Metabolic Gatekeepers of Pathological B Cell Activation.

Unlike other cell types, B cells undergo multiple rounds of V(D)J recombination and hypermutation to evolve high-affinity antibodies. Reflecting high frequencies of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. In addition, the vast majority of newly generated B cells express an autoreactive B cell receptor (BCR). Thus, B cells are under intense selective pressure to remove autoreactive and premalignant clones. Despite stringent negative selection, B cells frequently give rise to autoimmune disease and B cell malignancies. In this review, we discuss mechanisms that we term metabolic gatekeepers to eliminate pathogenic B cell clones on the basis of energy depletion. Chronic activation signals from autoreactive BCRs or transforming oncogenes increase energy demands in autoreactive and premalignant B cells. Thus, metabolic gatekeepers limit energy supply to levels that are insufficient to fuel either a transforming oncogene or hyperactive signaling from an autoreactive BCR.

Loss of anergic B cells in prediabetic and new-onset type 1 diabetic patients.

Although dogma predicts that under normal circumstances, potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, islet antigen-reactive B lymphocytes are known to play a crucial role in the development of autoimmunity in type 1 diabetes (T1D). Thus, participation of these cells in T1D may reflect escape from silencing mechanisms. Consistent with this concept, we found that in healthy subjects, high-affinity insulin-binding B cells occur exclusively in the anergic naive IgD(+), IgM(-) B-cell (BND) compartment. Antigen receptors expressed by these cells are polyreactive and have N-region additions, Vh usage, and charged complementarity-determining region 3 consistent with autoreactivity. Consistent with a potential early role in autoimmunity, these high-affinity insulin-binding B cells are absent from the anergic compartment of some first-degree relatives and all prediabetic and new-onset (<1 year) T1D patients tested, but return to normal levels in individuals diabetic for >1 year. Interestingly, these changes were correlated by transient loss of the entire BND compartment. These findings suggest that environmental events such as infection or injury may, by disrupting B-cell anergy, dispose individuals toward autoimmunity, the precise nature of which is specified by genetic risk factors, such as HLA alleles.

Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes.

T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells "measure" Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide-MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated "measurements" of Ag dose that determine subsequent functional outcomes.

Toll-like receptors 7, 8, and 9 expression and function in primary human cervical cancer Langerhans cells: evidence of anergy.

OBJECTIVE: Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. METHODOLOGY: Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a*CD207* cells. Acute monocytic leukemia cell line THP-1-derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1ß, IL-6, IL-10, IL-12p70, interferon (IFN) α, interferon γ, and tumor necrosis factor (TNF) α by Luminex multiplex bead array. Human papillomavirus was genotyped. RESULTS: We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1ß, and tumor necrosis factor α to TLR8 ligand and interferon α in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. CONCLUSIONS: Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells.

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.

The evolving role of mTOR inhibition in transplantation tolerance.

The mammalian target of rapamycin (mTOR) plays a key role in the immune response. mTOR inhibitors suppress T cell activation and proliferation and are effective immunosuppressants. Today there is growing interest in their potential role in inducing tolerance after transplantation. mTOR inhibitors induce anergy in naïve T cells, promote the expansion of regulatory T cells, and inhibit the maturation of dendritic cells, thus promoting immunologic tolerance. Here we review the mechanisms by which mTOR inhibitors promote tolerance. We discuss the clinical relevance of these mechanisms and suggest how they might be used in the design of future protocols to induce tolerance.

Prolongation of renal allograft survival by anergic cells: advantages and limitations.

Even in the era of pharmacological calcineurin inhibitors, a current major challenge in organ transplantation remains the development of immunosuppressive regimens that protect against rejection. One potentially effective procedure is the use of donor-specific anergic T cells generated ex vivo and adoptively transferred back into the recipient after transplantation. In our own work, we first investigated the effect of anergic cells on the prolongation of graft survival in non-human primates. In six animals, half of the recipients survived for over one yr (all animals died or were killed within eight yr). The cause of death was acute renal failure because of cellular rejection (one), uncontrolled bleeding after renal biopsy (two), hydronephrosis probably because of ureteral stenosis (one), and chronic rejection (one). The remaining animal was killed at the end of the study. No infection, malignancy, or signs of graft versus host disease (GVHD) was observed in any of these monkeys. Except for the one animal that died of acute cellular rejection, there was no evidence of tubular infiltration by mononuclear cells, glomerular damage, or parenchymal necrosis. In all animals surviving for more than one yr, a mild grade of interstitial fibrosis, an increase in mesangial matrix, or glomerulopathy was noted. In two of three monkeys, no vascular narrowing of the luminal area caused by fibrointimal thickening of arteries was noted, and arteriosclerotic change was dominant. In this chapter, we summarize the efficacy and limitations of our strategy.

Gene related to anergy in lymphocytes (GRAIL) expression in CD4+ T cells impairs actin cytoskeletal organization during T cell/antigen-presenting cell interactions.

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death.

The balance of T-cell proliferation, anergy and apoptosis is central to immune function. In this regard, co-receptor CTLA-4 is needed for the induction of anergy and tolerance. One central question concerns the mechanism by which CTLA-4 can induce T-cell non-responsiveness without a concurrent induction of antigen induced cell death (AICD). In this study, we show that CTLA-4 activation of the phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. CTLA-4 ligation induced PI 3K activation as evidenced by the phosphorylation of PKB/AKT that in turn inactivated GSK-3. The level of activation was similar to that observed with CD28. CTLA-4 induced PI 3K and AKT activation also led to phosphorylation of the pro-apoptotic factor BAD as well as the up-regulation of BcL-XL. In keeping with this, CD3/CTLA-4 co-ligation prevented apoptosis under the same conditions where T-cell non-responsiveness was induced. This effect was PI 3K and PKB/AKT dependent since inhibition of these enzymes under conditions of anti-CD3/CTLA-4 co-ligation resulted in cell death. Our findings therefore define a mechanism by which CTLA-4 can induce anergy (and possibly peripheral tolerance) by preventing the induction of cell death.

Induction of CD4+ T-cell anergy and apoptosis by activated human B cells.

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Inducing an anergic state in mast cells and basophils without secretion.

BACKGROUND: IgE-mediated secretion from mast cells or basophils depends on the activity of both spleen tyrosine kinase (syk) and phosphatidyl inositol 3' kinase (PI3K), but several specific downregulatory pathways (eg, loss of syk expression) do not. OBJECTIVE: We tested whether stimulation with antigen in the presence of a syk inhibitor (NVP-QAB205) would ablate secretion while simultaneously allowing anergy. METHODS: The anergic or desensitized state in human basophils, cultured-derived mast cells, and in situ stimulated airway mast cells (in organ baths) was assessed after stimulation with antigen in the presence of syk inhibitor. RESULTS: Antigen caused 35 +/- 7% and 62 +/- 10% histamine release from basophils and mast cells, respectively, and it caused an 87 +/- 5% histamine/leukotriene D(4)-dependent contraction of human isolated bronchi. All of these responses were blocked >95% by the syk inhibitor. Rechallenging the preparations with antigen, after first washing out the syk inhibitor and antigen, revealed that near complete anergy (92% to 100%) occurred in each case. A similar result was found when using a PI3K inhibitor, LY294002, in studies of basophils. CONCLUSION: Although the syk inhibitor nearly abolished the antigen-induced secretion from mast cells and basophils, it had little effect on the pathways involved in anergy. These results suggest that syk and PI3K are not involved in downregulation leading to anergy.

FoxP3 maintains Treg unresponsiveness by selectively inhibiting the promoter DNA-binding activity of AP-1.

Regulatory T cells (Tregs) have been shown to play a crucial role in maintaining self-tolerance and suppressing autoimmunity. The forkhead transcription factor, FoxP3, is a key molecule necessary and sufficient for Tregs development and function. However, the molecular mechanisms by which FoxP3 regulates the phenotypic (anergic) and the functional (suppressive) characteristics of Tregs are not well defined. Here we found that the promoter DNA-binding activity of AP-1 transcription factors is selectively inhibited in the naturally occurring CD4+ CD25+ Tregs from mice. The impaired AP-1 DNA binding is not the result of the decreased nuclear translocation of AP-1 family transcription factors, including c-Jun, JunB, and c-Fos. FoxP3 significantly suppresses both the transcriptional activity and promoter DNA-binding of AP-1 by interacting with c-Jun. The N-terminus of FoxP3, but not its C-terminus forkhead domain, specifically interacts with phosphorylated c-Jun and alters c-Jun subnuclear distribution. This N-terminus of FoxP3 with nuclear localization signals (FoxP3N/NLS) is able to suppress AP-1 transcriptional activity. Ectopic expression of FoxP3N/NLS sufficiently induces the unresponsiveness of mouse primary CD4+ CD25- T cells, whereas the full-length FoxP3 is required for the suppressive functions of Tregs. These findings uncover one of the mechanisms underlying how FoxP3 maintains the unresponsiveness of Tregs.

The impact of cell-bound antigen transport on mucosal tolerance induction.

Mucosal surfaces are exposed continuously to a flood of foreign antigens demanding a tightly controlled balance between immunity and tolerance induction. Tolerance toward food and inhaled antigens, known as oral and respiratory tolerance, respectively, evokes a body-wide nonresponsiveness against the plethora of environmental antigens. Key issues in understanding the induction of mucosal tolerance relate to the site of antigen entrance, the mechanisms of antigen transport, and the exact anatomical location where lymphocytes meet their cognate antigens. In this regard, opposing ideas have been put forward: In one scenario, antigens taken up at mucosal surfaces are considered to spread throughout the body, thus potentially evoking tolerogenic immune responses in all secondary lymphoid organs. Alternatively, tolerance induction might be confined to the draining regional lymph nodes (LN). Recent observations strongly supported the latter scenario, emphasizing the importance of regional LN and their network of afferent lymphatics in this process. In this model, air-borne and intestinal antigens are captured at mucosal sites by dendritic cells, which then migrate exclusively in a CCR7-dependent way to draining regional LN. Tolerance is then induced actively by the activation of antigen-specific T cells, which are subsequently deleted, become anergic, or alternatively, differentiate into regulatory T cells. Thus, the concept of local induction of immune responses seems to hold true for the majority of immune reactions, regardless of whether they are tolerogenic or defensive in their outcome.

Regulation of peripheral T cell tolerance by the E3 ubiquitin ligase Cbl-b.

The family of the Casitas B-lineage Lymphoma (Cbl) proteins, c-Cbl, Cbl-b, and Cbl-3, function as E3 ubiquitin ligases and molecular adaptors. In particular, Cbl-b acts as a gatekeeper in T cell activation that controls activation thresholds and the requirement for co-stimulation. Loss of Cbl-b expression renders animals susceptible to antigen-triggered autoimmunity suggesting that Cbl-b is a key autoimmunity gene. In addition, Cbl-b plays a critical role in T cell anergy and escape from regulatory T cells (Treg) suppression. Modulation of Cbl-b might provide us with a unique opportunity for future immune treatment of human disorders such as autoimmunity, immunodeficiency, or cancer.

Endothelial cell anergy is mediated by bFGF through the sustained activation of p38-MAPK and NF-kappaB inhibition.

Tumors escape from immune surveillance by, among other mechanisms, the down- regulation of endothelial adhesion molecules, such as ICAM-1, and by unresponsiveness to inflammatory signals, a process mediated by angiogenic factors that is called endothelial cell anergy. Here we present the cell biological regulation of these processes. The angiogenic basic fibroblast growth factor (bFGF/FGF-2) was found to inhibit tumor necrosis factor-alpha (TNF-alpha)- induced elevation of ICAM-1, at transcriptional level. Furthermore, we found that bFGF inhibits the TNF-mediated activation of NF-kappaB by blocking phosphorylation and degradation of IkappaBalpha. We also found that bFGF induces hyperphosphorylation of p38 MAPK on endothelial cells, whereas inhibition of such kinase abrogates the effect of bFGF on the TNF-mediated activation of NF-kappaB. Thus, we suggest that bFGF acts as an inhibitor of leukocyte adhesion in tumor vessels by decreasing the ICAM-1 expression through the sustained activation of p38-MAPK and via inhibition of NF-kappaB.

Maintenance of CD8+ T-cell anergy by CD4+CD25+ regulatory T cells in chronic graft-versus-host disease.

In a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD), donor CD8+ T cells rapidly fall into anergy to host cells, while donor CD4+ T cells hyperactivate B cells and break B-cell tolerance to self-Ags in the recipient mouse. The functional recovery of donor CD8+ T cells can result in the conversion of cGVHD to acute GVHD (aGVHD), indicating that donor CD8+ T-cell anergy is a restriction factor in the development of cGVHD. In this report, we present evidence that donor CD4+CD25+ regulatory T cells (Treg cells) are critical in maintaining the donor CD8+ T-cell anergy and thus suppressing the development of aGVHD in mice that are naturally prone to cGVHD. Our results provide a novel insight into the role of Treg cells in determining cGVHD versus aGVHD.

Primer: mechanisms of immunologic tolerance.

Successful adaptive immunity against a broad range of pathogens depends on the diversity of randomly generated T-lymphocyte and B-lymphocyte receptors. A subset of these receptors will be self-reactive and must be regulated to prevent autoimmunity. The process of immunologic tolerance addresses this problem by either purging autoreactive receptors from the system or tuning down their reactivity sufficiently to prevent disease. Immature lymphocytes generate a novel receptor during development in the thymus or bone marrow. Engagement of self antigens by these nascent receptors leads to their purging, either by the apoptotic death of the lymphocyte or by the initiation of receptor editing, a process in which the autoreactive receptor is replaced. If the lymphocytes mature further, the activation threshold of autoreactive cells can be tuned by the co-expression of inhibitory receptors or negative signaling molecules, allowing the persistence of the receptor without an increased risk of autoimmunity. T-cell and B-cell receptors that escape these checkpoints can still be regulated in the peripheral immune system by both purging and tuning mechanisms. A separate set of mechanisms, mediated by various regulatory cells, also operates to tune peripheral receptors in a cell-extrinsic fashion. The combined action of these processes ensures that the organism does not suffer autoimmune pathology, even if autoreactive receptors are generated and maintained in the immune system.

Requirement of high-affinity IL-2-IL-2R interaction for T cell anergy induction.

Incomplete T cell antigen receptor-mediated signaling induces an unresponsive state known as anergy. Previously, we had shown that anergy can be induced in antigen-primed but not naive T cells. In this report, we found that in vitro primed T cells from IL-2R alpha-deficient mice were resistant to anergy induction in contrast to comparably treated wild-type T cells. This resistance persisted even after proliferation of IL-2R alpha chain-deficient CD4 T cells with high-dose IL-2-IL-2R beta gamma chains interaction. Thus, antigen activation, and/or progression through cell cycle are not sufficient to induce anergy susceptibility in T cells. The high-affinity IL-2-IL-2R interaction appears to play a critical role in this process.

Coculture of Th cells with interleukin (IL)-7 in the absence of antigenic stimuli induced T-cell anergy reversed by IL-15.

Interleukin-7 (IL-7) is an important survival factor for T cells. We report here for the first time that it has another important role, facilitating T-cell clonal unresponsiveness, or anergy. The anergy was induced by a 20-day coculture of activated-human CD4(+) T-cell clones with IL-7 and irradiated peripheral blood mononuclear cells without antigenic stimuli. T-cell survival, but not T-cell anergy induction, was dependent on direct cell contacts between T cells and irradiated peripheral blood mononuclear cells. The anergic T cells exhibited no or very low expression of IL-7 receptor alpha chain (IL-7Ralpha), IL-2 receptor alpha chain (IL-2Ralpha), and common gamma chain (gammac), and did not express cytotoxic T-lymphocyte-associated protein 4, but expressed IL-15Ralpha. Coculture for 3 to 9 days of anergic T cells with a T-cell-activating cytokine IL-15, but not IL-2, restored the responsiveness of IL-7-induced anergic T cells together with reexpressions of IL-7Ralpha, IL-2Ralpha, and gammac. The anergy induction by IL-7 and restoration of responsiveness by IL-15 suggest novel mechanisms for regulation of helper T-cell responses, induction of peripheral tolerance, and breakdown of T-cell self-tolerance.