Correction: The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

The endocytic adaptor Eps15 controls marginal zone B cell numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

Eps15 has multiple roles in the immune system.

<p><b>A.</b> Scheme depicting the outline of the competitive bone marrow transplantation experiment. Bone marrow cells from Eps15-WT or -KO mice were mixed with WT bone marrow cells at a 1∶1 ratio and injected into sub-lethally irradiated recipient mice. The origin of the cells in the recipient mice was traced taking advantage of isogenic variation of the CD45 locus: CD45.2 is present in Eps15-WT and Eps15-KO mice, while CD45.1 is present in the wildtype (WT) mice used as competitor donor and as recipient mice. <b>B.–F.</b> Pie charts depicting the relative contribution of CD45.2 (Eps15-WT or Eps15-KO) versus WT CD45.1 positive competitor cells in the bone marrow (<b>B</b>), spleen (<b>C</b>), peritoneum (<b>D</b>), thymus (<b>E</b>) and lymph nodes (<b>F</b>) of recipient mice, as determined by FACS analysis. Significant differences (*p<0.05, **p<0.01) in the competition between Eps15-WT (n = 4) and Eps15-KO (n = 5) CD45.2⁺ cells with CD45.1⁺ WT cells, are indicated by red lines in the pie charts.</p

Eps15-KO mice are viable and show an immunological defect. A.

<p>Scheme depicts the region within the Eps15 wildtype (WT) locus that was altered to generate the knockout (KO) locus. The first coding exon of Eps15 and 2 kb of the 5′ promoter region were replaced by gene targeting with a neomycin (NEO) cassette. The location of the primers (a, b, c) used for genotyping is indicated (not to scale). <b>B.</b> Three primer PCR on tail biopsies gave the expected bands for the WT and KO allele: 515 and 642 bp, respectively. <b>C.</b> Western blot of total spleen lysates from Eps15-WT (WT) or Eps15-KO (KO) mice decorated with an anti-Eps15 antibody, or anti-vinculin antibody as loading control, shows lack of Eps15 protein in Eps15-KO mice. <b>D.</b> Bar graph depicting the percent of mice that have been weaned (black bars) for the indicated genotypes (wildtype (WT), heterozygous (Het), knockout (KO)) from the breeding of Eps15 heterozygous mice. The expected frequency for each genotype is depicted by the grey bars. A total of 231 pups were analyzed. <b>E.</b> Western blot analysis of multiple tissues from Eps15-WT (WT) and Eps15-KO (KO) mice reveals ubiquitous expression of Eps15 and Eps15L1. Total tissue lysates (10 µg protein) were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed for Eps15, Eps15L1 or vinculin, as loading control, as indicated. For brain lysates obtained from Eps15-WT (WT) or Eps15-KO (KO) mice, 10 and 20 µg protein were loaded to allow a better comparison of Eps15 and Eps15L1 protein levels. <b>F.–G.</b> Bar graphs displaying cell surface receptor levels (<b>F</b>) and internalization rate constants Ke (<b>G</b>) for the transferrin receptor (TfR) and the EGF receptor (EGFR) in Eps15-WT (white bars) and Eps15-KO (black bars) primary mouse fibroblasts. <b>H.</b> Bar graph depicting the number of white blood cells (WBC), lymphocytes (Lym), monocytes (Mo), granulocytes (Gr) and eosinophils (Eo) in the peripheral blood of 3-month-old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by hematological analysis. <b>I.</b> Bar graph depicting the number of B cells (B), T cells (T), granulocytes (Gr), monocytes (Mo) and natural killer cells (NK) in the peripheral blood of 3 month old Eps15-WT (WT, white bars, n = 10) and Eps15-KO (KO, black bars, n = 10) mice as determined by FACS analysis. Statistical significance was assessed using Student’s t-test. Significant differences are indicated as * = p<0.05, *** = p<0.005.</p

FACS analysis of the cellular populations present in the thymus three months after competitive bone marrow transplantation.

<p>Distribution of CD45.1+ and CD45.2+ cells in the thymus of recipient mice 3 months after bone marrow transplantation. CD45.1+ WT and CD45.2+ WT or CD45.2+ KO donor cells were mixed at a 1∶1 ratio prior to injection into recipient CD45.1+ mice. The percentage of total cells gated and the percentage of CD45.1+ or CD45.2+ cells for any given gate are shown. Significance was assessed using Student’s t-test and p-values are indicated as *p<0.05, **p<0.01, ***p<0.001.</p

The MZ B cell numbers increase over time in Eps15-KO mice. A.–B.

<p>Bar graphs depicting the total number of Fo, MZ and T1 B cells in the spleen of 2 month old (<b>A</b>) and 24 month old (<b>B</b>) Eps15-WT (WT, white bars) and Eps15-KO (KO, black bars) mice. <b>C.–D.</b> Bar graphs depicting the total and CD19+ cell number in the spleen of 2-month-old (<b>A</b>) and 24-month-old (<b>B</b>) (Eps15-WT (WT, white bars) and Eps15-KO (KO, black bars) mice. Values are depicted as mean±standard error mean. N = 4 per two month old and n = 5 per 24 month old mice. Statistical significance was assessed using Student’s t-test and significant differences are indicated as * = p<0.05, ** = p<0.01.</p

Normal motor activity in aged Eps15-KO mice.

<p>Motoractivity was measured in aged (>20-month old) mice according to the modified SHIRPA protocol.</p

FACS analysis of the B cell populations present in the bone marrow three months after competitive bone marrow transplantation.

<p>Distribution of CD45.1+ and CD45.2+ cells in the bone marrow of recipient mice 3 months after bone marrow transplantation. CD45.1+ WT and CD45.2+ WT or CD45.2+ KO donor cells were mixed at a 1∶1 ratio prior to injection into recipient CD45.1+ mice. The percentage of total cells gated and the percentage of CD45.1+ or CD45.2+ cells for any given gate are shown. Significance was assessed using Student’s t-test and p-values are indicated as. *p<0.05, **p<0.01, ***p<0.001.</p

FACS analysis of the B cell populations present in the peritoneal cavity three months after competitive bone marrow transplantation.

<p>Distribution of CD45.1+ and CD45.2+ cells in the peritoneal cavity of recipient mice 3 months after bone marrow transplantation. CD45.1+ WT and CD45.2+ WT or CD45.2+ KO donor cells were mixed at a 1∶1 ratio prior to injection into recipient CD45.1+ mice. The percentage of total cells gated and the percentage of CD45.1+ or CD45.2+ cells for any given gate are shown. Significance was assessed using Student’s t-test and p-values are indicated as *p<0.05, **p<0.01, ***p<0.001.</p

Eps15-KO mice show normal pre-MZ B and transitional B cell numbers. A.

<p>Photomicrographs of spleen sections from adult Eps15-WT and Eps15-KO mice. Sections were stained for IgD (PE, red) and IgM (FITC, green) to visualize MZ B (IgM⁺IgD^low/−) and Fo B cells (IgD⁺IgM⁻). <b>B.</b> Bar graph representing the quantification of the ratio of the area occupied by MZ B cells over the area occupied by Fo B cells in Eps15-WT and Eps15-KO spleens (n = 12 per genotype). <b>C.</b> Dot plots of splenocytes from Eps15-WT and Eps15-KO mice stained for CD19 and CD1d to identify the pre-MZ B plus MZ B (pre+MZB) cell populations. This mixed CD19⁺CD1d⁺⁺ population was further analyzed for CD23 to distinguish pre-MZ B (CD19⁺/CD1d⁺⁺/CD23⁺) from mature MZ B (CD19⁺/CD1d⁺⁺/CD23⁻) cells. <b>D.</b> Bar graph depicting total cell numbers of pre+MZB cells, mature MZ B and pre-MZ B cells in the spleens of Eps15-WT (WT, white bars) and Eps15-KO (KO, black bars) mice (n = 5). <b>E.</b> Dot plots of splenocytes from Eps15-WT and Eps15-KO mice stained for CD19, CD21 and CD23 to identify Fo B (CD19⁺/CD21⁺/CD23⁺), MZ B (CD19⁺/CD21⁺/CD23⁻) and T1 transitional B (CD19⁺/CD21^−/CD23⁻) cells. <b>F.</b> Bar graph depicting total cell numbers of Fo B, MZ B and T1 transitional B cells in the spleens of Eps15-WT (WT, white bars) and Eps15-KO (KO, black bars) mice (n = 5). <b>G.</b> Dot plots of splenocytes from Eps15-WT and Eps15-KO mice stained for CD19, IgD and IgM to identify Fo B (CD19⁺/IgD+IgM^Low), MZ B plus T1 transitional B (MZ B+T1, CD19⁺/IgD^low/−IgM⁺) and T2 transitional B (CD19⁺/IgD⁺IgM⁺) cells. <b>H.</b> Bar graph depicting total cell numbers of Fo B, MZ B+T1, and T2 transitional B cells in the spleens of Eps15-WT (WT, white bars) and Eps15-KO (KO, black bars) mice (n = 5). Values are depicted as mean±standard error mean. Statistical significance was assessed using Student’s t-test and significant differences are indicated as *p<0.05, **p<0.01, ***p<0.005.</p