STOCK Ptprc^a Tcrb^Ln1Bdb Tcra^Ln1Bdb H2^d/J

Stock number: 028062
Product name: Jedi (Just EGFP Death Inducing)
Strain synonyms: Jedi
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Jedi (Just EGFP Death Inducing) mice are a GFP-specific TCR mouse line that produces T cells that recognize the immunodominant epitope of enhanced GFP (GFP_200-208) presented on H-2K^d; enabling GFP to be used as a model antigen. Activation of Jedi CD8⁺ T cells rapidly and stably eliminates GFP⁺ cell populations. Jedi are the first antigen-specific TCR mice with T cells recognizing a florescent protein. The ability for direct fluorescent visualization of the target antigen is a novel and invaluable aspect of the Jedi technology.

Detailed description

The GFP-specific TCR mouse line Jedi (Just EGFP Death Inducing) produces T cells that recognize the immunodominant epitope of enhanced GFP (GFP_200-208) presented on H-2K^d. In every Jedi mouse, at least 50% of CD8⁺ T cells are specific for the GFP_200-208-H-2K^d pentamer and their phenotype is naïve (CD44^- CD62L⁺).
Specificity is high: Jedi CD8⁺ T-cells target and destroy GFP⁺ cells, but not YFP⁺ cells (despite that YFP has >95% homology to GFP but differs by one amino acid in the immunodominant epitope). Similarly, when co-cultured with dendritic cells expressing GFP or YFP, the Jedi CD8⁺ T cells proliferate in the presence of GFP but not YFP.

Jedi CD8⁺ T cells rapidly and stably eliminate GFP⁺ cell populations. Whether GFP mice are designed to express GFP on the cell surface or in the cell interior (cytoplasm, or bound to the inner side of membranes, etc.), all GFP expressing cells (with normal MHC class I function) present some GFP epitopes on their MHC class I molecules - that random occurrence of "self GFP_200-208 epitope" presentation on those cells is enough for Jedi T cells to identify them. Some examples are listed below.
When Jedi CD8⁺ T cells are injected into mice expressing GFP in pancreatic β-cells (MIP-EGFP; see Stock No. 006864), subsequent vaccination with GFP activates the transferred cells - resulting in rapid β-cell destruction and hyperglycemia. This is a model of inducible type I diabetes that also allows direct visualization of the islet-antigen. [Agudo et al. 2015 Nat Biotechnol 33:1287]
When Jedi CD8⁺ T cells are injected into mice engineered to express GFP in microglia (CX3CR1-EGFP; see Stock No. 005582) but RFP in peripheral hematopoietic cells, the resulting elimination of the GFP⁺ microglia occurred rapidly both with or without GFP vaccination/activation. This is a model for studying T cell trafficking and cytotoxic lymphocyte clearance. [Agudo et al. 2015 Nat Biotechnol 33:1287]
Jedi CD8⁺ T cells are able to stably deplete a population of renewing cells. For example, when injected into mice expressing GFP in Foxp3⁺ regulatory T cells (Foxp3-EGFP; similar to Stock Nos. 006772 or 018628), subsequent vaccination with GFP activates the transferred cells - resulting in rapid and stable depletion of Foxp3⁺GFP⁺ Tregs. Specifically, when the host is a female heterozygous for the X-linked Foxp3-EGFP allele, only ~half of the Foxp3⁺ Tregs are GFP⁺ due to random X inactivation. While those cells are stably depleted, the Foxp3⁺GFP^- cells remained and proliferated to compensate for the loss of the GFP⁺ Tregs. When the host is a male hemizygous for the X-linked Foxp3-EGFP allele, complete depletion of Foxp3⁺ Tregs resulted in immune dysregulation and illness. [Agudo et al. 2015 Nat Biotechnol 33:1287]
Jedi CD8⁺ T cells are able to eliminate rare cell populations in a non-lymphoid, non-inflamed organ. For example, when injected into mice expressing GFP in an extremely rare population of pacemaker heart cells (Hcn4-EGFP), subsequent vaccination with GFP activates the transferred cells - resulting in rapid and stable depletion of Hcn4⁺ cells. Unique from other depletion technologies, this allowed assessment of target cell depletion at single cell resolution via flow cytometry. [Agudo et al. 2015 Nat Biotechnol 33:1287]

When homozygous for the CD45.1 allele (Ptprc^a) on chromosome 1, heterozygous for the two Jedi TCR alleles (Tcrb^Ln1Bdb on chromosome 6 and Tcra^Ln1Bdb on chromosome 14) and homozygous for the H-2K^d allele (H2^d) on chromosome 17, the Jedi mice are viable and fertile. The donating investigator reports that when they breed heterozygous Jedi mice together they obtain the expected number of litters, but their genotyping protocol cannot properly distinguish heterozygous from homozygous to date (August 2018).

By November 2019, The Jackson Laboratory has genotyping assays in place for the two Jedi TCR alleles to distinguish heterozygous from homozygous. Additionally, the donating investigator suggests that mice may be immunocompromised when homozygous for the two Jedi TCR alleles.

In 2020, The Jackson Laboratory has generated mice homozygous for Ptprc^a, the two Jedi TCR alleles (Tcrb^Ln1Bdb and Tcra^Ln1Bdb) and H-2K^d. Between ~4-5 months of age, the quadruple homozygotes may appear hunched with labored breathing and the underside of their neck appears swollen - incidence ~50% of females and ~25% of males. As such, quadruple homozygous breeding units should be closely monitored and replaced by 4 months of age as needed.

Development

Jedi (Just EGFP Death Inducing) mice harbor four alleles that are not linked and are expected to segregate independently: the CD45.1 allele (Ptprc^a) on chromosome 1, the two Jedi TCR alleles (Tcrb^Ln1Bdb on chromosome 6 and Tcra^Ln1Bdb on chromosome 14) and the H-2K^d allele (H2^d) on chromosome 17. Specific details are below.

The two Jedi TCR alleles were created by Dr. Brian D. Brown (Icahn School of Medicine at Mount Sinai) using a somatic cell nuclear transfer (SCNT) approach. First, a lentivirus encoding GFP was injected into (BALB/cJ x C57BL/6J)F1 mice (CB6F1; H-2K^b/d). After 2 weeks, GFP_200-208-H-2K^d tetramer⁺ CD8⁺ T cells were isolated. The donating investigator reports the identity of the T cell receptor Vα and Vβ chains was found to be trav7-4, traj30 and trbv2, trbj1.6 (Vα1-J30 and Vβ4-D1-J1.6-C1). The T cells nucleus was injected into an enucleated oocyte, and the resulting cell was used to generate embryonic stem (ES) cells. ES cells were injected into recipient blastocysts, and blastocysts implanted into psuedopregnant females. The F1 mice were then bred for several generations to the C5-functional B10D2 mice (originally from Stock No. 000463; B10.D2-Hc¹ H2^d H2-T18^c/nSnJ) and selected to retain the H2^d allele (H-2K^d) on chromosome 17. Next, Jedi mice were bred to B6.SJL-Ptprc^a Pepc^b/BoyJ mice (originally from Stock No. 002014) to obtain the Ptprc^a allele (CD45.1) on chromosome 1. The Jedi mice were then bred approximately four more generations to the C5-functional B10D2 mice (originally from Stock No. 000463) and selected to retain the H-2K^d and CD45.1 alleles - resulting in the Jedi H-2K^d CD45.1 colony (on a predominantly B10.D2 genetic background). In 2018, both female and male mice that were homozygous for CD45.1, heterozygous or homozygous for the Jedi TCR alleles and homozygous for H-2K^d were sent to The Jackson Laboratory Repository. Upon arrival, our live colony was generated using those females and males.

Allele

Symbol: Tcrb^Ln1Bdb
Name: recombination line 1, Brian Brown
MGI accession ID: MGI:6194858
Generation method: Other
Attributes: Not Specified
Marker symbol: Tcrb
Marker name: T cell receptor beta chain
Marker synonyms: TCRbeta; Tib
Chromosome: 6
Strain of origin: (BALB/cJ x C57BL/6J)F1
Molecular note: This allele is generated using somatic cell nuclear transfer (SCNT). First, a lentivirus encoding GFP was injected into (BALB/cJ x C57BL/6J)F1 mice (CB6F1; H-2KH-2K^b/d). After 2 weeks, GFP-200-208- H-2K^d tetramer⁺ CD8⁺ T cells were isolated. The T cell nucleus was injected into an enucleated oocyte, and the resulting cell was used to generate embryonic stem (ES) cells. ES cells were injected into recipient blastocysts, and blastocysts implanted into pseudopregnant females. PCR analysis and Sanger sequencing reveal that the rearranged TCR is Vbeta4-D1-J1.6-C1 (trbv2-trbj1.6-trbc1).

Allele

Symbol: H2^d
Name: d variant
MGI accession ID: MGI:3579320
Generation method: Spontaneous
Marker symbol: H2
Marker name: histocompatibility-2, MHC
Marker synonyms: H-2; MHC-II
Chromosome: 17
Strain of origin: various
General note: The d variant has been observed in the following strains: DBA/2, DBA/2J BALB/c, BALB/cByJ, BALB/cJ, C57BLKS, NZB.

Allele

Symbol: Ptprc^a
Name: a variant
MGI accession ID: MGI:4819849
Generation method: Not Applicable
Synonyms: CD45.1; Ly5^a; Ptprc^SJL
Marker symbol: Ptprc
Marker name: protein tyrosine phosphatase, receptor type, C
Marker synonyms: B220; CD45; CD45R; GP180; IMD105; LCA; L-CA; LY5; Ly-5; Lyt-4; RT7; T200
Chromosome: 1
Strain of origin: Not Applicable
Site of expression: Widely expressed on all adaptive and innate immune cells.
Molecular note: Ptprc^a is found in strains SJL/J, STS/A, and DA. Ptprc^b is found in strains C57BL/6, C3H/An, DBA/2, AKR, and many others (J:13367, J:12054, J:12077, J:8603). Twelve nucleotide differences between the a and b alleles have been identified. These base substitutions correspond to five amino-acid changes within the extracellular domain of the encoded protein. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat sequences (J:22485).

Note that the allele designations originally described were reversed in 1987 (J:8603); all publications prior to 1987 show SJL/J, STS/A, and DA as having the b allele and the C57BL/6J group as having the a allele (J:22341).

Allele

Symbol: Tcra^Ln1Bdb
Name: recombination line 1, Brian Brown
MGI accession ID: MGI:6194869
Generation method: Other
Attributes: Not Specified
Marker symbol: Tcra
Marker name: T cell receptor alpha chain
Marker synonyms: Tcr alpha; Tcralpha
Chromosome: 14
Strain of origin: (BALB/cJ x C57BL/6J)F1
Molecular note: This allele is generated using somatic cell nuclear transfer (SCNT). First, a lentivirus encoding GFP was injected into (BALB/cJ x C57BL/6J)F1 mice (CB6F1; H-2K^b/d). After 2 weeks, GFP-200-208 H-2K^d tetramer⁺ CD8⁺ T cells were isolated. The T cell nucleus was injected into an enucleated oocyte, and the resulting cell was used to generate embryonic stem (ES) cells. ES cells were injected into recipient blastocysts, and blastocysts implanted into pseudopregnant females. PCR analysis and Sanger sequencing reveal that the rearranged TCR is Valpha1-J30 (trav7-4, traj30, and trac).