Agammaglobulinemia with normal B-cell numbers in a patient lacking Bob1
Authors: Patrick Kury, Julian Staniek, Oliver Wegehaupt, Iga Janowska, Matthias Eckenweiler, Rudolf Korinthenberg, Natia Japaridze, Manuela Pendziwiat, Ingo Helbig, Els Verhoeyen, Johannes Jung, Andres Caballero Garcia de Oteyza, Michele Proietti, Tamar Phirtskhalaishvili, Irakli Rtskhiladze, Peter J. Nielsen, Stephan Ehl, Carsten Speckmann, Marta Rizzi
Journal: Journal of Allergy and Clinical Immunology (2021)

We describe a 3-year-old boy (P-FR1) from consanguineous white parents lacking immunoglobulins despite normal total B-cell numbers. Healthy until the age of 18 months, he developed recurrent respiratory infections, followed by a progressive central nervous system disease with progressive spastic tetraparesis from 24 months onward. Cerebral magnetic resonance imaging revealed enlarged outer cerebral spinal fluid spaces and hypomyelination of the white matter. A chronic viral disease was suspected. Cerebral spinal fluid analysis showed moderate pleocytosis but negative microbial PCR results (for further clinical details, see the Methods section of the Online Repository at www.jacionline.org). A biopsy for brain tissue PCRs was declined by the parents. P-FR1's brother died of Zellweger syndrome caused by a homozygous mutation in PEX16 (p.His231Arg) at the age of 14 years. P-FR1 and his sister were heterozygous for this mutation (Fig 1, A) and did not present clinical or laboratory findings of Zellweger syndrome. Exome sequencing performed on P-FR1 and his parents (see Tables E1 and E2 in the Online Repository at www.jacionline.org) revealed the frameshift mutation c.233delC in POU2AF1 (p.Thr78Lysfs∗63 [see Fig E1, A in the Online Repository at www.jacionline.org]) that was homozygous in P-FR1 and heterozygous in his parents and sister (Fig 1, A). POU2AF1 encodes Bob1 (also called OBF-1 or OCA-B). Although POU2AF1 transcripts were detected (Fig 1, B), Bob1 protein was absent in a P-FR1 EBV B-cell line (Fig 1, C and see Fig E1, B) and in HEK293T cells transfected with the mutated Bob1 compared with the wild-type Bob1 control (see Fig E1, C), indicating instability of the aberrant fusion protein. In B cells, Bob1 is a transcriptional coactivator that confers octamer-dependent specificity to the transcription factors Oct-1 and Oct-2.1Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.Nature. 1996; 383: 538-542Crossref PubMed Scopus (237) Google Scholar P-FR1 had normal B-cell numbers but disturbed B-cell differentiation, with decreased class-switched memory cells and a relative increase in atypical-memory cells (IgG+ or IgA+ CD27– [Fig 1, D and E]). His T cells were normal in terms of distribution and activation (Fig 1, D and see Fig E1, D and E) except for a reduction in circulating T-follicular helper (TFH) cells (Fig 1, D and E). P-FR1–naive B cells showed an abnormal expression pattern of surface receptors and signaling molecules, with low levels of IgD, IgM, CD79α, CD79β, and Syk (Fig 2, A and see Fig E1, F). Indeed, Bob1 can directly regulate expression of B-cell receptor (BCR) signaling molecules by interacting with octamer motifs in their promoter regions, as described for CD79β.2Malone C.S. Wall R. Bob1 (OCA-B/OBF-1) differential transactivation of the B cell-specific B29 (Ig beta) and mb-1 (Ig alpha) promoters.J Immunol. 2002; 168: 3369-3375Crossref PubMed Scopus (20) Google Scholar Low CD79β may contribute to low BCR and CD79α expression, as shown in B-cell lines.3Minuzzo S. Indraccolo S. Tosello V. Piovan E. Cabrelle A. Trentin L. et al.Heterogeneous intracellular expression of B-cell receptor components in B-cell chronic lymphocytic leukaemia (B-CLL) cells and effects of CD79b gene transfer on surface immunoglobulin levels in a B-CLL-derived cell line.Br J Haematol. 2005; 130: 878-889Crossref PubMed Scopus (10) Google Scholar Additionally, interaction with Bob1 has been reported to control the stability of Syk.4Siegel R. Kim U. Patke A. Yu X. Ren X. Tarakhovsky A. et al.Nontranscriptional regulation of SYK by the coactivator OCA-B is required at multiple stages of B cell development.Cell. 2006; 125: 761-774Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Also, BAFF-R expression and CD22 expression were reduced in P-FR1 B cells. The abnormal pattern of surface expression was confirmed in a P-FR1–derived EBV B-cell line (see Fig E2, A in the Online Repository at www.jacionline.org). Their lentiviral reconstitution with wild-type Bob1 increased expression of CD22, BAFF-R, and IgD (see Fig E2, B and C). In P-FR1 B cells, the response to BCR stimulation, as measured by Syk and PLCγ2 phosphorylation (Fig 2, B), and response to CD40 stimulation, as measured with nuclear factor-κB and mTORC1 phosphorylation (pS6) (Fig 2, C), were compromised. Accordingly, activated P-FR1–derived B cells failed to upregulate CD86 (see Fig E3, A in the Online Repository at www.jacionline.org). Ex vivo, the P-FR1–derived B cells did not show signs of intrinsic activation, with the exception of increased S6 phosphorylation (see Fig E3, B). The formation of antibody-secreting plasmablasts in response to CD40L and IL-21 was abolished in Bob1-deficient B cells (Fig 2, D and see Fig E3, C-F), and class switch to IgG was delayed and less efficient (Fig E3, D and G), showing a dissociation between class switch and immunoglobulin secretion. The addition of IL-4 or T cell–independent activation with a Toll-like receptor 9 (TLR9) agonist did not compensate for this defective response (see Fig E3, D-G). Marginal zone–like B-cell (CD27+IgD+) formation in response to TLR9 agonist was also defective (see Fig E3, D and G). Consistent with the absence of plasmablasts, secreted immunoglobulins were not detected in the supernatant of cultured P-FR1 B cells (Fig 2, E and see Fig E3, H). Lentiviral reconstitution of P-FR1–naive B cells with wild-type Bob1 (see Fig E4, A in the Online Repository at www.jacionline.org) restored plasmablast formation and immunoglobulin secretion in response to CD40L and IL-21 (Fig 2, F and G and see Fig E4, B and C), strongly suggesting that the POU2AF1 mutation was causative for the impaired B-cell development. The immunologic manifestations of P-FR1 showed some overlap with the Bob1 knockout (Bob1–/–) mice, including dysregulated surface expression of BCR, Syk, CD79β, and BAFF-R2Malone C.S. Wall R. Bob1 (OCA-B/OBF-1) differential transactivation of the B cell-specific B29 (Ig beta) and mb-1 (Ig alpha) promoters.J Immunol. 2002; 168: 3369-3375Crossref PubMed Scopus (20) Google Scholar,5Hess J. Nielsen P.J. Fischer K.D. Bujard H. Wirth T. The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.Mol Cell Biol. 2001; 21: 1531-1539Crossref PubMed Scopus (57) Google Scholar,6Samardzic T. Marinkovic D. Nielsen P.J. Nitschke L. Wirth T. BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.Mol Cell Biol. 2002; 22: 8320-8331Crossref PubMed Scopus (41) Google Scholar; defect in B-cell activation,6Samardzic T. Marinkovic D. Nielsen P.J. Nitschke L. Wirth T. BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.Mol Cell Biol. 2002; 22: 8320-8331Crossref PubMed Scopus (41) Google Scholar and impaired in vivo T-dependent responses, with a lack of germinal centers and strongly reduced IgG.1Schubart D.B. Rolink A. Kosco-Vilbois M.H. Botteri F. Matthias P. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.Nature. 1996; 383: 538-542Crossref PubMed Scopus (237) Google Scholar However, some significant differences were also observed. Bob1–/– mice showed a 2-fold reduction in B-cell numbers, which were normal in the patient. Although serum IgM antibodies were absent in P-FR1, IgM levels were only slightly reduced in Bob1–/– mice, which was attributed to their unaffected B1 B-cell compartment,5Hess J. Nielsen P.J. Fischer K.D. Bujard H. Wirth T. The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.Mol Cell Biol. 2001; 21: 1531-1539Crossref PubMed Scopus (57) Google Scholar the existence of which is debated in humans. Furthermore, expression of CD22, which negatively regulates BCR activation, was increased in Bob1–/– mice and low in P-FR1.7Samardzic T. Gerlach J. Muller K. Marinkovic D. Hess J. Nitschke L. et al.CD22 regulates early B cell development in BOB.1/OBF.1-deficient mice.Eur J Immunol. 2002; 32: 2481-2489Crossref PubMed Scopus (21) Google Scholar This may have resulted from the different source of the B cells examined, the spleen in Bob1–/– mice, and the blood in P-FR1. Interestingly, stimulation of Bob1–/– murine B cells with LPS resulted in plasmablast formation and immunoglobulin secretion in vitro.8Corcoran L.M. Hasbold J. Dietrich W. Hawkins E. Kallies A. Nutt S.L. et al.Differential requirement for OBF-1 during antibody-secreting cell differentiation.J Exp Med. 2005; 201: 1385-1396Crossref PubMed Scopus (50) Google Scholar TLR4 is not expressed in human B cells, but stimulation with TLR9 agonist in P-FR1 was not able to restore plasmablast formation. This is the first report of a patient with a Bob1 deficiency caused by a homozygous POU2AF1 null mutation resulting in a severe B-cell–intrinsic defect. The paucity in TFH cells may derive from dysregulated Bcl-69Stauss D. Brunner C. Berberich-Siebelt F. Hopken U.E. Lipp M. Muller G. The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6.EMBO J. 2016; 35: 881-898Crossref PubMed Scopus (22) Google Scholar and/or from an altered B-cell–T-cell interaction that is essential in the first phase of TFH cell differentiation. Bob1-deficient B cells respond poorly to activating signals. BCR signaling was reduced and plasmablast development and immunoglobulin secretion seemed abrogated in the patient. These functional impairments are Bob1 dependent because reconstitution experiments restored plasmablast development, immunoglobulin secretion, dysregulated surface expression of BCR complex molecules and costimulatory and survival signaling molecules. Overall, these data point to a role for Bob1 in the late stages of human B-cell development, regulating not only activation but also survival, possibly by controlling the expression of key mediators such as BAFF-R and CD79β. In addition, Bob1 deficiency represents a unique example of agammaglobulinemia with normal B-cell numbers. As Bob1 downregulation has been found in non–immunoglobulin-secreting Hodgkin lymphoma, a careful clinical follow-up for the development of lymphoma should be performed in P-FR1. Whether the neurodegenerative symptoms are the consequence of an infection, as is frequently observed in Bruton agammaglobulinemia, or related to an additional genetic disorder remains to be determined, also because central nervous system involvement was not reported in Bob1–/– mice. We wish to thank the patient and his family, as well as the healthy volunteers, for consenting to this work. We thank the CCI Advanced Diagnostic Unit, the Genetics and Genomics Unit, and the Freeze-Biobank, University Medical Center Freiburg, for technical assistance.