Immunologic Profiling of Immune-Related Cutaneous Adverse Events with Checkpoint Inhibitors Reveals Polarized Actionable Pathways.

PURPOSE: Immune-related cutaneous adverse events (ircAEs) occur in ≥50% of patients treated with checkpoint inhibitors (CPI), but mechanisms are poorly understood. EXPERIMENTAL DESIGN: Phenotyping/biomarker analyses were conducted in 200 patients on CPIs (139 with ircAEs, 61 without, control) to characterize their clinical presentation and immunologic endotypes. Cytokines were evaluated in skin biopsies, skin tape strip (STS) extracts and plasma using real-time PCR and Meso Scale Discovery multiplex cytokine assays. RESULTS: Eight ircAE phenotypes were identified: pruritus (26%), maculopapular rash (MPR; 21%), eczema (19%), lichenoid (11%), urticaria (8%), psoriasiform (6%), vitiligo (5%), and bullous dermatitis (4%). All phenotypes showed skin lymphocyte and eosinophil infiltrates. Skin biopsy PCR revealed the highest increase in IFN-gamma mRNA in patients with lichenoid (p<0.0001) and psoriasiform dermatitis (p<0.01) as compared to patients without ircAEs, while the highest IL-13 mRNA levels were detected in the eczema (p<0.0001, compared to control). IL-17A mRNA was selectively increased in psoriasiform (p<0.001), lichenoid (p<0.0001), bullous dermatitis (p<0.05) and MPR (p<0.001), compared to control. Distinct cytokine profiles were confirmed in STS and plasma. Analysis determined increased skin/plasma IL-4 cytokine in pruritus, skin IL-13 in eczema, plasma IL-5 and IL-31 in eczema and urticaria, and mixed-cytokine pathways in MPR. Broad inhibition via corticosteroids or type 2-cytokine targeted inhibition resulted in clinical benefit in these ircAEs. In contrast, significant skin upregulation of type 1/type 17 pathways was found in psoriasiform, lichenoid, bullous dermatitis, and type 1 activation in vitiligo. CONCLUSIONS: Distinct immunologic ircAE endotypes suggest actionable targets for precision medicine-based interventions.

Skin biomarkers predict the development of food allergy in early life.

BACKGROUND: Food allergy (FA) often occurs in early childhood with and without atopic dermatitis (AD). FA can be severe and even fatal. For primary prevention, it is important to find early biomarkers to predict the future onset of FA before any clinical manifestations. OBJECTIVE: Our aim was to find early predictors of future onset of FA in the stratum corneum (SC). METHODS: Skin tape strips were collected from the forearm of newborns (n = 129) at age 2 months, before any signs of clinical FA or AD. Children were clinically monitored until they reached age 2 years to confirm the presence or absence of FA and AD. Skin tape strips were subjected to lipidomic analyses by liquid chromatography-tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. RESULTS: Overall, 9 of 129 infants (7.0%) developed FA alone and 9 of 129 infants (7.0%) developed FA concomitantly with AD. In the stratum corneum of children with future FA and concomitant AD and FA, absolute amounts of unsaturated (N24:1)(C18-sphingosine)ceramide and (N26:1)(C18-sphingosine)ceramide and their relative percentages within the molecular group were increased compared with the amounts and percentages in healthy children, with P values ranging from less than .01 to less than .05 according to ANOVA. The children with future AD had normal levels of these molecules. IL-33 level was upregulated in those infants with future FA but not in those with future AD, whereas thymic stromal lymphopoietin was upregulated in those with future AD but not in those with future FA. Logistic regression analysis revealed strong FA predicting power for the combination of dysregulated lipids and cytokines, with an odds ratio reaching 101.4 (95% CI = 5.4-1910.6). CONCLUSION: Noninvasive skin tape strip analysis at age 2 months can identify infants at risk of FA in the future.

Dupilumab Inhibits Vascular Leakage of Blood Proteins Into Atopic Dermatitis Skin.

BACKGROUND: Atopic dermatitis (AD) skin lesions are associated with oozing, bleeding, and erythema. This suggests that AD is associated with vascular changes. Dupilumab is an antibody to the alpha subunit of IL-4 receptor that demonstrates strong efficacy in the treatment of AD. IL-4 is known to reduce the permeability barrier function of vascular endothelium. OBJECTIVE: To examine the effects of dupilumab on vascular barrier function in AD skin. METHODS: Using proteomic analysis, we evaluated the plasma protein composition in skin tapes of lesional and nonlesional skin of adults and adolescents with moderate to severe AD over the course of a 16-week treatment with dupilumab and compared those with matched healthy subjects. RESULTS: At baseline, 115 plasma proteins were detected in AD skin and globally increased (1.5-fold or greater) compared with healthy skin. Functionally, these proteins included immunoglobulins, proteins involved in the coagulation process, enzymes, protease inhibitors, transport proteins, acute-phase proteins, complement proteins, and other pleiotropic proteins. Noteworthy, fibrinogens, fibronectin, and heme-binding proteins haptoglobin and hemopexin were among the top proteins originating from plasma and were increased in AD lesional versus healthy skin at baseline (P < .0001). Dupilumab treatment resulted in significantly reduced levels of plasma proteins in AD skin (P < .0001), with most dropping to levels seen in healthy skin or no longer detectable at week 16. CONCLUSIONS: Inhibition of IL-4/IL-13 action by dupilumab significantly reduces the efflux of plasma proteins into AD skin. Several of these proteins, such as fibrinogens and fibronectin, are known to enhance Staphylococcus aureus colonization and are associated with AD skin severity.

Stratum corneum lipid and cytokine biomarkers at age 2 months predict the future onset of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) commonly occurs in children and can progress into severe phenotypes or atopic march, causing significant impairment in quality of life. It is important to find early biomarkers of future onset of AD before any clinical manifestations. OBJECTIVE: We sought to find early predictors of future onset of AD in skin stratum corneum (SC). METHODS: Skin tape strips were collected from the forearm of newborns (n = 111) with and without family history of atopic diseases at the age of 2 months before any signs of clinical AD. Children were clinically monitored until they reached age 2 years to ensure the presence or absence of AD. Skin tape strips were subjected to lipidomic analyses by the liquid chromatography electrospray ionization tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. RESULTS: Overall, 22 of 74 (29.7%) and 5 of 37 (13.5%) infants developed AD in the risk group and the control group, respectively. In the SC of future AD children, protein-bound ceramides were decreased (P < .001), whereas unsaturated sphingomyelin species (P < .0001) and "short-chain" nonhydroxy fatty acid sphingosine and alpha-hydroxy fatty acid sphingosine ceramides were elevated (P < .01 and .05, respectively) as compared with healthy children. Thymic stromal lymphopoietin and IL-13 levels were increased in the SC of future AD subjects (by 74.5% and 78.3%, P = .0022 and P < .0001, respectively). Multivariable logistic regression analysis revealed strong AD predicting power of the combination of family history, type 2 cytokines, and dysregulated lipids, with an odds ratio reaching 54.0 (95% CI, 9.2-317.5). CONCLUSIONS: Noninvasive skin tape strip analysis at age 2 months can identify asymptomatic children at risk of future AD development with a high probability.

Transient receptor potential vanilloid 1 plays a major role in low temperature-mediated skin barrier dysfunction.

BACKGROUND: Children born in the fall and winter are at increased risk for developing atopic dermatitis and food allergy. Because these seasons are associated with low temperatures, we hypothesized that exposure to low temperatures may compromise keratinocyte differentiation and contribute to skin barrier dysfunction. OBJECTIVE: We examined whether low temperature causes skin barrier dysfunction. METHODS: Primary human epidermal keratinocytes (HEK) were differentiated in 1.3 mmol CaCl2 media and cultured at different temperatures. The cells were transfected with transient receptor potential cation channel subfamily V member 1 (TRPV1) or STAT3 small interfering RNA (siRNA) to examine the effects of these gene targets in HEK exposed to low temperature. Gene expression of TRPV1, epidermal barrier proteins, and keratinocyte-derived cytokines were evaluated. Organotypic skin equivalents were generated using HEK transfected with control or TRPV1 siRNA and grown at 25°C or 37°C. Transepidermal water loss (TEWL) and levels of epidermal barrier proteins were evaluated. RESULTS: Filaggrin (FLG) and loricrin (LOR) expression, but not keratin (KRT)-1 and KRT-10 expression, was downregulated in HEK incubated at 25°C, while TRPV1 silencing increased intracellular Ca2+ influx (keratinocyte differentiation signal) and enhanced the expression of epidermal differentiation proteins. IL-1ß and thymic stromal lymphopoietin induced by low temperature inhibited FLG expression in keratinocytes through the TRPV1/STAT3 pathway. Moreover, low temperature-mediated inhibition of FLG and LOR was recovered, and TEWL was decreased in organotypic skin transfected with TRPV1 siRNA. CONCLUSION: TRPV1 is critical in low temperature-mediated skin barrier dysfunction. Low temperature exposure induced thymic stromal lymphopoietin, an alarmin implicated in epicutaneous allergen sensitization.

Our current understanding of checkpoint inhibitor therapy in cancer immunotherapy.

OBJECTIVE: Treatments with Food and Drug Administration-approved blocking antibodies targeting inhibitory cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD-1) receptor, or programmed cell death ligand 1 (PD-L1), collectively named checkpoint inhibitors (CPIs), have been successful in producing long-lasting remissions, even in patients with advanced-stage cancers. However, these treatments are often accompanied by undesirable autoimmune and inflammatory side effects, sometimes bringing severe consequences for the patient. Rapid expansion of clinical applications necessitates a more nuanced understanding of CPI function in health and disease to develop new strategies for minimizing the negative side effects, while preserving the immunotherapeutic benefit. DATA SOURCES: This review summarizes a new paradigm-shifting approach to cancer immunotherapy with the focus on the mechanism of action of immune checkpoints (CTLA4, PD-1, and its ligands). STUDY SELECTIONS: We performed a literature search and identified relevant recent clinical reports, experimental research, and review articles. RESULTS: This review highlights our understanding of the CPI mechanism of action on cellular and molecular levels. The authors also discuss how reactivation of T cell responses through the inhibition of CTLA4, PD-1, and PD-L1 is used for tumor inhibition in cancer immunotherapy. CONCLUSION: Mechanisms of PD-1 and CTLA4 blockade and normal biological functions of these molecules are highly complex and require additional studies that will be critical for developing new approaches to dissociate the benefits of checkpoint blockade from off-target effects of the immune reactivation that leads to immune-related adverse events.

SMOC1 and IL-4 and IL-13 Cytokines Interfere with Ca2+ Mobilization in Primary Human Keratinocytes.

Immunoregulatory effects of IL-4 and IL-13 and alterations of keratinocyte (KC) differentiation are important factors in the pathogenesis of atopic dermatitis. This study investigated the role of IL-4 and IL-13 in KC responses to changes in extracellular calcium (Ca2+) and analyzed differentiation signals elicited via a Ca2+ sensor, SMOC1. Real-time dynamics of transmembrane Ca2+ influx were assessed in live KCs by flow cytometry and microscopy. Exposure of KCs to a high Ca2+ environment (1.3 mM) triggered a rapid intracellular Ca2+ influx, whereas IL-4- and IL-13-treated cells exhibited a significant decrease in the peak amplitude of Ca2+ influx (P < 0.01). IL-17A and IL-22 did not elicit such responses. Evaluation of intracellular Ca2+ dynamics by microscopy confirmed these observations and revealed heterogeneity of individual KC responses. IL-4 and IL-13 significantly inhibited the expression of Ca2+-binding protein SMOC1 (P < 0.001). Inhibition of epidermal differentiation markers were also observed in SMOC1 small interfering RNA-transfected KCs. Concurrently, the deletion of SMOC1 increased the amplitude of Ca2+ peak response (P < 0.05). In conclusion, our results provide innovative data that IL-4 and IL-13 regulate KC sensitivity to microenvironmental Ca2+ changes and inhibit Ca2+-induced KC differentiation signals. SMOC1 inhibition by IL-4 and IL-13 alters Ca2+ transport in KCs and inhibits differentiation, suggesting a new target for treatment of atopic dermatitis.

Skin tape sampling technique identifies proinflammatory cytokines in atopic dermatitis skin.

BACKGROUND: Monitoring the effects of biologic therapies in skin diseases will benefit from alternative noninvasive skin sampling techniques to evaluate immune pathways in diseased tissue early and longitudinally. OBJECTIVE: To establish a minimally invasive profiling of skin cytokines for diagnosis, therapeutic response monitoring, and clinical research in atopic dermatitis (AD) and other skin diseases, particularly in pediatric cohorts. METHODS: We developed a novel method for cytokine profiling in the epidermis using skin tape strips (STSs) in a setting designed to maximize the efficiency of protein extraction from STSs. This method was applied to analyze STS protein extracts from the lesional skin of children having AD (n = 41) and normal, healthy controls (n = 22). A total of 20 cytokines were probed with the ultrasensitive Mesoscale multiplex cytokine assay. RESULTS: A significant increase in interleukin (IL)-1b (P < .01), IL-18 (P < .001), and IL-8 (P < .001) with a decrease in IL-1a (P < .001) in the stratum corneum of AD lesional skin was found. Concurrently, an increase in markers associated with type 2 inflammatory response was readily detectable in AD lesional skin, including C-C motif chemokine ligand (CCL) 22, CCL 17, and thymic stromal lymphopoietin (TSLP). The levels of IL-1b, IL-18, and TSLP exhibited positive correlations with the AD severity index (Scoring AD index) and skin transepidermal water loss (TEWL), whereas an inverse correlation between IL-1a and Scoring AD index and IL-1a and TEWL was found. The levels of CCL17, CCL22, TSLP, IL-22, and IL-17a correlated with skin TEWL measurements. CONCLUSION: Using minimally invasive STS analysis, we identified cytokine profiles easily sampled in AD lesional skin. The expression of these markers correlated with disease severity and reflected changes in TEWL in lesional skin. These markers suggest new response assessment targets for AD skin. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03168113.

Targeting the Immune Complex-Bound Complement C3d Ligand as a Novel Therapy for Lupus.

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment-derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2-/- mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d-CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/lpr model of SLE.

Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells.

Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and ß-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.

B cell receptor signaling-based index as a biomarker for the loss of peripheral immune tolerance in autoreactive B cells in rheumatoid arthritis.

This study examines the loss of peripherally induced B cell immune tolerance in Rheumatoid arthritis (RA) and establishes a novel signaling-based measure of activation in a subset of autoreactive B cells--the Induced tolerance status index (ITSI). Naturally occurring naïve autoreactive B cells can escape the "classical" tolerogenic mechanisms of clonal deletion and receptor editing, but remain peripherally tolerized through B cell receptor (BCR) signaling inhibition (postdevelopmental "receptor tuning" or anergy). ITSI is a statistical index that numerically determines the level of homology between activation patterns of BCR signaling intermediaries in B cells that are either tolerized or activated by auto antigen exposure, and thus quantifies the level of peripheral immune tolerance. The index is based on the logistic regression analysis of phosphorylation levels in a panel of BCR signaling proteins. Our results demonstrate a new approach to identifying autoreactive B cells based on their BCR signaling features.

Rheumatoid arthritis is associated with signaling alterations in naturally occurring autoreactive B-lymphocytes.

Immune tolerance established during the development of B lymphocytes can be subverted in mature cells and lead to autoimmunity. This study focuses on the recently discovered subset of CD19(+)CD27(-)IgD(+)IgM(low/-) B cells that recognize self-antigens and have the capacity to produce autoantibodies, but under normal conditions do not generate autoimmune response due to intrinsic signaling inhibition (a condition known as clonal anergy and characterized by impaired antigen receptor signaling). Phosphorylation of intracellular signaling proteins and Ca(2+) responses in anergic B cells were measured by multicolor flow cytometry. Our results demonstrate a distinct phosphorylation pattern for major signal transduction proteins, which distinguishes anergic B cells. Comparison of B cell signaling properties in Rheumatoid Arthritis patients and healthy controls revealed a reversal of pTyr and Ca(2+) anergic signaling features in patients, accompanied by phosphorylation decreases of Blnk, Syk, SHP2, CD19. We identified BCR signaling pathway alterations associated with the loss of anergic B cell tolerance in Rheumatoid Arthritis.

Complement activation and toll-like receptor-2 signaling contribute to cytokine production after renal ischemia/reperfusion.

The innate immune system causes tissue inflammation and injury after renal ischemia/reperfusion (I/R). The complement system is activated on ischemic tubular epithelial cells (TECs) and induces the cells to produce pro-inflammatory chemokines. TECs also express toll-like receptors (TLRs)-2 and -4. Signaling through the TLRs induces TECs to produce a variety of chemokines, some of which can also be induced by complement activation fragments. We sought to determine whether the effects of complement activation and TLR signaling in TECs are redundant, or whether additive protection can be achieved by blocking both of these innate immune systems. To confirm that the complement system, TLR-2 signaling, and TLR-4 signaling induce production of a similar repertoire of inflammatory chemokines, we stimulated TECs with complement sufficient serum or with TLR-2 and TLR-4 ligands in vitro. We found that all three of these stimuli induce TECs to produce KC, MIP-2, IL-6, and TNF-α, and that there was a trend toward greater production of KC in cells exposed to two stimuli. Based upon these results, we hypothesized that mice deficient in both complement activation and TLR-2 signaling would demonstrate greater protection from I/R than mice deficient only in the complement system. To test this hypothesis we induced ischemic acute kidney injury (AKI) in wild-type mice, mice with targeted deletion of complement factor B (fB(-/-) mice), or mice with targeted deletion of factor B and TLR-2 (fB(-/-)TLR2(-/-) mice). Surprisingly, we found that fB(-/-)TLR2(-/-) mice developed more severe injury than those with single deficiency of factor B. Our results indicate that blockade of the complement system may be more protective than simultaneous blockade of both the complement system and TLR-2 in ischemic AKI.

Potentially autoreactive naturally occurring transitional T3 B lymphocytes exhibit a unique signaling profile.

B lymphocytes exhibit phenotypic differences that correlate with their developmental or functional stages and affect humoral immune responses. One recently described subset of naturally occurring immature transitional type 3 (T3) B lymphocytes is believed to consist of potentially autoimmune cells whose signaling properties have not been studied in detail. This study characterizes intracellular signaling in T3 B cells in wildtype C57BL/6 mice. Protein phosphorylation and Ca(2+) responses upon B-cell antigen receptor (BCR) engagement were measured by multicolor flow cytometry. We observed high baseline signaling activity and reduced BCR-mediated responses in T3 cells, which confirmed their anergy - a functional state in which lymphocytes recognize chronically present self-antigens but cannot produce immune response due to intrinsic signaling inhibition. Our results also revealed a previously unknown T3-specific phosphorylation pattern of 24 key signaling molecules involved in BCR signal transduction. These characteristics reflect the balance between stimulatory and inhibitory BCR signaling pathways in anergy. Results obtained in the collagen-induced arthritis model demonstrate the loss of anergy in T3 B cells during the onset of the disease. Our findings provide rationale for further investigating alterations in B-cell signaling patterns as earliest functional biomarkers of changes in the immune tolerance of autoreactive B cells.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Ca²+ signaling in B cells.

An increase in intracellular Ca²+ concentration is one of the major initial steps in B-cell activation that occurs within minutes after antigen receptor (BCR) engagement. In recent years, significant advances have been made in characterizing molecular mechanisms of Ca²+ signaling in lymphocytes, although the majority of work was done on T cells. This mini-review discusses several underexplored areas of Ca²+ signaling in B cells: (1) Ca²+ signaling in immune synapse and multifaceted Ca²+ responses within a single cell, (2) source of Ca²+ involved in Ca²+-dependent protein phosphorylation events and the role of store-operated influx, (3) role of BCR coreceptors in Ca²+ signaling, and (4) Ca²+ signaling and maintenance of B-cell tolerance and clinical significance of Ca²+ signaling alterations.

Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling.

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca(2+)) influx occurs via highly selective Ca(2+) release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca(2+)-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca(2+) levels in areas adjacent to the immune synapse differ from those in the rest of the cytoplasm. Finally, a comparison of phosphorylation patterns of BCR-triggered signaling proteins in the presence or absence of Ca(2+) revealed the unanticipated finding that initial BCR-triggered, Ca(2+)-dependent tyrosine phosphorylation events involve predominantly Ca(2+) released from intracellular stores and that influx-derived Ca(2+) is not essential. This suggests a different role for this phase of Ca(2+) influx.

Cutting edge: Complement (C3d)-linked antigens break B cell anergy.

C3dg adducts of Ag can coligate complement receptor type 2 (CR2; CD21) and the B cell Ag receptor. This interaction significantly amplifies BCR-mediated signals in Ag-naive wild-type mice, lowering the threshold for B cell activation and the generation of humoral immune responses as much as 1000-fold. In this study we demonstrate that CR2-mediated complementation of BCR signals can also overcome B cell anergy. Unlike Ag alone, BCR/CR2 costimulation (Ars-CCG/C3dg complexes) of anergic Ars/A1 B cells led to Ca(2+) mobilization in vitro and the production of autoantibodies in vivo. Interestingly, the in vivo immune response of anergic cells occurs without the formation of germinal centers. These results suggest that the Ag unresponsiveness of anergic B cells can be overcome by cross-reactive (self-mimicking) Ags that have been complement-opsonized. This mechanism may place individuals exposed to complement-fixing bacteria at risk for autoimmunity.

Intrinsic B cell hypo-responsiveness in mice prematurely expressing human CR2/CD21 during B cell development.

We previously reported that human CR2 (hCR2) prematurely expressed under a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene during the CD43+ CD25- late pro-B cell stage of development results in peripheral B cells with impaired responses to immunization with T-dependent antigens. Herein, we show that hCR2 transgenic (Tg) mice also demonstrate a severe defect in T-independent antigen responses and are substantially protected from clinical arthritis, synovitis and cartilage/bone destruction in a collagen-induced arthritis model. This outcome is found despite the apparently normal development of autoreactive T cells with equivalent cytokine and proliferative responses to antigen when compared to non-Tg control mice. These data suggest the presence of an intrinsic B cell defect in the hCR2 Tg mice. We also show that an hCR2-dependent Ca2+ influx can be generated in both developing and mature Tg B cells, but with different rates of decay as compared to control wild-type (WT) mice. In addition, although analysis of tyrosine-phosphorylated proteins in WT and Tg B cells following B cell receptor (BCR)-induced activation revealed the presence of distinctly different phosphorylation patterns, no differences were identified in several candidate protein targets. Overall, these data suggest that premature hCR2 expression and the consequences thereof during B cell development intrinsically alters the way mature B cells develop and subsequently respond to antigen through the BCR signaling complex.