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 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
I. Essential revisions 1. Control for the impact of Cre expression in LIS1flox+/-. Responses: We did not detect defects associated to the partial loss of LIS1 in CD2-Cre and CD4-Cre LIS1flox/+ mice (Figure 1 - Figure Supplement 1B and Figure 3 - Figure Supplement 1E) 2. Assess the ablation of Lis1 to ensure that the effects are not an artefact of the experimental approach to deletion. Responses: We show in the revised version that LIS1 is efficiently deleted in both subsets (Figure 3 - Figure Supplement 2B). 3. More experimentation to clarify the step in beta-selection that are disrupted by LIS deletion (expression of Notch and IL7R signalling components, cell cycle analysis, assess DN3a and DN3b stages). Responses: * DN3a versus DN3b stages: we analyzed the intracytoplasmic expression of the TCR chain versus the cell surface expression of CD27, a cell surface marker that discriminate DN3a (CD27low) from DN3b (CD27hi) thymocytes (Figure 1C of the revised manuscript). The percentages of TCR hiCD27hi thymocytes in DN3 cells were decreased in the absence of LIS1, suggesting a defect upon or after the beta-selection checkpoint. * Notch and IL-7R signaling: We now show that the loss of LIS1 does not affect the average cell-size of post- -selection thymocytes and the expression level of CD71 in these cells, suggesting that Notch signaling is preserved in the absence of LIS1 (Figure 1E). This was confirmed in vitro following stimulation of DN3a thymocytes with OP9-dl1 cells (Figure 2D of the revised manuscript). 4. Extend analysis of CD4 responses to later timepoints. Responses: CD4+ T-cell proliferation was analyzed at a later timepoint 7 days after immunization. We confirmed that LIS1 defiency leads to a major defect of CD4+ T-cell expansion at this stage of T-cell responses (Figure 3 - Figure Supplement 2C). 5. Incorporate findings In the literature into the discussion on ACD as an explanation of the differences in proliferative effects between CD4 and CD8 cells. Response: A new paragraph was introduced p18 of the manuscript to discuss this point in greater details. II. Revisions addressing specific reviewers' requests Analyze cell cycle in CD8+ T cells (requested by reviewer-1): we now show that LIS1 deficiency does not affect the percentages of cells in the G2/M phase in CD8+ T cells. Analyze p53 expression in CD8+ T cells (requested by reviewer-2): we now show that p53 expression is comparable in wild-type and LIS1-deficient CD8+ T cells.