Limits...
Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs.

de Winde CM, Zuidscherwoude M, Vasaturo A, van der Schaaf A, Figdor CG, van Spriel AB - Histochem. Cell Biol. (2015)

Bottom Line: CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen.In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs.This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein-Zuid 26, 6525 GA, Nijmegen, The Netherlands.

ABSTRACT
Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

No MeSH data available.


Expression of CD37 and CD53 on immune cell subsets in blood. a Flow cytometry analysis of expression of CD37 or CD53 (black line) on CD4 and CD8 T cells, B cells, monocytes and NK cells versus isotype control (gray line). Gating strategy is presented in Supplementary Figure 1. Expression levels of CD37 (b) and CD53 (c) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from three healthy donors. Data present mean ± SD. d Flow cytometry analysis of expression of CD37 or CD53 (black line) on mDCs (BDCA1+CD19−) or pDCs (BDCA2+) versus isotype control (gray line). Expression levels of CD37 (e) and CD53 (f) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from two healthy donors. Data present mean ± SD
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4522275&req=5

Fig1: Expression of CD37 and CD53 on immune cell subsets in blood. a Flow cytometry analysis of expression of CD37 or CD53 (black line) on CD4 and CD8 T cells, B cells, monocytes and NK cells versus isotype control (gray line). Gating strategy is presented in Supplementary Figure 1. Expression levels of CD37 (b) and CD53 (c) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from three healthy donors. Data present mean ± SD. d Flow cytometry analysis of expression of CD37 or CD53 (black line) on mDCs (BDCA1+CD19−) or pDCs (BDCA2+) versus isotype control (gray line). Expression levels of CD37 (e) and CD53 (f) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from two healthy donors. Data present mean ± SD

Mentions: To investigate cell surface expression of tetraspanins CD37 and CD53 on the plasma membrane of different immune cell subsets in blood, PBLs were stained for CD4 (T cells), CD8/CD3/CD56− (T cells), CD20 (B cells), CD14 (monocytes), CD56/CD3−/CD8− (NK cells) and BDCA1/CD19− (myeloid DC (mDC)) and BDCA2 (plasmacytoid DC (pDC)) (Supplementary Fig. 1). We observed the highest CD37 expression on B cells and low to medium expression on T cells, monocytes and NK cells (Fig. 1a, b).CD53 was expressed on all subsets, with highest expression on B cells and monocytes (Fig. 1a, c). CD37 and CD53 were expressed on both mDCs and pDCs, with no apparent differences in expression level between the two DC subsets (Fig. 1d–f). It has been reported that tetraspanins can be expressed at intracellular membranes (Kobayashi et al. 2000; Xu et al. 2009), which stimulated us to investigate the subcellular localization of CD37 and CD53. Monocytes were double stained with CD37 or CD53 antibodies in combination with antibodies specific for the endoplasmatic reticulum (ER), endosomes or lysosomes. Next to the expression on the plasma membrane, both CD37 and CD53 were abundantly expressed in the endosomes, in contrast to the ER (Fig. 2a, b). In the lysosomes, we observed only CD53 to be present.Fig. 1


Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs.

de Winde CM, Zuidscherwoude M, Vasaturo A, van der Schaaf A, Figdor CG, van Spriel AB - Histochem. Cell Biol. (2015)

Expression of CD37 and CD53 on immune cell subsets in blood. a Flow cytometry analysis of expression of CD37 or CD53 (black line) on CD4 and CD8 T cells, B cells, monocytes and NK cells versus isotype control (gray line). Gating strategy is presented in Supplementary Figure 1. Expression levels of CD37 (b) and CD53 (c) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from three healthy donors. Data present mean ± SD. d Flow cytometry analysis of expression of CD37 or CD53 (black line) on mDCs (BDCA1+CD19−) or pDCs (BDCA2+) versus isotype control (gray line). Expression levels of CD37 (e) and CD53 (f) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from two healthy donors. Data present mean ± SD
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522275&req=5

Fig1: Expression of CD37 and CD53 on immune cell subsets in blood. a Flow cytometry analysis of expression of CD37 or CD53 (black line) on CD4 and CD8 T cells, B cells, monocytes and NK cells versus isotype control (gray line). Gating strategy is presented in Supplementary Figure 1. Expression levels of CD37 (b) and CD53 (c) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from three healthy donors. Data present mean ± SD. d Flow cytometry analysis of expression of CD37 or CD53 (black line) on mDCs (BDCA1+CD19−) or pDCs (BDCA2+) versus isotype control (gray line). Expression levels of CD37 (e) and CD53 (f) were normalized for isotype staining by background subtraction. Experiments were performed with PBLs from two healthy donors. Data present mean ± SD
Mentions: To investigate cell surface expression of tetraspanins CD37 and CD53 on the plasma membrane of different immune cell subsets in blood, PBLs were stained for CD4 (T cells), CD8/CD3/CD56− (T cells), CD20 (B cells), CD14 (monocytes), CD56/CD3−/CD8− (NK cells) and BDCA1/CD19− (myeloid DC (mDC)) and BDCA2 (plasmacytoid DC (pDC)) (Supplementary Fig. 1). We observed the highest CD37 expression on B cells and low to medium expression on T cells, monocytes and NK cells (Fig. 1a, b).CD53 was expressed on all subsets, with highest expression on B cells and monocytes (Fig. 1a, c). CD37 and CD53 were expressed on both mDCs and pDCs, with no apparent differences in expression level between the two DC subsets (Fig. 1d–f). It has been reported that tetraspanins can be expressed at intracellular membranes (Kobayashi et al. 2000; Xu et al. 2009), which stimulated us to investigate the subcellular localization of CD37 and CD53. Monocytes were double stained with CD37 or CD53 antibodies in combination with antibodies specific for the endoplasmatic reticulum (ER), endosomes or lysosomes. Next to the expression on the plasma membrane, both CD37 and CD53 were abundantly expressed in the endosomes, in contrast to the ER (Fig. 2a, b). In the lysosomes, we observed only CD53 to be present.Fig. 1

Bottom Line: CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen.In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs.This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein-Zuid 26, 6525 GA, Nijmegen, The Netherlands.

ABSTRACT
Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

No MeSH data available.