Noncanonical binding of Lck to CD3Œµ promotes TCR signaling and CAR function
Authors: Esmeralda Beck-Garc√¨a
Gina J Fiala
Pankaj Mishra
Nico Hoefflin
Kristian Schweimer
Maja K√∂hn
Anna Morath
Julia Zimmermann
Simon M Brandl
Lea J Flachsmann
Wolfgang W Schamel
Susana Minguet
Sanaz Taromi
Frederike A Hartl
O Sascha Yousefi
Rub√≠ M-H Velasco C√°rdenas
Robert Zeiser
Birgitta M W√∂hrl
Stefan G√ºnther
Nadine M Woessner
Journal: Nature Immunology (2020)
Discipline: IMMUNOLOGY
Keywords: T cell antigen receptor; CD3 cytoplasmic tails; Lck; RK motif; SH3 domain
Affiliations: Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany., Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany., Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, Freiburg, Germany., Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Freiburg, Germany., Department of Medicine I, Medical Center, University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany., Faculty of Medical and Life Sciences, Furtwangen University, Campus VS-Schwenningen, Furtwangen, Germany., Pharmaceutical Bioinformatics, Albert-Ludwigs-University of Freiburg, Freiburg, Germany., Biopolymers, University of Bayreuth, Bayreuth, Germany., Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany. ., Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany. ., Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, Freiburg, Germany. .

Abstract:  Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3Œµ cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer. 

Retracted: No

PubPeer comments:
 Dear Authors, We have read with great interest and discussed your paper u201cNoncanonical binding of Lck to CD3e promotes TCR signaling and CAR functionu201d during our weekly literature meeting. However, as we have noticed a few things that we would have liked to see explained (or corrected) in future publications, we permit ourselves to present some suggestions for improvements: General comments: 1.tAlthough, the figures are readable and informative, we have spotted a few recurring improvement points: a.tCell types used for the experiment could have been named in the figure panels. b.tPLA staining and ligation targets should have been described in the figures. c.tImmunoblots could have been framed to improve visibility (for example in ext. data fig. 2d it is impossible to tell if blots on the right are empty or absent). d.tNot all pull-downs are labelled in the figures (for example in fig. 2a and b pull-downs are not labelled, while in fig. 2c it is named Lck(SH3)-PD). e.tNumber of replicates are not indicated (in the figure description) in several experiments. If the experiments were done only once, it should be repeated or should have been commented on. It is especially important for peptide pull-downs, where it is impossible to monitor the level of bait (peptides) in the sample. f.tIn fig. 4e and f, u201c-u201c symbols should have been written in all appropriate places (above all samples that have not been stimulated with the given reagent) or not written at all. g.tIn fig. 6b and 6e, the quadrants could have been labelled with population names used in the figure description (i.e. fig. 6b: DN, DP, CD8, CD4 and fig 6e: DN1, DN2, DN3, DN4) to facilitate reading. h.tGraphs in fig. 7f and g are unnecessarily sophisticated which makes it difficult to compare markers across samples. i.tSome immunoblots in the extended data figures lack indication of the molecular size. 2.tThe number of cells quantified in PLA experiments is not stated anywhere. 3.tShowing data as mean with SEM is not wrong, but it is not a really honest way of data presentation either. It would be better for the reader to judge results by seeing all the data points. Presenting medians with range would have been better. 4.tWhy are CD3udb40udd64u03b6 and CD3u03b5 shown to have different molecular size on different blots? (for example in fig. 2c CD3udb40udd64u03b6 is indicated at 34 kDa, in fig. 5a at 15 kDa, and in fig. 4b at 17+ kDa). As both molecules size strongly depend on post-translational modifications, it is advised to always present immunoblots ranging from 15 to 35 kDa. 5.tFrom our extensive IP experience, we have learnt that experiment presented in fig. 5a is most likely not reliable. Firstly, performing antibody-based pull down, one has to expect finding them also in the western blot samples. The light chains appear at 25kDa, heavy chains appear at 50kDa and some other forms of degradation appear at higher molecular size (for example 75kDa). They are so abundant, that even using light-chain specific secondary antibodies is not helping the specific signal to emerge from underneath. Therefore, probing for co-IP molecules at those sizes, in any standard-antibody based IP samples, is doomed. The only exception is the bait itself u2013 direct antibody target is usually abundant enough to give a reliable signal. We have recently tested nanobodies to overcome this problem and it worked very well. However, the nanobodies choice is extremely poor, which strongly decreases their usefulness. Secondly, we have observed that the antibody used for activation prior to lysis can also affect the IP. Protein G based IP will pulldown any IgG present in the sample (with varying efficiency). For that sake, we are using only IgM C305 for stimulation of Jurkat cells prior to IP. Therefore, there is a high chance that the results observed in the fig. 5a is actually 2C11+4G10 IP. A good control that helps judging on that kind of problems is a sample that contains only the beads covered with abs. If the pattern of unspecific bands is similar (not necessarily of equal strength, though) to any strong signal present in other samples, it should be a warning sign that it could be unspecific (coming from abs). 6.tIt would be interesting to see the authorsu2019 explanation hypothesis about the lack of observed exhaustion in their novel CAR T cells. Specific comments: 1.tFig. 4c is unnecessary and could be wrongly interpreted. Firstly, as the maximum phosphorylation is not known, it should not be called 100% phosphorylation. Secondly, presenting side by side results from two antibodies creates a false impression that they can be compared to each other. None two antibodies can be compared to each other directly as their signal depends on their individual specificity, which does not equal to absolute antigen concentration. Thirdly, fig. 4d presents the same data in a much better fashion. If authors wanted to preserve the increased phosphorylation of Lck pY394 in steady state, they could have divided pY signal by the signal from the total amount of Lck protein detected (it probably could have been measured with anti-CD3u03b6 antibody at 43 kDa size of the reporter construct) and present both u201c-u201c and u201c+u201d samples on the same graph. 2.tSequence conservation (in ext. data fig. 3a) could have been presented better. Firstly, there is a slight misalignment of the sequences, possibly due to use of non-monospaced font. Secondly, the conservation is not properly indicated (for example with color or highlighting). It would have been helpful if authors used Clustal to generate a real conservation alignment. 3.tIt seems that ext. data fig. 7g is missing SEM bars. , 
 This paper is very intriguing and the data is quite exciting! However, I found some points that somehow confused me. Some points overlap with #1 : Fig 2c. As I understand, u03b6 can be 17-23 kDa due to different ITAM phosphorylation in Jurkat cells...but how u03b6 is indicated at 34 kDa? Is it an exogenous u03b6? Fig 3b. In the figure legend, the authors showed the pooled data were from 4 independent experiments, but there are only 3 dots in the graph. Fig 3C. The authors showed that the binding between Lck SH3 domain and CD3u03b5 RKAA was abrogated upon CD3 stimulation. The PD western blot showed the RKAA group has a higher binding ability than the DOM group, but it s not that consistent with the graph data. Fig 4b. According to Fig 4e,f and Extended data 4h, pRep (Y394) should be about 95kDa, and it s labeled as 43kDa (Or maybe I misunderstood something). Fig 5a. A little bit confused about which bands should be u03b5 in the lysate panel? I m also wondering is it okay to have different levels of mutated u03b5 in the input and used them to compare their phosphorylation. Fig 6c. It is quite strange that a huge difference in DN DP groups only gets a star, and almost no difference in the CD8 group also gets a star. Fig 6d. is reasonable. Extended data Fig 4b,f, and h: some panels did not indicate the molecular weight. I , 
 Corrected [19 November 2020] .  In the version of this article initially published, in Fig. 4, the graph in panel f duplicated the graph in panel e. The correct Fig. 4e,f are shown below. The statement about data collection and analysis at the end of the Fig. 4 caption was also incorrect. The correct statement is u201cData from at least three independent experiments were pooled and analyzed using an ANOVA test.u201d The errors have been corrected in the HTML and PDF versions of the article.  ,