CD3EDG: Signaling in Tregs for Mechanism and Therapeutic Potential

• Gene Information: The CD3E, CD3D, and CD3G genes, located in a cluster on chromosome 11q23, encode the three invariant polypeptide chains that assemble into the CD3εδ and CD3εγ heterodimers of the T-cell receptor (TCR) complex.
• Protein Expression: These proteins are exclusively and constitutively expressed on the surface of mature T lymphocytes and thymocytes, serving as definitive lineage markers for the T-cell population.
•  Signaling Pathway: Upon TCR engagement by an antigen-MHC complex, the CD3 chains transduce activation signals through their cytoplasmic Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), which recruit ZAP-70 to trigger downstream MAPK, NF-κB, and calcium signaling pathways.
•  Therapeutic Inhibition: Therapeutic strategies utilize monoclonal antibodies or bispecific T-cell engagers (TCEs) to either modulate TCR signaling for immunosuppression in transplantation or to bypass MHC restriction by directly linking T cells to tumor cells for targeted cytotoxicity.

Targeting the TRAC/TRBC1/TRBC2: Biological Roles and Therapeutic Strategies

•  Gene Information: TRAC, TRBC1, and TRBC2 encode the constant regions of the T-cell receptor (TCR) alpha and beta chains, where mutually exclusive TRBC1 or TRBC2 selection establishes a distinct genetic marker for clonal T-cell populations. 
•  Protein Expression: These proteins form the structural backbone of the surface αβTCR-CD3 complex, which is universally present on healthy T cells but exhibits strict monotypic restriction (either TRBC1 or TRBC2) on malignant T-cell clones. 
•  Signaling Pathway: Antigen engagement shifts the TCR constant domain to trigger downstream CD3 phosphorylation cascades, activating essential transcription factors for T-cell proliferation and survival. 
•  Therapeutic Inhibition: Targeting the specific TRBC subunit expressed by the lymphoma allows CAR-T cells or antibodies to selectively eradicate the malignancy while preserving the reciprocal, healthy T-cell compartment to maintain immunity.

CD3EDG

•  The chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out in B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice.

TRAC

•  The partial exon 1, exon 2, partial exon 3 and introns 1-2 of mouse Trac were replaced by partial exon 1, exon 2, partial exon 3 and introns 1-2 of human TRAC in the B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice.

TRBC1/TRBC2

•  The exons 1-2, partial exon 3 and introns 1-2 of mouse Trbc1 and Trbc2 were replaced by human exons 1-2, partial exon 3 and introns 1-2 of human TRBC1 and TRBC2 in B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice.

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by flow cytometry. Splenocytes and blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, 10-week-old, n=3).Protein expression was analyzed with anti-mouse CD3 antibody (Biolegend, 100312) and anti-humanCD3 antibody (BD, 556612) by flow cytometry.

Strain specific analysis of TRAC mRNA expression in wild-type C57BL/6JNifdc mice and B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by RT-PCR. Spleen RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TRAC primers.

Strain specific TRAC expression analysis in homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by flow cytometry. Splenocytes and blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, 10-week-old, n=3).TRAC expression was analyzed by flow cytometry using anti- TRAC antibody (Invitrogen, PA5-95587).

Strain specific TRBC1 expression analysis in homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by flow cytometry. Splenocytes and blood cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, 10-week-old, n=3).TRBC1 expression was analyzed by flow cytometry using anti-human TRBC1 antibody (Biolegend, 383515).

Strain specific TRBC2 expression analysis in homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by flow cytometry. Splenocytes were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, 10-week-old, n=3). TRBC2 expression was analyzed by flow cytometry using anti-human TRBC2 antibody (BD, 571191).

Strain-specific TCR α/β expression analysis in homozygous B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice by flow cytometry. Splenocytes were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, 10-week-old, n = 1). TCR α/β expression was analyzed by flow cytometry using anti-human TCR α/β antibody (BioLegend, 306717).

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes and thymus were isolated from male C57BL/6JNifdc and B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, n = 3, 10-week-old). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, lymph nodes and thymus were isolated from male C57BL/6JNifdc and B-hCD3EDG/hTRAC/hTRBC1/hTRBC2 mice (male, n = 3, 10-week-old). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8/hTRAC/hTRBC1/hTRBC2 mice (24h). T cells were isolated from splenocytes of C57BL/6JNifdc (male, 9-week-old, n = 3) and B-hCD3EDG/hCD8/hTRAC/hTRBC1/hTRBC2 mice (male, 9-week-old, n = 2), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 24h.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hCD3EDG/hCD8/hTRAC/hTRBC1/hTRBC2 mice (48h). T cells were isolated from splenocytes of C57BL/6JNifdc (male, 9-week-old, n = 3) and B-hCD3EDG/hCD8/hTRAC/hTRBC1/hTRBC2 mice (male, 9-week-old, n = 2), and incubated in the presence of anti-mCD3ε antibody (2 ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5 ug/ml, BioXcell, BE0015-1) for 48h.