CD3/CD28: A pivotal dual-receptor axis for complete T cell activation and its therapeutic engagement.

• Gene Information: CD3 is a multi-protein complex functioning as the primary T cell receptor (TCR) co-receptor. CD28 is a critical costimulatory receptor. Together, they form the essential dual-signal axis required for complete T cell activation and survival.
• Protein Expression: Both CD3 and CD28 are constitutively and highly expressed on the surface of mature naive and memory T lymphocytes. This ubiquitous co-expression profile makes them ideal foundational targets for universally engaging and activating T cells.
• Signaling Pathway: CD3 engagement by antigen initiates primary TCR phosphorylation cascades. Concurrent CD28 binding by costimulatory ligands triggers synergistic PI3K/AKT pathways. This combined interaction promotes robust cellular proliferation, enhanced cytokine secretion, and prolonged effector T cell survival.
• Therapeutic Engagement: Engaging the CD3/CD28 axis with bispecific antibodies or engineered CAR constructs specifically redirects and supercharges T cell cytotoxicity against malignant cells. This overcomes tumor immunosuppression, driving robust anti-tumor efficacy and demonstrating significant potential in advanced cancer immunotherapy.

• Humanized CD3D and CD3G mRNA were detectable only in thymus of homozygous B-hCD3EDG/hCD28 mice (H/H, H/H), but not in wild-type C57BL/6 mice (+/+).
• Mouse Cd3d and Cd3g mRNA were detectable only in thymus of wild-type C57BL/6 mice (+/+).

Mouse and human CD3D and CD3G analysis in thymus by RT-PCR and sequencing. Thymus RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (H/H, H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human CD3D and CD3G primers.

• Mouse CD3E was detected on T cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/hCD28 mice.
• Human CD3E was detected on T cells populations in B-hCD3EDG/hCD28 mice, but not in wild-type C57BL/6 mice.

Mouse and human CD3E expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD28 mice (female, 8-week-old, n = 1). CD3E expression on T cells was analyzed by flow cytometry using species-specific anti-CD3E antibodies (anti-human CD3E antibody, BD Horizon, 562426; anti-mouse CD3E antibody, Biolegend, 100312 ).

• Mouse CD28 was detected on T cells populations in wild-type C57BL/6 mice, but not in B-hCD3EDG/hCD28 mice.
• Human CD28 was detected on T cells populations in B-hCD3EDG/hCD28 mice, but not in wild-type C57BL/6 mice.

Mouse and human CD28 expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hCD3EDG/hCD28 mice (female, 8-week-old, n = 1). CD28 expression on T cells was analyzed by flow cytometry using species-specific anti-CD28 antibodies (anti-human CD28 antibody, Biolegend, 302912; anti-mouse CD28 antibody, Biolegend, 102105).

• The percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, and macrophages in homozygous B-hCD3EDG/hCD28 mice are similar to those in C57BL/6 mice.
• Humanization of CD3EDG/CD28 does not affect normal immune cell development or distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hCD3EDG/hCD28 mice are comparable to those in C57BL/6 mice.
• Humanization of CD3EDG/CD28 does not affect normal T cell development, differentiation or distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and B-hCD3EDG/hCD28 mice (female, 8-week-old, n = 3). Single live cells were gated on the TCR+ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• T cell activation in B-hCD3EDG/hCD28 mice was significantly up-regulated by Anti-CD3ε antibody and Anti-CD28 antibody.

Analysis of T-cell activation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 24 hours. T-cell activation was analyzed by flow cytometry using anti-mouse CD25 antibody (BioLegend, 104514) and anti-mouse CD69 antibody (BioLegend, 102034).

Analysis of T-cell activation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 48 hours. T-cell activation was analyzed by flow cytometry using anti-mouse CD25 antibody (BioLegend, 104514) and anti-mouse CD69 antibody (BioLegend, 102034).

Analysis of T-cell activation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 72 hours. T-cell activation was analyzed by flow cytometry using anti-mouse CD25 antibody (BioLegend, 104514) and anti-mouse CD69 antibody (BioLegend, 102034).

Analysis of T-cell proliferation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 48 hours. T-cell proliferation was analyzed by flow cytometry using CFSE (Invitrogen, C34554).

Analysis of T-cell proliferation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 72 hours. T-cell proliferation was analyzed by flow cytometry using CFSE (Invitrogen, C34554).

• Anti-human CD28 therapies led to changed cytokine profile in B-hCD3EDG/hCD28 mice

Analysis of T-cell activation stimulated with anti-CD3 and anti-CD28 antibodies in vitro. T cells were isolated from the splenocytes of wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3EDG/hCD28 mice (7-week-old, n = 3). Cells were incubated in the presence of anti-mouse CD3ε antibody (2 μg/mL, BioXcell, BE0001-1), anti-human CD3ε antibody (2 μg/mL, BioXcell, BE0001-2), anti-mouse CD28 antibody (5 μg/mL, BioXcell, BE0015-1), and anti-human CD28 antibody (5 μg/mL, BioXcell, BE0248) for 24, 48, and 72 hours. IFN-γ and IL-2 production was then analyzed using the SLU Testing System (from SOLUTION). Values are expressed as mean ± SEM. Significance was determined by a two-way ANOVA test. *p < 0.05, p < 0.01, ***p < 0.001, ****p < 0.0001.