Low-affinity, Smith antigen-specific B cells are tolerized by dendritic cells and macrophages

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.     

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.     

T cell-intrinsic and -extrinsic contributions of the IFNAR/STAT1-axis to thymocyte survival

STAT1 is an essential part of interferon signaling, and STAT1-deficiency results in heightened susceptibility to infections or autoimmunity in both mice and humans. Here we report that mice lacking the IFNα/β-receptor (IFNAR1) or STAT1 display impaired deletion of autoreactive CD4(+)CD8(+)-T-cells. Strikingly, co-existence of WT T cells restored thymic elimination of self-reactive STAT1-deficient CD4(+)CD8(+)-T cells. Analysis of STAT1-deficient thymocytes further revealed reduced Bim expression, which was restored in the presence of WT T cells. These results indicate that type I interferons and STAT1 play an important role in the survival of MHC class I-restricted T cells in a T cell intrinsic and non-cell intrinsic manner that involves regulation of Bim expression through feedback provided by mature STAT1-competent T cells.     

Induction of neonatal tolerance to the Mls-1a self-super-antigen. Time kinetics and MHC restriction.

We examined the accessibility of the thymus to a self-super-Ag encoded by the Mls-1a region of chromosome 1 and the process by which this Ag establishes immunologic tolerance. Intravenously administered Mls-1a Ag accumulates quickly in peripheral organs of adult or newborn Mls-1a- recipients, where it mounts an immune response. The Ag does not enter the thymus in detectable amounts and does not induce an immune response of Mls-1a-responsive T cells present in this organ. Instead, the thymus of newborn Mls-1a- recipients of Mls-1a+ lymphoid cells continues for several days to export Mls-1a-reactive T cells, which respond to Mls-1a Ag when they encounter it in peripheral organs. This response peaks around day 3 or day 4 and declines very rapidly thereafter. The deletion of intrathymic Mls-1a-reactive T cells ensues simultaneously with this decline. It has previously been shown that Mls-1a Ag causes deletion or anergy of Mls-1a-reactive peripheral T cells, subsequent to their activation. We see the same time kinetics in producing deletion or anergy of Mls-1a-reactive T cells in the thymus of newborn animals, with the exception that the activation phase that precedes the deletion of Mls-1a-reactive T cells occurs in the periphery and not in the thymus. This observation indicates that thymic Mls-1a-specific T cells are not deleted through activation. Whether their deletion depends on a feed-back from the peripheral activation of Mls-1a-reactive cells, as the time relationship could suggest, is not clear. The finding establishes, however, that the deletion of functionally mature Mls-1a-reactive T cells and the activation of such cells are not necessarily related events, which may or may not utilize a common trigger mechanism, such as the engagement of the TCR. Concerning the trigger mechanism, we report that Mls-1a-specific deletion of T cells is an MHC-restricted process, whereas Mls-1a-specific activation of T cells is not MHC restricted.     

Autoimmunity: basic mechanisms and implications in endocrine diseases. Part II

Regulation of the immune response to self-antigens is a complex process that involves maintaining self-tolerance while preserving the capacity to exert an effective immune response. The primary mechanism that leads to self-tolerance is central tolerance. However, potential pathogenic autoreactive lymphocytes are normally present in the periphery of all individuals. This suggests the existence of mechanisms of peripheral tolerance that prevent the initiation of autoimmune diseases by limiting the activation of autoreactive lymphocytes. If these mechanisms of peripheral tolerance are impaired, the autoreactive lymphocytes may be activated and autoimmune diseases can develop. Several processes are involved in the maintenance of peripheral tolerance: the active suppression mediated by regulatory T cell populations, the different maturation state of antigen-presenting cells presenting the autoantigen to autoreactive lymphocytes, inducing tolerance instead of cell activation, the characteristics of B cell populations. A deeper comprehension of these mechanisms may lead to important therapeutic applications, such as the development of cellular vaccines for organ-specific autoimmune diseases. In addition, autoimmunity does not always have pathological consequences, but may exert a protective function, as suggested by several observations on the beneficial role of autoreactive T cells in central nervous system injury.     

The dendritic cell-specific CC-chemokine DC-CK1 is expressed by germinal center dendritic cells and attracts CD38-negative mantle zone B lymphocytes

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA(+) T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38(-)) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.     

Activation-induced apoptosis of autoreactive and alloreactive T lymphocytes in the target organ as a major mechanism of tolerance.

Normal individuals have mature T lymphocytes that are capable of reacting to self-antigens and can be activated by cross-reacting environmental antigens. The mechanism that maintains immune tolerance and prevents these activated autoreactive T cells from causing autoimmune disease is unclear. We have previously hypothesized that activation-induced apoptosis of previously activated autoreactive T cells in the target organ is a major mechanism for maintaining tolerance. Here I review the current evidence to support this hypothesis. It is proposed that when activated autoreactive T cells enter the target organ, they are reactivated mainly by non-professional antigen-presenting cells (APC) and deleted by activation-induced apoptosis through the Fas (CD95) pathway before producing significant target organ damage. This apoptosis occurs because the reactivated T cells do not receive sufficient costimulation from the non-professional APC to up-regulate their expression of Bcl-2-related anti-apoptotic proteins, which inhibit the CD95 pro-apoptotic pathway. This is in contrast to the situation in peripheral lymphoid organs, where reactivation of T cells by professional APC results in sufficient costimulation-induced up-regulation of Bcl-2-related proteins to inhibit the CD95 pathway and allow T cell proliferation and survival as memory T cells. Activation-induced apoptosis of alloreactive T cells in allografts can similarly account for spontaneous allograft acceptance, as occurs after MHC-mismatched liver transplantation.     

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.     

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.     

The interface between biochemical signaling and cell mechanics shapes T lymphocyte migration and activation

tT cells migrate to lymphoid organs to become activated through specific contacts with antigen-presenting cells bearing foreign antigens. During migration and activation, T lymphocytes are exposed not only to diverse biochemical inputs, but also to different mechanical conditions. Passage from the blood or lymph to solid tissues involves lymphocyte rolling, firm arrest and diapedesis through endothelial monolayers. Throughout this process, cells are subjected to diverse fluid flow regimes. After extravasation, T lymphocytes crawl through viscoelastic media of different biochemical and mechanical properties and geometries. In lymph nodes, T cell contact with antigen-presenting cells is guided by rigidity cues and ligand-receptor interactions. T lymphocyte adaptation to diverse mechanical regimes involves multiple signaling and morphological modifications, many of which enable the conversion of mechanical forces into biochemical signals and vice-versa. These components enable T lymphocyte survival, homing and activation. Here, we review the mechanisms that enable T lymphocytes to survive and thrive under the different mechanical conditions they encounter during their life cycle. These processes require the integration of diverse signaling networks that convert extracellular mechano-chemical cues into force, movement and activation.     

The proapoptotic Bcl-2 family member Bim plays a central role during the development of virus-induced hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.     

Constitutive production of NF-kappaB2 p52 is not tumorigenic but predisposes mice to inflammatory autoimmune disease by repressing Bim expression

Normal development of the immune system requires regulated processing of NF-kappaB2 p100 to p52, which activates NF-kappaB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-kappaB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-kappaB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-kappaB2 mutants and p52 and suggest a causal role for sustained NF-kappaB2 activation in the pathogenesis of autoimmunity.     

Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

Humans and mice deficient in Fas, a tumor necrosis factor (TNF)-receptor family member, cannot induce apoptosis of autoreactive cells, and consequently develop progressive lymphoproliferative disorders and lupus-like autoimmune diseases. Previous studies have shown that short-term administrations of agonistic monoclonal antibodies against CD137, another TNF-receptor family member, activate T cells and induce rejection of allografts and established tumors. Here we report that treatment with an agonistic monoclonal antibody to CD137 (2A) blocks lymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ultimately leading to their prolonged survival. Notably, 2A treatment rapidly augments IFN-gamma production, and induces the depletion of autoreactive B cells and abnormal double-negative T cells, possibly by increasing their apoptosis through Fas- and TNF receptor-independent mechanisms. This study demonstrates that agonistic monoclonal antibodies specific for costimulatory molecules can be used as novel therapeutic agents to delete autoreactive lymphocytes and block autoimmune disease progression.     

Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4(+)CD25(+) regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4(+)CD25(+) regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4(+)CD25(+) thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.     

Human follicular dendritic cells express prostacyclin synthase: a novel mechanism to control T cell numbers in the germinal center

Stromal cells in the lymphoid organs provide a microenvironment where lymphocytes undergo various biological processes such as development, homing, clonal expansion, and differentiation. Follicular dendritic cells (FDCs) in the primary and secondary follicles of the peripheral lymphoid tissues interact with lymphocytes by contacting directly or producing diffusible molecules. To understand the biological role of human FDC at the molecular level, we developed a mAb, 3C8, that recognizes FDC but not bone marrow-derived cells. Through expression cloning and proteome analysis, we identified the protein that is recognized by 3C8 mAb, which revealed that FDC expresses prostacyclin synthase. The 3C8 protein purified from FDC-like cells indeed displayed the enzymatic activity of prostacyclin synthase and converted PGH2 into prostacyclin. In addition, prostacyclin significantly inhibited proliferation of T cells but delayed their spontaneous apoptosis. These findings may help explain why T cells constitute only a minor population compared with B cells in the germinal center.     

Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery

Autophagy is implicated in regulating cell death in activated T cells, but the underlying mechanism is unclear. Here, we show that inhibition of autophagy via Beclin 1 gene deletion in T cells leads to rampant apoptosis in these cells upon TCR stimulation. Beclin 1-deficient mice fail to mount autoreactive T-cell responses and are resistant to experimental autoimmune encephalomyelitis. Compared with Th17 cells, Th1 cells are much more susceptible to cell death upon Beclin 1 deletion. Cell death proteins are highly increased in Beclin 1-deficient T cells and inhibition of caspases and genetic deletion of Bim reverse apoptosis. In addition, p62/sequestosome 1 binds to caspase-8 but does not control levels of procaspase-8 or other cell death-related proteins. These results establish a direct role of autophagy in inhibiting the programmed cell death through degradation of apoptosis proteins in activated T cells.     

A pathogenic role for CD8+ T cells in a spontaneous model of demyelinating disease

Transgenic (Tg) mice that overexpress the costimulatory ligand B7.2/CD86 on microglia spontaneously develop a T cell-mediated demyelinating disease. Characterization of the inflammatory infiltrates in the nervous tissue revealed a predominance of CD8+ T cells, suggesting a prominent role of this T cell subset in the pathology. In this study, we show that the same neurological disease occurred in Tg mice deficient in the generation of CD4+ T cells, with an earlier time of onset. Analysis of the CD8+ T cell repertoire at early stage of disease revealed the presence of selected clonal expansions in the CNS but not in peripheral lymphoid organs. We further show that Tg animals deficient in IFN-gamma receptor expression were completely resistant to disease development. Microglia activation that is an early event in disease development is IFN-gamma dependent and thus appears as a key element in disease pathogenesis. Collectively, our data indicate that the spontaneous demyelinating disease in this animal model occurs as a consequence of an inflammatory response initiated through the activation of CNS-specific CD8+ T cells by Tg expression of B7.2 within the target organ. Thus, autoreactive CD8+ T cells can contribute directly to the pathogenesis of neuroinflammatory diseases such as multiple sclerosis.     

Colocalization of antigen-specific B and T cells within ectopic lymphoid tissue following immunization with exogenous antigen

Chronic inflammation promotes the formation of ectopic lymphoid tissue morphologically resembling secondary lymphoid tissues, though it is unclear whether this is a location where Ag-specific immune responses develop or merely a site of lymphocyte accumulation. Ectopic lymphoid tissue formation is associated with many humoral autoimmune diseases, including lupus induced by tetramethylpecadentane in mice. We examined whether an immune response to 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin (NP-KLH) and NP-OVA develops within ectopic lymphoid tissue ("lipogranulomas") induced by tetramethylpecadentane in C57BL/6 mice. Following primary immunization, NP-specific B cells bearing V186.2 and related heavy chains as well as lambda-light chains accumulated within ectopic lymphoid tissue. The number of anti-NP-secreting B cells in the ectopic lymphoid tissue was greatly enhanced by immunization with NP-KLH. Remarkably, the H chain sequences isolated from individual lipogranulomas from these mice were diverse before immunization, whereas individual lipogranulomas from single immunized mice had unique oligo- or monoclonal populations of presumptive NP-specific B cells. H chain CDR sequences bore numerous replacement mutations, consistent with an Ag-driven and T cell-mediated response. In mice adoptively transferred with OT-II or DO11 T cells, there was a striking accumulation of OVA-specific T cells in lipogranulomas after s.c. immunization with NP-OVA. The selective colocalization of proliferating, Ag-specific T and B lymphocytes in lipogranulomas from tetramethylpecadentane-treated mice undergoing primary immunization implicates ectopic lymphoid tissue as a site where Ag-specific humoral immune responses can develop. This has implications for understanding the strong association of humoral autoimmunity with lymphoid neogenesis, which may be associated with deficient censoring of autoreactive cells.     

T cell differentiation in the thymus

T lymphocytes arise in the thymus and seed to peripheral lymphoid organs as fully functional cells at the time of exit. In humans, the thymus begins to function very early in ontogeny and releases large numbers of T cells before the time of birth. However, the vast majority of developing thymocytes (>95%) die within the thymus as a result of stringent selection processes. Positive selection imposes self-MHC-restriction on thymocytes and dictates the MHC-restricted repertoire of post-thymic T cells. Negative selection results in deletion of autoreactive cells. Both types of selection depend on cell to cell contracts and on the presence of appropriate growth factors which are still largely undetermined. Cell to cell contacts occur between developing thymocytes and cells of the thymic microenvironment (accessory cells), and are mediated by several receptor/ligand interactions which subserve the function of establishing and stabilizing these contacts. Besides MHC-TCR interactions, adhesion molecules are important for thymocyte maturation, selection and activation, and for the export and peripheral homing of mature T cells produced in the thymus. Here we describe a novel integrin involved in thymocyte-thymic epithelial cell interactions.     

Apoptosis in Activated T Cells – What Are the Triggers,and What the Signal Transducers?

At the end of an immune response, apoptosis drastically reduces the numbers of activated T cells. It has been a matter of intense research how this form of apoptosis is regulated and initiated, and a number of proteins have been identified that contribute to this process. The present, widely accepted model assumes that the interplay of pro- and anti-apoptotic Bcl-2 family members determines the onset of activated T cell death, with the BH3-only protein Bim activating pro-apoptotic Bax/Bak. In the search for up-stream signals, factors from other immune cells have been shown to play a role, and the NF-κB family member Bcl-3 has been implicated as a signalling-intermediate in T cells. Recent work has tested the interrelation of these factors and has suggested that Bcl-3 acts as a regulator of Bim activation, that the induction of apoptosis through Bim can be complemented by its relative Puma, and that the presence of certain cytokines during T cell activation delays the activation of Bim and Puma. Here we discuss these recent insights and provide a view on how the regulation of activated T cell death is achieved and how extrinsic signals may translate into the activation of the apoptotic pathway.