Editorial
Editorial: Btk—friend or foe in autoimmune diseases? By Patricia Barral1 Peter Gorer Department of Immunobiology, King’s College London, United Kingdom RECEIVED MAY 15, 2013; REVISED MAY 28, 2013; ACCEPTED JUNE 2, 2013. DOI: 10.1189/jlb.0513279
‹ SEE CORRESPONDING ARTICLE ON PAGE 865
B
cells are critical players in the development of a broad range of autoimmune diseases, including systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, and multiple sclerosis. In addition to the secretion of autoantibodies, B cells can potentiate autoimmunity by several additional means, such as their function as APCs and the secretion of proinflammatory cytokines. Also, the successful use of B cell-depletion therapies for the treatment of autoimmune disorders has supported the fundamental role of B cells in the development and sustaining of autoimmunity. All of these factors lead to a growing interest in understanding how autoreactive B cells develop and how their activation is controlled in health and disease. In the current issue of the Journal of Leukocyte Biology, Nündel and colleagues [1] provide new insight into these questions by exploring the role of Btk signaling in autoreactive B cell development and activation in response to nucleic acidassociated autoantigens. Systemic autoimmune diseases are frequently associated with the production of autoantibodies that recognize nuclear or cytosolic cellular components. Cellular debris derived from dying cells is likely to induce activation of autoreactive B cells and subsequent production of autoantibodies. In this context, the activation of autoreactive B cells that express low-affinity receptors
Abbreviations: Btk⫽Bruton’s tyrosine kinase, MZ⫽marginal zone, RF⫽rheumatoid factor, xid⫽X-linked immunodeficiency, XLA⫽X-linked agammaglobulinemia
0741-5400/13/0094-859 © Society for Leukocyte Biology
for nucleic acids (or nucleic acid-associated autoantigens) is prompted by signals that originate from the engagement of TLRs with nucleic acids [2]. Hence, in addition to BCR stimulation, the binding of RNA- or DNA-containing antigen to the BCR leads to receptor internalization and delivery of antigen to intracellular compartments containing TLR7 and TLR9. As a model system to understand development and activation of these autoreactive B cells, Nündel and colleagues [1] take advantage of mice expressing a prototypic RF BCR, encoded by heavy and light-chain, sitedirected transgenes (AM14 mice). These mice possess a highly homogeneous B cell pool, in which essentially all B cells express the same BCR. RF⫹ B cells have a low-affinity receptor and proliferate in response to IgG2a immune complexes that incorporate DNA or RNA. Therefore, AM14 mice allow evaluation of the effect of individual signaling molecules in the homeostasis, differentiation, and activation of autoreactive B cells, irrespectively of BCR specificity. Among the many molecules involved in BCR signaling, Btk is known to be essential for B cell activation and differentiation in both mouse and humans. As such, mutations in Btk result in XLA in humans and xid in mice. Importantly, xid mice, Btk-deficient mice, and XLA patients show a reduction in mature peripheral B cells [3]. In general, mutations in components of the BCR– Btk pathway result in a loss of follicular B cells and comparative preservation of MZ B cells. Accordingly, Nündel and colleagues [1] show that whereas BtkVolume 94, November 2013
sufficient RF⫹ B cells develop into a homogenous follicular B cell population, Btk deficiency leads to an increased proportion of RF⫹ B cells with a MZ phenotype. Therefore, the loss in signal strength derived from Btk deficiency favors differentiation of the MZ compartment. Interestingly, AM14-xid mice show a marked increase in the number of MZ precursors relative to the number of MZ B cells, suggesting that Btk could be required for survival of MZ RF⫹ B cells. Nevertheless, it is worth mentioning that some clones of transgenic B cells that recognize self-antigens have shown a requirement for Btk for differentiation to MZ B cells [4 – 6]. Consequently, although MZ B cells seem to be able to differentiate in the absence of Btk, it is possible that some clones (maybe with very low BCR affinity) might need Btk signaling to enter and/or survive in the MZ B cell pool. In addition to the altered splenic B cell populations, xid and Btk-deficient mice show a severe drop in the numbers of CD5⫹ B1 peritoneal B cells and remarkably low levels of circulating IgM and IgG3 [3]. Interestingly, Btk-sufficient AM14 mice lack CD5⫹ peritoneal B cells and display low levels of serum IgM compared with WT counterparts. Depletion of Btk in AM14 mice induces a further drop in IgM, suggesting that Btk could affect circulating IgM levels separately from the contribution of B1 cells.
1. Correspondence: Peter Gorer Dept. of Immunobiology, King’s College London, SE19RT, London, UK. E-mail: [email protected]
Journal of Leukocyte Biology 859
xid mice and Btk-deficient mice fail to respond to immunization with type 2 thymus-independent antigens, and accordingly, peripheral B cells from these animals are unresponsive to BCR crosslinking [3]. The same holds true for RF⫹ Btk-deficient B cells that remain unresponsive to anti-IgM(Fab)2 stimulation in vitro. However, stimulation of B cells from AM14-xid mice with nucleic acid-containing IgG2a-immune complexes resulted in several rounds of B cell proliferation(Fig. 1). In line with this, Btk-deficient B cells from mice carrying the 56R anti-DNA Ig transgene have been shown to be able to proliferate in response to CpG-containing DNA fragments [4]. In summary, these data indicate that coengagement of the BCR and TLR can bypass the need of Btk for
B cell proliferation, suggesting a Btkindependent mechanism for activation of autoreactive B cells. In this regard, it would be interesting to address whether other parameters, resulting from autoreactive B cell activation (i.e., cytokine secretion, antigen presentation capacity), are affected by Btk expression and how this may condition the development of autoimmune pathologies. The precise role of Btk in autoimmune processes is difficult to decipher, as Btk takes part in myriad signaling pathways, and it has a broad cellular expression within the hematopoietic compartment. Nonetheless, several lines of evidence suggest that regulation of Btk levels and/or function can affect the development of autoimmunity. For instance, when autoimmune-prone MLR
WT auto-reactive B cell BCR-crosslink (crosslinking antibody)
BCR
mice are crossed with xid animals, the resulting offspring show delayed disease development, although the xid gene does not prevent the autoimmune phenotype completely [7]. In line with this, administration of Btk inhibitors reduces the levels of autoantibodies and the development of kidney disease in MLR mice [8]. It is worth mentioning that in those settings, Btk function is abolished in the different APCs, and the precise contribution of Btk, specifically on B cells, to the development of autoimmunity is difficult to interpret. Notably, a transgenic mouse model that targets overexpression of Btk to B cells has been reported recently [9]. These animals display spontaneous B cell activation, secretion of antinuclear antibodies, and development of a lupus-like autoim-
Btk-deficient auto-reactive B cell
Nucleic-acid associated antigen
BCR
BCR-crosslink (crosslinking antibody)
BCR
Nucleic-acid associated antigen
BCR
Endosome
Endosome
TLR
TLR
Figure 1. Effect of Btk expression in autoreactive B cell activation. WT B cells proliferate in response to anti-IgM stimulation in vitro, whereas Btkdeficient B cells remain unresponsive to such stimulation. However, both Btk-sufficient and -deficient B cells proliferate in response to nucleic acidcontaining autoantigen, although Btk-deficient cells show a less-robust response than their WT counterparts.
860 Journal of Leukocyte Biology
Volume 94, November 2013
www.jleukbio.org
EDITORIAL Barral Btk: friend or foe in autoimmune diseases?
mune pathology, implying a direct role for Btk in setting the threshold for autoreactive B cell activation and/or differentiation. Additionally, it should be borne in mind that on top of its role in BCR signaling, Btk becomes activated by a broad range of receptors, and it has been proposed to participate in many different processes, including cytokine signaling, TLR signaling, and integrinmediated adhesion. In this line, Btkdeficient cells show decreased IL-10 production following stimulation with multiple TLR ligands, suggesting a potential regulatory role for Btk [10]. Further studies will help in the understanding of the requirement for Btk function in specific signaling pathways (within different cell types) and their precise contribution to the development of autoimmunity. Finally, an interesting remark of the present manuscript is the observation that activation of autoreactive B cells in response to BCR/TLR7 and BCR/TLR9 results in different degrees of B cell proliferation and survival. These data point to disparities in the signaling cascades and/or regulatory mechanism underlying coengagement of BCR and TLR7/TLR9. Although the molecular mechanisms that explain these differences will require further investigation, these results are in line with the contrasting roles that have been suggested for TLR7 and TLR9 in the development of autoimmunity [11]. Indeed, whereas both receptors are located in endosomal
www.jleukbio.org
compartments and signal through MyD88, they seem to convey opposite effects in murine models of autoimmunity, with TLR7 playing a pathogenic and TLR9 a protective role. Although the data provided by Nündel et al. [1] provide insight into this issue, a more complex picture could be happening in vivo. Many different cell types express TLRs, can respond to nucleic acid-containing immune complexes, and affect the production of autoantibodies. Further research is needed to decipher the complex cellular interactions involved in the activation of autoreactive B cells and to understand the outcome of such processes. Finally, it is important to keep in mind that mouse and human B cells show differences in the expression and function of several TLRs, and the phenotype of xid mice is milder than that of XLA patients. Therefore, in the future, it will be central to address how the findings presented by Nündel et al. [1] can be translated to human autoimmune pathologies. REFERENCES
1.
Nündel, K., Busto, P., Debatis, M., MarshakRothstein, A. (2013) The roll of Bruton’s tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells. J. Leukoc. Biol. 94, XXX–XXX. 2. Marshak-Rothstein, A. (2006) Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6, 823–835. 3. Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Müller, S., Kantor, A. B., Herzen-
4.
5.
6.
7.
8.
9.
10.
11.
berg, L. A. (1995) Defective B cell development and function in Btk-deficient mice. Immunity 3, 283–299. Halcomb, K. E., Musuka, S., Gutierrez, T., Wright, H. L., Satterthwaite, A. B. (2008) Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells. Mol. Immunol. 46, 233–241. Kanayama, N., Cascalho, M., Ohmori, H. (2005) Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone. J. Immunol. 174, 1438 –1445. Martin, F., Kearney, J. F. (2000) Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and Btk. Immunity 12, 39 –49. Steinberg, E. B., Santoro, T. J., Chused, T. M., Smathers, P. A., Steinberg, A. D. (1983) Studies of congenic MRL-Ipr/Ipr.xid mice. J. Immunol. 131, 2789 –2795. Honigberg, L. A., Smith, A. M., Sirisawad, M., Verner, E., Loury, D., Chang, B., Li, S., Pan, Z., Thamm, D. H., Miller, R. A., Buggy, J. J. (2010) The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. USA 107, 13075–13080. Kil, L. P., de Bruijn, M. J., van Nimwegen, M., Corneth, O. B., van Hamburg, J. P., Dingjan, G. M., Thaiss, F., Rimmelzwaan, G. F., Elewaut, D., Delsing, D., van Loo, P. F., Hendriks, R. W. (2012) Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice. Blood 119, 3744 –3756. Schmidt, N. W., Thieu, V. T., Mann, B. A., Ahyi, A. N., Kaplan, M. H. (2006) Bruton’s tyrosine kinase is required for TLR-induced IL-10 production. J. Immunol. 177, 7203–7210. Christensen, S. R., Shupe, J., Nickerson, K., Kashgarian, M., Flavell, R. A., Shlomchik, M. J. (2006) Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity 25, 417–428.
KEY WORDS: systemic lupus erythematosus 䡠 Toll-like receptor 䡠 X-linked immunodeficiency
Volume 94, November 2013
Journal of Leukocyte Biology 861
Editorial: Btk—friend or foe in autoimmune diseases? By Patricia Barral1 Peter Gorer Department of Immunobiology, King’s College London, United Kingdom RECEIVED MAY 15, 2013; REVISED MAY 28, 2013; ACCEPTED JUNE 2, 2013. DOI: 10.1189/jlb.0513279
‹ SEE CORRESPONDING ARTICLE ON PAGE 865
B
cells are critical players in the development of a broad range of autoimmune diseases, including systemic lupus erythematosus, type I diabetes, rheumatoid arthritis, and multiple sclerosis. In addition to the secretion of autoantibodies, B cells can potentiate autoimmunity by several additional means, such as their function as APCs and the secretion of proinflammatory cytokines. Also, the successful use of B cell-depletion therapies for the treatment of autoimmune disorders has supported the fundamental role of B cells in the development and sustaining of autoimmunity. All of these factors lead to a growing interest in understanding how autoreactive B cells develop and how their activation is controlled in health and disease. In the current issue of the Journal of Leukocyte Biology, Nündel and colleagues [1] provide new insight into these questions by exploring the role of Btk signaling in autoreactive B cell development and activation in response to nucleic acidassociated autoantigens. Systemic autoimmune diseases are frequently associated with the production of autoantibodies that recognize nuclear or cytosolic cellular components. Cellular debris derived from dying cells is likely to induce activation of autoreactive B cells and subsequent production of autoantibodies. In this context, the activation of autoreactive B cells that express low-affinity receptors
Abbreviations: Btk⫽Bruton’s tyrosine kinase, MZ⫽marginal zone, RF⫽rheumatoid factor, xid⫽X-linked immunodeficiency, XLA⫽X-linked agammaglobulinemia
0741-5400/13/0094-859 © Society for Leukocyte Biology
for nucleic acids (or nucleic acid-associated autoantigens) is prompted by signals that originate from the engagement of TLRs with nucleic acids [2]. Hence, in addition to BCR stimulation, the binding of RNA- or DNA-containing antigen to the BCR leads to receptor internalization and delivery of antigen to intracellular compartments containing TLR7 and TLR9. As a model system to understand development and activation of these autoreactive B cells, Nündel and colleagues [1] take advantage of mice expressing a prototypic RF BCR, encoded by heavy and light-chain, sitedirected transgenes (AM14 mice). These mice possess a highly homogeneous B cell pool, in which essentially all B cells express the same BCR. RF⫹ B cells have a low-affinity receptor and proliferate in response to IgG2a immune complexes that incorporate DNA or RNA. Therefore, AM14 mice allow evaluation of the effect of individual signaling molecules in the homeostasis, differentiation, and activation of autoreactive B cells, irrespectively of BCR specificity. Among the many molecules involved in BCR signaling, Btk is known to be essential for B cell activation and differentiation in both mouse and humans. As such, mutations in Btk result in XLA in humans and xid in mice. Importantly, xid mice, Btk-deficient mice, and XLA patients show a reduction in mature peripheral B cells [3]. In general, mutations in components of the BCR– Btk pathway result in a loss of follicular B cells and comparative preservation of MZ B cells. Accordingly, Nündel and colleagues [1] show that whereas BtkVolume 94, November 2013
sufficient RF⫹ B cells develop into a homogenous follicular B cell population, Btk deficiency leads to an increased proportion of RF⫹ B cells with a MZ phenotype. Therefore, the loss in signal strength derived from Btk deficiency favors differentiation of the MZ compartment. Interestingly, AM14-xid mice show a marked increase in the number of MZ precursors relative to the number of MZ B cells, suggesting that Btk could be required for survival of MZ RF⫹ B cells. Nevertheless, it is worth mentioning that some clones of transgenic B cells that recognize self-antigens have shown a requirement for Btk for differentiation to MZ B cells [4 – 6]. Consequently, although MZ B cells seem to be able to differentiate in the absence of Btk, it is possible that some clones (maybe with very low BCR affinity) might need Btk signaling to enter and/or survive in the MZ B cell pool. In addition to the altered splenic B cell populations, xid and Btk-deficient mice show a severe drop in the numbers of CD5⫹ B1 peritoneal B cells and remarkably low levels of circulating IgM and IgG3 [3]. Interestingly, Btk-sufficient AM14 mice lack CD5⫹ peritoneal B cells and display low levels of serum IgM compared with WT counterparts. Depletion of Btk in AM14 mice induces a further drop in IgM, suggesting that Btk could affect circulating IgM levels separately from the contribution of B1 cells.
1. Correspondence: Peter Gorer Dept. of Immunobiology, King’s College London, SE19RT, London, UK. E-mail: [email protected]
Journal of Leukocyte Biology 859
xid mice and Btk-deficient mice fail to respond to immunization with type 2 thymus-independent antigens, and accordingly, peripheral B cells from these animals are unresponsive to BCR crosslinking [3]. The same holds true for RF⫹ Btk-deficient B cells that remain unresponsive to anti-IgM(Fab)2 stimulation in vitro. However, stimulation of B cells from AM14-xid mice with nucleic acid-containing IgG2a-immune complexes resulted in several rounds of B cell proliferation(Fig. 1). In line with this, Btk-deficient B cells from mice carrying the 56R anti-DNA Ig transgene have been shown to be able to proliferate in response to CpG-containing DNA fragments [4]. In summary, these data indicate that coengagement of the BCR and TLR can bypass the need of Btk for
B cell proliferation, suggesting a Btkindependent mechanism for activation of autoreactive B cells. In this regard, it would be interesting to address whether other parameters, resulting from autoreactive B cell activation (i.e., cytokine secretion, antigen presentation capacity), are affected by Btk expression and how this may condition the development of autoimmune pathologies. The precise role of Btk in autoimmune processes is difficult to decipher, as Btk takes part in myriad signaling pathways, and it has a broad cellular expression within the hematopoietic compartment. Nonetheless, several lines of evidence suggest that regulation of Btk levels and/or function can affect the development of autoimmunity. For instance, when autoimmune-prone MLR
WT auto-reactive B cell BCR-crosslink (crosslinking antibody)
BCR
mice are crossed with xid animals, the resulting offspring show delayed disease development, although the xid gene does not prevent the autoimmune phenotype completely [7]. In line with this, administration of Btk inhibitors reduces the levels of autoantibodies and the development of kidney disease in MLR mice [8]. It is worth mentioning that in those settings, Btk function is abolished in the different APCs, and the precise contribution of Btk, specifically on B cells, to the development of autoimmunity is difficult to interpret. Notably, a transgenic mouse model that targets overexpression of Btk to B cells has been reported recently [9]. These animals display spontaneous B cell activation, secretion of antinuclear antibodies, and development of a lupus-like autoim-
Btk-deficient auto-reactive B cell
Nucleic-acid associated antigen
BCR
BCR-crosslink (crosslinking antibody)
BCR
Nucleic-acid associated antigen
BCR
Endosome
Endosome
TLR
TLR
Figure 1. Effect of Btk expression in autoreactive B cell activation. WT B cells proliferate in response to anti-IgM stimulation in vitro, whereas Btkdeficient B cells remain unresponsive to such stimulation. However, both Btk-sufficient and -deficient B cells proliferate in response to nucleic acidcontaining autoantigen, although Btk-deficient cells show a less-robust response than their WT counterparts.
860 Journal of Leukocyte Biology
Volume 94, November 2013
www.jleukbio.org
EDITORIAL Barral Btk: friend or foe in autoimmune diseases?
mune pathology, implying a direct role for Btk in setting the threshold for autoreactive B cell activation and/or differentiation. Additionally, it should be borne in mind that on top of its role in BCR signaling, Btk becomes activated by a broad range of receptors, and it has been proposed to participate in many different processes, including cytokine signaling, TLR signaling, and integrinmediated adhesion. In this line, Btkdeficient cells show decreased IL-10 production following stimulation with multiple TLR ligands, suggesting a potential regulatory role for Btk [10]. Further studies will help in the understanding of the requirement for Btk function in specific signaling pathways (within different cell types) and their precise contribution to the development of autoimmunity. Finally, an interesting remark of the present manuscript is the observation that activation of autoreactive B cells in response to BCR/TLR7 and BCR/TLR9 results in different degrees of B cell proliferation and survival. These data point to disparities in the signaling cascades and/or regulatory mechanism underlying coengagement of BCR and TLR7/TLR9. Although the molecular mechanisms that explain these differences will require further investigation, these results are in line with the contrasting roles that have been suggested for TLR7 and TLR9 in the development of autoimmunity [11]. Indeed, whereas both receptors are located in endosomal
www.jleukbio.org
compartments and signal through MyD88, they seem to convey opposite effects in murine models of autoimmunity, with TLR7 playing a pathogenic and TLR9 a protective role. Although the data provided by Nündel et al. [1] provide insight into this issue, a more complex picture could be happening in vivo. Many different cell types express TLRs, can respond to nucleic acid-containing immune complexes, and affect the production of autoantibodies. Further research is needed to decipher the complex cellular interactions involved in the activation of autoreactive B cells and to understand the outcome of such processes. Finally, it is important to keep in mind that mouse and human B cells show differences in the expression and function of several TLRs, and the phenotype of xid mice is milder than that of XLA patients. Therefore, in the future, it will be central to address how the findings presented by Nündel et al. [1] can be translated to human autoimmune pathologies. REFERENCES
1.
Nündel, K., Busto, P., Debatis, M., MarshakRothstein, A. (2013) The roll of Bruton’s tyrosine kinase in the development and BCR/TLR-dependent activation of AM14 rheumatoid factor B cells. J. Leukoc. Biol. 94, XXX–XXX. 2. Marshak-Rothstein, A. (2006) Toll-like receptors in systemic autoimmune disease. Nat. Rev. Immunol. 6, 823–835. 3. Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Müller, S., Kantor, A. B., Herzen-
4.
5.
6.
7.
8.
9.
10.
11.
berg, L. A. (1995) Defective B cell development and function in Btk-deficient mice. Immunity 3, 283–299. Halcomb, K. E., Musuka, S., Gutierrez, T., Wright, H. L., Satterthwaite, A. B. (2008) Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells. Mol. Immunol. 46, 233–241. Kanayama, N., Cascalho, M., Ohmori, H. (2005) Analysis of marginal zone B cell development in the mouse with limited B cell diversity: role of the antigen receptor signals in the recruitment of B cells to the marginal zone. J. Immunol. 174, 1438 –1445. Martin, F., Kearney, J. F. (2000) Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and Btk. Immunity 12, 39 –49. Steinberg, E. B., Santoro, T. J., Chused, T. M., Smathers, P. A., Steinberg, A. D. (1983) Studies of congenic MRL-Ipr/Ipr.xid mice. J. Immunol. 131, 2789 –2795. Honigberg, L. A., Smith, A. M., Sirisawad, M., Verner, E., Loury, D., Chang, B., Li, S., Pan, Z., Thamm, D. H., Miller, R. A., Buggy, J. J. (2010) The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. USA 107, 13075–13080. Kil, L. P., de Bruijn, M. J., van Nimwegen, M., Corneth, O. B., van Hamburg, J. P., Dingjan, G. M., Thaiss, F., Rimmelzwaan, G. F., Elewaut, D., Delsing, D., van Loo, P. F., Hendriks, R. W. (2012) Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice. Blood 119, 3744 –3756. Schmidt, N. W., Thieu, V. T., Mann, B. A., Ahyi, A. N., Kaplan, M. H. (2006) Bruton’s tyrosine kinase is required for TLR-induced IL-10 production. J. Immunol. 177, 7203–7210. Christensen, S. R., Shupe, J., Nickerson, K., Kashgarian, M., Flavell, R. A., Shlomchik, M. J. (2006) Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity 25, 417–428.
KEY WORDS: systemic lupus erythematosus 䡠 Toll-like receptor 䡠 X-linked immunodeficiency
Volume 94, November 2013
Journal of Leukocyte Biology 861
