52P The inducible Medigene T-cell receptor (iM-TCR) controls cytotoxicity of tumor-specific TCR-modified T-cells with improved avidity through control of TCR surface expression

Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.

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Expression of T-cell receptor alpha-chain genes in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5459-5469.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5459-5469

Author(s):

L J Berg ◽

B Fazekas de St Groth ◽

F Ivars ◽

C C Goodnow ◽

S Gilfillan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Specific Monoclonal Antibody ◽

Alpha Chain ◽

Receptor Alpha Chain ◽

Cell Receptor Alpha

To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).

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Differential contribution of the FcRγ chain to the surface expression of the T cell receptor among T cells localized in epithelia: analysis of FcRγ-deficient mice

European Journal of Immunology ◽

10.1002/eji.1830250746 ◽

1995 ◽

Vol 25 (7) ◽

pp. 2107-2110 ◽

Cited By ~ 35

Author(s):

Seung Yong Park ◽

Hisashi Arase ◽

Keisuke Wakizaka ◽

Nakami Hirayama ◽

Shigehiro Masaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Differential Contribution ◽

Deficient Mice

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Surface expression of the beta T cell receptor (TCR) chain in the absence of other TCR or CD3 proteins on immature T cells.

The EMBO Journal ◽

10.1002/j.1460-2075.1991.tb07924.x ◽

1991 ◽

Vol 10 (1) ◽

pp. 93-100 ◽

Cited By ~ 80

Author(s):

H. Kishi ◽

P. Borgulya ◽

B. Scott ◽

K. Karjalainen ◽

A. Traunecker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression

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A novel method to generate T-cell receptor–deficient chimeric antigen receptor T cells

Blood Advances ◽

10.1182/bloodadvances.2017012823 ◽

2018 ◽

Vol 2 (5) ◽

pp. 517-528 ◽

Cited By ~ 14

Author(s):

Takahiro Kamiya ◽

Desmond Wong ◽

Yi Tian Png ◽

Dario Campana

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Chimeric Antigen Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Key Points ◽

Novel Method

Key Points Newly designed PEBLs prevent surface expression of T-cell receptor in T cells without affecting their function. Combined with chimeric antigen receptors, PEBLs can rapidly generate powerful antileukemic T cells without alloreactivity.

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Expression of T-cell receptor alpha-chain genes in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5459 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5459-5469 ◽

Cited By ~ 42

Author(s):

L J Berg ◽

B Fazekas de St Groth ◽

F Ivars ◽

C C Goodnow ◽

S Gilfillan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Specific Monoclonal Antibody ◽

Alpha Chain ◽

Receptor Alpha Chain ◽

Cell Receptor Alpha

To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).

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Playing the Game Together: Coexpression of a Single Chain T Cell Receptor and a T Cell Receptor Constant-Alpha Domain Triggers Tumor Reactivity.

Blood ◽

10.1182/blood.v108.11.3716.3716 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3716-3716

Author(s):

Simone Thomas ◽

Ralf H. Voss ◽

Ratna Intan ◽

Renate Engel ◽

Juergen Kuball ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Target Cells ◽

Single Chain ◽

Double Chain ◽

Human T Cells

Abstract Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2- and p53-specific self-tolerance. In contrast to the use of HuCD8×A2Kb transgenic mice to generate an MDM2-specific CD8-dependent TCR, we generated a high-affinity, CD8-independent p53-specific TCR in single human A2.1 transgenic mice. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR α/β chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1-restricted peptide-specific murine single chain (sc) TCRs were constructed (Vα-Li-VβCβ) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2- or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Cα) comprising solely the TCRα signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Cα would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Cα domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Cα constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant β-domain of the scTCR was replaced by murine Cβ. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Cα demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets. In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains.

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An Endoplasmic Reticulum Retention Function for the Cytoplasmic Tail of the Human Pre–T Cell Receptor (Tcr) α Chain

Journal of Experimental Medicine ◽

10.1084/jem.193.9.1045 ◽

2001 ◽

Vol 193 (9) ◽

pp. 1045-1058 ◽

Cited By ~ 21

Author(s):

Yolanda R. Carrasco ◽

Almudena R. Ramiro ◽

César Trigueros ◽

Virginia G. de Yébenes ◽

Marina García-Peydró ◽

...

Keyword(s):

T Cells ◽

Endoplasmic Reticulum ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Cytoplasmic Domain ◽

Retention Function ◽

Expression Levels ◽

Er Retention

The pre-T cell receptor (TCR), which consists of a TCR-β chain paired with pre–TCR-α (pTα) and associated with CD3/ζ components, is a critical regulator of T cell development. For unknown reasons, extremely low pre-TCR levels reach the plasma membrane of pre-T cells. By transfecting chimeric TCR-α–pTα proteins into pre-T and mature T cell lines, we show here that the low surface expression of the human pre-TCR is pTα chain dependent. Particularly, the cytoplasmic domain of pTα is sufficient to reduce surface expression of a conventional TCR-α/β to pre-TCR expression levels. Such reduced expression cannot be attributed to qualitative differences in the biochemical composition of the CD3/ζ modules associated with pre-TCR and TCR surface complexes. Rather, evidence is provided that the pTα cytoplasmic tail also causes a reduced surface expression of individual membrane molecules such as CD25 and CD4, which are shown to be retained in the endoplasmic reticulum (ER). Native pTα is also observed to be predominantly ER localized. Finally, sequential truncations along the pTα cytoplasmic domain revealed that removal of the COOH-terminal 48 residues is sufficient to release a CD4-pTα chimera from ER retention, and to restore native CD4 surface expression levels. As such a truncation in pTα also correlates with enhanced pre-TCR expression, the observed pTα ER retention function may contribute to the regulation of surface pre-TCR expression on pre-T cells.

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