affinity to any of the class II molecules tested in the peptide binding assays. The in vitro patterns of peptide/HLA interactions correlated with immunogenicity in vivo. A multiple antigen peptide (MAP) containing the aa 326-345 epitope elicited responses in eight inbred strains (H-2(a,b,d,k,p,q,r,s)), while the T1 MAP was recognized by only a single haplotype, H-2b. The combination of the universal aa 326-345 T cell epitope and the T1 repeat in a di-epitope MAP overcame the genetic restriction to the P. falciparum CS repeat region and elicited antisporozoite Ab responses in all of the MAP-immunized mice. Synthetic peptide malaria vaccines containing the aa 326-345 universal T cell epitope would be expected to elicit parasite-specific immune responses in both sporozoite-primed and naive individuals of diverse genetic backgrounds.     

CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C-) to TCR/CD8 signaling in response to antigen

Memory CD8+ T cells from mice previously primed with alloantigen (alloAg) can respond in vitro to IL-2 and purified class I alloAg presented on microspheres, while no response can be detected using cells from naive mice. Similar results have been obtained using cells from OT-1 mice expressing a transgenic TCR that is specific for OVA(257-264) (SIINFEKL) peptide bound to H-2Kb. A population of resting memory cells (defined on the basis of low forward scatter and CD44high, Ly-6C+, CD25-, CD69-surface phenotype) that is present in the OT-1 mice exhibits a substantially higher sensitivity to Ag-stimulation than do naive cells (CD44low, Ly-6C-) expressing the same TCR. CD44high cells respond vigorously to H-2Kb immobilized on microspheres and pulsed with peptide, while CD44low cells respond weakly and only at high class I density and peptide concentration. The Ag-presenting surface only has ligands for TCR and CD8 (class I and peptide), thus ruling out the possibility that differences are due to ligand binding by other adhesion or costimulatory receptors that are expressed at high levels on the memory cells. Experiments using anti-TCR mAb as the stimulus and coimmobilized non-Ag class I as a ligand for CD8 suggest that the difference between naive and memory cells may be at the level of stimulation through the TCR. Thus, in addition to expressing increased levels of adhesion receptors that may enhance responses to Ag on APCs, memory CD8+ T cells appear to be intrinsically more sensitive than naive cells to stimulation through the TCR/CD8 complex.     

HLA-independent heterogeneity of CD8+ T cell responses to MAGE-3, Melan-A/MART-1, gp100, tyrosinase, MC1R, and TRP-2 in vaccine-treated melanoma patients

An important element in melanoma vaccine construction is to identify peptides from melanoma-associated Ags that have immunogenic potential in humans and are recognized by CD8+ T cells in vivo. To identify such peptides, we evaluated HLA-A*02+ melanoma patients immunized to a polyvalent vaccine containing multiple Ags, including MAGE-3, Melan-A/MART-1, gp100, tyrosinase, melanocortin receptor (MC1R), and dopachrome tautomerase (TRP-2). Using a filter spot assay, we measured peripheral blood CD8+ T cell responses, before and after immunization, to a panel of 45 HLA-A*0201-restricted peptides derived from these Ags. The peptides were selected for immunogenic potential based on their strong binding affinity in vitro to HLA-A*0201. Vaccine treatment induced peptide-specific CD8+ T cell responses to 22 (47.8%) of the peptides. The most striking finding was the HLA-independent heterogeneity of responses to both peptides and Ags. All responding patients reacted to different combination of peptides and Ags even though the responding patients were all A*0201+ and the peptides were all A*0201-restricted. From 9 to 27% of patients developed a CD8+ T cell response to at least one peptide from each Ag, but no more than 3 (14%) reacted to the same peptide from the same Ag. This heterogeneity of responses to individual peptides and Ags in patients with the same haplotype points to the need to construct vaccines of multiple peptides or Ags to maximize the proportion of responding patients.     

T cell responses to heat-shock protein 60: differential responses by CD4+ T cell subsets according to their expression of CD45 isotypes

We demonstrate that human T lymphocytes proliferate in vitro to highly purified human heat-shock protein 60 (Hu.hsp60). The response to this self Ag was confined to the CD45RA+ RO- T cell subset, with minimal responses by adult CD45RA- RO+ T cells. Experiments using keyhole limpet hemocyanin as a prototypic novel Ag, or tetanus toxoid as a recall Ag, were consistent with the notion that CD45RA+ RO- and CD45RA- RO+ T cell subsets can be designated as naive and memory cells, respectively; thus, responses to Hu.hsp60 were confined to the putative naive subset. In contrast, both CD45RA+ RO- and CD45RA- RO+ T cell populations proliferated to bacterial hsp60 from Mycobacterium leprae, Escherichia coli, or Chlamydia trachomatis. However, only CD45RA- RO+ (memory) T cells responded to a mycobacterial hsp60-derived peptide previously defined as a major bacteria-specific epitope. Experiments with cord blood T cells, which are CD45RA+ RO- and can be considered truly naive, showed that the peptide could elicit responses from naive T cells in vitro; cord blood cells also responded to Hu.hsp60. Since bacterial hsp60 Ags contain both conserved and nonconserved epitopes, we speculate that in vivo challenge with bacterial hsp60 will activate T cells capable of seeing either type of epitope, but only those that see nonconserved epitopes maintain the CD45RA- RO+ memory phenotype. However, T cells recognizing conserved epitopes, while not apparently being recruited to the memory pool, may nevertheless play a role in immunoregulation, particularly in the context of inflammation, when expression of Hu.hsp60 is increased.     

Chronic intravenous injections of antigen induce and maintain tolerance in T cell receptor-transgenic mice

Antigen-specific T cell tolerance can be induced by systemic injection of high-dose antigen. In particular, a single intravenous (i.v.) injection of influenza virus hemagglutinin peptide in HNT-TCR transgenic mice induces T cell tolerance through thymocyte apoptosis as well as anergy and deletion of peripheral CD4+ T cells. We now show that this tolerance is reversed after 8 weeks probably due to the short in vivo half-life of the peptide. Since durable tolerance is required for this strategy to be of therapeutic value, we tested whether weekly i.v. injections of peptide (up to 12 weeks) could maintain the CD4+ T cell tolerance. Each injection induces a profound deletion of thymocytes, although their level recovers before the next injection. Therefore, during the treatment period, the thymus undergoes cycles of contraction/expansion. In the periphery, the number of CD4+ T cells is stably decreased and the persisting CD4+ T cells are hyporeactive both in vitro and in vivo. This tolerance is essentially peripheral since comparable results were obtained in thymectomized HNT-TCR mice injected weekly. Our data show that stable antigen-specific tolerance can be induced by repeated i.v. injections of antigen. These findings might have implications for the treatment of T cell-mediated autoimmune diseases.     

Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa

We found that feeding keyhole limpet hemocyanin (KLH) to CD8-deficient (CD8-/-) mice induced oral tolerance that was comparable in both magnitude and quality to that induced in wild-type (wt) mice. The tolerance was dose dependent, and only higher doses of KLH caused significant reduction in specific Ab and T cell responses. Both Th1 and Th2 CD4+ T cell functions were affected. Feeding KLH together with cholera toxin (CT) adjuvant, however, abrogated the induction of oral tolerance equally well in CD8-/- and wt mice. On the contrary, CT adjuvant was unable to abrogate already established oral tolerance in both CD8-/- and wt mice. Most importantly, whereas Ag feeding induced hyporesponsiveness in systemic as well as in local gut IgA responses in wt mice, a lack of local suppression was evident in orally tolerant CD8-/- mice following oral immunizations. Thus, contrary to the situation in wt mice, Ag feeding induces systemic, but not local, gut IgA hyporesponsiveness in CD8-/- mice, suggesting that CD8+ T cells in the normal gut mucosa exert an important down-regulatory function. In wt mice the local suppression extended to an unrelated Ag, OVA, given together with KLH and CT adjuvant, i.e., bystander suppression. Based on these results we propose that tolerance induced by feeding Ag is highly compartmentalized, requiring CD8+ T cells for local suppression of IgA responses, whereas systemic tolerance may affect CD4+ T cells of both Th1 and Th2 types independently of CD8+ T cells. Finally, the adjuvant effect of CT abrogates induction, but not established, oral tolerance through a mechanism that does not require CD8+ T cells.     

Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway

By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II-deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II-deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency.