TIM‐3 and CEACAM1 do not interact in<i>cis</i>and in<i>trans</i>

Linhares, Annika De Sousa; Kellner, Florian; Jutz, Sabrina; Zlabinger, Gerhard J.; Gabius, Hans‐Joachim; Huppa, Johannes B.; Leitner, Judith; Schmitz, Peter

doi:10.1002/eji.201948400

Cited by 26 publications

(16 citation statements)

References 53 publications

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“…The Jurkat leukemic T-cell line is a widely used model system for the study of TCR function, 21 and we previously developed a triple parameter TCR signalling reporter cell line (TPR) based on the Jurkat line E6.1. 22 These reporter cells have been proven to be highly suitable to evaluate costimulatory pathways and the function of chimeric antigen receptors, [23][24][25] but to date, their potential to evaluate transgenically expressed TCRs in a high-throughput manner that still reflects physiological T-cell biology as seen in primary human T cells had not been tested. To facilitate highly sensitive and unbiased TCR functional characterisation, we introduced two additional modifications in the TPR cell line.…”

Section: Introductionmentioning

confidence: 99%

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

et al. 2020

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Objective Transgenic re‐expression enables unbiased investigation of T‐cell receptor (TCR)‐intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large‐scale transgenic TCR re‐expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re‐expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat‐based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read‐out of TCR functionality and facilitates high‐throughput screening approaches. Methods As a proof‐of‐concept, we transgenically re‐expressed 59 human cytomegalovirus‐specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells. Results We demonstrate that the TPRKO cell line facilitates antigen‐HLA specificity screening via sensitive peptide‐MHC‐multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co‐receptor. Conclusion Overall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high‐throughput investigation of TCR biology.

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Section: Introductionmentioning

confidence: 99%

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

et al. 2020

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“…Further, using various cellular, biochemical and biophysical methods (Gandhi et al, 2018;Haidar et al, 2019;Y.-H. Huang et al, 2016;Sabatos-Peyton et al, 2018;Zhang et al, 2020), we and others have demonstrated a conserved role of the GFCC' faces of hCEACAM1 and hTIM-3 in the heterodimerization between these two proteins (K D of ~ 2-3 μM) (Figure 7-figure supplement 12). Despite these findings, a recent paper (De Sousa Linhares et al, 2020) suggested a lack of appreciable binding of hCEACAM1 by hTIM-3. This is surprising and likely due to a number of factors, including use of incompletely characterized Fc fusion proteins that do not take into account the monomer-dimer equilibrium or structural state of the proteins used or consideration of the relative affinities of the CEACAM1 homodimer (450 nM) and CEACAM-TIM-3 heterodimer (2-3 M).…”

Section: Discussionmentioning

confidence: 94%

“…interactions with its various ligands, a note of caution given the recent studies of others (De Sousa Linhares et al, 2020) .…”

Section: Discussionmentioning

confidence: 99%

“…Further, there is an absence of titration experiments in the micromolar range to probe the binding and there are confounding results that show that the strongest ligand binding to the hCEACAM1-Ig used was with galectin-9 in the absence of galectin-9 binding to hTIM-3. Importantly, galectin-9 has never been described as a ligand for hCEACAM1 and numerous groups have unambiguously identified an interaction between hTIM-3 and galectin-9 (Cao et al, 2007;van de Weyer et al, 2006;Zhang et al, 2020;Zhu et al, 2005), raising important concerns about the interpretation of these and other results contained therein (De Sousa Linhares et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

Gandhi

Sun

Kim

et al. 2020

Preprint

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Human (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5 and hTIM-3, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC face including V39, I91, N97 and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A monomer. These studies reveal the flexibility of the GFCC face and its important role in governing the formation of hCEACAM1 dimers and potentially heterodimers.

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“…Furthermore, transplantation and peripheral tolerance was shown to be critically dependent on an undisturbed Tim-3 function [ 43 ]. Whether galectin-9 or CEACAM1 as Tim-3 ligands play essential interactive roles is still discussed controversially [ 44 – 47 ]. The putative role of Tim-3 for Treg cell function has been characterized by Wang et al .…”

Section: Discussionmentioning

confidence: 99%

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma

et al. 2021

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T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

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TIM‐3 and CEACAM1 do not interact incisand intrans

Cited by 26 publications

References 53 publications

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma

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