In vivo behavior of peptide-specific T cells during mucosal tolerance induction: antigen introduced through the mucosa of the conjunctiva elicits prolonged antigen-specific T cell priming followed by anergy

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11. 10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.     

Extinction of IL-12 signaling promotes Fas-mediated apoptosis of antigen-specific T cells.

In previous studies we have shown that peripheral tolerance achieved by high dose feeding of OVA to intact OVA-TCR transgenic mice was enhanced when endogenous IL-12 was neutralized simultaneously. To generalize this phenomenon, in the present study we investigated the tolerogenic mechanisms underlying the blockade of IL-12 signaling following oral and systemic Ag delivery. We found that the numbers of Ag-specific T cells in several lymphoid organs were significantly reduced due to T cell apoptosis following oral OVA or systemic OVA administration when combined with anti-IL-12 injection, but there was no decrease in T cell numbers for OVA-fed, OVA-injected, or anti-IL-12 alone-treated mice compared with those in untreated control mice. In addition, mostly Fas+ T cells were subject to apoptotic deletion in the OVA- plus anti-IL-12-treated groups, and an enhanced cell death of T cells upon OVA restimulation in vitro could be partially reversed by blockade of the Fas/Fas ligand interaction. Finally, in a murine model of superantigen-driven T cell expansion and deletion, we observed no deletional effects of anti-IL-12 treatment on CD4+ cells in Fas-deficient (MRL/lpr) mice, but did find these effects in MRL wild-type mice. In summary, our data suggest that in the course of Ag-induced cell proliferation of Th1 cells, signaling through IL-12 is required to prevent an induction of Fas-mediated apoptosis. Thus, the use of anti-IL-12 may be potentially useful in modulating peripheral immune responses by promotion of Fas-mediated cell death.     

Inhibition of specific immune responses by feeding protein antigens. V. Induction of the tolerant state in the absence of specific suppressor T cells

The effect of CY pretreatment on the ability of OVA feeding to induce both tolerance and active suppression was examined in mice. CY-pretreated, OVA fed mice were fully unresponsive in both OVA-specific DTH and antibody responses, but, in contrast to untreated OVA-fed mice, did not transfer suppression to normal recipients via splenic lymphocytes. Restoration of Ts activity in CY-pretreated mice was accomplished by reconstitution with normal T cells before antigen feeding, indicating that the CY effect was at the Ts precursor level. In addition, it was found that certain OVA-specific immune parameters (DTH and splenic PFC responses) in recipient mice were susceptible to suppression by transfer of spleen cells from OVA-fed donors, whereas other measures (antigen-induced T cell proliferation and serum antibody titers) were not. The data suggest that CY-sensitive Ts are not necessary for either induction or maintenance of specific tolerance after OVA feeding.     

Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4(+) T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4(+) T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-gamma, cytokine production by OVA-specific CD4(+) T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH(4)Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4(+) T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4(+) T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic "inflammatory" professional APCs able to activate primary CD4(+) T cell responses in regional LNs.     

Regulation of the primary in vitro response to TNP-polymerized ovalbumin by T suppressor cells induced by ovalbumin feeding

Spleen cells from DBA/2 mice that received a single feeding of 20 mg of ovalbumin (OVA) 7 days previously were specifically hyporesponsive to primary in vitro challenge with the thymic-dependent antigen TNP-polymerized ovalbumin (TNP-POL-OVA). The tolerance observed in spleen cells from OVA-fed animals was dependent upon OVA-specific T suppressor cells, because splenic T cells from OVA-fed mice suppressed the primary response to TNP-POL-OVA of cultures containing normal T and B cells. The tolerance and suppression was OVA specific, because spleen cells from OVA-fed animals responded well to other antigens (including TNP on another carrier), and splenic T cells from OVA-fed mice did not affect the response of normal T and B cells to sheep erythrocytes. These data confirm the existence of T suppressor cells after OVA feeding and provide a direct means of assaying their activity in a primary in vitro response.     

CTLA-4 regulates expansion and differentiation of Th1 cells following induction of peripheral T cell tolerance

Intravenous treatment with Ag (peptide)-coupled, ethylene carbodiimide-fixed syngeneic splenocytes (Ag-SP) is a powerful method to induce anergy in vitro and peripheral T cell tolerance in vivo. In this study, we examined the effects of Ag-SP administration on T cell activity ex vivo and in vivo using OVA-specific DO11.10 TCR transgenic T cells. Although treatment with OVA323-339-SP resulted in a strong inhibition of peptide-specific T cell recall responses in vitro, examination of the immediate effects of Ag-SP treatment on T cells in vivo demonstrated that tolerogen injection resulted in rapid T cell activation and proliferation. Although there was an increase in the number of OVA-specific DO11.10 T cells detected in the lymphoid organs, these previously tolerized T cells were strongly inhibited in mounting proliferative or inflammatory responses upon rechallenge in vivo with peptide in CFA. This unresponsiveness was reversible by treatment with anti-CTLA-4 mAb. These results are consistent with the hypothesis that Ag-SP injection induces a state of T cell anergy that is maintained by CTLA-4 engagement.     

The adjuvant effects of Mycobacterium tuberculosis heat shock protein 70 result from the rapid and prolonged activation of antigen-specific CD8+ T cells in vivo

Heat shock protein 70 (hsp70) is a potent adjuvant that links innate and adaptive immune responses. To study how hsp70 activates naive CD8(+) T cells in vivo, we tracked Ag-specific CD8(+) T cells in mice immunized with a fusion protein containing chicken OVA linked to hsp70 derived from Mycobacterium tuberculosis (OVA.TBhsp70). On a molar basis, OVA.TBhsp70 was several hundred times more effective than OVA peptide plus CFA in eliciting specific CD8(+) T cell responses. Immunization with OVA.TBhsp70 activated >90% of detectable OVA-specific CD8(+) T cells within 3 days and led to the persistence of cytotoxic effectors for at least 17 days. These studies demonstrate that the potent adjuvant effect of M. tuberculosis hsp70 results from the relatively complete, rapid, and durable activation of Ag-specific CD8(+) T cells.     

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.     

Inhibition of specific immune responses by feeding protein antigens. IV. Evidence for tolerance and specific active suppression of cell-mediated immune responses to ovalbumin.

We studied the effect of a single intragastric administration of ovalbumin (OVA) on the subsequent development of OVA-specific cell-mediated immune (CMI) responses in BDF1 mice. In animals fed OVA 7 days before subcutaneous sensitization with OVA-CFA, we observed a concomitant dose-dependent decrease in both the humoral and CMI responses specific for OVA. The CMI tolerance was found to be antigen-specific when assayed in vivo by ear swelling or in vitro by an antigen-induced T cell proliferation assay because OVA-fed mice responded normally to sensitization with horse gamma-globulin. It was also shown that either spleen or lymph node cells, but not serum, from OVA-fed donors transferred suppression to normal recipients. The transfer was mediated by antigen-specific suppressor T cells (Ts) that appeared to inhibit the induction phase (afferent limb) of the CMI response, since the Ts were only effective when transferred before or shortly after the onset of sensitization.     

Cell cycle block in anergic T cells during tolerance induction

The induction of anergy, or T cell unresponsiveness to antigen, is preceded by T cell activation and cell division in response to fed antigens. These events parallel the activation observed in T cells following sensitization with antigen and adjuvant. The events that distinguish eventual sensitization versus tolerance remain unclear. Using a T lymphocyte transfer model specific to OVA, we demonstrated previously that oral encounter with antigen leads to functional anergy. Antigen-specific CD4+ T cells nevertheless become activated and cycle briefly after encounter with antigen. In this study, we measured the extent of cell cycling of antigen-specific T cells after oral encounter with their antigen. Whereas T cells cycle on the average of eight times in 4 days after conventional immunization, an abortive proliferation was observed in the draining LN T cells after oral encounter with antigen; OVA-specific T cells divided fewer times after exposure to fed OVA, compared to T cells in mice immunized with OVA. This abortive proliferation is antigen specific and not due to bystander suppression, as coadministration of an unrelated antigen that was previously used as a tolerogen does not alter the degree of abortive proliferation. Measurement of BrdU incorporation in mice that were previously fed ovalbumin indicates that up to 3 days following feeding, OVA-specific cells are actively cycling in vivo. However, by day 4, they have stopped cycling while identical T cells in OVA-sensitized mice continue to cycle. Our results indicate either that tolerance is a default pathway and a secondary stimulus is required at day 3 to progress to sensitization, or that elements that limit cell cycle progression are provided for tolerance induction.     

Tolerance induction by transcutaneous immunization through ultraviolet-irradiated skin is transferable through CD4+CD25+ T regulatory cells and is dependent on host-derived IL-10

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4(+)CD25(+) T regulatory (T(reg)) cells. These Ag-specific T(reg) cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of T(reg) cells through UV-irradiated skin as IL-10-deficient T(reg) cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated T(reg) cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced T(reg) cells mediate their effects in part through the modulation of IL-10.     

Intestinal epithelial antigen induces mucosal CD8 T cell tolerance, activation, and inflammatory response

Intestinal autoimmune diseases are thought to be associated with a breakdown in tolerance, leading to mucosal lymphocyte activation perhaps as a result of encounter with bacterium-derived Ag. To study mucosal CD8(+) T cell activation, tolerance, and polarization of autoimmune reactivity to self-Ag, we developed a novel (Fabpl(4x at -132)-OVA) transgenic mouse model expressing a truncated form of OVA in intestinal epithelia of the terminal ileum and colon. We found that OVA-specific CD8(+) T cells were partially tolerant to intestinal epithelium-derived OVA, because oral infection with Listeria monocytogenes-encoding OVA did not elicit an endogenous OVA-specific MHC class I tetramer(+)CD8(+) T cell response and IFN-gamma-, IL-4-, and IL-5-secreting T cells were decreased in the Peyer's patches, mesenteric lymph nodes, and intestinal mucosa of transgenic mice. Adoptive transfer of OVA-specific CD8(+) (OT-I) T cells resulted in their preferential expansion in the Peyer's patches and mesenteric lymph nodes and subsequently in the epithelia and lamina propria but failed to cause mucosal inflammation. Thus, CFSE-labeled OT-I cells greatly proliferated in these tissues by 5 days posttransfer. Strikingly, OT-I cell-transferred Fabpl(4x at -132)-OVA transgenic mice underwent a transient weight loss and developed a CD8(+) T cell-mediated acute enterocolitis 5 days after oral L. monocytogenes-encoding OVA infection. These findings indicate that intestinal epithelium-derived "self-Ag" gains access to the mucosal immune system, leading to Ag-specific T cell activation and clonal deletion. However, when Ag is presented in the context of bacterial infection, the associated inflammatory signals drive Ag-specific CD8(+) T cells to mediate intestinal immunopathology.     

IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.     

T regulatory cells 1 inhibit a Th2-specific response in vivo

We recently described a new population of CD4(+) regulatory T cells (Tr1) that inhibits proliferative responses of bystander T cells and prevents colitis induction in vivo through the secretion of IL-10. IL-10, which had been primarily described as a Th2-specific cytokine inhibiting Th1 responses, has displayed in several models a more general immune suppression on both types of effector T cell responses. Using an immediate hypersensitivity model in which BALB/c mice immunized with OVA (alum) normally generate Th2-dominated responses, we examined the ability of OVA-specific Tr1 T cell clones to inhibit OVA-specific cytokines and Ab responses. In contrast to Th2 or Th1 T cell clones, transfer of Tr1 T cell clones coincident with OVA immunization inhibited Ag-specific serum IgE responses, whereas IgG1 and IgG2a synthesis were not affected. This specific inhibition was mediated in part through IL-10 secretion as anti-IL-10 receptor Abs treatment reverted the inhibitory effect of Tr1 T cell clones. Although specifically targeted to IgE responses, Tr1 clones' inhibitory effects were more profound as they affected Ag-specific Th2 cell priming both in term of proliferative responses and cytokine secretion. These results suggest that regulatory T cells may play a fundamental role in maintaining the balance of the immune system to prevent allergic disorders.     

Antigenic epitopes regulate the phenotype of CD8+ CTL primed by exogenous antigens

We previously reported that insulin-specific, MHC class I-restricted CTL precursors can be primed by injecting C57BL/6 mice with bovine insulin in CFA. These bovine insulin-primed CTL displayed a type 0 CTL phenotype, producing IL-4, IL-5, IL-10, low levels of IFN-gamma, but no TNF-alpha. By contrast, CTL generated from C57BL/6 mice primed with OVA in CFA produced IFN-gamma and TNF-alpha but no IL-4, IL-5, or IL-10 and therefore were classified as type 1 CTL. Although CD4+ T cell subsets have been compared extensively in the literature, CTL subsets are less well characterized. Here, the phenotype, function, and requirements for the in vivo activation of type 1 and type 0 CTL cells were studied. Although both types of CTL express many of the same cell-surface Ags, OVA-specific CTL but not bovine insulin-primed CTL expressed CT-1, a carbohydrate epitope of CD45, and bovine insulin-primed CTL but not OVA-specific CTL expressed Fas constitutively. Priming of CTL was abrogated by depletion of phagocytic cells but not CD4+ T cells, whereas depletion of CD4+ T cells but not phagocytic cells inhibited Ab responses in the same mice. Neither endogenous IL-4 nor the dose of priming Ag altered the CTL phenotypes, but the antigenic peptides of OVA and bovine insulin were key to determining the differentiation of either type 1 or type 0 CTL. To our knowledge, this is the first time that antigenic epitopes have been demonstrated to influence the phenotype of Ag-specific CTL responses. These results may be relevant to the development of peptide vaccines in which a particular type of CTL response is desired.     

Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

Loss of the surface antigen 3G11 characterizes a distinct population of anergic/regulatory T cells in experimental autoimmune encephalomyelitis

T cell anergy is an important mechanism in the induction of peripheral tolerance against autoimmune diseases, yet no surface marker unique to anergic T cells in these diseases has been identified. In this study we induced in vivo anergy by i.v. tolerance against experimental autoimmune encephalomyelitis in myelin basic protein TCR transgenic mice, and showed that the hyporesponsiveness of autoantigen-reactive T cells from tolerized mice was associated with a dramatic loss of 3G11, a cell surface molecule on the surface of CD4+ T cells. Purified 3G11-CD4+ T cells lost autoantigen-induced proliferation and IL-2 production, whereas 3G11+CD4+ T cells retained responsiveness. Furthermore, 3G11- T cells actively suppressed proliferation and Th1 cytokine production of 3G11+ T cells and splenocytes of nontolerized mice. Active suppression by 3G11- T cells was at least partially due to soluble immunoregulatory factors, including IL-10. The T regulatory property of 3G11- T cells was confirmed in vivo because the transfer of purified 3G11- T cells effectively suppressed clinical experimental autoimmune encephalomyelitis. We conclude that loss of the surface molecule 3G11 characterizes a distinct population of anergic/regulatory T cells. This is the first demonstration of the ability to identify and purify anergic T cells by a distinct cell surface marker in an autoimmune disease and paves the way for a better understanding of the mechanism of tolerance in autoimmune diseases.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.