Volume 26, Issue 3, Pages 323-333 (March 2007) Conventional Light Chains Inhibit the Autonomous Signaling Capacity of the B Cell Receptor  Sonja Meixlsperger, Fabian Köhler, Thomas Wossning, Michael Reppel, Markus Müschen, Hassan Jumaa  Immunity  Volume 26, Issue 3, Pages 323-333 (March 2007) DOI: 10.1016/j.immuni.2007.01.012 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Tight Control of SLP-65 Function in ERT2-SLP-65-Expressing Cells (A) Schematic overview of the ERT2-SLP-65 construct and FACS analysis of pre-BCR expression. On the left, μHC versus GFP plots are shown with GFP being a marker for ERT2-SLP-65-positive cells. Oct cells were transduced with ERT2-SLP-65 and incubated with EtOH (−OHT, first row) or with 1 μM OHT (+OHT, second row) for 2 days. The gates indicate the populations that were compared in the histograms for the expression of μHC. In the histograms, GFP-negative cells are shown in black versus GFP-positive cells in gray. (B) FACS analysis of pre-B cell differentiation in Oct cells sorted for the expression of ERT2-SLP-65. On top, μHC versus GFP profiles and a histogram for μHC expression are shown for cells cultured either without or with 1 μM OHT for 2 days. On the bottom, κLC versus GFP FACS profiles and a histogram for κLC expression are shown for cells cultured without (left) or with (right) 1 μM OHT for 4 days in medium lacking IL-7. The numbers indicate the percentage of κ-positive cells. In histograms, cells cultured with EtOH are shown in black versus OHT treated cells in gray. Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 SLP-65 Induces Kappa Transcription RT-PCR analysis for the transcription of the indicated genes in ERT2-SLP-65-positive Oct cells incubated without or with 1 μM OHT for the indicated time. Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 SLP-65 Activation Induces Ca2+ Flux in Pre-B Cells (A) Ca2+ measurements of ERT2-SLP-65-positive Oct cells without and with addition of 1 μM OHT. In the sample without OHT, the respective amount of EtOH, the solvent of OHT, was added as a mock stimulus. (B) Ca2+ measurement of ERT2-SLP-65-positive Oct cells that were preincubated overnight (ON) with the respective amount of EtOH and then mock stimulated with EtOH (left). The ERT2-SLP-65-positive Oct cells shown in the next three samples were preincubated with 1 μM OHT ON. Ca2+ flux was measured without (second from left) and with (second from right) addition of 1 μM OHT or with stimulation of the pre-BCR with 10 μg anti-μHC (right). Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Distinct Requirements for Ca2+ Flux in Pre-BCR- and BCR-Positive Cells (A) FACS analysis for μHC expression and Ca2+ measurement of ERT2-SLP-65-positive SLP-65, RAG2, λ5 triple-deficient cells without and with addition of 1 μM OHT (first row). FACS analysis for μHC expression of these ERT2-SLP-65-positive triple-deficient cells that were transduced and sorted for μHC expression (gray) as compared to the untransduced cells (black), and Ca2+ measurement of the μHC-positive cells either without or with addition of 1 μM OHT (second row). (B) FACS analysis and Ca2+ measurement of ERT2-SLP-65, μHC-positive triple-deficient cells (shown in [A], second row) that were transduced with λ5 and sorted for receptor expression. FACS staining for pre-BCR expression (μHC versus SLC) and Ca2+ measurement without or with addition of 1 μM OHT is shown (first row). The same is shown for ERT2-SLP-65, μHC-positive triple-deficient cells transduced with λ5 with a deleted non-Ig portion (second row). (C) As in [B], but FACS staining (μHC versus λLC) and Ca2+ measurement for cells expressing BCR (first row). Ca2+ measurement without or with addition of 1 μM OHT in combination with stimulation with 10 μg/ml NIP-BSA is shown for the same cells (second row). Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 ERT2-SLP-65 Is Phosphorylated after Addition of OHT (A) Immunoblot analysis of whole-cell lysates of SLP-65, RAG2, λ5 triple-deficient cells (tko, lane 1), of ERT2-SLP-65, pre-BCR-positive triple-deficient cells (pre-BCR, lane 2), of ERT2-SLP-65, BCR-positive triple-deficient cells (BCR, lane 3), of SLP-65-deficient Oct pre-B cells (lane 4), and of WEHI231 B cells as control (lane 5) on the same blot. μHC, SLP-65, and Ig-α expression were tested with the respective antibodies. Immunoblotting with anti-actin served as loading control. (B) Immunoblot analysis of total cellular lysates (TCL, lanes 1–6) or IPs for ERT2-SLP-65 (lanes 8–13) of ERT2-SLP-65, pre-BCR-positive triple-deficient cells (lanes 1–3 and 8–10) and of ERT2-SLP-65, BCR-positive triple-deficient cells (lanes 4–6 and 11–13). Cells were incubated for 3 min at 37°C in complemented Iscove's medium containing 1 μM OHT or the respective amount of EtOH (-), α-μHC was added at 10 μg/ml where indicated. Tyrosine phosphorylation was detected with 4G10 antibody. Immunoblotting with anti-HA (tag for ERT2-SLP-65) and anti-actin (bottom) served as controls. The asterisk marks yet uncharacterized proteins. M, marker. Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Ligand Independence of Pre-BCR but Not BCR Signaling in Human B Lymphoid Cell Lines (A) Human SLP-65-deficient HPB null pre-B cells were transiently transfected with MIG-hSLP-65 (gray) or MIG-GFP (black), and single isolated cells were subjected to Ca2+ measurement in the absence of stimulating antibody. (B) Human SLP-65-deficient Karpas-422 B cells were transiently transfected with MIG-hSLP-65 or MIG-GFP and subjected to Ca2+ measurement in the presence or absence of stimulating antibody. Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Model for Autoselection of B Cells The μHC may possess a weak intrinsic aggregation capacity. The SLC enhances this aggregation capacity and induces strong autonomous signaling of the pre-BCR, resulting in proliferation and LC recombination. A conventional LC (cLC), which abolishes the μHC interaction and prevents the aggregation of the BCR, allows stable BCR expression on the surface of immature B cells that may then be selected to exit the bone marrow. B cells with a conventional LC, which fails to prevent BCR auto-aggregation (cLC∗) or allows recognition of autoantigens, may proliferate and undergo further LC recombination until an aggregation-preventing LC is produced. Immunity 2007 26, 323-333DOI: (10.1016/j.immuni.2007.01.012) Copyright © 2007 Elsevier Inc. Terms and Conditions