The Impact of Single Amino Acid Substitutions in CD3γ on the CD3ϵγ Interaction and T-Cell Receptor–CD3 Complex Formation

Thomassen, E.A.J.; Dekking, E.H.A.; Thompson, Allan; Franken, Kees L. M. C.; Sanal, Özden; Abrahams, Jan Pieter; Tol, Maarten J.D. van; Koning, Frits

doi:10.1016/j.humimm.2006.04.015

Cited by 9 publications

(11 citation statements)

References 41 publications

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“…The first involved hCD3␥-deficient patient T cells as recipients for retroviral transduction while the second exploited an in vitro transcription/translation system (42,43). Unlike in the mouse system, the introduction of hCD3␥ C82S or hCD3␥ C85S failed to support association with CD3 or T cell surface expression (42). These data are consistent with earlier studies showing the striking effect of cysteine mutations of hCD3 stalk residues on both hCD3␥ and hCD3␦ in heterodimer formation (44).…”

Section: Discussionsupporting

confidence: 78%

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Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Touma

Sun

Clayton

et al. 2007

The Journal of Immunology

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CD3εγ and CD3εδ are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal β-strands that are linked to individual subunit membrane proximal stalk segments. CD3ε, CD3γ, and CD3δ stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal β-strand, we created a CD3γ double mutant CD3γC82S/C85S and a CD3γ β-strand triple mutant CD3γQ76S/Y78A/Y79A for use in retroviral transduction of lymphoid progenitors for comparison with CD3γwt. Although both mutant CD3γ molecules reduced association with CD3ε in CD3εγ heterodimers, CD3γQ76S/Y78A/Y79A abrogated surface TCR expression whereas CD3γC82S/C85S did not. Furthermore, CD3γC82S/C85S rescued thymic development in CD3γ−/− fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3γC82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3γ−/− thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3γwt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3εγ, as evidenced by the loss of reactivity with CD3γ N terminus-specific antisera. Single histidine substitution of either CD3γ stalk cysteine failed to restore CD3εγ association and conformation in transient COS-7 cell transfection studies. Thus, CD3γC82 and CD3γC85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3εC80 and CD3εC83. The implications of these results for CD3εγ and TCR structure and signaling function are discussed.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Touma

Sun

Clayton

et al. 2007

The Journal of Immunology

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“…It is known that in digitonin lysis buffer all components of the TCR-CD3 complex remain complexed, whereas in NP40 lysis buffer the TCR-CD3 complex dissociates into the TCR-␣␤ heterodimer, -homodimer, a CD3␥⑀ heterodimer, and a CD3␦⑀ heterodimer. 17,19 RCD cell line P1 was found to express all TCR-CD3 subunits intracellularly ( Figure 3A). The TCR-␤ antibody immunoprecipitated 2 specific bands that appeared to be glycosylation variants of each other as, in agreement with previous observations, 23 Figure 3A).…”

Section: Org Frommentioning

confidence: 99%

“…19 For 35 S metabolic labeling, 10 ϫ 10 6 cells were washed 3 times in PBS and resuspended in 5 mL methionine-and cysteine-free RPMI (Sigma-Aldrich, Zwi ¨ndrecht, The Netherlands) containing 0.5% fetal calf serum (FCS), and 10 ng/mL recombinant human IL-15; 1 mCi 35 Smethionine/cysteine was added and cells were incubated overnight at 37°C. After incubation, cells were washed in PBS and lysed overnight at 4°C in 1 to 2 mL lysis buffer containing either 0.5% Nonidet P40 (NP40; Pierce Chemical, Rockford, IL) or 1% digitonin (Sigma Chemie).…”

Section: S Metabolic Labeling and Cell Surface Iodinationmentioning

confidence: 99%

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor–CD3 expression in enteropathy-associated T-cell lymphoma

Tjon

Verbeek

Kooy–Winkelaar

et al. 2008

Blood

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IntroductionCeliac disease (CD) is an inflammatory disorder of the small intestine caused by a dysregulated immune response to ingested wheat gluten, which typically leads to villous atrophy and increased numbers of intraepithelial lymphocytes (IELs) in the intestinal mucosa. Whereas most patients recover on a gluten-free diet, a small proportion of patients fails to improve and develops a condition called refractory celiac disease (RCD). RCD is characterized by persisting or recurring villous atrophy with crypt hyperplasia and an increase of IELs despite a gluten-free diet. Two types of RCD are currently recognized: RCD I, without aberrant IELs; and RCD II, with aberrant IELs. [1][2][3] The aberrant IELs in RCD II lack CD3, CD4, CD8, and the T-cell receptor (TCR) on the surface but express CD3 intracellularly and display monoclonal TCR-␥ gene rearrangement. 2,4,5 Furthermore, it has been shown that interleukin-15 (IL-15) is up-regulated in the lamina propria and epithelial cells of RCD patients, which induces growth and activation of these clonal IELs. 6,7 Because an expansion of IELs under the influence of IL-15 may eventually give rise to overt enteropathy-associated T-cell lymphoma (EATL), the presence of such a clonal IEL population is thought to be a premalignant condition. 1,2,6,8 It is not known what drives lymphoma development and the associated loss of TCR expression in RCD II. In the present study, we report the isolation of a cell line from a duodenal biopsy of a patient with RCD II. This cell line has the characteristic CD4 Ϫ , CD8 Ϫ , intracellular CD3⑀ ϩ , surface TCR-CD3 Ϫ phenotype of RCD II-associated IELs and proliferates in the presence of IL-15. In addition, these IELs express CD30 on the cell surface, as is typically seen in EATL. 9 We have used this cell line and subunit-specific antibodies to analyze the expression and assembly of the TCR and CD3 subunits. The results indicate that, whereas all TCR-CD3 subunits were present intracellularly, proper assembly of the TCR-␣␤ dimer was defective. Functional cell surface expression of the complex could be restored by the introduction of an exogenous TCR-␤ chain. A similar analysis of cell lines isolated from 2 additional RCD II patients indicated defects in the synthesis of the TCR chains. Defective synthesis or defective association of TCR chains thus causes loss of functional surface TCR-CD3 expression on IELs in RCD II, a process that is probably important in escape from immune regulation and progression into EATL. Methods Patient historiesPatient 1 (P1) was typed as human leukocyte antigen (HLA)-A1/A2, -B8, -Cw7/Cw12, DR3/7, DQ2 and developed CD at the age of 51 years. At age 67, RCD type II with aberrant IELs was diagnosed. Until the present study, no EATL has developed. 10 Patient 2 (P2) was typed as HLA-A3/32, -B8, -Cw7, DR3, DQ2 and patient 3 (P3) as HLA-A1, -B8, -Cw7, DR3, DQ2. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and ...

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“…Studies on murine CD3γ (Touma et al, 2007) and human CD3γ (Thomassen et al, 2006; Xu et al, 2006) attest to the importance of the cysteines in the CxxC motif for TCR function and/or assembly. Similar findings have been shown for CD3ε (Martínez-Martín et al, 2009; Wang et al, 2009).…”

Section: The Cxxc Motif In Cd3ε Cd3γ and Cd3δ: A Potential Role In mentioning

confidence: 99%

TCR Mechanobiology: Torques and Tunable Structures Linked to Early T Cell Signaling

Kim¹,

Shin²,

Brazin³

et al. 2012

Front. Immun.

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Mechanotransduction is a basis for receptor signaling in many biological systems. Recent data based upon optical tweezer experiments suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into biochemical signals upon specific peptide-MHC complex (pMHC) ligation. Tangential force applied along the pseudo-twofold symmetry axis of the TCR complex post-ligation results in the αβ heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell–APC interface. Accompanying TCR quaternary change likely fosters signaling via the lipid bilayer predicated on the magnitude and direction of the TCR–pMHC force. TCR glycans may modulate quaternary change, thereby altering signaling outcome as might the redox state of the CxxC motifs located proximal to the TM segments in the heterodimeric CD3 subunits. Predicted alterations in TCR TM segments and surrounding lipid will convert ectodomain ligation into the earliest intracellular signaling events.

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The Impact of Single Amino Acid Substitutions in CD3γ on the CD3ϵγ Interaction and T-Cell Receptor–CD3 Complex Formation

Cited by 9 publications

References 41 publications

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Defective synthesis or association of T-cell receptor chains underlies loss of surface T-cell receptor–CD3 expression in enteropathy-associated T-cell lymphoma

TCR Mechanobiology: Torques and Tunable Structures Linked to Early T Cell Signaling

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