B-cell anergy induces a Th17 shift in a novel B lymphocyte transgenic NOD mouse model, the 116C-NOD mouse

B lymphocyte hyperactivity plays a pathogenic role in systemic lupus erythematosus (SLE), and spliced X box-binding protein 1 (XBP1s) has been implicated in B cell maturation and differentiation. We hypothesized that blockade of the XBP1s pathway inhibits the B cell hyperactivity underlying SLE and lupus nephritis (LN) development. In the present study, we systematically evaluated the changes in B cell activation induced by the Xbp1 splicing inhibitor STF083010 in a pristane-induced lupus mouse model. The lupus mouse model was successfully established, as indicated by the presence of LN with markedly increased urine protein levels, renal deposition of Ig, and mesangial cell proliferation. In lupus mice, B cell hyperactivity was confirmed by increased CD40 and B cell-activating factor levels. B cell activation and plasma cell overproduction were determined by increases in CD40-positive and CD138-positive cells in the spleens of lupus mice by flow cytometry and further confirmed by CD45R and Ig light chain staining in the splenic tissues of lupus mice. mRNA and protein expression of XBP1s in B cells was assessed by real-time PCR, Western blot analysis, and immunofluorescence analysis and was increased in lupus mice. In addition, almost all changes were reversed by STF083010 treatment. However, the expression of XBP1s in the kidneys did not change when mice were exposed to pristane and STF083010. Taken together, these findings suggest that expression of XBP1s in B cells plays key roles in SLE and LN development. Blockade of the XBP1s pathway may be a potential strategy for SLE and LN treatment.

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Uncoupling of BCR Internalization and Signaling in Treatment Naive and Btki-Treated Patients with Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.191.191 ◽

2016 ◽

Vol 128 (22) ◽

pp. 191-191

Author(s):

Eve M Coulter ◽

Andrea Buggins ◽

Silvia Mele ◽

Najeem Folarin ◽

William Townsend ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Intracellular Signaling ◽

Statistical Significance ◽

High Surface ◽

Lymphocytic Leukemia ◽

Receptor Complexes ◽

Bcr Signaling ◽

Cell Anergy ◽

B Cell Anergy

Abstract Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi). BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p<0.0001, n=40) although there was considerable inter-patient variation. We observed a significant correlation between pHrodo-αIgM uptake and high surface IgM (sIgM) expression (r2=0.529, p=0.0001, n=40), and an association with markers of adverse prognosis including, CD38 positivity (p=0.001, n=40), and the capacity to mobilize calcium ions following BCR ligation (p=0.001, n=26). Cases of CLL with unmutated IGHV genes showed higher uptake of pHrodo-αIgM than mutated cases however this did not reach statistical significance (p=0.053, n=40). Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression. When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy. Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy. Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL. Disclosures Patten: Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.

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Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process

International Immunology ◽

10.1093/intimm/11.5.765 ◽

1999 ◽

Vol 11 (5) ◽

pp. 765-776 ◽

Cited By ~ 64

Author(s):

Hooman Noorchashm ◽

Anh Bui ◽

Hsiu-Ling Li ◽

Ashlyn Eaton ◽

Laura Mandik-Nayak ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Reversible Process ◽

Cell Anergy ◽

B Cell Anergy

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A Molecular Signature of Anergy Detected in a Subset of CLL Patients.

Blood ◽

10.1182/blood.v110.11.742.742 ◽

2007 ◽

Vol 110 (11) ◽

pp. 742-742

Author(s):

Paolo Ghia ◽

Marta Muzio ◽

Benedetta Apollonio ◽

Cristina Scielzo ◽

Michela Frenquelli ◽

...

Keyword(s):

B Cell ◽

Clinical Course ◽

Molecular Signature ◽

Leukemic Cells ◽

Quiescent State ◽

Akt Activation ◽

Cell Anergy ◽

B Cell Anergy ◽

Functional Unresponsiveness

Abstract It has been long assumed that BCR signaling plays a relevant role in the natural history of Chronic Lymphocytic Leukemia (CLL), by regulating various and possibly opposing mechanisms. A proportion of CLL cases carry a surface Immunoglobulin (sIg) which is responsive to crosslinking, resulting in effective activation of leukemic cells in vitro. In clear contrast, the remaining cases show a typical functional unresponsiveness through the Immunoglobulin, as indicated by a lack of tyrosine phosphorylation following BCR cross-linking. This feature together with the characteristic decreased expression of the BCR, have led to the time-honored hypothesis that these cells might have been anergized in vivo upon their encounter with a (self-) antigen. Of note, this functional feature tends to correlate with an indolent clinical course of the disease, which may derive from the quiescent state of the functionally silenced leukemic lymphocytes. Despite extensive studies, the concept of B cell anergy in the human immune system and in particular of the functional unresponsiveness in CLL lacks a molecular and biochemical definition. These observations prompted us to examine the expression and activation of key molecules involved in the signaling pathways originating from the BCR in 37 CLL patients and to investigate potential association of the distinct signaling patterns with clinical prognostic features. Our studies showed that in those CLL patients characterized by cellular unresponsiveness to sIg ligation, the MAP kinases ERK1/2 were constitutively phosphorylated and remained unchanged after stimulation with anti-IgM. These cases also displayed constitutive phosphorylation of MEK1/2 and Raf-1 and increased NF-AT transactivation; interestingly, these biochemical features were associated with a lack of AKT activation. In contrast, PMA that bypasses BCR proximal events was able to induce phosphorylation of ERK of these CLL cases indicating that the MAPK signaling machinery is intact but is selectively silenced and unresponsive to BCR-mediated signals. Notably, CLL cases with a constitutive ERK1/2 activation showed a more favorable clinical presentation as compared to the ERK1/2 negative cases. Constitutive activation of MAPK, along with NF-AT transactivation and lack of AKT activation has been previously described as a hallmark of anergic B lymphocytes in the murine system. Taking together all the above, we propose that the “molecularly anergic” subset of CLL described in our work may represent a human cellular model of anergic human B cells aberrantly expanded as part of the malignant process. These molecular features of constitutive BCR-mediated activation may provide evidence for BCR engagement by a putative anergizing antigen occurring in vivo. In addition, these data raise the intriguing hypothesis that this molecular signature of in vivo anergization may also be responsible for the quiescent state of the leukemic cells resulting in the indolent clinical course. In conclusion, our results may provide a molecular basis for the concept of human B-cell anergy and for the distinct functional and clinical behavior observed in a subset of CLL patients.

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B-Cell Anergy Underlies Indolent Clinical Behavior Of CLL Stereotyped Subset #4

Blood ◽

10.1182/blood.v122.21.4115.4115 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4115-4115

Author(s):

Stavroula Ntoufa ◽

Nikos Papakonstantinou ◽

Benedetta Apollonio ◽

Maria Gounari ◽

Achilles Anagnostopoulos ◽

...

Keyword(s):

B Cell ◽

Biochemical Characterization ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Luciferase Reporter ◽

Down Regulation ◽

Immune Signaling ◽

Erk Phosphorylation ◽

Cell Anergy ◽

B Cell Anergy

Abstract Mounting evidence suggests that specific modalities of B-cell receptor (BcR) and Toll-like receptor (TLR) collaboration and/or regulation may exist in chronic lymphocytic leukemia (CLL), eventually impacting on the behavior of the malignant clones. CLL patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BcRs, SS4), the largest subset within mutated CLL, are characterized clinically by early age at diagnosis and indolent disease course and genetically by few lesions, with deletion of chromosome 13q representing the exclusive recurrent aberration. Examination of the IG sequences of SS4 clonotypic BcRs allows for parallels to be drawn with pathogenic anti-DNA antibodies since both carry long positively charged VH CDR3s. However, SS4 IGs also display distinctive somatic hypermutation (SHM) patterns, the most notable concerning the obligatory introduction of negatively charged residues in either the heavy or the light chains or both, alluding to editing of an autoreactive progenitor. Moreover, SS4 BcRs continue to acquire SHMs within their IG genes overtime, implying antigen selection in the clonal evolution of CLL SS4, albeit without a negative clinical impact. Altogether, these findings suggest that CLL SS4 cells may exist in an anergic state showing attenuated responses to selecting (auto)antigenic elements. To test this hypothesis, after BcR engagement in vitro we analysed basal phosphorylated ERK (pERK) levels and calcium mobilization, both known to be features of B-cell anergic state. Nine of 10 SS4 cases expressed high pERK levels and were unable to increase intracellular Ca2+ upon BcR crosslinking; moreover, stimulation through the BcR had no effect on pERK levels. Primary cell culture for 50min caused a statistically significant reduction of pERK, while culture for 6-24 hours restored the ability of the cells to respond to BcR activation by inducing ERK phosphorylation. Furthermore, analysis of serial samples of 4 SS4 cases over a period spanning 5-7 years revealed consistently positive pERK expression. On these grounds, we conclude that SS4 CLL clones are anergic through the BcR possibly because of chronic (auto)antigen activation. To determine if this profile is SS4-biased or linked to the usage of the IGHV4-34 gene, we also performed similar experiments in 6 cases using this gene in mutated non-stereotyped rearrangements of the MD isotype and found a more heterogeneous pattern: 3/4 cases were pERK negative, while 3/6 cases showed Ca2+ fluxes after BcR crosslinking. Given that SS4 cells are responsive to TLR1/2 ligands, we next investigated if TLR signals can modulate BcR anergy. To this end, SS4 CLL cells pre-stimulated through TLR1/2 with Pam3CSK4 for 50min were then subjected to BcR crosslinking for 10min. TLR1/2 stimulation up-regulated pERK levels, confirming our previous finding that CLL SS4 cells are responsive to TLR1/2 signalling. Notably, BcR crosslinking in TLR1/2-pretreated cells induced even higher pERK levels, indicating that TLR1/2 triggering restores BcR functionality, likely breaching the anergic state. We have previously demonstrated that TLR1/2 stimulation induces up-regulation of the miR-17∼92 cluster eventually leading to down-regulation of critical BcR and/or TLR signaling molecules (i.e. MAP3K1, MAP2K3, MAPK8, CASP8, IKBKB) which are potential targets of these miRNAs. In order to experimentally validate the predicted mRNA-miRNA interactions, we performed 3’ UTR luciferase reporter assay experiments in HeLa cells and documented 5 novel interactions between miR-17∼92 cluster members and MAP3K1, MAP2K3 and MAPK8. To ensure that these interactions are valid also in CLL, we performed transfection experiments with miRNA mimics in negatively selected CLL B cells and obtained identical results. In conclusion, we demonstrate that SS4 CLL cells are anergic through the BcR and that stimulation through TLR1/2 may break B-cell anergy. Moreover, we propose a regulatory mechanism whereby TLR1/2 signals induce miR-17∼92 cluster up-regulation, leading to down-regulation of MAP3K1, MAP2K3 and MAPK8 and eventual modulation of ERK phosphorylation, a key to the anergic state. On a broader scale, the detailed molecular and biochemical characterization of immune signaling in SS4, a prototype for clinically indolent, good prognosis CLL, is also relevant for the advancement of therapeutic strategies incorporating immune signaling inhibitors. Disclosures: Stamatopoulos: Roche: Research Funding.

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