Pathogen‐specific B‐cell receptors drive chronic lymphocytic leukemia by light‐chain‐dependent cross‐reaction with autoantigens
2017; Springer Nature; Volume: 9; Issue: 11 Linguagem: Inglês
10.15252/emmm.201707732
ISSN1757-4684
AutoresNereida Jiménez de Oya, Marco De Giovanni, Jessica Fioravanti, Rudolf Übelhart, Pietro Di Lucia, Amleto Fiocchi, Stefano Iacovelli, Dimitar G. Efremov, Federico Caligaris‐Cappio, Hassan Jumaa, Paolo Ghia, Luca G. Guidotti, Matteo Iannacone,
Tópico(s)Lymphoma Diagnosis and Treatment
ResumoReport12 September 2017Open Access Source DataTransparent process Pathogen-specific B-cell receptors drive chronic lymphocytic leukemia by light-chain-dependent cross-reaction with autoantigens Nereida Jiménez de Oya Nereida Jiménez de Oya Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marco De Giovanni Marco De Giovanni Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Jessica Fioravanti Jessica Fioravanti Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Rudolf Übelhart Rudolf Übelhart Institute of Immunology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Pietro Di Lucia Pietro Di Lucia Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Amleto Fiocchi Amleto Fiocchi Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Stefano Iacovelli Stefano Iacovelli Molecular Hematology Unit, International Centre for Genetic Engineering & Biotechnology, Trieste, Italy Search for more papers by this author Dimitar G Efremov Dimitar G Efremov Molecular Hematology Unit, International Centre for Genetic Engineering & Biotechnology, Trieste, Italy Search for more papers by this author Federico Caligaris-Cappio Federico Caligaris-Cappio Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Hassan Jumaa Hassan Jumaa orcid.org/0000-0003-3383-141X Institute of Immunology, University Hospital Ulm, Ulm, Germany Department of Molecular Immunology, Faculty of Biology, Albert-Ludwigs University of Freiburg, Freiburg, Germany Search for more papers by this author Paolo Ghia Paolo Ghia Vita-Salute San Raffaele University, Milan, Italy Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Luca G Guidotti Luca G Guidotti Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Matteo Iannacone Corresponding Author Matteo Iannacone [email protected] orcid.org/0000-0002-9370-2671 Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Nereida Jiménez de Oya Nereida Jiménez de Oya Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Marco De Giovanni Marco De Giovanni Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy Search for more papers by this author Jessica Fioravanti Jessica Fioravanti Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Rudolf Übelhart Rudolf Übelhart Institute of Immunology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Pietro Di Lucia Pietro Di Lucia Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Amleto Fiocchi Amleto Fiocchi Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Stefano Iacovelli Stefano Iacovelli Molecular Hematology Unit, International Centre for Genetic Engineering & Biotechnology, Trieste, Italy Search for more papers by this author Dimitar G Efremov Dimitar G Efremov Molecular Hematology Unit, International Centre for Genetic Engineering & Biotechnology, Trieste, Italy Search for more papers by this author Federico Caligaris-Cappio Federico Caligaris-Cappio Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Hassan Jumaa Hassan Jumaa orcid.org/0000-0003-3383-141X Institute of Immunology, University Hospital Ulm, Ulm, Germany Department of Molecular Immunology, Faculty of Biology, Albert-Ludwigs University of Freiburg, Freiburg, Germany Search for more papers by this author Paolo Ghia Paolo Ghia Vita-Salute San Raffaele University, Milan, Italy Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Luca G Guidotti Luca G Guidotti Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Matteo Iannacone Corresponding Author Matteo Iannacone [email protected] orcid.org/0000-0002-9370-2671 Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita-Salute San Raffaele University, Milan, Italy Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Author Information Nereida Jiménez de Oya1,‡, Marco De Giovanni1,2,‡, Jessica Fioravanti1,‡, Rudolf Übelhart3,‡, Pietro Di Lucia1, Amleto Fiocchi1, Stefano Iacovelli4, Dimitar G Efremov4, Federico Caligaris-Cappio5,8, Hassan Jumaa3,6, Paolo Ghia2,5, Luca G Guidotti1 and Matteo Iannacone *,1,2,7 1Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy 2Vita-Salute San Raffaele University, Milan, Italy 3Institute of Immunology, University Hospital Ulm, Ulm, Germany 4Molecular Hematology Unit, International Centre for Genetic Engineering & Biotechnology, Trieste, Italy 5Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy 6Department of Molecular Immunology, Faculty of Biology, Albert-Ludwigs University of Freiburg, Freiburg, Germany 7Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy 8Present address: Associazione Italiana Ricerca sul Cancro, Milan, Italy ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as second authors *Corresponding author. Tel: +39 (02) 2643 6359; E-mail: [email protected] EMBO Mol Med (2017)9:1482-1490https://doi.org/10.15252/emmm.201707732 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Several lines of evidence indirectly suggest that antigenic stimulation through the B-cell receptor (BCR) supports chronic lymphocytic leukemia (CLL) development. In addition to self-antigens, a number of microbial antigens have been proposed to contribute to the selection of the immunoglobulins expressed in CLL. How pathogen-specific BCRs drive CLL development remains, however, largely unexplored. Here, we utilized mouse models of CLL pathogenesis to equip B cells with virus-specific BCRs and study the effect of antigen recognition on leukemia growth. Our results show that BCR engagement is absolutely required for CLL development. Unexpectedly, however, neither acute nor chronic exposure to virus-derived antigens influenced leukemia progression. Rather, CLL clones preferentially selected light chains that, when paired with virus-specific heavy chains, conferred B cells the ability to recognize a broad range of autoantigens. Taken together, our results suggest that pathogens may drive CLL pathogenesis by selecting and expanding pathogen-specific B cells that cross-react with one or more self-antigens. Synopsis Prior studies have associated infections to chronic lymphocytic leukemia (CLL), but the impact on CLL development and progression is unclear. It is here shown that pathogens may drive CLL by selecting and expanding pathogen-specific B cells that cross-react with self-antigens. B cell receptor expression is required for CLL development. Acute or chronic exposure to virus-derived antigens does not lead to faster leukemia development or progression. Malignant clones preferentially select light chains that confer broad auto-reactivity. Introduction Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western World, is characterized by the clonal expansion of CD5+ B cells in blood and peripheral tissues (Zhang & Kipps, 2014). BCR signaling plays a critical role in CLL pathogenesis (Burger & Chiorazzi, 2013), and, accordingly, inhibitors targeting BCR-associated kinases [Bruton's tyrosine kinase (BTK), phosphoinositide 3-kinase (PI3K) δ] have shown great clinical efficacy in patients (Burger & Chiorazzi, 2013; Furman et al, 2014). Patient-derived CLL cells with either unmutated or mutated immunoglobulin genes often express similar, if not identical, BCRs with common stereotypic features and/or structural similarities (Burger & Chiorazzi, 2013; Stevenson et al, 2014; Zhang & Kipps, 2014). This marked restriction in the immunoglobulin gene repertoire of CLL cells suggests that binding to restricted sets of antigenic epitopes is key to the selection and the expansion of those normal B-cell clones that eventually enter the CLL pathogenic process (Burger & Chiorazzi, 2013; Stevenson et al, 2014; Zhang & Kipps, 2014). The nature of such antigens and the mechanisms of BCR stimulation during CLL remain, however, incompletely understood. The majority of unmutated CLLs express low-affinity BCRs that are polyreactive to several autoantigens (Burger & Chiorazzi, 2013; Stevenson et al, 2014; Zhang & Kipps, 2014). The BCR specificity of mutated CLLs is less characterized, although a number of self-antigens, as well as microbial or virus-associated antigens, have been identified (Zhang & Kipps, 2014). Indeed, epidemiological studies indicate that several infections are associated with CLL development and CLL-associated immunoglobulins are known to react with various viruses and other pathogens (Landgren et al, 2007; Lanemo Myhrinder et al, 2008; Kostareli et al, 2009; Steininger et al, 2009, 2012; Hoogeboom et al, 2013; Hwang et al, 2014). If and how pathogen-specific BCRs drive CLL development and progression is largely unexplored. Results and Discussion To begin addressing these issues, we took advantage of a well-established CLL mouse model, the Eμ-TCL1 transgenic mouse, where the oncogene Tcl1 is expressed in both immature and mature B cells (Bichi et al, 2002). As such, Eμ-TCL1 transgenic mice develop a lymphoproliferative disorder that entails the clonal expansion of CD5+ IgM+ B cells (Bichi et al, 2002). Like in human CLL, the immunoglobulin rearrangements from different Eμ-TCL1 leukemic mice can be structurally very similar and closely resemble antibodies reactive to self and to microbial antigens (Yan et al, 2006). We started out by generating Eμ-TCL1 mice that either expressed defined virus-specific BCRs or that lacked the BCR entirely. To this end, we bred Eμ-TCL1 mice against the following mouse lineages: (i) KL25 mice (Hangartner et al, 2003), which carry a gene-targeted immunoglobulin heavy chain expressing a neutralizing specificity for lymphocytic choriomeningitis virus (LCMV) strain WE; (ii) VI10YEN mice (Hangartner et al, 2003), which carry a gene-targeted immunoglobulin heavy chain (VI10) and a transgenic non-targeted immunoglobulin light chain (YEN) expressing a neutralizing specificity for vesicular stomatitis virus (VSV) serotype Indiana; and (iii) DHLMP2A mice (Casola et al, 2004), which carry a targeted replacement of Igh by the Epstein–Barr virus protein LMP2A and develop B cells lacking surface-expressed and secreted immunoglobulins. Of note, the choice of these particular virus-specific transgenic BCR lineages rests on the notion that they have been extremely useful at characterizing the role of humoral immunity in the pathogenesis of acute and chronic viral infections (Hangartner et al, 2003, 2006; Sammicheli et al, 2016) and that, being heavy-chain knock-in lineages, they allow for examining the eventual role of light chains in CLL development. B cells isolated from the resulting progeny were first characterized with regard to Tcl1 expression, subset development, and BCR responsiveness. As shown in Fig EV1A, 8-week-old pre-leukemic KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice showed levels of splenic Tcl1 expression that were comparable to those of Eμ-TCL1 mice expressing a polyclonal BCR repertoire. We next enumerated CD5+ B cells in blood, serosal cavities, secondary lymphoid organs and liver of the above-mentioned mouse strains, again at 8 weeks of age. The number of CD5+ B cells in blood, spleen and lymph nodes of KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice was similar to that detected in the same districts of Eμ-TCL1 mice (Fig EV1B and C); performing a similar comparison in liver and peritoneum, however, indicated that CD5+ B cells were reduced in KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice (Fig EV1B and C). While the molecular basis for this selective reduction of CD5+ B cells in liver and peritoneum of pre-leukemic KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice are unclear, it is worth noting that the extent of such reduction was similar in all the 3 above-mentioned mouse lineages (Fig EV1B and C). We finally confirmed that both KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice express the respective virus-specific BCR in all B-cell subsets and appropriately respond to cognate antigen stimulation (Fig EV1D–G). Click here to expand this figure. Figure EV1. Characterization of KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice A. TCL1 expression in the spleen of 8-week-old WT (gray), Eμ-TCL1 (black), KL25 × Eμ-TCL1 (red), VI10YEN × Eμ-TCL1 (blue), and DHLMP2A × Eμ-TCL1 (green) male mice analyzed by RT–qPCR. n = 3. Data are representative of more than three independent experiments. B. Concentration of CD5+ CD19+ cells per ml of blood and peritoneal wash of 8-week-old WT (gray), Eμ-TCL1 (black), KL25 × Eμ-TCL1 (red), VI10YEN × Eμ-TCL1 (blue), and DHLMP2A × Eμ-TCL1 (green) male mice. n = 6 (WT), 8 (Eμ-TCL1), 9 (KL25 × Eμ-TCL1), 8 (VI10YEN × Eμ-TCL1), and 9 (DHLMP2A × Eμ-TCL1). C. Absolute numbers of CD5+ CD19+ cells in the indicated organs of the same mice described in (B). D. Representative flow cytometry plots showing transgenic BCR expression in B1a (CD19+ CD5+ CD23−), B1b (CD19+ CD5− CD23−), and B2 (CD19+ CD5− CD23+) cells from 8-week-old WT (top panel) and KL25 × Eμ-TCL1 (bottom panel) male mice. Results are representative of more than 10 independent experiments. E. Representative flow cytometry plots showing transgenic BCR expression in B1a (CD19+ CD5+ CD23−), B1b (CD19+ CD5− CD23−), and B2 (CD19+ CD5− CD23+) cells from 8-week-old WT (top panel) and VI10YEN × Eμ-TCL1 (bottom panel) male mice. Results are representative of more than 10 independent experiments. F, G. Representative flow cytometry plots showing proliferation (assessed by CFSE dilution, left panels) and activation (assessed by CD25 and CD69 upregulation, middle and right panels, respectively) of KL25 × Eμ-TCL1 (F) or VI10YEN × Eμ-TCL1 (G) B cells exposed or not to inactivated LCMV or VSV, respectively. Results are representative of three independent experiments. Data information: Results are expressed as mean + SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Exact P-values for each experiment are reported in Appendix Table S1. One-way ANOVA (Bonferroni's multiple comparison) was used in (A–C). Source data are available online for this figure. Download figure Download PowerPoint We next compared KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice to Eμ-TCL1 mice expressing a polyclonal BCR repertoire with regard to leukemia development at steady state (in the absence of cognate antigen challenge). Disease progression was monitored by quantifying the frequency of CD5+ B cells in peripheral blood (Fig 1A), and we arbitrarily defined leukemic those mice that had ≥ 20% CD5+ cells among total CD19+ B cells (a frequency never reached by WT mice in the 36-week-long observation period) (Fig 1B). As previously reported (Bichi et al, 2002), Eμ-TCL1 mice showed an expansion of circulating CD5+ B cells as early as 16 weeks of age, and by 36 weeks of age 100% of them were frankly leukemic, with accumulation of large numbers of CD5+ B cells in blood, serosal cavities and lymphoid as well as non-lymphoid organs (Fig 1A–C). DHLMP2A × Eμ-TCL1 mice showed a profound impairment in leukemia development (Fig 1A–C), indicating that BCR expression is required for leukemia growth and that the tonic signal provided by the LMP2A protein is not sufficient to support leukemic expansion. We then assessed whether pathogen-specific BCRs sustained cancer development. Both KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice developed CLL, even though they differed in regard to disease incidence and leukemic cell accumulation. Whereas CLL development in KL25 × Eμ-TCL1 mice occurred at a rate that was indistinguishable from that of Eμ-TCL1 mice, VI10YEN × Eμ-TCL1 mice had a more indolent course of disease (Fig 1A–C). These results indicate that the BCR shapes CLL incidence and behavior in vivo. Figure 1. Leukemia development in KL25 × Eμ-TCL1, VI10YEN × Eμ-TCL1, and DHLMP2A × Eμ-TCL1 mice Percentage of CD5+ cells (out of total CD19+ peripheral blood leukocytes) in WT (gray), Eμ-TCL1 (black), KL25 × Eμ-TCL1 (red), VI10YEN × Eμ-TCL1 (blue), and DHLMP2A × Eμ-TCL1 (green) male mice at the indicated time points. Two-way ANOVA (Bonferroni's multiple comparison). Incidence of leukemia (defined as ≥ 20% CD5+ cells out of total CD19+ peripheral blood leukocytes) over time in the same mice described in (A). n = 4–20 (WT), 16–45 (Eμ-TCL1), 8–29 (KL25 × Eμ-TCL1), 14–34 (VI10YEN × Eμ-TCL1), 19–35 (DHLMP2A × Eμ-TCL1). Log-rank (Mantel-Cox). Percentage of CD5+ cells (out of total CD19+ cells) in the indicated organs of the same mice described in (A) at 36 weeks of age. n = 6 (WT), 9 (Eμ-TCL1), 6 (KL25 × Eμ-TCL1), 8 (VI10YEN × Eμ-TCL1), 12 (DHLMP2A × Eμ-TCL1). One-way ANOVA (Bonferroni's multiple comparison). Data information: Results are expressed as mean + SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Exact P-values for each experiment are reported in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 1A [emmm201707732-sup-0006-SDataFig1A.pdf] Source Data for Figure 1B [emmm201707732-sup-0007-SDataFig1B.pdf] Source Data for Figure 1C [emmm201707732-sup-0008-SDataFig1C.pdf] Download figure Download PowerPoint Before attempting to pinpoint the mechanisms underlying the difference in CLL incidence between KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice, we sought to determine whether cognate antigen recognition, prior to disease onset, influenced CLL development and progression in Eμ-TCL1 mice expressing virus-specific BCRs. To this end, we infected 8-week-old VI10YEN × Eμ-TCL1 mice (and Eμ-TCL1 controls) with 106 p.f.u. of VSV Indiana. VSV infection induced B cells in VI10YEN × Eμ-TCL1 mice to get activated, proliferate, and differentiate into Ab-secreting cells (Fig EV2A), but it did not alter the kinetics of leukemia development or progression (Figs 2A and B, and EV2B). We then infected 8-week-old KL25 × Eμ-TCL1 mice and Eμ-TCL1 controls with 106 f.f.u. of LCMV WE. Unexpectedly, LCMV infection of both KL25 × Eμ-TCL1 mice and Eμ-TCL1 controls (that express a polyclonal BCR repertoire) abrogated CLL development (Fig EV3). The cellular and molecular underpinnings of this intriguing observation extend beyond the scope of this study and will be the subject of a future report. To avoid potentially confounding effects, we henceforth decided to test the role of antigenic stimulation in this setting by repetitively immunizing 8-week-old KL25 × Eμ-TCL1 mice (and Eμ-TCL1 controls) with the purified LCMV WE glycoprotein [which contains the antigenic determinant recognized by KL25 B cells (Sammicheli et al, 2016)]. Although LCMV immunization induced B cells in KL25 × Eμ-TCL1 mice to get activated, proliferate and differentiate into Ab-secreting cells (Fig EV4A), it did not alter the kinetics of leukemia development or progression (Figs 2C and D, and EV4B and C). Together, these results suggest that high-affinity recognition of pathogen-derived antigens does not affect CLL development or progression, and they prompted us to investigate whether virus-specific BCRs may drive CLL pathogenesis by mechanisms that are unrelated to pathogen specificity. Click here to expand this figure. Figure EV2. VSV infection does not affect CLL development or progression VSV neutralizing antibody (nAb) titers in the serum of VI10YEN × Eμ-TCL1 9-week-old male mice that were infected (open bars) or not (closed bars) with VSV 7 days earlier. n = 6 (VI10YEN × Eμ-TCL1) and 9 (VI10YEN × Eμ-TCL1 + VSV). Percentage of CD5+ cells (out of total CD19+ cells) in the indicated organs of Eμ-TCL1 (black) and VI10YEN × Eμ-TCL1 (blue) male mice that were infected (open bars) or not (closed bars) with 106 p.f.u. of VSV Indiana at 8 weeks of age and sacrificed 28 weeks later. n = 9 (Eμ-TCL1), 7 (Eμ-TCL1 + VSV), 8 (VI10YEN × Eμ-TCL1), 7 (VI10YEN × Eμ-TCL1 + VSV). One-way ANOVA (Bonferroni's multiple comparison). Data information: Results are expressed as mean + SEM. *P < 0.05, **P < 0.01. Exact P-values for each experiment are reported in Appendix Table S1. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. High-affinity antigen recognition does not affect CLL development or progression Percentage of CD5+ cells (out of total CD19+ peripheral blood leukocytes) over time in Eμ-TCL1 (black) and VI10YEN × Eμ-TCL1 (blue) male mice that were infected (open symbols) or not (closed symbols) with 106 p.f.u. of VSV Indiana at 8 weeks of age. Incidence of leukemia (defined as ≥ 20% CD5+ cells out of total CD19+ peripheral blood leukocytes) over time in the same mice described in (A). n = 16–45 (Eμ-TCL1), 9 (Eμ-TCL1 + VSV), 14–34 (VI10YEN × Eμ-TCL1), 7–9 (VI10YEN × Eμ-TCL1 + VSV). Percentage of CD5+ cells (out of total CD19+ peripheral blood leukocytes) over time in Eμ-TCL1 (black) and KL25 × Eμ-TCL1 (red) mice that were immunized (open symbols) or not (closed symbols) with LCMV-GP + Addavax at the indicated time points. Incidence of leukemia (defined as ≥ 20% CD5+ cells out of total CD19+ peripheral blood leukocytes) over time in the same mice described in (C). n = 16–45 (Eμ-TCL1), 7 (Eμ-TCL1 + LCMV-GP + Addavax), 8–29 (KL25 × Eμ-TCL1), 10–11 (KL25 × Eμ-TCL1 + LCMV-GP + Addavax). Data information: Results are expressed as mean + SEM. *P < 0.05, ***P < 0.001. Exact P-values for each experiment are reported in Appendix Table S1. Two-way ANOVA (Bonferroni's multiple comparison) was used in (A, C); Log-Rank (Mantel-Cox) was used in (B, D). Source data are available online for this figure. Source Data for Figure 2A [emmm201707732-sup-0009-SDataFig2A.pdf] Source Data for Figure 2B [emmm201707732-sup-0010-SDataFig2B.pdf] Source Data for Figure 2C [emmm201707732-sup-0011-SDataFig2C.pdf] Source Data for Figure 2D [emmm201707732-sup-0012-SDataFig2D.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. LCMV infection prevents CLL development in Eμ-TCL1 micePercentage of CD5+ cells (out of total CD19+ peripheral blood leukocytes) over time in WT (gray) and in Eμ-TCL1 (black) male mice that were infected (open symbols) or not (closed symbols) with LCMV. n = 4–20 (WT), 16–45 (Eμ-TCL1), 6–10 (Eμ-TCL1 + LCMV), 8–29 (KL25 × Eμ-TCL1), 9–11 (KL25 × Eμ-TCL1 + LCMV). Please note that the uninfected WT, Eμ-TCL1, and KL25 × Eμ-TCL1 controls are the same as the ones reported in Fig 1A. Results are expressed as mean + SEM. **P < 0.01, ***P < 0.001. Exact P-values for each experiment are reported in Appendix Table S1. Two-way ANOVA (Bonferroni's multiple comparison) was used. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Immunization with LCMV-GP does not affect CLL development or progression LCMV neutralizing antibody (nAb) titers over time in the serum of Eμ-TCL1 (black) and KL25 × Eμ-TCL1 (red) male mice that were immunized (open symbols) or not (closed symbols) with LCMV-GP + Addavax. n = 6 (Eμ-TCL1), 7 (Eμ-TCL1 + LCMV-GP + Addavax), 6 (KL25 × Eμ-TCL1), 11 (KL25 × Eμ-TCL1 + LCMV-GP + Addavax). Percentage of CD5+ cells (out of total CD19+ peripheral blood leukocytes) over time in control Eμ-TCL1 mice (black symbols) or in mice that were injected intramuscularly with Addavax (gray) or with LCMV-GP + Addavax (open symbols). Percentage of CD5+ cells (out of total CD19+ cells) in the indicated organs of Eμ-TCL1 (black) and KL25 × Eμ-TCL1 (red) mice that were immunized (open bars) or not (closed bars) with LCMV-GP + Addavax and sacrificed 24 weeks after the first immunization. n = 9 (Eμ-TCL1), 5 (Eμ-TCL1 + LCMV-GP + Addavax), 6 (KL25 × Eμ-TCL1), 10 (KL25 × Eμ-TCL1 + LCMV-GP + Addavax). Data information: Results are expressed as mean + SEM. Source data are available online for this figure. Download figure Download PowerPoint To begin investigating such potential pathogenic mechanisms, we analyzed the BCR repertoire of leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice and compared it to that of age-matched KL25 and VI10YEN mice. Since both KL25 and VI10YEN are knock-in for the BCR heavy chain (Hangartner et al, 2003), there is no alternative heavy chain that these mice can express. Accordingly, all analyzed leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice expressed the expected transgenic heavy chain as an IgM (Tables EV1 and EV2, and Fig EV5). As per the light-chain repertoire, we noticed that, when compared with age-matched KL25 and VI10YEN mice, leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice had a biased light-chain usage. Specifically, among the light chains preferentially expressed by leukemic KL25 × Eμ-TCL1 mice, we found the IGKV12-44*01 F/IGKJ2*01 F gene associated with the LCDR3 motif CQH-HYGTPY-TF and the IGKV6-32*01 F/IGKJ2*01 F gene associated with the LCDR3 motif CQQ-DYSS-TF (Fig 3A and Table EV1). Similarly, leukemic VI10YEN × Eμ-TCL1 mice preferentially expressed the IGKV6-32*01 F/IGKJ2*01 F gene associated with the LCDR3 motif CQQ-DYSS-TF and the IGKV6-32*01 F/IGKJ2*01 F gene associated with the LCDR3 motif CQQ-DYSSPY-TF (the YEN transgenic light chain, Fig 3A and Table EV2). This preferential light-chain usage is reminiscent to what has been described for polyclonal Eμ-TCL1 (Yan et al, 2006) and suggested that leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice selected BCRs capable of cross-reacting with one or more autoantigens. One particular case in point is the capacity of CLL-derived BCRs to recognize an internal epitope of the BCR itself, a feature referred to as cell autonomous signaling (Dühren-von Minden et al, 2012). We therefore set out to test whether BCRs derived from leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice possessed cell autonomous signaling activity. To this end, we introduced the corresponding heavy and light chains in the BCR-deficient murine B-cell line TKO, which expresses an inactive B-cell linker (BLNK) adaptor protein that becomes functional in the presence of 4-hydroxytamoxifen (4-OHT) (Dühren-von Minden et al, 2012). Addition of 4-OHT to TKO cells with an autonomously active BCR results in signal activation and propagation, ultimately resulting in an increase in intracellular Ca++ levels that is detectable by flow cytometry (Dühren-von Minden et al, 2012; Fig 3B). The BCR composed of the KL25 transgenic heavy chain coupled with the light chain that was most frequently expressed in non-leukemic 9-month-old KL25 mice (IGKV3-10*01 F/IGKJ1*01 F gene associated with the LCDR3 motif CQQ-NNEDPW-TF) was not autonomously active (Fig 3C, left panel); similarly, the BCR composed of the VI10 transgenic heavy chain coupled to the transgenic YEN light chain (the most frequently expressed light chain in non-leukemic 9-month-old VI10YEN mice) did not possess autonomous signaling capability (Fig 3D, left panel). We next evaluated BCRs composed of the same KL25 and VI10 heavy chains coupled to the light chains that were most frequently expressed in 9-month-old leukemic KL25 × Eμ-TCL1 and VI10YEN × Eμ-TCL1 mice, respectively. When expressed together with the KL25 heavy chain, two of the light chains that were most frequently selected in leukemic KL25 × Eμ-TCL1 mice endowed TKO cells with autonomous signalin
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