A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

  1. Jérémy Argenty
  2. Nelly Rouquié
  3. Cyrielle Bories
  4. Suzanne Mélique
  5. Valérie Duplan-Eche
  6. Abdelhadi Saoudi
  7. Nicolas Fazilleau
  8. Renaud Lesourne

  • Curated by eLife

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    Evaluation Summary:

    The authors study the requirement for Lis1, a dynein binding protein, in T cells, and present solid evidence that the requirement differs between different T cell lineages, suggesting cell division mechanisms differ across these cell lineages. This work is valuable for cell biologists and immunologists interested in mechanisms that contribute to cell proliferation and differentiation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after β-selection and of peripheral CD4+ T-cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1 deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.

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  1. Version published to 10.7554/elife.80277 on eLife
  2. eLife

    Author Response

    Reviewer #3 (Public Review):

    Argenty et al. investigated the role of Lissencephaly gene 1 (LIS1), a dynein-binding protein, in thymic development and T cell proliferation. They find that LIS1 is essential for the early stages of T and B cell development, and demonstrate that loss of LIS1 has a negative impact on the transition from DN3 to DN4 thymocytes and on the maturation of pre-pro-B cells into pro-B cells in the bone marrow. Using a CD2Cre Lis1fl/fl murine model, they observe that in thymocytes LIS1 is critical for DN3 proliferation and completion of cell division. Then, using a CD4Cre Lisfl/fl model (Cd4 promoter is up-regulated just in later stages of thymic development and, thus, does not impact DN3 thymocytes) they show that LIS1-deficient CD4 T cells have proliferation defects following both TCR-dependent or -independent stimulation, which results in apoptosis. They also confirm previous reports that show that LIS1-deficient CD8 T cells do not have their proliferation impaired upon TCR stimulation, which suggests that these two cell types rely on different mechanisms to regulate the cell cycle. Finally, the authors make efforts to determine how LIS1 regulates proliferation in thymocytes and CD4 T cells. Interestingly, they show that LIS1 is important for chromosome alignment and centrosome integrity and provide data that support a model where LIS1 would facilitate the assembly of active dyneindynactin complexes. These data provide interesting insights into how different cell types use distinct strategies to undergo mitosis and how this can impact on their proliferation and fate decisions. The conclusions of the manuscript are mostly supported by the provided data, although certain aspects can be further investigated and clarified.

    Strengths of the paper:

    By combining a re-assessment of previous reports with new findings, the data from this manuscript convincingly demonstrates that LIS1 is crucial for cell proliferation in certain development steps/cell types. Furthermore, the manuscript provides clear evidence of how LIS1 loss causes proliferation defects by disrupting centrosome integrity and chromosome alignment both in CD4+ T cells and thymocytes.

    Weakness of the paper:

    Although authors successfully address the mechanistic role of LIS in thymocyte and CD4+ T cell division, the manuscript would be strengthened by both providing further evidence to support some of their conclusions and a review of some speculations raised in the discussion.

    In Figure 1, the authors claim that LIS1 is not required for pre-TCR assembly, but for expansion/proliferation of DN3 thymocytes as a step prior to reaching the DN4 stage. However, authors indeed observe increased expression of CD5 (which is a downstream event of Notch and IL-7R signalling). Thus, from the data provided it is not clear whether signalling through Notch or IL-7R is definitely not affected, which could be clarified by assessing the expression of other downstream targets of these molecules.

    CD5 is a downstream target of the pre-TCR signaling but to our knowledge, it is not a downstream target of Notch or IL-7R signaling. The sentence p7 of the initial manuscript was re-formulated since we understand that it could be misleading. However, we fully agree with the reviewer’s comment on Notch and IL-7R signaling and included new data in the revised version of the manuscript to address this point. Notch signaling stimulates metabolic changes which lead to the increase of thymocyte cell-size following the b-selection checkpoint (Ciofani M. et al., Nature Immunology, 2005; Maillard I. et al., The Journal of Experimental Medicine, 2006) and to the up-regulation of the transferrin receptor CD71 (Kelly, A.P. et al., The EMBO journal, 2007). We now show in Figure 1E of the revised manuscript that the loss of LIS1 does not affect the average cell-size of post-b-selection thymocytes and the expression level of CD71 in these cells, suggesting that Notch signaling is preserved in the absence of LIS1. This was confirmed in vitro following stimulation of DN3a thymocytes with OP9-dl1 cells (Figure 2D of the revised manuscript). In this Figure, we also analyzed the expression level of Bcl-2, which is regulated by IL-7R signaling (von Freeden-Jeffry, U. et al., Immunity, 1997). We show that Bcl-2 is comparable in abundance in LIS1 wild-type and LIS1-deficient thymocytes following stimulation with OP-9dl1, suggesting that Il-7R signaling is not affected by the absence of LIS1.

    In Figure 3, the authors mostly confirm previous data from Ngoi, Lopez, Chang, Journal of Immunology, 2016 (reference 34), but also provide evidence of a role of LIS1 in CD4+ T cell proliferation in more physiological setups, using OT2-CD4-Cre Lis1flox/flox (or OT2 Lisflox/flox as controls) in adoptive transfer experiments followed by antigen-specific immunization. However, the evidence provided by the authors about proliferation defects in LIS1-deficient cells in this context is limited by the early timepoint chosen: day 3 post-immunization.

    We choose to analyze CD4+ T cells at day 2 and 3 after immunization because we sought to catch early cell-division waves through CTV dilution. We also wanted to show that LIS1 deficient CD4+ T cells could normally survive and migrate to lymph nodes before they start to proliferate. Given the dramatic effect of LIS1 on CD4+ T-cell proliferation at day 3, we anticipated that very low numbers of LIS1 deficient cells would survive at later time points after immunization. To address the reviewer’s comment, we transferred OT2+CD45.1+ CD4+ T cells stained with CTV in C57BL/6 mice and analyzed the percentages and numbers of CD45.1+ T cells as well as the dilution of CTV in those cells at day 7 after immunization. As expected, all CD45.1+ cells were negative for CTV at this time of analysis (data not shown). The percentages and numbers of CD45.1+ T cells were strongly decrease in the absence of LIS1 in comparison to wild-type controls (Figure 3 - Figure Supplement 2C), confirming results obtained at day 3 after immunization.

    In the discussion, the authors speculate about the differences observed between CD4 and CD8 T cells, as the latter do now show proliferative defects upon TCR-triggered stimulation, and come up with the hypothesis that LIS1 might be important for symmetric cell divisions, but not for asymmetric cell divisions. However, the arguments used by the authors have few caveats, especially because CD4+ T cells can also undergo asymmetric cell division following TCR-triggered stimulation upon the first cognate antigen encounter (Chang et al., Science, 2007, Ref. 8).

    We agree that CD4+ T cells can undergo asymmetric division (actually, this is mentioned and referenced p3 and p18 of the manuscript). However, it is unknown whether these divisions occur systematically or whether they occur with variable frequency which could be context-dependent. It is also unclear whether CD4+ and CD8+ T cells have similar rates of asymmetric division. The literature is lacking of comparative studies in which cellular events associated to mitosis would be investigated side-by-side in those two subsets. As mentioned to reviewer-1, only one study to our knowledge performed a comparative analysis of T-bet repartition in daughter cells after a first round of cell division in CD4+ and CD8+ T cells (Chang, J. T. et al., Immunity, 2011). They found that T-bet segregates unequally in daughter cells in both CD4+ and CD8+ T cells. However, the disparity between daughter cells was higher in CD8+ T cells as compared to that in CD4+ T cells (5- versus 3-fold). This suggests that key molecules are either more equally (or less unequally) distributed in daughter cells from the CD4+ lineage or that the rate of symmetric divisions is higher in CD4+ T cells than in the CD8+ T cells. Those results are in accordance with our interpretation and previous findings (Yingling, J. et al., Cell, 2008; Zimdahl, B. et al, Nature Genetics, 2014), suggesting that LIS1 is predominantly involved in mitosis associated to symmetric divisions. Another possibility to explain this difference is that asymmetrical division might occur at different stages in CD4+ and CD8+ T cells. Although some asymmetrical divisions have been detected early after antigen encounter in CD4+ T cells, a more recent study from the same group suggest that asymmetric division might occur mainly later after several rounds of divisions of CD4+ T cells to enable self-renewal to be coupled to production of differentiated effector CD4+ T cells (Nish, S. A., Journal of Experimental Medicine, 2017). It is therefore possible that LIS1 could be critical early in CD4+ T cell expansion, when cells mainly divide through symmetrical process, and less critical later when cells are engaged in asymmetrical division. This is now discussed in greater details p18 of the revised version of the manuscript.

    Finally, the authors discuss that mono-allelic LIS1 defects might contribute to malignancies. Certainly not all points raised in the discussion need to be experimentally addressed, but for this particular hypothesis the authors would likely have the tools to achieve that, which would broaden the relevance of understanding LIS1 function.

    We have addressed this point experimentally in the revised version of the manuscript. We show that mono-allelic LIS1 deficiency does not have a significant impact on the percentages of thymocyte populations in Cd2-Cre Lis1flox/+ mice (Figure 1 - Figure Supplement 1B) and on the numbers of peripheral T cells in Cd4-Cre Lis1flox/+ (Figure 3 - Figure Supplement 1E), suggesting that LIS1 does not operate in a dose-dependent fashion in the context of T-cell development and T-cell homeostatic maintenance. Additionally, Cd4-Cre Lis1flox/+ CD4+ T cells proliferate effectively following TCR and CD28 stimulation (Figure 3 - Figure Supplement 2A), indicating further that mono-allelic LIS1 dosage is sufficient to support cell division of CD4+ T cells. The part of the discussion related to Lis1 haplo-deficiency has been rephrased according to this new set of data.

  3. Version published to 10.1101/2022.05.23.493045v2 on bioRxiv
  8. eLife

    Evaluation Summary:

    The authors study the requirement for Lis1, a dynein binding protein, in T cells, and present solid evidence that the requirement differs between different T cell lineages, suggesting cell division mechanisms differ across these cell lineages. This work is valuable for cell biologists and immunologists interested in mechanisms that contribute to cell proliferation and differentiation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  9. eLife

    Reviewer #1 (Public Review):

    The authors initially demonstrated that the deletion of LIS1 using an inducible Cre mouse model hindered the maturation of T cells, as evidenced by a reduction in the number of DPs. Furthermore, it reduced early T cell and B cell development, specifically during β selection and prepro to pro B cells in the case of T and B cells, respectively. This correlated with an increase in cells at the G2/M stage. The authors then sorted for DN3 cells and seeded them onto OP9-DL1 stromal cells. In this model, the deletion of Lis1 reduced proliferation and lead to an accumulation of the cells at G2/M, similar to the results in vivo.

    The authors then switch to examining the role of Lis1 at later stages of T cell development by deleting Lis1 at the DP stage. The deletion of Lis1 at this stage resulted in a reduction in CD4+ and CD8+ cells, which correlated with a drop in proliferation in CD4+, after the first division and a slight reduction in CD8+ cells. The drop in proliferation and increase in cells at the G2/M stage was shown to be due to an inability to correct condense the DNA at metaphase, resulting in aberrant numbers of centromeres and upregulation of apoptosis, which was also confirmed in DN3 cells. Finally, they demonstrate that this is due to an ineffective interaction between dynein and dynactin. Overall, this was an interesting study into the role of Lis1 in T cell division.

  10. eLife

    Reviewer #2 (Public Review):

    The authors study the requirement for Lis1 in T lineage cells. The approach is to genetically delete the gene for Lis1, which encodes a dynein binding protein likely to be important in mitosis. The authors delete Lus1 at different stages of T cell development and examine the consequences in T cell precursors, as well as mature CD4 T cells and CD8 T cells. The requirement is shown to be different between CD4 and CD8 T cells.

  11. eLife

    Reviewer #3 (Public Review):

    Argenty et al. investigated the role of Lissencephaly gene 1 (LIS1), a dynein-binding protein, in thymic development and T cell proliferation. They find that LIS1 is essential for the early stages of T and B cell development, and demonstrate that loss of LIS1 has a negative impact on the transition from DN3 to DN4 thymocytes and on the maturation of pre-pro-B cells into pro-B cells in the bone marrow. Using a CD2Cre Lis1fl/fl murine model, they observe that in thymocytes LIS1 is critical for DN3 proliferation and completion of cell division. Then, using a CD4Cre Lisfl/fl model (Cd4 promoter is up-regulated just in later stages of thymic development and, thus, does not impact DN3 thymocytes) they show that LIS1-deficient CD4 T cells have proliferation defects following both TCR-dependent or -independent stimulation, which results in apoptosis. They also confirm previous reports that show that LIS1-deficient CD8 T cells do not have their proliferation impaired upon TCR stimulation, which suggests that these two cell types rely on different mechanisms to regulate the cell cycle. Finally, the authors make efforts to determine how LIS1 regulates proliferation in thymocytes and CD4 T cells. Interestingly, they show that LIS1 is important for chromosome alignment and centrosome integrity and provide data that support a model where LIS1 would facilitate the assembly of active dynein-dynactin complexes. These data provide interesting insights into how different cell types use distinct strategies to undergo mitosis and how this can impact on their proliferation and fate decisions. The conclusions of the manuscript are mostly supported by the provided data, although certain aspects can be further investigated and clarified.

    Strengths of the paper:

    By combining a re-assessment of previous reports with new findings, the data from this manuscript convincingly demonstrates that LIS1 is crucial for cell proliferation in certain development steps/cell types. Furthermore, the manuscript provides clear evidence of how LIS1 loss causes proliferation defects by disrupting centrosome integrity and chromosome alignment both in CD4+ T cells and thymocytes.

    Weakness of the paper:

    Although authors successfully address the mechanistic role of LIS in thymocyte and CD4+ T cell division, the manuscript would be strengthened by both providing further evidence to support some of their conclusions and a review of some speculations raised in the discussion.

    In Figure 1, the authors claim that LIS1 is not required for pre-TCR assembly, but for expansion/proliferation of DN3 thymocytes as a step prior to reaching the DN4 stage. However, authors indeed observe increased expression of CD5 (which is a downstream event of Notch and IL-7R signalling). Thus, from the data provided it is not clear whether signalling through Notch or IL-7R is definitely not affected, which could be clarified by assessing the expression of other downstream targets of these molecules.

    In Figure 3, the authors mostly confirm previous data from Ngoi, Lopez, Chang, Journal of Immunology, 2016 (reference 34), but also provide evidence of a role of LIS1 in CD4+ T cell proliferation in more physiological setups, using OT2-CD4-Cre Lis1flox/flox (or OT2 Lisflox/flox as controls) in adoptive transfer experiments followed by antigen-specific immunization. However, the evidence provided by the authors about proliferation defects in LIS1-deficient cells in this context is limited by the early timepoint chosen: day 3 post-immunization.

    In the discussion, the authors speculate about the differences observed between CD4 and CD8 T cells, as the latter do now show proliferative defects upon TCR-triggered stimulation, and come up with the hypothesis that LIS1 might be important for symmetric cell divisions, but not for asymmetric cell divisions. However, the arguments used by the authors have few caveats, especially because CD4+ T cells can also undergo asymmetric cell division following TCR-triggered stimulation upon the first cognate antigen encounter (Chat et al., Science, 2007, Ref. 8).

    Finally, the authors discuss that mono-allelic LIS1 defects might contribute to malignancies. Certainly not all points raised in the discussion need to be experimentally addressed, but for this particular hypothesis the authors would likely have the tools to achieve that, which would broaden the relevance of understanding LIS1 function.

  12. Version published to 10.1101/2022.05.23.493045v1 on bioRxiv