CD45: A receptor-type protein tyrosine phosphatase broadly expressed on leukocytes

• Gene Information: Protein tyrosine phosphatase receptor type C (PTPRC, also known as CD45) is a protein-coding gene located on chromosome 1q31.3-q32.1. CD45, a leukocyte common antigen, is pivotal in the hematological and immunological systems.
• Protein Expression: CD45, a receptor tyrosine phosphatase, functions as a cell-surface antigen expressed by all immune and hematopoietic cells—such as hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs)—excluding platelets and erythrocytes. As one of the most highly expressed cell-surface glycoproteins, CD45 occupies as much as 10% of the total cellular surface area. Its cytoplasmic domain exhibits an extraordinarily high sequence homology of 95% among all examined mammalian species, whereas the extracellular region merely shares 35% sequence identity.
• Signaling Pathway: Unlike CD117, CD33 and CD123, CD45 represents one of the most highly abundant proteins on the plasma membrane of T cells. Concurrent expression of the same target antigen by CAR‑T cells and their intended target cells poses a critical obstacle that hinders the clinical efficacy of CAR‑T cell therapy.
• Therapeutic Inhibition: Anti-CD45 antibody-drug conjugate (ADC) is used for precise elimination of hematological tumors and pre-treatment before hematopoietic stem cell transplantation.

CD45

• The exons 2-15 of mouse Cd45 gene that encode extracellular domain and part of transmembrane domain are replaced by human counterparts in B-hCD45 mice.
• The genomic region of mouse Cd45 gene that encodes signal peptide, transmembrane domain and cytoplasmic portion are retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The CD45 expression is driven by endogenous mouse Cd45 promoter, while mouse Cd45 gene transcription and translation will be disrupted.

• Mouse CD45 was detected in wild-type C57BL/6 mice.
• Human CD45 was detected in B-hCD45 mice, but not in wild-type C57BL/6 mice.

Strain specific CD45 expression analysis in homozygous B-hCD45 mice by flow cytometry. Blood was collected from wild-type C57BL/6 (+/+) and homozygous B-hCD45 mice (H/H) and analyzed by flow cytometry with species-specific anti-CD45 antibody. Mouse CD45 was detectable in wild-type mice. Human CD45 was exclusively detectable in homozygous B-hCD45 mice but not in wild-type mice.

• The percentages of T cells, B cells, NK cells, DCs, granulocytes, monocytes, and macrophages in homozygous B-hCD45 mice were similar to those in C57BL/6 mice.
• Humanization CD45 does not affect normal immune cell development or splenic 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-hCD45 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-hCD45 mice were comparable to those in C57BL/6 mice.
• Humanization of CD45 does not affect normal T cell development, differentiation, or splenic 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-hCD45 mice (female, 8-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• Mouse CD45 was detected in megakaryocytes and mast cells but not erythrocytes from wild-type C57BL/6JNifdc mice.
• Human CD45 was detected in megakaryocytes and mast cells but not erythrocytes from B-hCD45 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD45 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD45 mice by flow cytometry. Bone marrow, peritoneal lavage fluid and blood were collected from wild-type C57BL/6JNifdc mice(+/+) (n=1, Female, 7-week-old) and homozygous B-hCD45 mice (H/H) (n=1, Female, 7-week-old). Protein expression was analyzed with anti-mouse CD45 antibody (Biolegend, 103138) and anti-human CD45 antibody (Biolegend, 304036) by flow cytometry. Mouse CD45 was only detectable in wild-type C57BL/6JNifdc mice, and human CD45 was only detectable in homozygous B-hCD45 mice.

• Mouse CD45 was detected in T cells, B cells and NK cells from wild-type C57BL/6JNifdc mice.
• Human CD45 was detected in T cells, B cells and NK cells from B-hCD45 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD45 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD45 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) (n=1, Female, 7-week-old) and homozygous B-hCD45 mice (H/H) (n=1, Female, 7-week-old). Protein expression was analyzed with anti-mouse CD45 antibody (Biolegend, 103138) and anti-human CD45 antibody (Biolegend, 304036) by flow cytometry. Mouse CD45 was only detectable in wild-type C57BL/6JNifdc mice, and human CD45 was only detectable in homozygous B-hCD45 mice.

• Mouse CD45 was detected in monocytes, macrophages, DC cells and neutrophils from wild-type C57BL/6JNifdc mice.
• Human CD45 was detected in monocytes, macrophages, DC cells and neutrophils from B-hCD45 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD45 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD45 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) (n=1, Female, 7-week-old) and homozygous B-hCD45 mice (H/H) (n=1, Female, 7-week-old). Protein expression was analyzed with anti-mouse CD45 antibody (Biolegend, 103138) and anti-human CD45 antibody (Biolegend, 304036) by flow cytometry. Mouse CD45 was only detectable in wild-type C57BL/6JNifdc mice, and human CD45 was only detectable in homozygous B-hCD45 mice.

• Mouse CD45 was detected in T cells, B cells, NK cells, monocytes, macrophages, DC cells and neutrophils from wild-type C57BL/6JNifdc mice.
• Human CD45 was detected in T cells, B cells, NK cells, monocytes, macrophages, DC cells and neutrophils from B-hCD45 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific CD45 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD45 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice(+/+) (n=1, Female, 7-week-old) and homozygous B-hCD45 mice (H/H) (n=1, Female, 7-week-old). Protein expression was analyzed with anti-mouse CD45 antibody (Biolegend, 103138) and anti-human CD45 antibody (Biolegend, 304036) by flow cytometry. Mouse CD45 was only detectable in wild-type C57BL/6JNifdc mice, and human CD45 was only detectable in homozygous B-hCD45 mice.

• Mouse CD45 was only detectable in megakaryocytes from wild-type C57BL/6JNifdc mice
• Human CD45 was only detectable in megakaryocytes but not platelets from homozygous B-hCD45 mice.

Strain specific CD45 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hCD45 mice by flow cytometry. Bone marrow and blood were collected from wild-type C57BL/6JNifdc mice(+/+) (n=1, Female, 7-week-old) and homozygous B-hCD45 mice (H/H) (n=1, Female, 7-week-old). Protein expression was analyzed with anti-mouse CD45 antibody (Biolegend, 103138) and anti-human CD45 antibody (Biolegend, 304036) by flow cytometry. Mouse CD45 was only detectable in megakaryocytes from wild-type C57BL/6JNifdc mice. Human CD45 was only detectable in megakaryocytes but not platelets from homozygous B-hCD45 mice.

• Human CD45 is expressed ubiquitously in tissues of female B-hCD45 mice.

Immunohistochemical (IHC) analysis was performed to evaluate CD45 protein expression in wild-type (WT) C57BL/6JNifdc mice and B-hCD45 mice. Tissues from eleven major organs were collected from WT and homozygous B-hCD45 mice (n=1, 8 weeks old) and analyzed by IHC using anti-CD45 antibody (CST, 70257). Human CD45 expression was detected in the heart, liver, spleen, lung, brain, stomach, colon, small intestine, uterus and ovary of homozygous B-hCD45 mice. The results demonstrated that the human CD45 is expressed ubiquitously in tissues of B-hCD45 mice. The red arrow indicates the positive cells.

• Human CD45 is expressed ubiquitously in tissues of male B-hCD45 mice.

Immunohistochemical (IHC) analysis was performed to evaluate CD45 protein expression in wild-type (WT) C57BL/6JNifdc mice and B-hCD45 mice. Tissues from eleven major organs were collected from WT and homozygous B-hCD45 mice (n=1, 8 weeks old) and analyzed by IHC using anti-CD45 antibody (CST, 70257). Human CD45 expression was detected in the heart, liver, spleen, lung, brain, stomach, colon, small intestine, and testis of homozygous B-hCD45 mice. The results demonstrated that the human CD45 is expressed ubiquitously in tissues of B-hCD45 mice . The red arrow indicates the positive cells.