Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions
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eLife Assessment
This valuable work investigates the role of protein N-glycosylation in regulating T-cell activation and function and suggests that B4GALT1 is a potential target for tumor immunotherapy. The strength of evidence is solid, and further mechanistic validation could be provided.
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Abstract
Cytotoxic CD8+ T-cells play central roles in tumor immunotherapy. Understanding mechanisms that regulate development, differentiation, and functions of cytotoxic CD8+ T-cells leads to development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8+ T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1, 4-galatosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8+ T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 CD8+ T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8+ T-cells. Finally, the expression levels of B4GALT1 normalized to tumor infiltrated CD8+ T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8+ T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.
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eLife Assessment
This valuable work investigates the role of protein N-glycosylation in regulating T-cell activation and function and suggests that B4GALT1 is a potential target for tumor immunotherapy. The strength of evidence is solid, and further mechanistic validation could be provided.
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Reviewer #1 (Public review):
Summary:
The study by Yu et al investigated the role of protein N-glycosylation in regulating T-cell activation and functions is an interesting work. By using genome-wide CRISPR/Cas9 screenings, the authors found that B4GALT1 deficiency could activate expression of PD-1 and enhance functions of CD8+ T cells both in vitro and in vivo, suggesting the important roles of protein N-glycosylation in regulating functions of CD8+ T cells, which indicates that B4GALT1 is a potential target for tumor immunotherapy.
Strengths:
The strengths of this study are the findings of novel function of B4GALT1 deficiency in CD8 T cells.
Weaknesses:
However, authors did not directly demonstrate that B4GALT1 deficiency regulates the interaction between TCR and CD8, as well as functional outcomes of this interaction, such as TCR …
Reviewer #1 (Public review):
Summary:
The study by Yu et al investigated the role of protein N-glycosylation in regulating T-cell activation and functions is an interesting work. By using genome-wide CRISPR/Cas9 screenings, the authors found that B4GALT1 deficiency could activate expression of PD-1 and enhance functions of CD8+ T cells both in vitro and in vivo, suggesting the important roles of protein N-glycosylation in regulating functions of CD8+ T cells, which indicates that B4GALT1 is a potential target for tumor immunotherapy.
Strengths:
The strengths of this study are the findings of novel function of B4GALT1 deficiency in CD8 T cells.
Weaknesses:
However, authors did not directly demonstrate that B4GALT1 deficiency regulates the interaction between TCR and CD8, as well as functional outcomes of this interaction, such as TCR signaling enhancements.
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Reviewer #2 (Public review):
Summary:
In this study, the authors identify the N-glycosylation factor B4GALT1 as an important regulator of CD8 T-cell function.
Strengths:
(1) The use of complementary ex vivo and in vivo CRISPR screens is commendable and provides a useful dataset for future studies of CD8 T-cell biology.
(2) The authors perform multiple untargeted analyses (RNAseq, glycoproteomics) to hone their model on how B4GALT1 functions in CD8 T-cell activation.
(3) B4GALT1 is shown to be important in both in vitro T-cell killing assays and a mouse model of tumor control, reinforcing the authors' claims.
Weaknesses:
(1) The authors did not verify the efficiency of knockout in their single-gene KO lines.
(2) As B4GALT1 is a general N-glycosylation factor, the phenotypes the authors observe could formally be attributable to indirect …
Reviewer #2 (Public review):
Summary:
In this study, the authors identify the N-glycosylation factor B4GALT1 as an important regulator of CD8 T-cell function.
Strengths:
(1) The use of complementary ex vivo and in vivo CRISPR screens is commendable and provides a useful dataset for future studies of CD8 T-cell biology.
(2) The authors perform multiple untargeted analyses (RNAseq, glycoproteomics) to hone their model on how B4GALT1 functions in CD8 T-cell activation.
(3) B4GALT1 is shown to be important in both in vitro T-cell killing assays and a mouse model of tumor control, reinforcing the authors' claims.
Weaknesses:
(1) The authors did not verify the efficiency of knockout in their single-gene KO lines.
(2) As B4GALT1 is a general N-glycosylation factor, the phenotypes the authors observe could formally be attributable to indirect effects on glycosylation of other proteins.
(3) The specific N-glycosylation sites of TCR and CD8 are not identified, and would be helpful for site-specific mutational analysis to further the authors' model.
(4) The study could benefit from further in vivo experiments testing the role of B4GALT1 in other physiological contexts relevant to CD8 T cells, for example, autoimmune disease or infectious disease.
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Author response:
Reviewer #1 (Public review):
Summary:
The study by Yu et al investigated the role of protein N-glycosylation in regulating T-cell activation and functions is an interesting work. By using genome-wide CRISPR/Cas9 screenings, the authors found that B4GALT1 deficiency could activate expression of PD-1 and enhance functions of CD8+ T cells both in vitro and in vivo, suggesting the important roles of protein N-glycosylation in regulating functions of CD8+ T cells, which indicates that B4GALT1 is a potential target for tumor immunotherapy.
Strengths:
The strengths of this study are the findings of novel function of B4GALT1 deficiency in CD8 T cells.
Weaknesses:
However, authors did not directly demonstrate that B4GALT1 deficiency regulates the interaction between TCR and CD8, as well as functional outcomes of this …
Author response:
Reviewer #1 (Public review):
Summary:
The study by Yu et al investigated the role of protein N-glycosylation in regulating T-cell activation and functions is an interesting work. By using genome-wide CRISPR/Cas9 screenings, the authors found that B4GALT1 deficiency could activate expression of PD-1 and enhance functions of CD8+ T cells both in vitro and in vivo, suggesting the important roles of protein N-glycosylation in regulating functions of CD8+ T cells, which indicates that B4GALT1 is a potential target for tumor immunotherapy.
Strengths:
The strengths of this study are the findings of novel function of B4GALT1 deficiency in CD8 T cells.
Weaknesses:
However, authors did not directly demonstrate that B4GALT1 deficiency regulates the interaction between TCR and CD8, as well as functional outcomes of this interaction, such as TCR signaling enhancements.
We are very sorry that we did not highlight our results in Fig. 5f-h enough. In those figures, we demonstrated the interaction between TCR and CD8 increased significantly in B4GALT1 deficient T-cells, by FRET assays. To confirm the important role of TCR-CD8 interaction in mediating the functions of B4GALT1 in regulating T-cell functions, such as in vitro killing of target cells, we artificially tethered TCR and CD8 by a CD8β-CD3ε fusion protein and tested its functions in both WT and B4GALT1 knockout CD8+ T-cell. Our results demonstrate that such fusion protein could bypass the effect of B4GALT1 knockout in CD8+T-cells (Fig. 5g-h). Together with the results that B4GALT1 directly regulates the galactosylation of TCR and CD8, those results strongly support the model that B4GALT1 modulates T-cell functions mainly by galactosylations of TCR and CD8 that interfere their interaction.
Reviewer #2 (Public review):
Summary:
In this study, the authors identify the N-glycosylation factor B4GALT1 as an important regulator of CD8 T-cell function.
Strengths:
(1) The use of complementary ex vivo and in vivo CRISPR screens is commendable and provides a useful dataset for future studies of CD8 T-cell biology.
(2) The authors perform multiple untargeted analyses (RNAseq, glycoproteomics) to hone their model on how B4GALT1 functions in CD8 T-cell activation.
(3) B4GALT1 is shown to be important in both in vitro T-cell killing assays and a mouse model of tumor control, reinforcing the authors' claims.
Weaknesses:
(1) The authors did not verify the efficiency of knockout in their single-gene KO lines.
Thank reviewer for reminding. We verified the efficiency of some gRNAs by FACS and Surveyor assay. We will add those data in supplementary results in revised version later.
(2) As B4GALT1 is a general N-glycosylation factor, the phenotypes the authors observe could formally be attributable to indirect effects on glycosylation of other proteins.
please see response to reviewer #1.
(3) The specific N-glycosylation sites of TCR and CD8 are not identified, and would be helpful for site-specific mutational analysis to further the authors' model.
Thank reviewer for suggestion! Unfortunately, there are multiple-sites of TCR and CD8 involved in N-glycosylation (https://glycosmos.org/glycomeatlas). We worry that mutations of all these sites may not only affect glycosylation of TCR and CD8 but also other essential functions of those proteins.
(4) The study could benefit from further in vivo experiments testing the role of B4GALT1 in other physiological contexts relevant to CD8 T cells, for example, autoimmune disease or infectious disease.
Thank reviewer for this great suggestion to expand the roles of B4GALT1 in autoimmune and infection diseases. However, since in current manuscript we are mainly focusing on tumor immunology, we think we should leave these studies for future works.
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