Activation of Ras overcomes B-cell tolerance to promote differentiation of autoreactive B cells and production of autoantibodies

Lenka Teodorovic

Chiara Babolin

Sarah Rowland

Sarah Greaves

David Baldwin

Raul Torres

Roberta Pelanda

Supplementary Material

Supporting Information:

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Department of Immunology, National Jewish Health and University of Colorado School of Medicine, Denver, CO, 80206

^{To whom correspondence should be addressed. E-mail:}ude.revnedcu@adnaleP.atreboR.

Edited by Michel C. Nussenzweig, The Rockefeller University, New York, NY, and approved May 28, 2014 (received for review February 4, 2014)

Author contributions: L.S.T., C.B., S.L.R., R.M.T., and R.P. designed research; L.S.T., C.B., S.L.R., S.A.G., and D.P.B. performed research; L.S.T., C.B., S.L.R., S.A.G., R.M.T., and R.P. analyzed data; and L.S.T., R.M.T., and R.P. wrote the paper.

^{L.S.T. and C.B. contributed equally to this work.}

Edited by Michel C. Nussenzweig, The Rockefeller University, New York, NY, and approved May 28, 2014 (received for review February 4, 2014)

Significance

Only a fraction of immature B cells enter the mature B-cell pool to produce antibodies. Autoreactive immature B cells expressing antibodies to self remain in the bone marrow to continue immunoglobulin gene rearrangements and are selected into the periphery only if they eliminate their autoreactive specificity. We show that the rat sarcoma (Ras)-Erk pathway, which leads to the generation of mature B cells, is not constitutively activated in autoreactive immature B cells. Furthermore, activation of Ras can alter the selection pattern of autoreactive cells, inhibiting immunoglobulin gene recombination via PI3K, promoting cell differentiation via Erk, and resulting in secretion of autoantibodies. This suggests that changes in the activation of the Ras–Erk/PI3K pathway have the potential to lead to autoimmune manifestations.

Keywords: Src, BAFF

Significance

Abstract

Newly generated immature B cells are selected to enter the peripheral mature B-cell pool only if they do not bind (or bind limited amount of) self-antigen. We previously suggested that this selection relies on basal extracellular signal-regulated kinase (Erk) activation mediated by tonic B-cell antigen receptor (BCR) signaling and that this signal can be replaced by an active rat sarcoma (Ras), which are small GTPase proteins. In this study we compared the activity of Ras and Erk in nonautoreactive and autoreactive immature B cells and investigated whether activation of Ras can break tolerance. Our results demonstrate lower levels of active Erk and Ras in autoreactive immature B cells, although this is evident only when these cells display medium/high avidity for self-antigen. Basal activation of Erk in immature B cells is proportional to surface IgM and dependent on sarcoma family kinases, whereas it is independent of B-cell activating factor, IFN, and Toll-like receptor signaling. Ectopic expression of the constitutively active mutant Ras form N-RasD12 in autoreactive cells raises active Erk, halts receptor editing via PI3 kinase, and promotes differentiation via Erk, breaking central tolerance. Moreover, when B cells coexpress autoreactive and nonautoreactive BCRs, N-RasD12 leads also to a break in peripheral tolerance with the production of autoantibodies. Our findings indicate that in immature B cells, basal activation of Ras and Erk are controlled by tonic BCR signaling, and that positive changes in Ras activity can lead to a break in both central and peripheral B-cell tolerance.

Abstract

B cells are generated in the bone marrow from progenitors and precursors that undergo random Ig variable gene rearrangements at the Ig heavy (H) and light (L) chain loci. Once the Ig H and L chains become expressed, they pair with the Igα (CD79a) and Igβ (CD79b) polypeptides to form the mature B-cell receptor (BCR), which is then transported onto the cell surface (initially in the form of IgM) where it can bind antigen and signal inside the cell. Despite representing the majority of newly formed clones (1, 2), immature B cells that bind self-antigen [i.e., autoreactive (A) cells] are not generally recruited into the primary mature B-cell pool and instead undergo negative selection via mechanisms of central tolerance. During tolerance, immature B cells arrest in differentiation and attempt to eliminate their autoreactivity by performing additional Ig gene rearrangements (receptor editing) or proceed to clonal deletion if the editing mechanism fails (reviewed in refs. 3 –6). In contrast to autoreactive cells, immature B cells that do not bind (or bind very limited amount of) antigen are positively selected into the mature B-cell population within peripheral lymphoid tissues. During this positive selection process, nonautoreactive (NA) immature B cells activate a developmental program that terminates Ig gene rearrangements, alters their tissue adhesion and migration, and promotes expression of novel surface proteins, such as CD21 and CD23, indicative of transitional and mature B-cell stages (reviewed in ref. 4). The analysis of mice and humans with defective B-cell maturation has shown that positive selection requires expression of a complete and functional BCR (reviewed in refs. 7, 8). This is supported by studies showing that the BCR mediates a ligand-independent signal termed basal or tonic that is necessary for the development of B lymphocytes (9 –11) and the survival of mature B cells (12, 13).

The discovery of tonic BCR signaling has prompted questions of whether and how it qualitatively differs from antigen-induced BCR signaling. Elegant studies have identified the phosphoinositide 3-kinase (PI3K) as one of the downstream mediators of tonic BCR signaling (reviewed in refs. 14, 15). The activity of PI3K in immature B cells is required to reduce levels of the Forkhead box protein O1 (FoxO1) transcription factor and, consequently, of recombination-activating gene (Rag) expression, Ig gene rearrangements, and receptor editing (16 –18). By comparing nonautoreactive immature B cells that express normal or subnormal levels of IgM, studies in our laboratory have indicated that tonic BCR signaling, directly or indirectly, positively regulates the activity of the mitogen-activated protein kinase (MAPK) Mek (MAPKK) –Erk (extracellular signal-regulated kinase) pathway and that this pathway mediates cell differentiation into the transitional/mature B-cell stages (19). Such a role for the Erk pathway has also been suggested by studies of CD19-deficient mice (20). Our studies have shown that in nonautoreactive immature B cells, inhibition of Mek decreases cell differentiation (19). Moreover, active Erk1/2 (phosphorylated Erk, pErk), when measured after pervanadate treatment, is present at significantly lower levels within cells that express subnormal (about 15%) amounts of BCR (BCR-low cells) and that are impaired in differentiation (19). Furthermore, expression of a constitutively active mutant form of the rat sarcoma protein N-Ras (N-RasD12, with a G to D amino acid substitution at position 12), a small GTPase known to activate the Erk pathway (21), restores the differentiation of BCR-low cells in a process that is dependent on the activity of Mek (19). Together with studies showing that Erk and Ras play an important role during the differentiation of pro-B cells into pre-B cells (22 –25), these findings suggest a role for Ras and Erk in both pre-BCR and mature BCR signaling.

PI3K, Ras, and Erk are also activated following antigen-induced BCR signaling, but this is a rapid event that is quickly quenched by phosphatases and other negative feedback mechanisms (26, 27). Thus, the chronic stimulation by antigen of autoreactive B cells might not necessarily result in higher activity of PI3K, Ras, and Erk relative to nonstimulated cells. Indeed, prolonged BCR stimulation in immature B cells reduces levels of downstream effectors of the PI3K pathway relative to nonstimulated cells (17). These findings are in line with an alternative model of immature B-cell selection advocated by Behrens and coworkers proposing that when immature B cells chronically bind self-antigen they revert to a phenotype similar to that of pro-B/pre-B cells and, therefore, to cells that experience neither antigen-induced nor tonic BCR signaling (28). This model is supported by finding that prolonged BCR engagement by antigen causes immature B cells to down-modulate their surface BCR (28 –31), express Rag at levels proportional to BCR down-modulation (28), and exhibit gene expression profiles similar to pre-B cells (28).

Resolving whether distinct signaling molecules, or levels of activation of those same molecules, regulate positive and negative B-cell selection in the bone marrow, and how the activities of these molecules are modulated, are of fundamental importance for understanding how the autoreactive capacity of the naive peripheral B-cell pool varies, depending on the genetic background of the individual and factors such as inflammation and infection (32, 33). In the case of distinct pathways, abnormal activation of mediators of the tonic BCR signaling cascade during B-cell development, like that of mediators of antigen-induced BCR signaling (34), can lead to positive selection of autoreactive immature B cells into the mature B-cell pool, raising the chance of autoantibody production and autoimmunity. In an attempt to investigate these matters, we used Ig H + L gene-targeted mice and other mouse models to determine whether Ras and Erk are differentially regulated in autoreactive and nonautoreactive immature B cells and if their basal activation depends on tonic BCR signaling. Furthermore, we explored whether chronic activation of the Ras pathway in autoreactive immature B cells, inhibits receptor editing and rescues cell differentiation despite antigen-induced BCR signaling.

We found that basal activation of both Erk and Ras is higher in nonautoreactive than autoreactive immature B cells, although only those with high avidity for self-antigen. Basal pErk levels depend on tonic BCR signaling and are not altered by chronic antigen-induced BCR signaling, B-cell activating factor (BAFF), IFN, or Toll-like receptor (TLR) signaling. Moreover, we show that chronic activation of the Ras pathway in autoreactive B cells leads to inhibition of receptor editing, cell differentiation, and production of circulating IgG autoantibodies.

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Acknowledgments

We thank Margot Kelly for technical assistance with cell preparation; Dr. Doug Everett (National Jewish Health, NJH) for assisting with statistical analyses; Janie Akerlund (John Cambier laboratory, NJH), Amy McKee (Andrew Fontenot laboratory, University of Colorado, Denver), and Laurel Lenz (NJH) for the gift of MD4/MD4 × ML5 mice, MYD88-deficient mice, and IFNαR/IFNγR-deficient mice, respectively; all laboratory members for the numerous useful discussions; Drs. Julie Lang, Lisa Peterson, and Andy Getahun for reading the manuscript and providing scientific and editorial suggestions; the NJH Flow Cytometry facility for assistance with cell sorting and analysis; and the Biological Resource Center for assistance with mouse husbandry. This work was supported by National Institutes of Health (NIH) Grants P01 AI022295, R01 AI052310, R01 AI052157, and the Cancer Center Support Grant P30CA046934 for shared resources. L.S.T. was supported by NIH T32-AI07405 Training Grant and C.B. was supported by the Viola Vestal Coulter Foundation.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1402159111/-/DCSupplemental.

Footnotes

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