Rapid identification and expression of human TCRs in retrogenic mice

Sprouse, Maran L.; Blahnik, Gabriele; Lee, Thomas; Tully, Natalie; Banerjee, Pinaki; James, Eddie A.; Redondo, María J.; Bettini, Matthew L.; Bettini, Maria

doi:10.1016/j.jim.2016.08.010

Cited by 7 publications

(17 citation statements)

References 45 publications

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“…We have overcome the limitation of TCR transgenic system by utilizing a TCR retrogenic approach that allows rapid functional analysis of multiple TCRs through retroviral gene delivery ( 110 , 145 , 146 ). Using this approach, we have expressed a GAD65 115–127 reactive TCR isolated from peripheral blood of an individual diagnosed with T1D ( 99 ). Although we observed robust development of GAD-reactive T cells in this system, similar to the transgenic expression, we detected a low level of islet infiltration.…”

Section: Hla-humanized Mice To Model T1d Antigen Responsesmentioning

confidence: 99%

Understanding Autoimmune Diabetes through the Prism of the Tri-Molecular Complex

Bettini

2017

Front. Endocrinol.

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The strongest susceptibility allele for Type 1 Diabetes (T1D) is human leukocyte antigen (HLA), which supports a central role for T cells as the drivers of autoimmunity. However, the precise mechanisms that allow thymic escape and peripheral activation of beta cell antigen-specific T cells are still largely unknown. Studies performed with the non-obese diabetic (NOD) mouse have challenged several immunological dogmas, and have made the NOD mouse a key experimental system to study the steps of immunodysregulation that lead to autoimmune diabetes. The structural similarities between the NOD I-Ag7 and HLA-DQ8 have revealed the stability of the T cell receptor (TCR)/HLA/peptide tri-molecular complex as an important parameter in the development of autoimmune T cells, as well as afforded insights into the key antigens targeted in T1D. In this review, we will provide a summary of the current understanding with regard to autoimmune T cell development, the significance of the antigens targeted in T1D, and the relationship between TCR affinity and immune regulation.

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Section: Hla-humanized Mice To Model T1d Antigen Responsesmentioning

confidence: 99%

Understanding Autoimmune Diabetes through the Prism of the Tri-Molecular Complex

Bettini

2017

Front. Endocrinol.

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“…Note: HEK293T cells are used to test successful TCR chain paring and cell surface expression ( Figure 3A) 8 . Peptide-MHC tetramer staining can also be performed on the transfected HEK293T cells to test for retention of antigenic specificity post TCR gene isolation ( Figure 3B) 4. In separate 1.5 mL tubes combine 1 µg of TCR-pMIA vector with 1 µg mouse CD3εγδζ-pMSCVII-GFP (pMIG) vector, or 1 µg of each vector separate for controls.…”

Section: Verify Tcr Cell Surface Expression and Specificitymentioning

confidence: 99%

“…In order to integrate the TCR sequence repertoire analysis with TCR functional assays, the sequencing approaches should be designed to be compatible with experimental systems and in vivo models utilized for subsequent functional analysis of select TCRs. We have developed an efficient approach for human TCR sequence isolation and streamlined sub-cloning into a chimeric human/mouse TCR template vector compatible with TCR expression in HLA-humanized mice 4 .…”

Section: Introductionmentioning

confidence: 99%

“…Although, several single-cell TCR cloning protocols have been developed and utilized, none so far have been easily compatible with high-throughput streamlined direct cloning of unknown Vα/Vβ TCRs into retroviral vectors necessary for re-expression in vivo 4,5,6,7 . Previous studies have utilized two major approaches, either to selectively amplify a limited portion of the TCR sufficient to extrapolate the sequence, or to amplify the entire TCR sequence 4,5,6,7 . Downstream functional analysis of TCR sequences obtained via the first approach requires costly in silico assembly and de novo construction of the TCR.…”

Section: Introductionmentioning

confidence: 99%

“…(DRB1*0401), HLA-DR1 (DRA*0101, DRB1*0101), HLA-A11 (A*11:01/K), HLA-B2705, HLA-A24, and HLA-B*0702 transgenic strains. This protocol can be successfully combined with the previously published protocol for retroviral mediated bone marrow stem cell expression of human TCRs 4,15,16 . We expect that the humanized TCR retrogenic system will be applicable and highly useful for functional analysis of human TCRs in various autoimmune, cancer, and infectious models.…”

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confidence: 99%

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Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for <em>In Vitro </em>and <em>In Vivo</em> Expression of Human TCRs

Sprouse

Blahnik²,

Lee

et al. 2017

JoVE

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Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

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Generation of T Cell Receptor Retrogenic Mice

Kong

Bettini

2019

CP in Immunology

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The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus‐mediated stem cell–based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor. After successful transduction, bone marrow is injected into recipient mice, in which T cell development is driven by expression of the vector‐encoded TCR. This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followed in most laboratories with standard biomedical equipment, and is supported by a troubleshooting guide that covers potential pitfalls and unexpected results. © 2019 by John Wiley & Sons, Inc.

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Rapid identification and expression of human TCRs in retrogenic mice

Cited by 7 publications

References 45 publications

Understanding Autoimmune Diabetes through the Prism of the Tri-Molecular Complex

Understanding Autoimmune Diabetes through the Prism of the Tri-Molecular Complex

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for <em>In Vitro </em>and <em>In Vivo</em> Expression of Human TCRs

Generation of T Cell Receptor Retrogenic Mice

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