Autoreactive B cells in lupus-prone New Zealand black mice exhibit aberrant survival and proliferation in the presence of self-antigen in vivo

To identify defects in B cell tolerance that may contribute to the production of autoantibodies in New Zealand Black (NZB) mice, we crossed soluble hen egg white lysozyme (sHEL) and anti-HEL Ig transgenes (Ig Tg) onto the NZB background. In this study, we have examined one of the first checkpoints involved in maintenance of peripheral B cell tolerance, follicular exclusion and elimination of self-reactive B cells in the absence of T cell help. Freshly isolated anti-HEL Ig Tg B cells were labeled with CFSE, adoptively transferred into sHEL recipients, and the fate of self-reactive anti-HEL Ig Tg B cells was followed using flow cytometry and immunofluorescence microscopy. Although anti-HEL Ig Tg B cells from NZB mice are appropriately excluded from B cell follicles in NZB sHEL recipient mice, they demonstrate aberrant survival, proliferation, and generation of anti-HEL Ab-producing cells. This abnormal response results from an intrinsic defect in NZB B cells, requires the presence of CD4(+) T cells, and is facilitated by the splenic environment in NZB mice. Thus, NZB mice have immune defects that interact synergistically to allow autoreactive B cells to become activated despite the presence of tolerizing autoantigens.     

T cell autoimmunity in Ig transgenic mice.

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.     

Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.     

Cutting edge: CD25+ regulatory T cells prevent expansion and induce apoptosis of B cells specific for tissue autoantigens

To study the role of CD25(+) regulatory T cells (T(regs)) in peripheral B cell tolerance, we generated transgenic rat insulin promoter RIP-OVA/HEL mice expressing the model Ags OVA and HEL in pancreatic islet beta cells (where RIP is rat insulin promoter and HEL is hen egg lysozyme). Adoptively transferred transgenic OVA-specific CD4(+) and CD8(+) T cells proliferated only in the autoantigen-draining pancreatic lymph node (PLN), demonstrating pancreas-specific Ag expression. Transferred HEL-specific transgenic B cells (IgHEL cells) disappeared within 3 wk from transgenic but not from nontransgenic mice immunized with autoantigen. Depletion of CD25(+) FoxP3(+) cells completely restored IgHEL cell numbers. T(reg) exerted an analogous suppressive effect on endogenous HEL-specific autoreactive B cells. T(regs) acted by inhibiting the proliferation of IgHEL cells in the spleen and PLN and by systemic induction of their apoptosis. Furthermore, they reduced BCR and MHC II surface expression on IgHEL cells in the PLN. These findings demonstrate that autoreactive B cells specific for a nonlymphoid tissue autoantigen are controlled by T(regs).     

IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production

Persistent cross-linking of hen egg lysozyme (HEL)-specific B cell membrane Ig (mIg) in double transgenic mice that express soluble HEL as a self Ag (HEL-Ig mice) decreases B cell mIgM expression, responsiveness, and life span. Because in vitro treatment with IL-4 inhibits T cell apoptosis through a Stat6-independent mechanism, increases mIg expression, and suppresses activation-induced B cell death, we studied IL-4 effects on B cell mIg expression, survival, and Ab secretion in Stat6-sufficient and deficient HEL-Ig mice. IL-4 treatment nearly normalized B cell number and greatly increased the percentage of mature B cells in HEL-Ig mice, but failed to normalize mIgM expression or spontaneous LPS-induced IgM secretion. IL-4 effects on B cell survival and maturation were CD4(+) T cell independent, but Stat6 dependent, and did not involve receptor editing. IL-4 had to be present while B cells were generated to have a detectable effect on autoreactive B cell survival; however, the survival of B cells generated in the presence of IL-4 was substantially increased even after IL-4 was withdrawn. These observations suggest that: 1) activation-induced B cell death and anergy are independent processes; 2) B cells that survive to maturity develop increased resistance to Ag-induced deletion; and 3) IL-4 promotes B and T cell survival through different mechanisms.     

Persistent T cell anergy in human type 1 diabetes

An anergic phenotype has been observed in nonobese diabetic (NOD) mice and some autoreactive T cells from patients with type I diabetes. To better understand this phenomenon, we measured T cell proliferative responses to 10 diabetes-associated and up to 9 control Ags/peptides in 148 new diabetic children, 51 age- and MHC (DQ)-matched siblings (sibs), 31 patients with longstanding diabetes, and 40 healthy controls. Most (78-91%) patient and sib responses to glutamate decarboxylase of 65 kDa (GAD65), islet cell cytoplasmic autoantibody (ICA) 69, diabetes-associated T cell epitopes in ICA69 (Tep69), and heat shock protein (Hsp) 60 involved anergic T cells that required exogenous IL-2 to proliferate. Responses to proinsulin, IA-2 (and tetanus toxoid) required no IL-2 and generated sufficient cytokine to rescue anergic T cell responses. Most new patients (85%) had autoreactive T cells, three quarters targeting more than half of the diabetes Ags. Only 7.8% of the sibs and none of the controls had such multiple T cell autoreactivities, which thus characterize overt disease. Multiple anergic and nonanergic T cell autoreactivities were sustained during 2 yr follow-up after onset and in patients with longstanding (3-26 yr) diabetes. Activated patient T cells survived severe IL-2 deprivation, requiring 20-100 times less IL-2 than normal T cells to escape apoptosis. Diabetic T cell anergy thus persists for decades and is Ag and host specific but not related to disease course. Rescue by IL-2 from bystander T cells and high resistance to apoptosis may contribute to this persistence. These data explain some of the difficulties in the routine detection of disease-associated T cells, and they emphasize challenges for immunotherapy and islet transplantation.     

Cellular competition independent of BAFF/B lymphocyte stimulator results in low frequency of an autoreactive clonotype in mature polyclonal B cell compartments

The peripheral B cell prosurvival cytokine BAFF/B lymphocyte stimulator (BLyS) has been proposed to participate in the regulation of immunological tolerance. Selective elimination or reconstitution of B cells expressing transgene-encoded, autoreactive BCRs upon systemic BLyS depletion or supplementation, respectively, was observed in two separate studies. Such findings led to a model positing a higher dependency of autoreactive B cells on BLyS. We tested this model by exploiting two targeted IgH transgenic mice (H chain knock-in [HKI]) that produce large numbers of follicular (FO) B cells that are either weakly or strongly autoreactive with nuclear autoantigens. Even though HKI B cells do not exhibit classical features of anergy, we found that mature, naive, autoreactive HKI B cells are outcompeted for representation in the periphery by a polyclonal B cell population. However, this is not due to a higher dependency of HKI B cells on BLyS for survival. Additionally, excess BLyS does not rescue HKI B cells from selective elimination. These findings suggest that some autoreactive FO B cells can fully develop while in competition with non-autoreactive cells for BLyS, but remain at a competitive disadvantage for other trophic factors that regulate peripheral stability. As such, our data indicate the existence of peripheral tolerance mechanisms that regulate the frequency of autoreactive FO B cells independent of the BLyS pathway.     

Autoreactive T cells revealed in the normal repertoire: escape from negative selection and peripheral tolerance

Self-reactive T cells are known to be eliminated by negative selection in the thymus or by the induction of tolerance in the periphery. However, developmental pathways that allow self-reactive T cells to inhabit the normal repertoire are not well-characterized. In this investigation, we made use of anti-small nuclear ribonucleoprotein particle (snRNP) Ig transgenic (Tg) mice (2-12 Tg) to demonstrate that autoreactive T cells can be detected and activated in both normal naive mice and autoimmune-prone MRL lpr/lpr mice. In contrast, autoreactive T cells of nonautoimmune Tg mice are tolerized by Tg B cells in the periphery. In adoptive transfer studies, autoreactive T cells from MRL lpr/lpr mice can stimulate autoantibody synthesis in nonautoimmune anti-snRNP Tg mice. Transferred CD4 T cells migrate to regions of the spleen proximal to the B cell follicles, suggesting that cognate B cell-T cell interactions are critical to the autoimmune response. Taken together, our studies suggest that anti-snRNP B cells are important APCs for T cell activation in autoimmune-prone mice. Additionally, we have demonstrated that anti-snRNP B cell anergy in nonautoimmune mice may be reversed by appropriate T cell help.     

Ig light chain receptor editing in anergic B cells

Receptor editing in the bone marrow (BM) serves to modify the Ag receptor specificity of immature self-reactive B cells, while anergy functionally silences self-reactive clones. Here, we demonstrate that anergic B cells in hen egg lysozyme Ig (HEL-Ig)/soluble HEL double transgenic mice show evidence of having undergone receptor editing in vivo, as demonstrated by the presence of elevated levels of endogenous kappa light chain rearrangements in the BM and spleen. In an in vitro IL-7-driven BM culture system, HEL-Ig BM B cells grown in the presence of soluble HEL down-regulated surface IgM expression and also showed induction of new endogenous kappa light chain rearrangements. Using a panel of soluble protein ligands with reduced affinity for the HEL-Ig receptor, the editing response was shown to correlate in a dose-dependent fashion with the strength of signaling through the B cell receptor. The finding that the level of B cell receptor cross-linking sufficient to induce anergy in B cells is also capable of engaging the machinery required for receptor editing suggests an intimate relationship between these two mechanisms in maintaining B cell tolerance.     

Autoreactive B cells escape clonal deletion by expressing multiple antigen receptors

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2-/-) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2-/- knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.     

B cell selection defects underlie the development of diabetogenic APCs in nonobese diabetic mice

One mechanism whereby B cells contribute to type 1 diabetes in nonobese diabetic (NOD) mice is as a subset of APCs that preferentially presents MHC class II-bound pancreatic beta cell Ags to autoreactive CD4 T cells. This results from their ability to use cell surface Ig to specifically capture beta cell Ags. Hence, we postulated a diabetogenic role for defects in the tolerance mechanisms normally blocking the maturation and/or activation of B cells expressing autoreactive Ig receptors. We compared B cell tolerance mechanisms in NOD mice with nonautoimmune strains by using the IgHEL and Ig3-83 transgenic systems, in which the majority of B cells recognize one defined Ag. NOD- and nonautoimmune-prone mice did not differ in ability to delete or receptor edit B cells recognizing membrane-bound self Ags. However, in contrast to the nonautoimmune-prone background, B cells recognizing soluble self Ags in NOD mice did not undergo partial deletion and were also not efficiently anergized. The defective induction of B cell tolerance to soluble autoantigens is most likely responsible for the generation of diabetogenic APC in NOD mice.     

Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4(+) Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.     

Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes

Loss of tolerance is considered to be an early event that is essential for the development of autoimmune disease. In contrast to this expectation, autoimmune (type 1) diabetes develops in NOD mice that harbor an anti-insulin Ig transgene (125Tg), even though anti-insulin B cells are tolerant. Tolerance is maintained in a similar manner in both normal C57BL/6 and autoimmune NOD mice, as evidenced by B cell anergy to stimulation through their Ag receptor (anti-IgM), TLR4 (LPS), and CD40 (anti-CD40). Unlike B cells in other models of tolerance, anergic 125Tg B cells are not arrested in development, and they enter mature subsets of follicular and marginal zone B cells. In addition, 125Tg B cells remain competent to increase CD86 expression in response to both T cell-dependent (anti-CD40) and T cell-independent (anti-IgM or LPS) signals. Thus, for anti-insulin B cells, tolerance is characterized by defective B cell proliferation uncoupled from signals that promote maturation and costimulator function. In diabetes-prone NOD mice, anti-insulin B cells in this novel state of tolerance provide the essential B cell contribution required for autoimmune beta cell destruction. These findings suggest that the degree of functional impairment, rather than an overt breach of tolerance, is a critical feature that governs B cell contribution to T cell-mediated autoimmune disease.     

In vivo survival of autoreactive B cells: characterization of long-lived B cells

To determine the effects of chronic Ag stimulation on B cell survival and phenotype, we compared survival and surface markers of hen egg lysozyme (HEL)-specific B cells in Ig transgenic (Tgn) mice, which lack HEL, and in HEL-Ig transgenic mice, which express soluble HEL. Serum HEL levels were maximized in HEL-Ig Tgn mice by feeding them zinc, which activates the metallothionein promoter that regulates HEL expression. B cell age was characterized by expression of heat-stable Ag, and B220 and B cell survival was studied by evaluating changes in B cell number when lymphopoiesis was suppressed with anti-IL-7 mAb and by identifying newly generated B cells through 5-bromo-2'-deoxyuridine incorporation. Our observations show that the mean B cell life span is considerably reduced in HEL-Ig Tgn compared with Ig Tgn mice, but also demonstrate that some HEL-Ig Tgn B cells survive to maturity. Some of these surviving B cells have undergone receptor editing (substitution of an endogenous Ig light chain for the transgenic Ig light chain), so that their ability to bind HEL is decreased or absent. Surviving HEL-Ig Tgn B cells that retain HEL specificity express decreased mIgD and little or no mIgM. mIgD expression progressively decreases with increasing HEL-Ig Tgn B cell age. These observations suggest that self Ag-specific B cells can survive in the presence of soluble self Ag by down-regulating mIg expression, which should limit B cell signaling by Ag that might otherwise cause deletion of these cells.     

Ig allotypic inclusion does not prevent B cell development or response

B cells expressing two different Ig kappa L chains (allotype included) have been occasionally observed. To determine frequency and function of these cells, we have analyzed gene-targeted mice that carry a human and a mouse Igk C region genes. Using different methodologies, we found that cells expressing two distinct kappa-chains were 1.4-3% of all B cells and that they were present in the follicular, marginal zone, and B1 mature B cell subsets. When stimulated in vitro with anti-IgM, dual kappa surface-positive cells underwent activation that manifested with cell proliferation and/or up-regulation of activation markers and similar to single kappa-expressing B cells. Yet, when activated by divalent reagents that bound only one of the two kappa-chains, dual kappa B cells responded suboptimally in vitro, most likely because of reduced Ag receptor cross-linking. Nonetheless, dual kappa B cells participated in a SRBC-specific immune response in vivo. Finally, we found that Ig allotype-included B cells that coexpress autoreactive and nonautoreactive Ag receptors were also capable of in vitro responses following BCR aggregation. In summary, our studies demonstrate that Ig kappa allotype-included B cells are present in the mouse mature B cell population and are responsive to BCR stimulation both in vitro and in vivo. Moreover, because in vitro activation in response to anti-IgM was also observed in cells coexpressing autoreactive and nonautoreactive Abs, our studies suggest a potential role of allotype-included B cells in both physiological and pathological immune responses.     

Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways

Although the existence of autoreactive T cells has been widely reported, the functional capacities of these populations have been less well defined. Studies were therefore carried out to characterize the relationship of autoreactive T cells to antigen-specific major histocompatibility complex (MHC)-restricted T cells in their ability to act as helper cells for the induction of immunoglobulin synthesis by B cells. A number of autoreactive T cell lines and clones were isolated from antigen-primed spleen and lymph node cell populations. Autoreactive T cells were found to proliferate in response to direct recognition of syngeneic I-A or I-E subregion-encoded antigens in the absence of any apparent foreign antigen. It was shown that cloned autoreactive T cells were capable of activating B cell responses through two distinct pathways. After appropriate stimulation by syngeneic cells, autoreactive T cells polyclonally activated primed or unprimed B cells to synthesize IgM antibodies. These activated T cells functioned in these responses through an MHC-unrestricted pathway in which polyclonal responses were induced in both syngeneic and allogeneic B cells. These cloned autoreactive T cells were also able to activate IgG responses by primed B cells through a different activation pathway. In contrast to the polyclonal activation of IgM responses, the induction of IgG antibodies by the same cloned T cells required primed B cells and stimulation with the priming antigen. The activation of B cells to produce IgG was strongly MHC restricted and required the direct recognition by the autoreactive T cells of self MHC determinants expressed on the B cell surface, with no bystander activation of allogeneic B cells. These results indicate that cloned autoreactive T cells resemble antigen-specific MHC-restricted T cells in their ability to function as T helper cells through distinct MHC-restricted and MHC-unrestricted pathways.     

Disruption of T cell tolerance to self-immunoglobulin causes polyclonal B cell stimulation followed by inactivation of responding autoreactive T cells

Scavenger receptor (SR)-specific delivery by maleylation of a ubiquitous self-protein, Ig, to SR-bearing APCs results in self-limiting induction of autoimmune effects in vivo. Immunization with maleyl-Ig breaks T cell tolerance to self-Ig and causes hypergammaglobulinemia, with increases in spleen weight and cellularity. The majority of splenic B cells show an activated phenotype upon maleyl-Ig immunization, leading to large-scale conversion to a CD138+ phenotype and to significant increases in CD138-expressing splenic plasma cells. The polyclonal B cell activation, hypergammaglobulinemia, and autoreactive Ig-specific T cell responses decline over a 2-mo period postimmunization. Following adoptive transfer, T cells from maleyl-Ig-immune mice taken at 2 wk postimmunization can induce hypergammaglobulinemia in the recipients, but those taken at 10 wk postimmunization cannot. Hypergammaglobulinemia in the adoptive transfer recipients is also transient and is followed by an inability to respond to fresh maleyl-Ig immunization, suggesting that the autoreactive Ig-specific T cells are inactivated peripherally following disruption of tolerance. Thus, although autoreactive T cell responses to a ubiquitous self-Ag, Ig, are induced by SR-mediated delivery to professional APCs in vivo resulting in autoimmune pathophysiological effects, they are effectively and rapidly turned off by inactivation of these activated Ig-specific T cells in vivo.     

Autoreactive T cells persist in rats protected against experimental autoimmune encephalomyelitis and can be activated through stimulation of innate immunity

Lewis rats can be rendered unresponsive to experimental autoimmune encephalomyelitis by immunization with myelin basic protein (MBP), or MBP68-86, the dominant encephalitogenic MBP epitope for this strain, administered in IFA. However, protected rats harbor potentially encephalitogenic T cells, which are maintained in an inactive state. We investigated whether these quiescent effector cells could be activated in vitro. Although these T cells respond poorly to MBP68-86, they proliferate vigorously whether cocultured with MBP68-86 and either IL-2 or IL-12, suggesting that the T cells are in a state of anergy. Moreover, we could activate these anergic T cells with peptide and cytosine-guanine dinucleotide (CpG) oligonucleotide, but not control oligonucleotide, suggesting that products of the innate immune response are capable of activating anergic autoreactive T cells. The activated T cells produced the proinflammatory cytokine, IFN-gamma in response to IL-12, and IL-6 was secreted in response to CpG oligonucleotide. IL-6 has been reported to play a role in T cell activation by blocking T regulatory/suppressor (Treg) cell-mediated suppression through a Toll-like receptor-dependent pathway. However, anti-IL-6 mAb did not block CpG activation of the anergized cells. In contrast, anti-TGF-beta(1) Ab released the unresponsive T cells from the anergic state in the presence of MBP68-86, whereas TGF-beta(1) inhibited proliferation of MBP68-86- plus CpG-activated T cells. Because TGF-beta(1) has previously been implicated in Treg activity, this finding is consistent with a role for Treg cells in maintaining autoreactive T cells in the anergic state.     

Break of neonatal Th1 tolerance and exacerbation of experimental allergic encephalomyelitis by interference with B7 costimulation

Ig-PLP1 is an Ig chimera expressing proteolipid protein-1 (PLP1) peptide corresponding to aa residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals comprise Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. In this work, studies were performed to determine whether costimulation through B7 molecules plays any role in the unusual form of splenic Th1 anergy. The results show that engagement of either B7.1 or B7.2 with anti-B7 Abs during induction of EAE in adult mice that were neonatally tolerized with Ig-PLP1 restores and exacerbates disease severity. At the cellular level, the anergic splenic T cells regain the ability to proliferate and produce IFN-gamma when stimulated with Ag in the presence of either anti-B7.1 or anti-B7.2 Ab. However, such restoration was abolished when both B7.1 and B7.2 molecules were engaged simultaneously, indicating that costimulation is necessary for reactivation. Surprisingly, both anti-B7.1 and anti-B7.2 Abs triggered splenic dendritic cells to produce IL-12, a key cytokine required for restoration of the anergic T cells. Thus, recovery from neonatally induced T cell anergy requires B7 molecules to serve double functions, namely, costimulation and induction of cytokine production by APCs.     

Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells

Anti-dsDNA B cells are actively tolerized in nonautoimmune BALB/c mice, as manifested by their developmental arrest, follicular exclusion, and rapid turnover rate. Previously, we have documented changes in the maturation status and follicular localization of anti-dsDNA B cells in autoimmune-prone MRL (+/+ and lpr/lpr) mice. To determine whether these differences in developmental status and follicular localization affect the functional capacity of anti-dsDNA B cells, we have now compared their in vivo life spans and their responses to in vitro stimuli. Our study shows that although anti-dsDNA B cells from both BALB/c and MRL-+/+ mice are localized to the T/B interface, only those in BALB/c mice have a rapid turnover rate. Therefore, the immature status and not the exclusion from the B cell follicle correlates with a shortened life span. Interestingly, apoptotic anti-dsDNA B cells were not detected at the T/B interface in BALB/c mice, suggesting that they are not dying there. This study also demonstrates that anti-dsDNA B cells, regardless of maturation status or follicular localization, are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40 ligand and IL-4. Therefore, one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ mice compared with MRL-lpr/lpr mice may be the availability of T cell help.