Protein expression analysis in spleen and blood T cells

Strain specific CD32A expression analysis in wild type C57BL/6 mice and homozygous humanized B-hCD32A mice by flow cytometry. Splenocytes(A) and blood(B) were collected from wild type C57BL/6 mice (+/+) and homozygous B-hCD32A mice (H/H), and analyzed by flow cytometry with species-specific anti-mCD16 antibody and anti-hCD32A antibody. mCD16 was not detectable in B cells of wild-type C57BL/6 mice. hCD32A was not detectable in B cells of homozygous B-hCD32A mice.

Protein expression analysis in spleen and blood NK cells

Protein expression analysis in spleen and blood DC cells

Strain specific CD32A expression analysis in wild type C57BL/6 mice and homozygous humanized B-hCD32A mice by flow cytometry. Splenocytes(A) and blood(B) were collected from wild type C57BL/6 mice (+/+) and homozygous B-hCD32A mice (H/H), and analyzed by flow cytometry with species-specific anti-mCD16 antibody and anti-hCD32A antibody. mCD16 was detectable in DC cells of wild-type C57BL/6 mice. hCD32A was detectable in DC cells of homozygous B-hCD32A mice.

Protein expression analysis in spleen and blood monocytes

Strain specific CD32A expression analysis in wild type C57BL/6 mice and homozygous humanized B-hCD32A mice by flow cytometry. Splenocytes(A) and blood(B) were collected from wild type C57BL/6 mice (+/+) and homozygous B-hCD32A mice (H/H), and analyzed by flow cytometry with species-specific anti-mCD16 antibody and anti-hCD32A antibody. mCD16 was detectable in monocytes of wild-type C57BL/6 mice. hCD32A was weakly detectable in monocytes of homozygous B-hCD32A mice.

Protein expression analysis in spleen and blood macrophages

Strain specific CD32A expression analysis in wild type C57BL/6 mice and homozygous humanized B-hCD32A mice by flow cytometry. Splenocytes(A) and blood(B) were collected from wild type C57BL/6 mice (+/+) and homozygous B-hCD32A mice (H/H), and analyzed by flow cytometry with species-specific anti-mCD16 antibody and anti-hCD32A antibody. mCD16 was detectable in macrophages of wild-type C57BL/6 mice. hCD32A was detectable in macrophages of homozygous B-hCD32A mice.

Protein expression analysis in spleen and blood neutrophils

Strain specific CD32A expression analysis in wild type C57BL/6 mice and homozygous humanized B-hCD32A mice by flow cytometry. Splenocytes(A) and blood(B) were collected from wild type C57BL/6 mice (+/+) and homozygous B-hCD32A mice (H/H), and analyzed by flow cytometry with species-specific anti-mCD16 antibody and anti-hCD32A antibody. mCD16 was detectable in neutrophils of wild-type C57BL/6 mice. hCD32A was detectable in neutrophils of homozygous B-hCD32A mice.

Summary of hCD32A expression in B-hCD32A mice

Frequency of leukocyte subpopulations in spleen of humanized B-hCD32A mice

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild type C57BL/6 mice and homozygous B-hCD32A mice (n=3, 7-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hCD32A mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD32A does not change the frequency or distribution of these cell types in spleen. The frequency of leukocyte subpopulations in lymph nodes and blood of B-Hc d32a mice were also comparable to wild-type C57BL/6 mice (Data not shown). Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001. 

Summary
Protein expression analysis:
Mouse CD16 was only detectable in wild type C57BL/6 mice. Human CD32A was detectable in homozygous B-hCD32A but not in wild type mice. 
Leukocytes cell subpopulation analysis:
Humanization of CD32A does not change the overall frequency or distribution of immune cell types in spleen, blood and lymph nodes.