LFA-1 signals to promote actin polymerization and upstream migration in T cells

Background

Migration of leukocytes from the vasculature into peripheral tissue is central to their role in fighting pathogens, promoting tissue repair, and attacking solid tumors. Leukocyte entry into inflamed tissue is a multi-step process that requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require integrins expressed on leukocytes to interact with their endothelial ligands. For T cell migration, LFA-1 and VLA-4 must interact with the endothelial ligands ICAM-1 and VCAM-1, respectively. Intravital microscopy has previously shown that T cells preferentially transmigrate against the direction of shear flow, which is surprising given the extra energy required for this process (1). However, in vitro studies have supported this observation, suggesting a link between migration against the direction of flow, and transmigration across the endothelium (2,3).

A striking complementary observation however, is that T cells migrating on ICAM-1-coated surfaces preferentially migrate against the direction of flow (upstream) while T cells migrating on VCAM-1-coated surfaces preferentially migrate with the direction of shear flow (downstream) (2-8), suggesting that these two ligands trigger distinct cellular responses, and are essential for directing leukocyte migration. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored.

Biophysical models have proposed that T cell migration on ICAM-1 and VCAM-1 is based on morphological changes induced on the leading or the back edges of the cell, favouring upstream or downstream migration (6). In their work, Roy et al  analyzed T cell migration and signaling in response to binding ICAM-1 or VCAM-1, to determine how these different integrin ligands control T cell migration (9).

Key findings and developments

Using primary mouse T cells, the authors confirmed previous observations (2-8) that T cells preferentially migrate against the direction of shear on ICAM-1 (upstream) and with the direction of shear on VCAM-1 (downstream). T cells on mixed surfaces displayed a phenotype similar to those on ICAM-1 alone, showing that ICAM-1 ligation induces the dominant phenotype.

The authors then went on to test whether upstream/downstream migration could be the result of large differences in LFA-1 or VLA-4 expression levels, or the ability of different integrins to promote firm adhesion. They showed that both proteins are significantly expressed on the T cell surface following activation, and that VCAM-1 supports more robust adhesion than ICAM-1, while mixed surfaces result in tighter adhesion than VCAM-1 alone. These assays allowed the authors to conclude that neither differences in integrin expression, nor adhesive patterns explain the upstream migration observed in T cells.

The authors then analysed the shape and behaviour of migrating T cells. They showed that T cells responded to ICAM-1 by spreading and forming an actin-rich leading edge (with increase in actin polymer), while they responded to VCAM-1 by forming an elongated shape, and a narrower leading edge which lacked robust F-actin accumulation. As expected, in mixed surfaces, the cellular response was indistinguishable from ICAM-1-coated surfaces. Live imaging on LifeAct-GFP cells supported these observations, and showed that T cells migrating on ICAM-1- and mixed surfaces migrated significantly faster and more directionally than T cells on VCAM-1. Moreover, on ICAM-1, T cells displayed smooth movement, while on VCAM-1 they showed only periodic bursts of movement which coincided with brief actin flares at the front of the cell. These data suggest that ICAM-1 triggers specific signaling events that lead to these responses.

Recent work by the authors had indicated that Crk adapter proteins (a family of 3 proteins) coordinate signalling that contributes to the cellular response to ICAM-1. To test this hypothesis, in the present work, the authors imaged WT and DKO T cells (T cells lacking all Crk proteins) in the presence of shear flow, and found that DKO T cells were unable to migrate upstream on ICAM-1, while they had no defect in downstream migration on VCAM-1. This suggests that Crk-dependent LFA-1 signaling drives T cell spreading, actin responses, and ultimately, upstream migration. The authors measured the activation state of PI3K and ERK pathways in T cells, as well as the singling intermediates CasL, cCb1 and Pyk2 in response to either ICAM-1 or VCAM-1. They showed that ICAM-1 stimulated robust phosphorylation of AKT, ERK, and cCbI, while VCAM-1 did not elicit such strong activation. Meanwhile, phosphorylation of CasL and Pyk2 was equally efficient after engagement of VCAM-1 and ICAM-1. This reinforced the suggestion that downstream signaling from LFA-1 and VLA-4 indeed, differs.

The authors then went on to test if and how specific signaling events downstream of LFA-1 determine upstream migration. For this, the authors implemented a CRISPR KO system to generate mutants for cCbI and PIK3δ, and analysed cell migration under static and flow conditions. They found that T cells expressing the PI3Kδ gRNA remained capable of migrating upstream on ICAM-1, while T cells expressing the cCbl gRNA failed to migrate upstream. Taken together, this study demonstrates novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behaviour.

What I like about this preprint

I like this preprint because it explores from various angles, a question which is important for many fields of research, namely immunology, parasitology, and cell biology to name a few. The authors explored the question of T cell migration from a cell biology point of view and a biophysics point of view. I think interdisciplinary research brings about interesting answers, and opens a whole new set of questions. I also like that the work is robust and concise, and easy to follow.

Open questions

1. You specifically used primary T cells for your work. Did you expect/observe overall differences in migration and overall behavior when compared to cell lines?
2. Inflammation causes important changes both to endothelial cells, chemokine presence, blood flow, and blood composition. How do you think these factors further contribute to the phenotype you observe related to T cell migration?
3. Upon inflammation not all cells will transmigrate a tissue. Based on your work, do you think the definitive decision to transmigrate or not, is guided to a large extent by the LFA1/VLA4 interaction with ICAM1/VCAM1?
4. These receptors in particular, have been shown to be relevant in the context of pathology, including tumour metastasis and parasite binding (eg. Plasmodium). Could the cell biological findings you did in your work help us understand better phenomena in the context of pathology?
5. This might be a naïve question, but are interactions of T cells with ICAM1/VCAM1 equal in all organs? Would equal rates of transmigration be observed in all organs during an inflammatory process?
6. Is there a minimum level of ICAM1 or LFA1 needed for upstream migration to occur?
7. Can DKO T cells still transmigrate, even though they cannot move in an upstream direction?
8. How do events happening before and after ICAM1 binding aid the process of upstream migration and/or transmigration? Namely, in a surface with high ICAM1 presence, is rolling adhesion (selectin-mediated), arrest and transmigration equal to surfaces with low ICAM1 presence?

References

1. Bartholomaus I et al. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature. 2009.
2. Steiner O et al. Differential roles for endothelial ICAM1, ICAM2 and VCAM1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium, Immunol. 2010.
3. Anderson NR et al. T lymphocytes migrate upstream after completing the leukocyte adhesion cascade. Cell Adh Migr.
4. Valignat MP et al. T lymphocytes orient against the direction of fluid flow during LFA1 mediated migration, Biophys J.
5. Valignat MP et al. Lymphocytes can self-steer passively with wind vane uropods, Nature Communications.
6. Hornung A et al. A bistable mechanism mediated by integrins controls mechanotaxis of leukocytes, Biophys J,
7. Dominguez GA et al. The direction of migration of T-lymphocytes under flow depends upon which adhesion receptors are engaged. Integr Biol, 2015.
8. Kim SHJ, et al. Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flow. Biol. 2019.
9. Roy NH et al. LFA1 signals to promote actin polymerization and upstream migration in T cells. 2020.