Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation Barbara Bertocci, Damiana Lecoeuche, Delphine Sterlin, Julius Kühn, Baptiste Gaillard, Annie De Smet, Frederique This information is current as Lembo, Christine Bole-Feysot, Nicolas Cagnard, Tatiana of September 28, 2021. Fadeev, Auriel Dahan, Jean-Claude Weill and Claude-Agnès Reynaud J Immunol 2017; 199:2408-2420; Prepublished online 14 August 2017; Downloaded from doi: 10.4049/jimmunol.1700708 http://www.jimmunol.org/content/199/7/2408 Supplementary http://www.jimmunol.org/content/suppl/2017/08/11/jimmunol.170070 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 60 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/199/7/2408.full#ref-list-1 Why The JI? Submit online. by guest on September 28, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation Barbara Bertocci,* Damiana Lecoeuche,* Delphine Sterlin,*,1 Julius Kuhn,€ † Baptiste Gaillard,*,2 Annie De Smet,* Frederique Lembo,‡ Christine Bole-Feysot,x Nicolas Cagnard,{ Tatiana Fadeev,* Auriel Dahan,* Jean-Claude Weill,* and Claude-Agne`s Reynaud* Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have Downloaded from passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt’s lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that http://www.jimmunol.org/ KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process. The Journal of Immunology, 2017, 199: 2408–2420. cell generation follows a stepwise process of differenti- guish T1 (CD93+CD232) from T2 B cells (CD93+CD23+) (6–8). It ation, first in the bone marrow and later in the periphery, has been observed that immature B cells can also differentiate into B where fully mature B cells reside. After a productive T1-like and T2-like B cells within the bone marrow (9, 10). T2 rearrangement and expression of a functional BCR at the surface, B cells, which emerge in spleen with a parallel but slightly B cells are still at an immature stage. Cross-linking of the BCR at delayed timing, could thus originate from both in situ T1 cell by guest on September 28, 2021 this stage induces cell death, a negative selection process purging differentiation and migration of T2-like cells from bone marrow the B cell repertoire from autoreactive specificities (1, 2). (11, 12). Interaction between the B cell–activating cytokine BAFF Immature B cells that survive past this checkpoint migrate to the and its specific receptor (BAFF-R) on B cells appears to play spleen and mature into follicular (FO) and marginal zone (MZ) different roles in this process, depending on the B cell differen- B cells (3). This maturation occurs in successive steps and implies tiation stage and the lymphoid organ concerned (13–19). Whereas the formation of two subsets of transitional B cells, transitional the immature to T1-like B cell progression in bone marrow seems type 1 (T1) and transitional type 2 (T2) (4), which have been to occur in a BAFF-independent mode, this is not the case in the proposed to be located in different areas of the spleen, the peri- spleen, where T1 cells require BAFF signals for differentiation arteriolar lymphoid sheath and the B cell follicles, respectively and/or survival, as shown in mixed bone marrow chimeras with (5). Several surface markers have been used to characterize T1 and BAFF-R–deficient and BAFF-R–competent cells (20). Conversely, T2 B cells, notably expression of CD93 and CD23, which distin- the differentiation of both splenic and bone marrow T1 into T2 *E´ quipe De´veloppement du Syste´me Immunitaire, Institut Necker-Enfant Malades, This work was supported by the Fondation Princesse Grace (to Team De´veloppement INSERM U1151-CNRS UMR8253, Faculte´ de Me´decine Paris Decartes, Universite´ du Syste`me Immunitaire) and the Ligue contre le Cancer (E´ quipe Labellise´e). Paris Descartes, Sorbone Paris Cite´, 75993 Paris Cedex 14, France; †Institute of The microarray data presented in this article have been submitted to the ArrayEx- Cellular and Molecular Immunology, Georg-August-University Medicine Go¨ttingen, press database (https://www.ebi.ac.uk/arrayexpress/browse.html) under accession 37073 Go¨ttingen, Germany; ‡Centre de Recherche en Cance´rologie de Marseille, x number E-MTAB-5928. INSERM U1068-CNRS UMR7258, 13273 Marseille Cedex 09, France; Plateforme de Ge´nomique, Imagine Institut des Maladies Ge´ne´tiques-Structure Fe´de´rative de Address correspondence and reprint requests to Dr. Barbara Bertocci, E´ quipe De´vel- Recherche Necker, INSERM 1163 and INSERM US24/CNRS UMS3633, 75015 oppement du Syste´me Immunitaire, Institut Necker-Enfant Malades, INSERM Paris, France; and {Plateforme de Bioinformatique, Universite´ Paris Descartes- U1151-CNRS UMR8253, Faculte´ de Me´decine Paris Decartes, Universite´ Paris Des- Structure Fe´de´rative de Recherche Necker, INSERM US24/CNRS UMS3633, cartes, Sorbone Paris Cite´, 14 rue Maria Helena Vieira Da Silva, Paris Cedex 14, 75993 Paris Cedex 14, France 75993 France. E-mail address: [email protected] 1Current address: Sorbonne Universite´s, Universite´ Pierre et Marie Curie/Universite´ The online version of this article contains supplemental material. Paris 6, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses Paris Abbreviations used in this article: AID, activation-induced cytidine deaminase; APC/ UMRS 1135, Assistance Publique – Hoˆpitaux de Paris, Groupement Hospitalier C, anaphase-promoting complex/cyclosome; CLL, chronic lymphocytic leukemia; Pitie´-Salpeˆtrie`re, De´partement d’Immunologie, Paris, France. Cul3, Cullin 3; FO, follicular; GC, germinal center; GSEA, gene set enrichment 2Current address: Service d’He´matologie, Hopital Robert Debre, Centre Hospitalier analysis; KO, knockout; MZ, marginal zone; PC, plasma cell; PLA, proximity liga- Universitaire de Reims, Reims, France. tion assay; T1, transitional type 1; T2, transitional type 2; wt, wild type. ORCIDs: 0000-0002-5532-9431 (T.F.); 0000-0002-4519-4473 (C.-A.R.). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Received for publication May 12, 2017. Accepted for publication July 17, 2017. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700708 The Journal of Immunology 2409 B cells is strongly dependent on BAFF signaling, because B cell Flow cytometry maturation in BAFF- and BAFF-R–deficient mice does not pro- Tissue samples were collected from the appropriate mice as described ceed beyond the T1 stage (13, 15, 19). in the text. After RBC lysis, cells were stained with a combination of The KLHL gene family encodes 42 different proteins, defined fluorophore-conjugated Abs (Supplemental Table I). Surface markers as being composed of an N-terminal BTB-POZ domain followed were detected and analyzed using a FACSCanto II flow cytometry ap- by a BTB and C-terminal kelch (BACK) domain and four to six paratus and the BD FACSDiva software (BD Biosciences). tandem Kelch motifs (21). The BTB domain, named by its ho- Cell culture mology with the Drosophila Bric-a-brac, Tramtrac, and Broad complex, is involved in protein–protein interaction and protein Splenic B cells, after purification (B cell isolation kit, MACS separation), were cultured in complete RPMI 1640 medium supplemented with 10% self-oligomerization (22). The BACK domain is very conserved, HyClone FetalClone I serum (Thermo Scientific), 100 U/ml penicillin and but its role is not unequivocally identified (23). The Kelch motif, 100 U/ml streptomycin, 1 mM sodium pyruvate, MEM nonessential which was discovered as a six-repeat element in the Drosophila amino acids (13), 25 mM HEPES (Invitrogen), 0.5 mM 2-ME (Sigma), kelch protein, is a segment of 44–56 aa that adopts a b-propeller and the indicated activating factors. All reagents, including anti-IgM (Jackson ImmunoResearch), LPS, CpG (Sigma), and recombinant BAFF structure and contains multiple potential protein contact sites protein (RD Biosystem), were prepared as per manufacturer’s instructions (24).