The inhibitory receptor Siglec‐G controls the severity of chronic lymphocytic leukemia

Abstract Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co‐receptor Siglec‐G and Siglec‐G‐deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec‐G expression influences the severity of CLL. Our results show that Siglec‐G deficiency leads to earlier onset and more severe course of the CLL‐like disease in the murine Eμ‐TCL1 model. In contrast, mice overexpressing Siglec‐G on the B cell surface are almost completely protected from developing CLL‐like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec‐10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec‐G in disease progression in mice, and suggest that a similar mechanism for Siglec‐10 in human CLL may exist.

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See also guide to authors: https://www.embopress.org/page/journal/14693178/authorguide#authorshipguidelines 12) Please add up to five keywords below the abstract and order the manuscript sections like this, using these names: Title page -Abstract -Keywords -Introduction -Results -Discussion -Materials and Methods -Data availability section -Acknowledgements (including funding information) -Disclosure and Competing Interests Statement -References -  Please use this link to submit your revision: https://embor.msubmit.net/cgi-bin/main.plex Yours sincerely, Achim Breiling Senior Editor EMBO Reports --------------Referee #1: The paper presented by Röder and colleagues focus on Siglec proteins in chronic lymphocytic leukemia (CLL). In the mouse Siglec-G inhibits B cell receptor signaling and thus impacts on B cell development and activation. In the Eμ-TCL1 tumor model recapitulating important aspects of CLL pathogenesis, the authors demonstrate that Siglec-G ablation has a positive effect on the onset and aggressiveness of the disease, whereas overexpression of the inhibitory receptor prevents/delays CLL development. Downregulation of Siglec-10, the human orthologue of Siglec-G, was detected in human CLL samples indicating the clinical relevance of the mouse studies. Overall, the results presented in the manuscript are well designed and conducted. The data in the mouse models confirm the hypothesis that downregulation of Siglec proteins is an oncogenic event in CLL pathogenesis. Thus, the study adds valuable insights into inhibitory receptor signaling in CLL biology and hints possible clinical implications of agonistic targeting of B cell inhibitory receptors.
The following comments/suggestions should be addressed to further strengthen the manuscript: Major comments: • The authors developed a novel mouse strain allowing the cell type specific overexpression of Siglec-G. In their Siglecg-R26ki/ki mb1cre mouse line Röder and colleagues focused on the B1a compartment, as the B cell population of interest for CLL development. However, as impaired antigen cell receptor signaling will impact on other B cell subtypes, the characterization of the B cell development and maturation in Siglecg-R26ki/ki mb1cre animals would be very interesting for the readers. • In the present study TCL1 x Siglecg-R26ki/ki mb1cre mice were analyzed for CLL development. The authors may want to comment on their choice of using homozygous Siglecg-R26ki mice in the analysis. Is there a threshold for Siglec-G overexpression to suppress the CLL-like cell expansion? Additionally, the survival curve of TCL1 x Siglecg-R26ki/ki mb1cre mice indicates that some aged animals develop signs of sickness ( Figure 5G). Do these animals suffer from CLL development as well? Maybe in these CLL-clones Siglec-G overexpression was less effective?! • The Ig repertoire analysis is elegantly showing the monoclonal expansion of the malignant cells in TCL1 x Siglecg-/-mice ( Figure 3). The input population for the analysis in these animals as well as in controls is the CD19+ pool isolated from the peripheral blood. However, this approach might mask or underestimate clonal expansion in TCL1 x Siglecg-R26ki/ki mb1cre mice at week 48 characterized by a low, but still increased population of B220low CD5+ cells ( Figure 6). The authors statement that "no leukemic clones developed"(Page 17) might be too optimistic. • Regarding the intracellular flow cytometry measurements of signaling proteins downstream of the BCR: the authors might want to show these data after gating on B220low CD5+ cells (instead of the entire B220 pool). Alternatively, an additional supplementary graph showing the percentages of CLL-like cells within the B220+ cells would be sufficient to better evaluate the Siglec-G effect on BCR signaling. • How much of the effects observed in BCR signaling between TCL1 x Siglecg-R26ki/ki and TCL1 x Siglecg-R26ki/ki mb1cre mice ( Figure 8B) is related to the "non-tumor vs tumor" comparison? As the authors did not detect significant differences in TCL1 vs TCL1 x Siglecg-/-tumor samples one might consider (and discuss) it.
Minor comments: • The roughly 10fold increase of the B220low CD5+ numbers in the TCL1 x Siglecg-R26ki/ki mice at week 36 ( Figure 5C) in comparison to the corresponding week in TCL1 ( Figure 2B) mice is surprising. Do the authors have any explanation?
• Please indicate the genetic background of the used mb1-cre line (as C57/BL6 and BALB/C mice are available). • In Figure 1B the progressive expansion of the B220lowCD5+ compartment could be depicted with an additional graph in which it is possible to follow the expansion of the CLL-like population in the individual mice over time (similarly in Figure 4B). • In Figure 2C and similarly in 5E, in the summary of B220lowCD5+ percentages in the liver, it would be more accurate to label the axis with 'leukocytes' instead of 'lymphocytes'. • In Figure 7A in the density plot the abscissa seems to be mislabeled, otherwise the CLL-like population is CD5 negative. • In Figure S2B the TCL1 x Siglecg-/-bars appear to be edited with variable patterns, unlike the solid purple color in the rest of the graphs throughout the manuscript.
• Please use a consistent nomenclature for the mouse strains throughout the manuscript (see page 11). This work by Nitschke and co-workers uses genetic models to demonstrate a convincing role for Siglec-G in the development of CLL. Both KO and over-expressing mouse models of Siglec-G are studied in the context of a mouse model of CLL. Results clearly and demonstrate that the kinetics of CLL development is inversely proportional to Siglec-G expression levels. Numerous measures are examined, including gross organ weight, survival, and cellular readouts. Moreover, it is demonstrated that Siglec-10 is down-regulated in human CLL. The manuscript is clear and well written and only requires a few considerations: 1) As this is the first study of this KI over-expressing mouse, a bit more characterization seems in order. Can the authors rule out that development impact given that MB1-Cre turns on very early in B cell development? Are lymphocyte proportions in the adult spleen roughly similar to WT mice?
2) Given the clear differences in Ca2+ flux, can they speculate why no differences were observed on downstream signaling molecules?
3) In the over-expressing mice, it was stated that they did not have enough CD5+ cells to perform cellular analyses. It would have been nice to see some readouts on the'total' B cells to complement the data have for the other models. If that data was acquired, it would be a useful addition.
--------------Referee #3: Major: 1. Results, page 6: "The size of these B1a cell populations in the two control mouse strains remained constant and did not increase until the age of 60 weeks (Fig.1B)." Is the increase at week 60 statistically significant? From the figure, this seems unlikely.
2. Results, page 6: "TCL1 mice showed an increase of the population of CLL-like cells (also characterized as B220low CD5+) in the blood from the age of 40 weeks, reaching a percentage of about 50% of all blood lymphocytes at week 60. In contrast, the CLL-like population in the blood of TCL1 x Siglecg-/-animals increased much earlier, already at the age of 20 weeks (on average 30 % of blood lymphocytes)." This text does not agree with the comparisons highlighted on the figure. The figure shows statistical comparisons of the two sets (WT vs Siglec6-/-; TCL1 vs TCL1x Siglec6-/-) to each other, whereas the text compares the same population to itself over time. This is the case for several other figures, e.g., Suppl Fig  3. Results, page 9: "This analysis revealed a significantly reduced binding of these antigens in TCL1 x Siglecg-/-mice in comparison to TCL1 littermates for most time points (Suppl Fig 3). This suggests an overall different BCR repertoire of CLL-like cells of TCL1 x Siglecg-/-mice, when compared to TCL1 mice. A similar shift in the BCR repertoire was detected previously in normal B1a cells of Siglecg-/-mice (Jellusova et al., 2010)." This finding is somewhat bothersome, as it suggests that the B cells that are being transformed in these mice are different than the standard model. Is the severity of the disease in these animals due to the development of a malignancy in B cells that do not usually transform rather than the transformation of same population a greater frequency? This is supported by the literature which I believe indicates that the major IgV gene families used by TCL1 mice and by B-1 cells are VH11 and VH12 (Baumgarth, 2011 cited; PMID: 32561570). This is compounded by the finding that these genes are identified when over expressing Siglec G (Fig 6). The authors appropriately address this issue in the Discussion but it remains a major concern. 4. Results, pages 14-15: Since the data in Figure 9B are not statistically significant, the authors should consider moving these to the Supplementary Section and bringing some of the data in Suppl Figs 6 and 7 to the main text?
5. Discussion, page 16: "The dependence of CLL on BCR signaling is not due to BCR-activating mutations in crucial signaling molecules as shown for diffuse B cell lymphoma (DLBCL) (Schmitz et al., 2018), but due to the ability of some CLL BCRs to cluster by binding to each other and in this way autonomously activating BCR signaling (Duhren-von Minden et al., 2012, Minici et al., 2017." Although this is an accurate statement, it implies that binding of antigens other than companion BCRs is irrelevant. This is not necessarily the case as shown by in vivo studies (PMID: 25564405).
Minor: 1. The following sentence on page 4 is confusing or possibly incomplete. In particular the comment about V families. "Murine CD5+ B1a cells share features of human CD5+ CLL cells in some aspects, including expression of a restricted Ig repertoire using similar V with B6) families."

Point-by-point responses
The paper presented by Röder and colleagues focus on Siglec proteins in chronic lymphocytic leukemia (CLL). In the mouse Siglec-G inhibits B cell receptor signaling and thus impacts on B cell development and activation. In the Eμ-TCL1 tumor model recapitulating important aspects of CLL pathogenesis, the authors demonstrate that Siglec-G ablation has a positive effect on the onset and aggressiveness of the disease, whereas overexpression of the inhibitory receptor prevents/delays CLL development. Downregulation of Siglec-10, the human orthologue of Siglec-G, was detected in human CLL samples indicating the clinical relevance of the mouse studies. Overall, the results presented in the manuscript are well designed and conducted. The data in the mouse models confirm the hypothesis that downregulation of Siglec proteins is an oncogenic event in CLL pathogenesis. Thus, the study adds valuable insights into inhibitory receptor signaling in CLL biology and hints possible clinical implications of agonistic targeting of B cell inhibitory receptors.
The following comments/suggestions should be addressed to further strengthen the manuscript: Major comments: • The authors developed a novel mouse strain allowing the cell type specific overexpression of Siglec-G. In their Siglecg-R26ki/ki mb1cre mouse line Röder and colleagues focused on the B1a compartment, as the B cell population of interest for CLL development. However, as impaired antigen cell receptor signaling will impact on other B cell subtypes, the characterization of the B cell development and maturation in Siglecg-R26ki/ki mb1cre animals would be very interesting for the readers.

Response
We thank the reviewer for the positive comments to our manuscript. We agree with the suggestion that a characterisation of the new SiglecG overexpressing mouse line in more detail is helpful for the reader. We have therefore performed more experiments to analyse this in detail. We find no impairment of B cell development or B cell subpopulations in the spleen in Siglecg-R26ki/ki mb1cre mice. These data are now shown in the new Fig EV 6. The only detected difference of this mouse line is a lower number of B1a cells (shown in Fig. EV 5).
• In the present study TCL1 x Siglecg-R26ki/ki mb1cre mice were analyzed for CLL development. The authors may want to comment on their choice of using homozygous Siglecg-R26ki mice in the analysis. Is there a threshold for Siglec-G overexpression to suppress the CLL-like cell expansion?

Response
The homozygous Siglecg-R26ki mice show an overexpression level of Siglec-G which is about 5 fold higher on B cells than in WT control mice. We chose this model, because we felt it still is a moderate higher expression pattern. Heterozygous mice were not tested in the TCL1 model, although it would be an interesting question to address. Since these long-term experiments for 1 year already involved 4 groups of mice, we did not want to include a further group. Therefore, we have not addressed this question.
Additionally, the survival curve of TCL1 x Siglecg-R26ki/ki mb1cre mice indicates that some aged animals develop signs of sickness ( Figure 5G). Do these animals suffer from CLL development as well? Maybe in these CLL-clones Siglec-G overexpression was less effective?!

Response
We can only speculate why some of the TCL1 x Siglecg-R26ki/ki mb1cre mice were also affected by a slightly lower survival. We have added one new figure (Fig. EV 1B), which shows the CLL progression in individual mice. In this figure you can see that the CLL development in this group is heterogenous. While most mice stay down with hardly any CLL-like cells in the blood until week 48, one or two mice develop some CLL cells, but to a lower cellular percentage than most controls.
• The Ig repertoire analysis is elegantly showing the monoclonal expansion of the malignant cells in 21st Mar 2023 1st Authors' Response to Reviewers TCL1 x Siglecg-/-mice (Figure 3). The input population for the analysis in these animals as well as in controls is the CD19+ pool isolated from the peripheral blood. However, this approach might mask or underestimate clonal expansion in TCL1 x Siglecg-R26ki/ki mb1cre mice at week 48 characterized by a low, but still increased population of B220low CD5+ cells ( Figure 6). The authors statement that "no leukemic clones developed"(Page 17) might be too optimistic.

Response
Yes, we agree and added the new figure (Fig. EV 1B), which shows the CLL progression in individual mice detects rising CLL-like populations in at least a few TCL1 x Siglecg-R26ki/ki mb1cre mice. We have modified the sentence on page 17 to: "no leukemic clones were detected by our analysis".
• Regarding the intracellular flow cytometry measurements of signaling proteins downstream of the BCR: the authors might want to show these data after gating on B220low CD5+ cells (instead of the entire B220 pool). Alternatively, an additional supplementary graph showing the percentages of CLLlike cells within the B220+ cells would be sufficient to better evaluate the Siglec-G effect on BCR signaling. • How much of the effects observed in BCR signaling between TCL1 x Siglecg-R26ki/ki and TCL1 x Siglecg-R26ki/ki mb1cre mice ( Figure 8B) is related to the "non-tumor vs tumor" comparison? As the authors did not detect significant differences in TCL1 vs TCL1 x Siglecg-/-tumor samples one might consider (and discuss) it.

Response
The suggestion of the reviewer to gate on B220low CD5+ cells was difficult to do, as this population was so small in the TCL1 x Siglecg-R26ki/ki mb1cre mice. However, we have added a supplementary graph showing the CLL-like cells of the TCL1 x Siglecg-R26ki/ki and TCL1 x Siglecg-R26ki/ki mb1cre mice of Figure 8B (new Fig EV 8). We have described this in the results section: "Although the percentage of CLL-like cells analyzed in Fig.8B was on average not different between TCL1 x Siglecg-R26 ki/ki and TCL1 x Siglecg-R26 ki/ki x mb1 cre mice, the latter had more widespread CLL-like numbers (Fig. EV 8). Therefore, their normal phopho-protein levels maybe also due to the higher percentage of normal B cells" (page 14).
Minor comments: • The roughly 10fold increase of the B220low CD5+ numbers in the TCL1 x Siglecg-R26ki/ki mice at week 36 ( Figure 5C) in comparison to the corresponding week in TCL1 ( Figure 2B) mice is surprising. Do the authors have any explanation?
• Please indicate the genetic background of the used mb1-cre line (as C57/BL6 and BALB/C mice are available).

Response
The stronger (and earlier) increase of CLL-like cells of the blood, spleen and other organs in controls of the second cohort (TCL1 x Siglecg-R26ki/ki mice), compared to the controls of the first cohort (TCL1 mice) was also noted by us. We cannot explain this, as all mice were on a pure BALB/c background. This information is now also added for the mb1-cre line in materials and methods. But of course, the TCL1 x Siglecg-R26ki/ki mice are the proper control for the same line with the additional mb1-cre allele, whereas the TCL1 line (on BALB/c) is the proper control for Siglecg-/-(on BALB/c). It is noticeable, but not easy to explain.
• In Figure 1B the progressive expansion of the B220lowCD5+ compartment could be depicted with an additional graph in which it is possible to follow the expansion of the CLL-like population in the individual mice over time (similarly in Figure 4B).

Response
Yes, as mentioned previously, these data are now shown in the new Fig. EV 1. • In Figure 2C and similarly in 5E, in the summary of B220lowCD5+ percentages in the liver, it would be more accurate to label the axis with 'leukocytes' instead of 'lymphocytes'. • In Figure 7A in the density plot the abscissa seems to be mislabeled, otherwise the CLL-like population is CD5 negative. • In Figure S2B the TCL1 x Siglecg-/-bars appear to be edited with variable patterns, unlike the solid purple color in the rest of the graphs throughout the manuscript. • Please use a consistent nomenclature for the mouse strains throughout the manuscript (see page 11). • In page 22 of the materials and methods please correct 'BZR'. • In page 30 caption of Fig 2C: please correct 'leucocytes'.

Response
All these points were corrected in the new manuscript. For Fig. 7A, we chose a new example. Due to the UV laser needed for Ca2+ measurements, another fluorochrome on the anti-CD5 antibody had to be chosen than in all other figures, which separates CD5-low cells not so well from CD5-negative cells.
--------------Referee #2: This work by Nitschke and co-workers uses genetic models to demonstrate a convincing role for Siglec-G in the development of CLL. Both KO and over-expressing mouse models of Siglec-G are studied in the context of a mouse model of CLL. Results clearly and demonstrate that the kinetics of CLL development is inversely proportional to Siglec-G expression levels. Numerous measures are examined, including gross organ weight, survival, and cellular readouts. Moreover, it is demonstrated that Siglec-10 is down-regulated in human CLL. The manuscript is clear and well written and only requires a few considerations: 1) As this is the first study of this KI over-expressing mouse, a bit more characterization seems in order. Can the authors rule out that development impact given that MB1-Cre turns on very early in B cell development? Are lymphocyte proportions in the adult spleen roughly similar to WT mice?

Response
We thank the reviewer for the positive comments. As in response to reviewer no.1, we agree with the suggestion that a characterisation of the new SiglecG overexpressing mouse line in more detail is helpful for the reader. We have therefore performed more experiments to analyse this in detail. We find no impairment of B cell development or B cell subpopulations in the spleen in Siglecg-R26ki/ki mb1cre mice. These data are now shown in the new Fig EV 6. The only detected difference of this mouse line is a lower number of B1a cells (shown in Fig. EV 5).
2) Given the clear differences in Ca2+ flux, can they speculate why no differences were observed on downstream signaling molecules? Response Yes, it is surprising that we cannot detect differences particularly in the phospho-proteins of the proximal signalling pathways between TCL1 and TCL1 x Siglecg-/-CLL-like cells. This has also been the case for the original description of the Siglecg-/-mice, where we detected the largely increased Ca2+ signalling in B1a cells, but could not find obvious increases in proximal signalling pathways in B1a cells by phospho-FACS (Hoffmann et al. Nat.Immunol.2007). So the TCL1 mice reflect also the normal Siglecg-/-mice. It maybe that we did not look at the right intracellular signalling proteins. For this, phopho-protein analysis is limited.
3) In the over-expressing mice, it was stated that they did not have enough CD5+ cells to perform cellular analyses. It would have been nice to see some readouts on the 'total' B cells to complement the data have for the other models. If that data was acquired, it would be a useful addition.

Response
Although we tried to do these additional experiments, we did not obtain conclusive results. Maybe because the total B cells are varying too much in their composition from mouse to mouse. We have added a sentence to the results section describing this: "Attempts to measure BCR-induced Ca 2+ responses in Siglec-G overexpressing mice were not successful due to the very low number of B220 low CD5 + cells in Siglecg-R26 ki/ki mb1 cre and TCL1 x Siglecg-R26 ki/ki mb1 cre mice and due to inconsistent results when gating on total B cells".
--------------Referee #3: Major: 1. Results, page 6: "The size of these B1a cell populations in the two control mouse strains remained constant and did not increase until the age of 60 weeks (Fig.1B)." Is the increase at week 60 statistically significant? From the figure, this seems unlikely.

Response
We thank the reviewer for the comments. The increase at week 60 between the two control mouse strains is not significant. All significant differences within the time points are shown.
2. Results, page 6: "TCL1 mice showed an increase of the population of CLL-like cells (also characterized as B220low CD5+) in the blood from the age of 40 weeks, reaching a percentage of about 50% of all blood lymphocytes at week 60. In contrast, the CLL-like population in the blood of TCL1 x Siglecg-/-animals increased much earlier, already at the age of 20 weeks (on average 30 % of blood lymphocytes)." This text does not agree with the comparisons highlighted on the figure. The figure shows statistical comparisons of the two sets (WT vs Siglec6-/-; TCL1 vs TCL1x Siglec6-/-) to each other, whereas the text compares the same population to itself over time. This is the case for several other figures, e.g., Suppl Fig 1A+B; Fig 2A, B; Fig 4B).
This is very confusing. The statistical analyses relating to the text should also be provided. The comparisons in Suppl Fig 1C serve as an example.

Response
We only show significant differences within each time point between the different mouse strains. We do not show differences over time, as this would be too confusing to show as well. We agree that the discussion of the findings in the results section was a bit focused on the changes during time. We have now changed the description of the results in all the respective parts. All the changes are marked in yellow. Parts describing the changes in time were deleted or shortened and more emphasis is now on the differences within the groups of mice.
3. Results, page 9: "This analysis revealed a significantly reduced binding of these antigens in TCL1 x Siglecg-/-mice in comparison to TCL1 littermates for most time points (Suppl Fig 3). This suggests an overall different BCR repertoire of CLL-like cells of TCL1 x Siglecg-/-mice, when compared to TCL1 mice. A similar shift in the BCR repertoire was detected previously in normal B1a cells of Siglecg-/mice (Jellusova et al., 2010)." This finding is somewhat bothersome, as it suggests that the B cells that are being transformed in these mice are different than the standard model. Is the severity of the disease in these animals due to the development of a malignancy in B cells that do not usually transform rather than the transformation of same population a greater frequency? This is supported by the literature which I believe indicates that the major IgV gene families used by TCL1 mice and by B-1 cells are VH11 and VH12 (Baumgarth, 2011 cited; PMID: 32561570). This is compounded by the finding that these genes are identified when over expressing Siglec G (Fig 6). The authors appropriately address this issue in the Discussion but it remains a major concern.
Response The group of Chiorazzi has analysed the CLL clones in TCL1 mice in detail (Yan et al 2006, cited in our paper). VH11 and VH12 were used more often in these clones than their normal representation in the Ig repertoire, but both in about 10% of the CLL clones. This means that 80% of the CLL clones contained other VH families. Therefore, we would argue that the shift in the Ig repertoire of CLL cells in TCL1 x Siglecg-/-mice is interesting, but does not necessarily mean that it is a different way of transformation. But it sheds light on the point that not only the VH11 and VH12 clones are the crucial VH families for CLL transformation. To clarify this point we have now added a sentence in the discussion: "Similarly, over-usage of the VH11, VH12, and VH4 gene segments has been demonstrated in TCL1 animals. Each of these three VH families was used in about 10% of leukemic clones of TCL1 transgenic mice, while the rest of leukemic clones used other VH families (Yan et al., 2006)" (page 18). 4. Results, pages 14-15: Since the data in Figure 9B are not statistically significant, the authors should consider moving these to the Supplementary Section and bringing some of the data in Suppl Figs 6 and 7 to the main text?

Response
Thank you for this suggestion, yes, we removed the data from Fig. 9B to the supplementary data and brought one figure from the supplementary data to the main Fig. 9B (differential mRNA expression).
5. Discussion, page 16: "The dependence of CLL on BCR signaling is not due to BCR-activating mutations in crucial signaling molecules as shown for diffuse B cell lymphoma (DLBCL) (Schmitz et al., 2018), but due to the ability of some CLL BCRs to cluster by binding to each other and in this way autonomously activating BCR signaling (Duhren-von Minden et al., 2012, Minici et al., 2017." Although this is an accurate statement, it implies that binding of antigens other than companion BCRs is irrelevant. This is not necessarily the case as shown by in vivo studies (PMID: 25564405).

Response
Thank you for this comment. We have added the ref. which you mentioned and changed the statement accordingly: "The induction of BCR signals by low affinity autoantigens also plays a role in CLL development (Iacovelli et al. 2015)". (page 14) Minor: 1. The following sentence on page 4 is confusing or possibly incomplete. In particular the comment about V families. "Murine CD5+ B1a cells share features of human CD5+ CLL cells in some aspects, including expression of a restricted Ig repertoire using similar V with B6) families." Response This was corrected.

27th Apr 2023 1st Revision -Editorial Decision
Dear Prof. Nitschke, Thank you for the submission of your revised manuscript to our editorial offices. I have now received the reports from the three referees that I asked to re-evaluate your study, you will find below. As you will see, the referees now fully support the publication of your study in EMBO reports.
Before proceeding with formal acceptance, I have these editorial requests I ask you to address in a final revised manuscript: -Please provide the abstract written in present tense.
-The figures of the manuscript need to be re-organized, and we need single figure files also for the EV figures. The Expanded View format, which will be displayed in the main HTML of the paper in a collapsible format, has replaced the Supplementary information. You can submit up to 5 images as Expanded View. Presently, there are 10! Looking through the figures, I think several figures could be combined. I would suggest doing that to have 6-7 (max. 8) main figures and 5 EV figures. Please follow the nomenclature Figure EV1, Figure EV2 Table EV1 as individual file using the file type 'Expanded View' (or add this to an Appendix file).
-We now use CRediT to specify the contributions of each author in the journal submission system. CRediT replaces the author contribution section. Please use the free text box to provide more detailed descriptions and remove the author contributions section from the manuscript text file. See also guide to authors: https://www.embopress.org/page/journal/14693178/authorguide#authorshipguidelines -We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests. Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary. Please name this section 'Disclosure and Competing Interests Statement' and put it after the Acknowledgements section.
-We could not find the source data for Fig. 7. Please provide the information on the deposited dataset for Fig. 7A in the data availability section and upload the SD for Fig. 7B.
-Please remove the referee token from the data availability section and make sure that the data is public latest when the manuscript is published online.
-Please order the manuscript sections like this (using these names as headings): Title page -Abstract -Keywords -Introduction -Results -Discussion -Materials and Methods -Data availability section -Acknowledgements -Disclosure and Competing Interests Statement -References -Figure legends -Expanded View Figure  Legends -Regarding data quantification and statistics, please make sure that the number "n" for how many independent experiments were performed, their nature (biological versus technical replicates), the bars and error bars (e.g. SEM, SD) and the test used to calculate p-values is indicated in the respective figure legends (also for potential EV figures and all those in the final Appendix). Please also check that all the p-values are explained in the legend, and that these fit to those shown in the figure. Please provide statistical testing where applicable. Please avoid the phrase 'independent experiment', but clearly state if these were biological or technical replicates. Please also indicate (e.g. with n.s.) if testing was performed, but the differences are not significant. In case n=2 please show the data as separate datapoints or bars without error bars and statistics. See also: http://www.embopress.org/page/journal/14693178/authorguide#statisticalanalysis If n<5, please show single datapoints for diagrams. Presently, there are still diagrams without or with only partial statistics (missing e.g. n.s.). Please fix this.
-Please make sure that all figure panels are called out separately and sequentially (main, EV and Appendix figures). Presently, there are no separate callouts for Figs. EV3A&B and EV4A&B. Please check.
-Please provide the author checklist with answers, i.e. select responses from the drop-down lists.
-Finally, please find attached a word file of the manuscript text (provided by our publisher) with changes we ask you to include in your final manuscript text and comments. Please use the attached file as basis for further revisions and provide your final manuscript file with track changes, in order that we can see any modifications done.
In addition, I would need from you: -a short, two-sentence summary of the manuscript (not more than 35 words).
-two to four short bullet points highlighting the key findings of your study (two lines each).
-a schematic summary figure as separate file that provides a sketch of the major findings (not a data image) in jpeg or tiff format (with the exact width of 550 pixels and a height of not more than 400 pixels) that can be used as a visual synopsis on our website. The manuscript is suitable for publication in EMBO reports without further revision.

14th Jun 2023 2nd Authors' Response to Reviewers
The authors have addressed all minor editorial requests.

22nd Jun 2023 2nd Revision -Editorial Decision
Prof. Lars Nitschke University of Erlangen Chair of Genetics, Department of Biology Staudtstr. 5 Erlangen 91058 Germany Dear Prof. Nitschke, I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal.
At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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