Clathrin-independent endocytosis and retrograde transport in cancer cells promote cytotoxic CD8 T cell activation

Reviewer #1 (Public review):

Summary:

This study by Xu et al. focuses on the impact of clathrin-independent endocytosis in cancer cells on T cell activation. In particular, by using a combination of biochemical approaches and imaging, the authors identify ICAM1, the ligand for T cell-expressed integrin LFA-1, as a novel cargo for EndoA3-mediated endocytosis. Subsequently, the authors aim to identify functional implications for T cell activation, using a combination of cytokine assays and imaging experiments.

They find that the absence of EndoA3 leads to a reduction in T cell-produced cytokine levels. Additionally, they observe slightly reduced levels of ICAM1 at the immunological synapse and an enlarged contact area between T cells and cancer cells. Taken together, the authors propose a mechanism where EndoA3-mediated endocytosis of ICAM1, followed by retrograde transport, supplies the immunological synapse with ICAM1. In the absence of EndoA3, T cells attempt to compensate for suboptimal ICAM1 levels at the synapse by enlarging their contact area, which proves insufficient and leads to lower levels of T cell activation.

Strengths:

The authors utilize a rigorous and innovative experimental approach that convincingly identifies ICAM1 as a novel cargo for Endo3A-mediated endocytosis.

Weaknesses:

The characterization of the effects of Endo3A absence on T cell activation appears incomplete. Key aspects, such as surface marker upregulation, T cell proliferation, integrin signalling and most importantly, the killing of cancer cells, are not comprehensively investigated.

As Endo- and exocytosis are intricately linked with the biophysical properties of the cellular membrane (e.g. membrane tension), which can significantly impact T-cell activation and cytotoxicity, the authors should address this possibility and ideally address it experimentally to some degree.

Crucially, key literature relevant to this research, addressing the role of ICAM1 endocytosis in antigen-presenting cells, has not been taken into consideration.

Reviewer #2 (Public review):

Summary:

The manuscript by Xu et al. studies the relevance of endophilin A3-dependent endocytosis and retrograde transport of immune synapse components and in the activation of cytotoxic CD8 T cells. First, the authors show that ICAM1 and ALCAM, known components of immune synapses, are endocytosed via endoA3-dependent endocytosis and retrogradely transported to the Golgi. The authors then show that blocking internalization or retrograde trafficking reduces the activation of CD8 T cells. Moreover, this diminished CD8 T cell activation resulted in the formation of an enlarged immune synapse with reduced ICAM1 recruitment.

Strengths:

The authors show a novel EndoA3-dependent endocytic cargo and provide strong evidence linking EndoA3 endocytosis to the retrograde transport of ALCAM and ICAM1.

Weaknesses:

The role of EndoA3 in the process of T cell activation is shown in a cell that requires exogenous expression of this gene. Moreover, the authors claim that their findings are important for polarized redistribution of cargoes, but failed to show convincingly that the cargoes they are studying are polarized in their experimental system. The statistics of the manuscript also require some refinement.

Reviewer #3 (Public review):

Summary:

Shiqiang Xu and colleagues have examined the importance of ICAM-1 and ALCAM internalization and retrograde transport in cancer cells on the formation of a polarized immunological synapse with cytotoxic CD8+ T cells. They find that internalization is mediated by Endophilin A3 (EndoA3) while retrograde transport to the Golgi apparatus is mediated by the retromer complex. The paper is building on previous findings from corresponding author Henri-François Renard showing that ALCAM is an EndoA3-dependent cargo in clathrin-independent endocytosis.

Strengths:

The work is interesting as it describes a novel mechanism by which cancer cells might influence CD8+ T cell activation and immunological synapse formation, and the authors have used a variety of cell biology and immunology methods to study this. However, there are some aspects of the paper that should be addressed more thoroughly to substantiate the conclusions made by the authors.

Weaknesses:

In Figure 2A-B, the authors show micrographs from live TIRF movies of HeLa and LB33-MEL cells stably expressing EndoA3-GFP and transiently expressing ICAM-1-mScarlet. The ICAM-1 signal appears diffuse across the plasma membrane while the EndoA3 signal is partially punctate and partially lining the edge of membrane patches. Previous studies of EndoA3-mediated endocytosis have indicated that this can be observed as transient cargo-enriched puncta on the cell surface. In the present study, there is only one example of such an ICAM-1 and EndoA3 positive punctate event. Other examples of overlapping signals between ICAM-1 and EndoA3 are shown, but these either show retracting ICAM-1 positive membrane protrusions or large membrane patches encircled by EndoA3. While these might represent different modes of EndoA3-mediated ICAM-1 internalization, any conclusion on this would require further investigation.

Moreover, in Figure 2C-E, uptake of the previously established EndoA3 endocytic cargo ALCAM is analyzed by quantifying total internal fluorescence in LB33-MEL cells of antibody labelled ALCAM following both overexpression and siRNA-mediated knockdown of EndoA3, showing increased and decreased uptake respectively. Why has not the same quantification been done for the proposed novel EndoA3 endocytic cargo ICAM-1? Furthermore, if endocytosis of ICAM-1 and ALCAM is diminished following EndoA3 knockdown, the expression level on the cell surface would presumably increase accordingly. This has been shown for ALCAM previously and should also be quantified for ICAM-1.

In Figure 4A the authors show micrographs from a live-cell Airyscan movie (Movie S6) of a CD8+ T cell incubated with HeLa cells stably expressing HLA-A*68012 and transiently expressing ICAM1-EGFP. From the movie, it seems that some ICAM-1 positive vesicles in one of the HeLa cells are moving towards the T cell. However, it does not appear like the T cell has formed a stable immunological synapse but rather perhaps a motile kinapse. Furthermore, to conclude that the ICAM-1 positive vesicles are transported toward the T cell in a polarized manner, vesicles from multiple cells should be tracked and their overall directionality should be analyzed. It would also strengthen the paper if the authors could show additional evidence for polarization of the cancer cells in response to T-cell interaction.

Finally, in Figures 4D-G, the authors show that the contact area between CD8+ T cells and LB33-MEL cells is increased in response to siRNA-mediated knockdown of EndoA3 and VPS26A. While this could be caused by reduced polarized delivery of ICAM-1 and ALCAM to the interface between the cells, it could also be caused by other factors such as increased cell surface expression of these proteins due to diminished endocytosis, and/or morphological changes in the cancer cells resulting from disrupted membrane traffic. More experimental evidence is needed to support the working model in Figure 4H.