Two-Dimensional Label-Free Affinity Analysis of Tumor-Specific CD8 T Cells with a Biomimetic Plasmonic Sensor

Maria Soler; Xiaokang Li; Aurelian John-Herpin; Julien Schmidt; George Coukos; Hatice Altug

doi:10.1021/acssensors.8b00523

Label Free Detection of CD4+ and CD8+ T Cells Using the Optofluidic Ring Resonator

Sensors ◽

10.3390/s100605798 ◽

2010 ◽

Vol 10 (6) ◽

pp. 5798-5808 ◽

Cited By ~ 34

Author(s):

John T. Gohring ◽

Xudong Fan

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ring Resonator ◽

Label Free ◽

Label Free Detection

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522 Metabolic requisites for T cell protein translation in tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0522 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A558-A558

Author(s):

Katie Hurst ◽

Megan Tennant ◽

Alex Andrews ◽

Lee Leddy ◽

David Neskey ◽

...

Keyword(s):

T Cells ◽

Protein Synthesis ◽

Glucose Metabolism ◽

Solid Tumors ◽

Cd8 T Cells ◽

Antitumor Immunity ◽

Protein Translation ◽

Cell Protein ◽

Label Free

BackgroundT cells are a secretory immune subset with the capacity to control solid tumors. Protein translation is of paramount importance in CD8 T cells, controlling proliferation, stimulation and lineage fate.MethodsHerein, we used both the fluorescent analogue of methionine homopropargylglycine (HPG) incorporation assay and O-propargyl-puromycin (OPP) method which enters the A-site of the ribosome and effectively labels and terminates nascent polypeptide chains to monitor protein synthesis in mouse and human tumors. Moreover, we employed label free quantitative proteomics (LFQ), lipidomics, metabolic analysis, and in vivo animal modeling to elucidate mechanisms of protein translation in antitumor immunity.ResultsWe found that canonical protein synthesis is restricted in endogenous CD8 tumor infiltrating lymphocytes (TILs) by the tumor microenvironment (TME). Proteomic analysis revealed that gluconeogenesis and B-oxidation of fatty acids (FAO) were upregulated in CD8 T cells under tumor stress but these metabolic sources were unable to support translation in the TME. Further, we discovered that glucose metabolism and mammalian target of rapamycin complex 1 (mTORC1) preferentially hinder protein synthesis in CD8 TILs. These data enabled the discovery that proteasomal protein degradation is the optimal source to fuel protein translation in T cells in the stress of solid tumors. We demonstrate that Rapamycin-primed T cells are preferentially powered by proteasomal proteolysis and are able to sustain protein translation in tumors and control tumor growth.ConclusionsOur data establish that canonical protein translation governed by mTORC1 and glucose metabolism is subject to inhibition in the TME and promotion of protein catabolism is a new strategy to support antitumor immunity.Ethics ApprovalAll animal experiments were in accordance with the MUSC Institutional Animal Care and Use Committee (IACUC), protocol # IACUC-2018-00422 and # IACUC-2018-00347

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Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4914-4922.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4914-4922

Author(s):

T Katagiri ◽

J P Ting ◽

R Dy ◽

C Prokop ◽

P Cohen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Tyrosine Phosphorylation ◽

Cd8 T Cells ◽

Fold Increase ◽

Cell Receptor ◽

Proteolytic Cleavage ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.

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Tyrosine phosphorylation of a c-Src-like protein is increased in membranes of CD4- CD8- T lymphocytes from lpr/lpr mice.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4914 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4914-4922 ◽

Cited By ~ 23

Author(s):

T Katagiri ◽

J P Ting ◽

R Dy ◽

C Prokop ◽

P Cohen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Tyrosine Phosphorylation ◽

Cd8 T Cells ◽

Fold Increase ◽

Cell Receptor ◽

Proteolytic Cleavage ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis

Mice homozygous for the autosomal recessive lpr gene have a disorder that results in autoimmunity and massive accumulation of T lymphocytes lacking CD4 and CD8 surface markers. These abnormal T cells exhibit constitutive tyrosine phosphorylation of a component of the CD3-T-cell receptor complex. We compared membrane tyrosine phosphorylation in lpr/lpr CD4- CD8- T cells and control T cells, lpr membranes exhibited a 7.3-fold increase (n = 16) in tyrosine phosphorylation of a 60-kilodalton protein. The increase was correlated with the Lpr but not the CD4- CD8- phenotype in that p60 phosphorylation was not increased in membranes from normal CD4- CD8- thymocytes. To identify the p60 in lpr cells, we examined the activity of several T-cell tyrosine-specific protein kinases. p56lck phosphorylation was only slightly increased in lpr membranes (2.2-fold; n = 16). Phorbol ester treatment of intact T cells before membrane isolation caused p56lck to migrate as pp60lck; however, pp60lck could be clearly distinguished from the pp60 in lpr cells by two-dimensional gel electrophoresis. The pp60 from lpr cells exhibited several isoforms at pH approximately 6.3 to 6.5. Although on two-dimensional gels pp60c-src had a pI (6.4 to 6.8) within a similar region, p60c-src mRNA, protein, and kinase activities were not increased in lpr cells. In addition, staphylococcal V8 proteolytic cleavage of the lpr pp60 isolated on two-dimensional gels yielded two major fragments, a pattern distinct from that of pp60c-src. However, by using an antiserum against the C-terminal sequence of c-Src and other related kinases, including p59fyn, the pp60 could be immunoprecipitated in greater amounts from lpr than from control T cells. When pp59(fyn) was selectively immunoprecipitated from T-cell membranes with specific antisera, its molecular weight, proteolytic cleavage pattern, and behavior on two-dimensional gels were identical to those of the pp60 from lpr cells. We conclude that p59(fyn) phosphorylation is increased in membranes from lpr/lpr CD4(-) CD8(-) T cells and that the increase is correlated with constitutive tyrosine phosphorylation and perhaps with the expansion of this unusual T-cell population.

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