Gene targeting strategy for B-hCD16B mice. The human gene coding sequence was inserted to mouse Hipp11(H11) allele in B-hCD16B mice.

Strain specific CD16B expression analysis in homozygous B-hCD16B mice by flow cytometry. Blood (A) and spleen (B) were collected from wild-type mice (+/+) and homozygous B-hCD16B mice (H/H), and analyzed by flow cytometry with species-specific anti-CD16 antibody, respectively. Human CD16B was exclusively detectable in granulocytes of homozygous B-hCD16B mice. Note: the clone of anti-human CD16B antibody is 3G8.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. B. Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Blood cells were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. B. Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of blood T cell subpopulations by FACS. Blood cells were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these T cell subtypes in blood. Values are expressed as mean ± SEM.

Analysis of lymph nodes leukocyte subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the lymph nodes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. B. Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these cell types in lymph nodes. Values are expressed as mean ± SEM.

Analysis of lymph nodes T cell subpopulations by FACS. Lymph nodes were isolated from female C57BL/6 and homozygous B-hCD16B mice (n=3, 7-week-old). Flow cytometry analysis of the lymph nodes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percentages of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCD16B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD16B does not change the overall development, differentiation or distribution of these T cell subtypes in lymph nodes. Values are expressed as mean ± SEM.