• CD5 is a type I transmembrane glycoprotein primarily expressed on T cells and a subset of B cells, where it functions as an important immune regulatory molecule. CD5 modulates T cell receptor signaling thresholds and plays a key role in immune tolerance, autoimmunity, and lymphocyte activation.
• The CD5 humanized mice were generated by replacing the extracellular domain of the mouse Cd5 gene with its human counterpart. This strategy preserves physiological expression patterns while enabling human CD5–specific antibody binding and signaling.
• This model provides a translational platform for studying human CD5 biology, evaluating CD5-targeting antibodies in vivo, and investigating T cell–mediated autoimmune and inflammatory diseases.

Key Advantages

• Physiological humanized CD5 expression under endogenous regulatory control
• Enables human-specific antibody binding without mouse cross-reactivity
• Preserved immune homeostasis suitable for chronic studies
• High translational relevance for autoimmune and immunotherapy research
• Compatible with multiple disease models, including inflammatory and immune dysregulation models

Validation

• Molecular Validation: Humanized CD5 mRNA is specifically detected in lymphoid tissues of CD5 humanized mice, while mouse Cd5 transcripts are absent.
• Protein Expression Validation: Flow cytometry confirms that humanized CD5 protein is selectively expressed on T cells and CD5-positive immune subsets in CD5 humanized mice, with no detectable mouse CD5 expression.
• Functional Validation: Humanized CD5 maintains normal immune cell development and T cell distribution, while enabling binding and functional assessment of human CD5–specific antibodies in vivo.

Applications

• Autoimmune disease research (e.g., rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus)
• T cell signaling and immune tolerance studies
• In vivo validation of anti-CD5 antibodies
• Immunotherapy mechanism-of-action studies
• Preclinical pharmacology and safety assessment

CD5A

• The exons 2-7 of mouse CD5 gene that encode signal peptide and extracellular domain are replaced by human counterparts in CD5 humanized mice.
• The genomic region of mouse CD5 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric CD5 expression is driven by endogenous mouse CD5 promoter, while mouse CD5 gene transcription and translation will be disrupted.

• Mouse CD5 mRNA was detectable in wild-type C57BL/6N mice.
• Human CD5 mRNA was detectable only in homozygous CD5 humanized mice but not in wild-type mice.

Strain-specific analysis of mouse and human CD5 mRNA expression in wild-type C57BL/6N mice and CD5 humanized mice by RT-PCR. Total RNA was isolated from thymus tissues of wild-type C57BL/6N mice (+/+) and homozygous CD5 humanized mice (H/H). cDNA was synthesized by reverse transcription, followed by PCR amplification using mouse- or human-specific CD5 primers.

• Mouse CD5 was detectable in wild-type mice.
• Human CD5 was exclusively detectable in homozygous CD5 humanized mice but not in wild-type mice.

Strain-specific analysis of mouse and human CD5 protein expression in wild-type C57BL/6N mice and CD5 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice (+/+) and homozygous CD5 humanized mice (H/H). Mouse and human CD5 protein expression was analyzed by flow cytometry using species-specific anti-mouse CD5 (BioLegend, 100625) and anti-human CD5 (BioLegend, 300607) antibodies.

• Mouse CD5 was detectable in cDC1 cells from wild-type mice.
• Human CD5 was exclusively detectable in cDC1 cells from homozygous CD5 humanized mice but not in wild-type mice.

Strain-specific analysis of mouse and human CD5 protein expression in wild-type C57BL/6 mice and CD5 humanized mice by flow cytometry. Spleens were collected from wild-type C57BL/6 mice (+/+) and homozygous CD5 humanized mice (H/H). Splenocytes were analyzed by flow cytometry using species-specific anti-CD5 antibodies. Mouse CD5 was detected in wild-type C57BL/6 mice, whereas human CD5 was detected exclusively in homozygous CD5 humanized mice.

• Mouse CD5 was detectable in cDC2 cells from wild-type mice.
• Human CD5 was exclusively detectable in cDC2 cells from homozygous CD5 humanized mice but not in wild-type mice.

Strain-specific analysis of mouse and human CD5 protein expression in wild-type C57BL/6 mice and CD5 humanized mice by flow cytometry. Spleens were collected from wild-type C57BL/6 mice (+/+) and homozygous CD5 humanized mice (H/H). Splenocytes were analyzed by flow cytometry using species-specific anti-CD5 antibodies.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous CD5 humanized mice are similar to those in C57BL/6mice.
• Humanization of CD5 does not affect normal immune cell development or distribution.

Flow cytometry analysis of leukocyte subpopulations in immune organs and peripheral blood of CD5 humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female wild-type C57BL/6 mice and CD5 humanized mice (8 weeks 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 CD5 humanized mice are comparable to those in C57BL/6 mice.
• Humanization of CD5 does not affect normal T cell development, differentiation, or distribution.

Flow cytometry analysis of T-cell subpopulations in immune organs and peripheral blood of CD5 humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female wild-type C57BL/6 mice and CD5 humanized mice (8 weeks old, n = 3). Single live cells were gated on the T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• MC38 tumor growth was comparable between CD5 humanized mice and wild-type C57BL/6 mice, suggesting that human CD5 expression does not significantly affect tumor growth in this model.

Subcutaneous MC38 homograft tumor growth in CD5 humanized mice. MC38 mouse colon carcinoma cells (5 × 10⁵) were subcutaneously implanted into wild-type C57BL/6 mice and CD5 humanized mice (female, 11 weeks old, n = 6). Tumor volumes were measured twice weekly and calculated using the formula: V = 0.5 × long diameter × (short diameter)². Tumor volumes are expressed in mm³, and data are presented as mean ± SEM. MC38 cells successfully established subcutaneous tumors in CD5 humanized mice, demonstrating the suitability of this model for in vivo antitumor efficacy studies.

• T cells from spleen, blood and LNs of B-hCD5 mice demonstrate specific delivery of CD5-tLNPs in vivo 

The efficient and specific delivery analysis of CD5-tLNP to CD5+ T cells in spleen of B-hCD5 mice. EGFP and CD5 expression in mCD19+ B cells , mCD11b+ myeloid cells, mCD3+ T cells,, mCD3+ mCD4+ T cells, and mCD3+ mCD8+ T cells 24 hours after intravenous dosing of 10 μg CD5-tLNP-EGFP (levostar) in B-hCD5 mice (n=3).

• T cells from spleen of B-hCD5 mice demonstrate specific delivery of CD5-tLNPs in vitro 
• CD5⁺ T cells show markedly decreased CD5 expression caused by internalization.

The efficient and specific delivery analysis of CD5-tLNP to CD5+ T cells in spleen of B-hCD5 mice. EGFP and CD5 expression in mCD19+ B cells , mCD11b+ myeloid cells, mCD3+ T cells,, mCD3+ mCD4+ T cells, and mCD3+ mCD8+ T cells 24 hours after intravenous dosing of CD5-tLNP-EGFP (levostar) in B-hCD5 mice (n=2). (A) Flow Cytometry. (B) Statistical Graph.

• CD5‑tLNP (CD19 CAR) can effectively eliminate B cells in B-hCD5 mice.

Evaluation of B cell depletion efficacy of CD5‑tLNP (mCD19 CAR) in B-hCD5 mice. B-hCD5 mice (n=3/group) were were injected via tail vein with different masses of CD5-tLNP encapsulating mCD19 CAR mRNAs. The Vector 1 group was injected with empty LNPs (not encapsulating RNA). On days 0, 1, 3, and 5, the expression efficiency of CAR in CD5⁺ T cells in peripheral blood (A) was measured; on days 0, 3, 5, and 8, changes in B cell counts (B) were assessed.

• CD5‑tLNP (CD19 CAR) can alleviate NMOSD disease in Humanized B-hCD5 mice. 
• It is demonstrated that Humanized B-hCD5 mice can serve as a robust model for evaluating in vivo CAR-T efficacy.

Efficacy Evaluation of CD5‑tLNP (mCD19 CAR) in NMOSD mouse model of Humanized B-hCD5 mice . Humanized B-hCD5 mice (n=5/group) were induced with NMOSD by MOG 35-55 and AQP4201-220, which were injected tail vein injection of corresponding CD5-tLNP formulations about one week after model induction. (A) Scoring criteria for NMOSD mice. (B) The statistical charts of motor impairment scores in NMOSD mice one week after treatment with different CD5-TLNPs. (C) The statistical charts of serum anti-AQP4 IgG antibodies in NMOSD mice one week after treatment with different CD5-TLNPs.