The Transmembrane Adaptor Protein LIME Is Essential for Chemokine-Mediated Migration of Effector T Cells to Inflammatiory Sites
Authors: Eunseon Ahn
Young-Woong Kim
Myoungsun Son
Inyoung Park
Yungdae Yun
Young-Yun Kong
Journal: Molecules and Cells (2020)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Lck-interacting transmembrane adaptor 1; chemokine; effector T cells; migration.
Affiliations: Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 03760, Korea., Department of Life Science, Ewha Womans University, Seoul 03760, Korea., School of Biological Sciences, Seoul National University, Seoul 08826, Korea.

Abstract:  Lck-interacting transmembrane adaptor 1 (LIME) has been previously identified as a raft-associated transmembrane protein expressed predominantly in T and B lymphocytes. Although LIME is shown to transduce the immunoreceptor signaling and immunological synapse formation via its tyrosine phosphorylation by Lck, a Src-family kinase, the in vivo function of LIME has remained elusive in the previous studies. Here we report that LIME is preferentially expressed in effector T cells and mediates chemokine-mediated T cell migration. Interestingly, in LIME-/- mice, while T cell receptor stimulation-dependent proliferation, differentiation to effector T cells, cytotoxic T lymphocyte (CTL) function and regulatory T lymphocyte (Treg) function were normal, only T cell-mediated inflammatory response was significantly defective. The reduced inflammation was accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites. 

Retracted: No

PubPeer comments:
 Dear Dr. Park and Dr. Yun, We have with great interest and excitement discussed your paper u201cThe Transmembrane Adaptor Protein LIME Is Essential for Chemokine-Mediated Migration of Effector T Cells to Inflammatory Sitesu201d during our weekly literature meeting. Recently we have started to use the PubPeer platform as a means to engage with the authors of papers we have discussed. This is particularly important now during the pandemic, since we are all unable to meet in person at scientific meetings. We too consider adaptor proteins to be an interesting component of membrane-proximal signalling. For TRAPs as well as other adaptor proteins, it has often been challenging to elucidate their functions, as you mentioned in the paper. In particular, in vitro observations of adaptor proteins are often difficult to replicate in vivo, as is the case for LIME. However, in this paper, you manage to show the importance of LIME for chemokine responses in T cells. We appreciated that the study was conducted using various experimental approaches. Specifically, we liked that in certain microscopic experiments, WT and LIME-deficient cells were assayed together and distinguished by labelling with different dyes. After reading your paper, we were left wondering about a few things that we think would be interesting to see explored or commented, either here, or in future publications: 1.tAs mentioned in the paper, LIME is expressed in both B and T lymphocytes, however, is it also expressed in other immune cell types or in non-hematopoietic cells? Are functions for LIME described in those cell types? This may be relevant to address, as the LIME-deficient mice were not conditional knockouts. For instance, the observations made in the EAE model, could it have been explained by altered B cell activity mediated by LIME? 2.tYou show that LIME may be implicated in chemokine signalling and consequently T cell migration, suggesting that LIME may be activated downstream of chemokine receptors. However, are there other known co-receptors that may activate LIME? Also, what are your thoughts on the mechanism of LIME in terms of migration in T cells? Do you expect it to be involved in membrane proximal-signalling or could it also directly be involved in mediating molecular processes of migration? 3.tIn Fig. 3, you report the cytokine production and cytolytic activity of CD4 and CD8 T cells, respectively and you conclude here that LIME is dispensable in these activities. Several adaptor proteins are regarded as dispensable or redundant in many studies, however we think that describing the role of these molecules as redundant should be done with caution. Although the assays performed here were carried out with standard differentiation and stimulation conditions, it may be that another type of stimulation combined with co-receptor stimulation is required to observe differences in the phenotype between WT and LIME-deficient T cells. As for the CD8 T cells, you use LCMV infection to analyse cytolytic capacity, but could it be that another type of immune challenge elicits a more important role for LIME in T cell signalling? 4.tContinuing with the LCMV infection model, did you analyse the immune cell populations in the spleen or liver of LCMV infected LIME-deficient mice? If so, were the Th1 CD4 and CD8 T cell numbers comparable to that of WT infected mice and similar to those observed in the in vitro assays? If not, this may be interesting to investigate and might support your other observations. 5.tIn Fig. 6D you presented a differential phosphorylation signalling between LIME WT and LIME KO T cells stimulated for a short time with CXCL10. What is your interpretation or molecular hypothesis of the differential kinetics of Pyk2 and Cas phosphorylation? Additionally, if you observed changes in phosphotyrosine signalling after such a short time, it suggests that LIME should have been involved in the cascade. For adaptor proteins activation usually means a post-translational modification (in this case, phosphorylation of tyrosines). It would strongly support your data if you could show that LIME is phosphotyrosylated upon CXCL10 stimulation. As your LIME antibody is polyclonal, the best way to do it would be through anti-pY immunoprecipitation followed by immunoblotting with anti-LIME antibody. We would greatly appreciate it if you could share your thoughts on our comments above, as this may aid us (and other readers) in better understanding your work and thus help us all contribute to a fruitful scientific discussion. Sincerely, Hanna Chan (on behalf of our weekly journal club) ,