scholarly journals An optimized workflow for CRISPR-Cas9 deletion of surface and intracellular factors in primary human T lymphocytes

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247232
Author(s):  
Cristina Leoni ◽  
Niccolò Bianchi ◽  
Lucia Vincenzetti ◽  
Silvia Monticelli
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Gene Editing ◽  
Protein A ◽  
Surface Protein ◽  
T Cell Responses ◽  
Cell Responses ◽  
Human T Lymphocytes ◽  
Human T Cell ◽  
Non Coding Rna

The appropriate regulation of T lymphocyte functions is key to achieve protective immune responses, while at the same time limiting the risks of tissue damage and chronic inflammation. Deciphering the mechanisms underpinning T cell responses in humans may therefore be beneficial for a range of infectious and chronic diseases. Recently, the development of methods based on CRISPR-Cas9 gene-editing has greatly expanded the available tool-box for the mechanistic studies of primary human T cell responses. While the deletion of a surface protein has become a relatively straightforward task, as long as an antibody for detection is available, the identification and selection of cells lacking an intracellular protein, a non-coding RNA or a protein for which no antibody is available, remain more problematic. Here, we discuss the options currently available to scientists interested in performing gene-editing in primary human T lymphocytes and we describe the optimization of a workflow for the screening and analysis of lymphocytes following gene-editing with CRISPR-Cas9 based on T cell cloning and T7 endonuclease I cleavage assay.

Antigen-activated human T lymphocytes express cell-surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK- cell lysis

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 606-615 ◽  
Author(s):  
Cristina Cerboni ◽  
Alessandra Zingoni ◽  
Marco Cippitelli ◽  
Mario Piccoli ◽  
Luigi Frati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Cell Activation ◽  
Nk Cell ◽  
Protein A ◽  
T Cell Responses ◽  
Activated T Cells ◽  
Cell Responses

AbstractRecent evidence indicates that natural killer (NK) cells can negatively regulate T-cell responses, but the mechanisms behind this phenomenon as a consequence of NK–T-cell interactions are poorly understood. We studied the interaction between the NKG2D receptor and its ligands (NKG2DLs), and asked whether T cells expressed NKG2DLs in response to superantigen, alloantigen, or a specific antigenic peptide, and if this rendered them susceptible to NK lysis. As evaluated by FACS, the major histocompatibility complex (MHC) class I chain-related protein A (MICA) was the ligand expressed earlier on both CD4+ and CD8+ T cells in 90% of the donors tested, while UL16-binding protein-1 (ULBP)1, ULBP2, and ULBP3 were induced at later times in 55%–75% of the donors. By carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, we observed that NKG2DLs were expressed mainly on T cells that had gone through at least one division. Real-time reverse-transcription polymerase chain reaction confirmed the expression of all NKG2DLs, except ULBP4. In addition, T-cell activation stimulated phosphorylation of ataxia-telangiectasia mutated (ATM), a kinase required for NKG2DLs expression after DNA damage, and ATM/Rad3-related kinase (ATR) inhibitors blocked MICA induction on T cells with a mechanism involving NF-κB. Finally, we demonstrated that activated T cells became susceptible to autologous NK lysis via NKG2D/NKG2DLs interaction and granule exocytosis, suggesting that NK lysis of T lymphocytes via NKG2D may be an additional mechanism to limit T-cell responses.

A BCR-ABL Oncoprotein p210b2a2 Fusion Region Sequence Is Recognized by HLA-DR2a Restricted Cytotoxic T Lymphocytes and Presented by HLA-DR Matched Cells Transfected With an Iib2a2 Construct

Blood ◽  
1999 ◽  
Vol 94 (3) ◽  
pp. 1038-1045 ◽  
Author(s):  
George J.A. ten Bosch ◽  
Jan H. Kessler ◽  
Antonia M. Joosten ◽  
Alexandra A. Bres-Vloemans ◽  
Annemieke Geluk ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
T Cell Responses ◽  
Myelogenous Leukemia ◽  
Invariant Chain ◽  
Specific Cell ◽  
Double Positive ◽  
Cell Responses ◽  
Human T Cell ◽  
Hla Dr

Peptides corresponding to the fusion site in 210 kD BCR-ABL protein b3a2 (p210b3a2) were previously shown to bind to several HLA class I and II alleles. We have found that b3a2 peptide-specific CD4-positive T-helper cells were able to recognize p210b3a2-positive chronic myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until now, there were no reports of b2a2 breakpoint-specific human T-cell responses. Here we show that repetitive stimulation of T lymphocytes with a 17mer peptide covering the fusion region in p210b2a2 also leads to specific T-cell responses. CD4 and CD4/CD8 double-positive clones obtained from a b2a2 peptide-specific cell line were cytotoxic and proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous Epstein-Barr virus (EBV) transformed cells, expressing BCR-ABLb2a2 on transfection, and allogeneic HLA-DR matched p210b2a2-positive cells from CML patients were, however, not lysed. BCR-ABL peptide-specific T-cell clones did respond to autologous EBV cells transfected with invariant chain (li) cDNA in which the HLA class II–associated invariant chain peptide (CLIP) was replaced by a BCR-ABL b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABLb2a2ligand.

Human T-cell responses to oral streptococci in human PBMC-NOD/SCID mice

2006 ◽  
Vol 21 (3) ◽  
pp. 169-176 ◽  
Author(s):  
M. A. Salam ◽  
R. Nakao ◽  
H. Yonezawa ◽  
H Watanabe ◽  
H. Senpuku
Keyword(s):  
T Cell ◽  
T Cell Responses ◽  
Scid Mice ◽  
Oral Streptococci ◽  
Human Pbmc ◽  
Cell Responses ◽  
Human T Cell

scholarly journals Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells

2001 ◽  
Vol 94 (6) ◽  
pp. 807-812 ◽  
Author(s):  
Emma J. Davidson ◽  
Michael D. Brown ◽  
Deborah J. Burt ◽  
Joanna L. Parish ◽  
Kevin Gaston ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Hpv 16 ◽  
Cell Responses ◽  
Human T Cell

Human T cell responses to the antigen ESAT-6 characterize vaccine candidate and potential diagnostic test for tuberculosis

1999 ◽  
Vol 39 (1) ◽  
pp. A9
Author(s):  
A. Lalvani ◽  
A. Pathan ◽  
R. Brookes ◽  
H. Pritchard ◽  
R. Wilkinson ◽  
...  
Keyword(s):  
T Cell ◽  
Diagnostic Test ◽  
Vaccine Candidate ◽  
T Cell Responses ◽  
Cell Responses ◽  
Human T Cell

scholarly journals BothCD4+andCD8+Lymphocytes Participate in the IFN-γ Response to Filamentous Hemagglutinin fromBordetella pertussisin Infants, Children, and Adults

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Violette Dirix ◽  
Virginie Verscheure ◽  
Françoise Vermeulen ◽  
Iris De Schutter ◽  
Tessa Goetghebuer ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Bacterial Infections ◽  
T Cell Responses ◽  
Cell Types ◽  
Production Flow ◽  
Mhc I ◽  
Cell Responses ◽  
Filamentous Hemagglutinin ◽  
Circulating Lymphocytes

Infant CD4+T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8+T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8+T-cell responses uponBordetella pertussisinfection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γsecretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4+and CD8+T lymphocytes are involved in IFN-γproduction. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4+lymphocytes were the major source of this cytokine. IFN-γsynthesis by CD8+cells was CD4+T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γsynthesis by CD4+cells was sometimes inhibited by CD8+lymphocytes, suggesting the presence of CD8+regulatory T cells. The role of this dual IFN-γsecretion by CD4+and CD8+T lymphocytes in pertussis remains to be investigated.

Human T cell responses to endogenously presented HLA-A*0201 restricted peptides of simian virus 40 large T antigen

2001 ◽  
Vol 82 (1) ◽  
pp. 155-162 ◽  
Author(s):  
Markwin P. Velders ◽  
M. Fatima Macedo ◽  
Maurizio Provenzano ◽  
Amira G. Elmishad ◽  
Hermann-Georg Holzhütter ◽  
...  
Keyword(s):  
T Cell ◽  
Simian Virus 40 ◽  
T Cell Responses ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Cell Responses ◽  
Human T Cell

Enhancement of human T cell responses to allogeneic stimuli by factor VIII concentrates

1992 ◽  
Vol 82 (1) ◽  
pp. 94-98 ◽  
Author(s):  
Alison Batchelor ◽  
C. Michael Steel ◽  
Christopher A. Ludlam
Keyword(s):  
T Cell ◽  
Factor Viii ◽  
T Cell Responses ◽  
Cell Responses ◽  
Human T Cell ◽  
Factor Viii Concentrates
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