|Coordinate||45,006,150 bp (GRCm38)|
|Base Change||G ⇒ T (forward strand)|
|Gene Name||CD3 antigen, epsilon polypeptide|
|Synonym(s)||T3e, CD3, CD3epsilon|
|Chromosomal Location||44,998,743-45,009,590 bp (-)|
|MGI Phenotype||Mice homozygous null for this mutation lack peripherial T cells and have a block of thymocyte development at the DN3 stage.|
|Amino Acid Change||Tyrosine changed to Stop codon|
|Institutional Source||Beutler Lab|
Y84* in Ensembl: ENSMUSP00000099896 (fasta)
|Gene Model||not available|
|Phenotypic Category||hematopoietic system, immune system|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2016-12-30 2:42 PM by Katherine Timer|
The tumormouse phenotype was identified in a screen for allogeneic sterility. G3 mice with homozygous ENU-induced mutations were injected intraperitoneally with L-929 fibroblasts of C3H background in order to pre-immunize against C3H alloreactive determinants. One animal developed an intra-abdominal tumor consisting of L-929 cells. The mutant phenotype was termed tumormouse.
Further analysis revealed that tumormouse animals have very small thymi with abnormal structure and no peripheral T cells. tumormouse mutants have no CD4+CD8+ double positive (DP) T cells, and thymocyte development was found to be arrested at the CD44+CD25- double negative (DN) 3 stage. Compared to wild type, tumormouse thymocytes display increased levels of apoptosis. B cells, macrophages and NK cells are found at normal levels in the periphery in tumormouse animals.
Some tumormouse animals older than 9 months of age appear to experience epileptic-like seizures.
|Nature of Mutation|
The tumormouse mutation was mapped to Chromosome 9 and corresponds to a C to A transversion at position 330 of the Cd3e transcript, in exon 5 of 8 total exons.
The mutated nucleotide is indicated in red lettering, and creates a premature stop codon in place of tyrosine 84 resulting in deletion of 106 amino acids from the C terminus of the protein.
The cytoplasmic domain of CD3ε contains a basic amino acid-rich region (4), a proline-rich region (5), and an immunoreceptor tyrosine-based activation motif (ITAM) (6). These motifs mediate interactions with downstream signaling molecules.
The tumormouse mutation replaces tyrosine 84 with a premature stop codon and truncates the CD3ε chain in the extracellular domain of the protein. The truncation results in a CD3ε null animal.
Cd3ε is expressed in immature thymocytes and T cells, and localizes to the plasma membrane.
T cell receptors (TCRs) are responsible for recognition of MHC/antigen ligands, with their different specificities generated by rearrangement of germline V, D, and J segments during development. The complete TCR consists of a complex including TCR α/β or γ/δ chains, several invariant CD3 chains, and ζ chains (see allia) (7). CD3γ, CD3δ and CD3ε constitute the three types of CD3 chains, and combine to form TCRs with stoichiometry TCRαβ or TCRγδ /CD3γε/CD3δε/ζζ.
Development of thymocytes into mature T cells occurs in the thymus, where thymocytes follow a program of differentiation characterized by expression of distinct combinations of cell surface proteins including CD4, CD8, CD44 and CD25. The most immature thymocytes are CD4-CD8- double negative (DN). This group can be further subdivided into 4 groups that differentiate in the following order: CD44+CD25- (DN1) to CD44+CD25+ (DN2) to CD44-CD25+ (DN3) to CD44-CD25- (DN4). During this process, expression of pre-TCRα (pTα), TCRα, TCRβ and CD3 proteins is activated in temporal sequence to promote T cell development. Transcripts of all the CD3 chains are expressed from the earliest identifiable thymic precursor stage (8). Studies of CD3ε-deficient mice demonstrate that CD3ε specifically contributes to formation of a pre-TCR complex with TCRβ and pTα at the CD44-CD25+ DN3 stage, and these animals have no mature T cells (9-11). Interestingly, one group reported that CD3ε null thymocytes were severely, but not completely arrested at the DN3 stage, suggesting that expression of the other components of the pre-TCR may assemble a partial complex that can weakly induce transition out of the DN3 stage (11).
In addition to the signal transduction cascade mediated by Lck and Fyn, another independent pathway involving recruitment of Nck to CD3ε may activate TCR signaling (5;15). This type of activation involves a conformational change in CD3ε (16), exposing a proline-rich region where Nck binds. Little data on the nature of the conformational change is available. Interestingly, this method of TCR complex activation does not require TCR crosslinking or tyrosine phosphorylation.
Recent work identified interactions between the membrane-proximal basic amino acid-rich region of CD3ε and G-protein-coupled receptor kinase 2 (GRK2) in thymocytes, which may be regulated by T cell activation and chemokines (4).
As in other CD3ε mutants, thymocyte development arrests at the DN3 stage resulting in a complete lack of mature T cells in tumormouse mutants. The absence of mature T cells describes the condition of severe combined immunodeficiency (SCID). Several mechanisms causing SCID have been identified, including defective pre-TCR/TCR signaling. CD3ε mutations cause SCID in this way (17) (OMIM +186830). Likewise, mice deficient in VDJ recombination due to deletion of recombination activating gene-1 (Rag-1) (18) or Rag-2 (19) are devoid of T cells and contain only immature DN3 thymocytes. In addition, mutations in any of the components of DNA-dependent protein kinase (DNA-PK) (20-22), CD3δ (19) or Artemis, a DNA double-strand break repair protein (23), results in a lack of T cells. As in mice, CD3e mutations in humans lead to immunodeficiency (17), and patients with such mutations develop serious infections for which antibiotics are poorly effective (24).
The immune system, being capable of recognizing and destroying developing tumors, and controlling the tumorigenicity of cancer cells, plays a strong protective role against cancer. Tumors derived from immunodeficient Rag2-/- mice are more immunogenic than those derived from immunocompetent mice (25). NK cells recognize invading cells, and together with dendritic cells, prime and activate CD4+ and CD8+ T cells to eliminate neoplastic cells. Immunotherapy using antigen-specific splenocytes confers protection from established and subsequent tumor burdens in mice (26). Tumor development in the tumormouse mutant can likely be attributed to a deficiency in ability to reject foreign cells due to lack of T cells.
|Primers||Primers cannot be located by automatic search.|
Tumormouse genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
Tumor(F): 5’- GGACACCTCTCTACCAAGCAAACCTCG -3’
Tumor(R): 5’- GGTAGTGGCCCTGAGTCTCTCTGGATG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 59°C 0:30
4) 68°C 1:00
5) repeat steps (2-4) 35X
6) 68°C 5:00
7) 4°C ∞
Primers for sequencing
Tumor_seq(F): 5’- CGACACCCAGCGATAAGTAACTTC -3’
Tumor_seq(R): 5’- TAGACAACTCCAGGCCACACCG -3’
The following sequence of 678 nucleotides (from Genbank genomic region NC_000075 for linear genomic sequence of Cd3e) is amplified:
7093 ggacacct ctctaccaag caaacctcga cacccagcga taagtaactt
7141 cctgtaatct agttgcctct cacagcacta atttggcatt tgtgaaactt ccctagagtt
7201 tccccttcaa tccccttccc ttttcttctt ttcccagaat acaaagtctc catctcagga
7261 accagtgtag agttgacgtg ccctctagac agtgacgaga acttaaaatg ggaaaaaaat
7321 ggccaagagc tgcctcagaa gcatgataag cacctggtgc tccaggattt ctcggaagtc
7381 gaggacagtg gctactacgt ctgctacaca ccagcctcaa ataaaaacac gtacttgtac
7441 ctgaaagctc gaggtaactc gggctcctcc caaatcagcc ttctcaagaa ccctatcatc
7501 tctcagctgc tcctgcactc caccccaggt tctccggggc cacacattca gtattttctg
7561 aaaaatagac tgcacggtgt ggcctggagt tgtctaggta attccattgg taccaggtgt
7621 acaacagcaa cttcccacag atgaaggctt gggtcacctg ccttgtaacg tacccagaat
7681 caaggcttac tactatcatg agttgatggg gtcacttatt cagagaccca ttttattaca
7741 aggacatcca gagagactca gggccactac c
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated C is highlighted in red.
1. Gold, D. P., Clevers, H., Alarcon, B., Dunlap, S., Novotny, J., Williams, A. F., and Terhorst, C. (1987) Evolutionary relationship between the T3 chains of the T-cell receptor complex and the immunoglobulin supergene family, Proc. Natl. Acad. Sci. U. S. A 84, 7649-7653.
2. Arnett, K. L., Harrison, S. C., and Wiley, D. C. (2004) Crystal structure of a human CD3-epsilon/delta dimer in complex with a UCHT1 single-chain antibody fragment, Proc. Natl. Acad. Sci. U. S. A 101, 16268-16273.
3. Call, M. E., Pyrdol, J., Wiedmann, M., and Wucherpfennig, K. W. (2002) The organizing principle in the formation of the T cell receptor-CD3 complex, Cell 111, 967-979.
4. Ford-Watts, L. M., Young, J. A., Pitcher, L. A., and van Oers, N. S. (2007) The membrane-proximal portion of CD3 epsilon associates with the serine/threonine kinase GRK2, J. Biol. Chem. 282, 16126-16134.
5. Gil, D., Schamel, W. W., Montoya, M., Sanchez-Madrid, F., and Alarcon, B. (2002) Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation, Cell 109, 901-912.
6. Pitcher, L. A. and van Oers, N. S. (2003) T-cell receptor signal transmission: who gives an ITAM?, Trends Immunol. 24, 554-560.
7. Klausner, R. D., Lippincott-Schwartz, J., and Bonifacino, J. S. (1990) The T cell antigen receptor: insights into organelle biology, Annu. Rev. Cell Biol. 6, 403-431.
8. Wilson, A. and MacDonald, H. R. (1995) Expression of genes encoding the pre-TCR and CD3 complex during thymus development, Int. Immunol. 7, 1659-1664.
9. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E., and Malissen, B. (1995) Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene, EMBO J. 14, 4641-4653.
10. DeJarnette, J. B., Sommers, C. L., Huang, K., Woodside, K. J., Emmons, R., Katz, K., Shores, E. W., and Love, P. E. (1998) Specific requirement for CD3epsilon in T cell development, Proc. Natl. Acad. Sci. U. S. A 95, 14909-14914.
11. Wang, B., Wang, N., Whitehurst, C. E., She, J., Chen, J., and Terhorst, C. (1999) T lymphocyte development in the absence of CD3 epsilon or CD3 gamma delta epsilon zeta, J. Immunol. 162, 88-94.
12. Alarcon, B., Gil, D., Delgado, P., and Schamel, W. W. (2003) Initiation of TCR signaling: regulation within CD3 dimers, Immunol. Rev. 191, 38-46.
13. Boniface, J. J., Rabinowitz, J. D., Wulfing, C., Hampl, J., Reich, Z., Altman, J. D., Kantor, R. M., Beeson, C., McConnell, H. M., and Davis, M. M. (1998) Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected], Immunity. 9, 459-466.
14. Love, P. E. and Shores, E. W. (2000) ITAM multiplicity and thymocyte selection: how low can you go?, Immunity. 12, 591-597.
16. Kastrup, J., Pedersen, L. O., Dietrich, J., Lauritsen, J. P., Menne, C., and Geisler, C. (2002) In vitro production and characterization of partly assembled human CD3 complexes, Scand. J. Immunol. 56, 436-442.
17. de Saint Basile. G., Geissmann, F., Flori, E., Uring-Lambert, B., Soudais, C., Cavazzana-Calvo, M., Durandy, A., Jabado, N., Fischer, A., and Le, D. F. (2004) Severe combined immunodeficiency caused by deficiency in either the delta or the epsilon subunit of CD3, J. Clin. Invest 114, 1512-1517.
18. Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S., and Papaioannou, V. E. (1992) RAG-1-deficient mice have no mature B and T lymphocytes, Cell 68, 869-877.
19. Shinkai, Y., Rathbun, G., Lam, K. P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M., and . (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement, Cell 68, 855-867.
20. Bosma, G. C., Davisson, M. T., Ruetsch, N. R., Sweet, H. O., Shultz, L. D., and Bosma, M. J. (1989) The mouse mutation severe combined immune deficiency (scid) is on chromosome 16, Immunogenetics 29, 54-57.
21. Miller, R. D., Hogg, J., Ozaki, J. H., Gell, D., Jackson, S. P., and Riblet, R. (1995) Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus, Proc. Natl. Acad. Sci. U.S.A. 92, 10792-10795.
22. Hartley, K. O., Gell, D., Smith, G. C., Zhang, H., Divecha, N., Connelly, M. A., Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. P. (1995) DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product, Cell 82, 849-856.
23. Moshous, D., Callebaut, I., de, C. R., Corneo, B., Cavazzana-Calvo, M., Le, D. F., Tezcan, I., Sanal, O., Bertrand, Y., Philippe, N., Fischer, A., and de Villartay, J. P. (2001) Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency, Cell 105, 177-186.
25. Shankaran, V., Ikeda, H., Bruce, A. T., White, J. M., Swanson, P. E., Old, L. J., and Schreiber, R. D. (2001) IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity, Nature 410, 1107-1111.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Xin Du, Bruce Beutler|