INPP5E regulates CD3ζ enrichment at the immune synapse by phosphoinositide distribution control

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   eLife

   Jan 8, 2021

   This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on January 5 2021, follows.

   Summary

   Your work analyzes the impact of the INPP5E inositol lipid-5 phosphatase on immune synapse formation and function. INPP5E is a cilium enriched protein. Although T cells do not display primary cilia, previous work by several laboratories showed that several ciliary proteins are involved in immunological synapse formation and in T cell activation and your work intends to further this view. Although the work has potential for publication in eLife, it requires essential additional data to support the central claims of the paper. Each reviewer raised substantive concerns (see below) that need to be resolved experimentally. For instance, experiments involving knockout in primary T cells will need to be …

   This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on January 5 2021, follows.

   Summary

   Your work analyzes the impact of the INPP5E inositol lipid-5 phosphatase on immune synapse formation and function. INPP5E is a cilium enriched protein. Although T cells do not display primary cilia, previous work by several laboratories showed that several ciliary proteins are involved in immunological synapse formation and in T cell activation and your work intends to further this view. Although the work has potential for publication in eLife, it requires essential additional data to support the central claims of the paper. Each reviewer raised substantive concerns (see below) that need to be resolved experimentally. For instance, experiments involving knockout in primary T cells will need to be performed. A better time series will also help deciding in what process the INPP5E protein is involved in. Moreover, imaging data should be quantified more precisely to assess spatial and dynamic differences.

   Reviewer #1:

   An important aspect of mature synapse formation is signal termination and ... effector responses, such as secretion of cytokines, exosomes and CD40L on synaptic ectosomes (Huse et al, 2006; Mittelbrunn et al, 2011). The demonstration of ESRCT function in both TCR signal termination and CD40L release to B cells on synaptic ectosomes likely involves inositol lipids that lack phosphorylation on the 5' position.

   It might make sense for the author to investigate a synapse effector function like degranulation of CD8 or CD40L transfer of synaptic ectosome in CD4 T cells as these effector functions actually link into synapse formation more directly than bulk IL-2 secretion.

   The ESCRT machinery is also highly entwined with ciliary biology and several ESCRT components important for signal termination and effector function will also require PIP metabolism.

   Reviewer #2:

   Interesting similarities between the primary cilium and the immunological synapse have been noted and investigated extensively over the last few years. In this context and beyond, the role of phosphatidylinositol lipids in the organisation of the immunological synapse and T cell function has been extensively investigated. Here Chiu et al. add to these topics by investigating INPP5E, a primary cilium-associated 5' phosphatidylinositol lipid phosphatase that can use PIP3, PI(4,5P)P2 and PI(3,5)P2 as substrates, in T cell activation. The authors show that INPP5E is recruited to the interface of a T cell with an activating antigen presenting cell. INPP5E binds to TCRzeta, ZAP-70 and Lck. INPP5E knockdown reduces TCR recruitment to the T cell/APC interface, clearance of PI(4,5)P2 from the centre of the interface, and TCR and ZAP-70 phosphorylation. These findings are consistent with the large body of existing work on the role of phosphatidylinositol lipids in the organisation of the immunological synapse and T cell function and, therefore, don't constitute a conceptual advance. Nor do they provide new mechanistic insight into phosphatidylinositol lipids in T cell activation. The data add another molecule to the existing body of work.

   In the first two figures Chiu et al. show that a number of cilium-associated proteins, including INPP5E are recruited to the interface of a Jurkat cells with a Raji B cell presenting superantigen. Such recruitment is not surprising. On the contrary, because of the reorientation of the MTOC to the centre of the cellular interface and the accompanying shift of the nucleus to the back of the T cell to create more cytoplasmic space at the interface, most proteins associated with vesicular trafficking shift their subcellular distribution towards the interface. Only data showing spatial or temporal distinctions in such recruitment within the small cytoplasmic space underlying the T cell/APC interface could provide interesting new insight. Reduced detection of INPP5E interface recruitment after INPP5E knockdown could be trivially caused by the worse signal to staining background noise ratio (Fig. 2A-E). The STORM data showing that INPP5E interface recruitment occurs in the T cell not the APC are welcome. However, spatial and temporal features provided by the higher resolution of these experiments are not explored.

   In the investigation of the contribution of different INPP5E domains to its interface recruitment the representative imaging data in Fig. 3A suggest that substantial quantitative differences exist. The '% conjugate with recruitment' metric doesn't capture such differences. Some form of a recruitment index as used in other parts of the manuscript would be more powerful. A more complex picture of INPP5E domain contributions to INPP5E interface recruitment is likely to emerge.

   The immunological synapse is a highly dynamic structure. TCR interface recruitment and PI(4,5)P2 clearance in response to various manipulations of PI turnover are only analysed at a single time point. A dynamic picture should provide more insight. For example, interface recruitment of the TCR may be consistently impaired, delayed or shifted in time. Reduced interface recruitment of the TCR upon overexpression of PIP5Kgamma (Fig. 5D, E) has already been described in the cited Sun et al. reference. This should be acknowledged.

   In Fig. 6E, the authors show a small reduction in IL-2 secretion in Jurkat cells stimulated with anti-CD3/CD28 upon knockdown of INPP5E. As INPP5E is expected to exert its functional effects through the control of the spatiotemporal organisation of the immunological synapse, activation of Jurkat cells with APCs would be more appropriate.

   The knockdown efficiency of INPP5E should be quantified.

   Reviewer #3:

   The work is fully performed in Jurkat cells, which a very good and widely used model to investigate T cell activation, yet, not perfect. Actually, in the case of events related with phosphoinositide function, Jurkat cells present a strong caveat. These cells lack the Phosphoinositide phosphatase PTEN, therefore having altered phosphoinositide turnover.

   Therefore, as a first critical point, the authors should confirm most of the central data of this work in primary T cells. They should also discuss this point, since it might bias some of their data.

   Additional points needing attention are detailed below.

   1. Regarding data in Fig 1D, the authors say the they find INPP5E localized with the centriole in the absence of SEB stimulation. The pattern shown is in the picture is very diffuse and blurry, not showing at all a centriole pattern.

   It seems to be more visible in Fig S1. The authors should replace Fig1D panel by a better "quality" picture if they wish to convey that message.

   1. What do the authors mean with "number of events" in the figures ? Please explain or replace by another term or means of quantification. If it means counting conjugates with INPP5E recruited "by visual observation", it would be much more appropriated to quantify fluorescence enrichment at the synapse making a ratio.

   It is also bizarre to plot "pairs" which are all at 100%. What does that mean?

   1. In Fig 2 D, E the authors observe by TIRF the presence of INPP5E at the planar pseudosynapse. They do in parallel TCRz. It would be interesting to better take advantage of that type of microscopy images to also quantify the impact of INPP5E on TCRz recruitment and to assess co-localization between INPP5E and TCRz using Pearse corelation on images with a very good resolution. From that image they look like they do not co-localize at all.

   2. The reasoning of the authors in Fig 2 H is somehow strange: "Since the distribution of INPP5E signals mostly appear at the T cell-APC contact site, it was necessary to examine whether INPP5E belonged to T or B cells" Although they use dSTORM the resolution of the image is not single molecule as they claim, but relatively large clusters. Moreover, they say that INPP5E is inside the T cell while TCRz is at the plasma membrane. In that image there are spots labelled far on the B cell. Moreover, it has been shown by several authors that TCRz largely occupies intracellular vesicular compartments. So the conclusion is not accurate. Finally, they claim that the overlap in some regions is suggestive possible interactions. The overlap is really minimal and in zones of clustering. So the comment is far from accurate. A proper colocalization analysis in TIRF_dSTORM images of INPP5E and TCRz quantified by Pearson correlation would be much more appropriate and accurate.

   By the way, the authors could use panel F of T cells transfected with Flag-INPP5E that relocalizes to the synapse to say that INPP5E in T cells relocalizes to the synapse.

   1. Fig 4A: The strongest interactor with INPP5E seems to be Lck, rather than TCRz. It would be interesting to also assess the effect of INPP5E silencing on Lck recruitment at the synapse.

   Is there a mistake in labeling IP in horizontal and IP in vertical. I guess one of them should be IB (immunoblotted / Western blot). Please clarify and correct if necessary. Same in B, there is labelled IP-Flag everywhere, is one of them input? Please clarify/correct if mistaken.

   The term INPP5E "interacted" with TCRz, ZAP and Lck in the text (line 168-169) is not fully correct here, since these molecules make complexes during TCR activation. The term "co-immunoprecipitated" would be more accurate here.

   Fig 4D Not clear here why the authors use cells transfected with TCRz-GFP while to conclude that INPP5E is required for exogenous CD3z clustering, they could just stain for endogenous TCR.

   Fig 6B: If the authors normalized the pProtein band density with respect to the total same protein, the Y axis should be expressed as band density ratio rather than "optical intensity (a.u.)"

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2. 

   Version published to 10.1101/2021.01.06.425541 on bioRxiv

   Jan 7, 2021