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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.


Localization and expression of CD53 in bone marrow (a–f), spleen (g–l) and appendix (m–r). a, g, m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a, g, m = 100 μm. b, h, n, Composite RGB image after spectral unmixing of original image (red CD20, blue CD53, green nuclei). c, i, o, Image showing scoring of CD20−CD53dim (blue), CD20+CD53dim (red), CD20+CD53bright (yellow) or CD20−CD53bright (green) cells. d, j, p, Optical density of CD53 on individual cells in human bone marrow (d), in B cell follicle (blue line) and in T cell zone and in red pulp (red line) in human spleen (j) and in B cell follicle (blue line) and in lamina propria (red line) in human appendix (p). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53dim and CD53bright cells in the CD20+ cell population (e) and in the CD3+ cell population (f) in human bone marrow. Percentage of CD53bright cells in the CD20+ cell population in the B cell follicle and red pulp (k), and in the CD3+ cell population in the T cell zone and red pulp (l) in human spleen. Percentage of CD53bright cells in the CD20+ cell population (q) and in the CD3+ cell population (r) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. *P < 0.05, ****P < 0.0001
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Fig7: Localization and expression of CD53 in bone marrow (a–f), spleen (g–l) and appendix (m–r). a, g, m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a, g, m = 100 μm. b, h, n, Composite RGB image after spectral unmixing of original image (red CD20, blue CD53, green nuclei). c, i, o, Image showing scoring of CD20−CD53dim (blue), CD20+CD53dim (red), CD20+CD53bright (yellow) or CD20−CD53bright (green) cells. d, j, p, Optical density of CD53 on individual cells in human bone marrow (d), in B cell follicle (blue line) and in T cell zone and in red pulp (red line) in human spleen (j) and in B cell follicle (blue line) and in lamina propria (red line) in human appendix (p). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53dim and CD53bright cells in the CD20+ cell population (e) and in the CD3+ cell population (f) in human bone marrow. Percentage of CD53bright cells in the CD20+ cell population in the B cell follicle and red pulp (k), and in the CD3+ cell population in the T cell zone and red pulp (l) in human spleen. Percentage of CD53bright cells in the CD20+ cell population (q) and in the CD3+ cell population (r) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. *P < 0.05, ****P < 0.0001

Mentions: We studied the localization and expression of tetraspanin CD37 in primary and secondary lymphoid organs. Since bone marrow does not contain different T and B cell areas, tissue segmentation was not applicable and only cell segmentation was performed (Figs. 6a–c, 7a–c). The intensity [optical density (OD)] of CD37 in bone marrow ranged from 0.05 to 0.85, with a mean of 0.29 (ODmean; Fig. 6d). We observed around 80 % of all bone marrow cells to be CD37bright (Fig. 6e) and 90 % of all T cells to be highly positive for CD37 (Fig. 6f). The scatter plots with the set thresholds to annotate cells with dim and bright expression of CD37 and CD53 are shown in Figs. 4j–l, 5i–l and Supplementary Figures 2 and 3. In human spleen, B cell follicles, T cell zones and red pulp areas were efficiently distinguished (Figs. 6g–i, 7g–i). CD37 showed highest expression in the B cell follicles (ODmean = 0.22) compared to the T cell zones and red pulp areas (ODmean = 0.15; Fig. 6j). In splenic B cell follicles, twice as many cells were CD37bright, compared to the red pulp where only 45 % of the cells was CD37bright (Fig. 6k). When focusing on CD37 expression on splenic T cells, we observed that significantly more T cells were CD37bright in the red pulp as compared to T cells in T cell zones (Fig. 6l). In the appendix, B cell follicles and lamina propria regions were located immediately below the crypts (Figs. 6m–o, 7m–o). CD37 showed highest expression in B cell follicles (ODmean = 0.12) compared to the lamina propria (ODmean = 0.09) in appendix (Fig. 6p) which is in line with CD37 expression in spleen. In the B cell follicles in human appendix, almost all cells were CD37bright, which was significantly more than in the lamina propria where around 80 % of total cells expressed high levels of CD37 (Fig. 6q). Similar to the red pulp in spleen, significantly more T cells in the lamina propria were CD37bright compared to the T cells within B cell follicles of the appendix (Fig. 6r). However, we need to be careful with interpreting these data, because the frequency of T cells in the appendix is very low.Fig. 6


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)

Localization and expression of CD53 in bone marrow (a–f), spleen (g–l) and appendix (m–r). a, g, m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a, g, m = 100 μm. b, h, n, Composite RGB image after spectral unmixing of original image (red CD20, blue CD53, green nuclei). c, i, o, Image showing scoring of CD20−CD53dim (blue), CD20+CD53dim (red), CD20+CD53bright (yellow) or CD20−CD53bright (green) cells. d, j, p, Optical density of CD53 on individual cells in human bone marrow (d), in B cell follicle (blue line) and in T cell zone and in red pulp (red line) in human spleen (j) and in B cell follicle (blue line) and in lamina propria (red line) in human appendix (p). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53dim and CD53bright cells in the CD20+ cell population (e) and in the CD3+ cell population (f) in human bone marrow. Percentage of CD53bright cells in the CD20+ cell population in the B cell follicle and red pulp (k), and in the CD3+ cell population in the T cell zone and red pulp (l) in human spleen. Percentage of CD53bright cells in the CD20+ cell population (q) and in the CD3+ cell population (r) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. *P < 0.05, ****P < 0.0001
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Fig7: Localization and expression of CD53 in bone marrow (a–f), spleen (g–l) and appendix (m–r). a, g, m Representative original multispectral image of lymphoid organ stained for CD20 (Warp Red), CD53 (True Blue) and cell nuclei (Nuclear Red). Scale bars in a, g, m = 100 μm. b, h, n, Composite RGB image after spectral unmixing of original image (red CD20, blue CD53, green nuclei). c, i, o, Image showing scoring of CD20−CD53dim (blue), CD20+CD53dim (red), CD20+CD53bright (yellow) or CD20−CD53bright (green) cells. d, j, p, Optical density of CD53 on individual cells in human bone marrow (d), in B cell follicle (blue line) and in T cell zone and in red pulp (red line) in human spleen (j) and in B cell follicle (blue line) and in lamina propria (red line) in human appendix (p). Optical densities were binned per 0.05 and normalized to % of max. Percentage of CD53dim and CD53bright cells in the CD20+ cell population (e) and in the CD3+ cell population (f) in human bone marrow. Percentage of CD53bright cells in the CD20+ cell population in the B cell follicle and red pulp (k), and in the CD3+ cell population in the T cell zone and red pulp (l) in human spleen. Percentage of CD53bright cells in the CD20+ cell population (q) and in the CD3+ cell population (r) in the B cell follicle and lamina propria in human appendix. Each dot represents data of one ×20 image from the lymphoid tissue. The red line represents the mean. *P < 0.05, ****P < 0.0001
Mentions: We studied the localization and expression of tetraspanin CD37 in primary and secondary lymphoid organs. Since bone marrow does not contain different T and B cell areas, tissue segmentation was not applicable and only cell segmentation was performed (Figs. 6a–c, 7a–c). The intensity [optical density (OD)] of CD37 in bone marrow ranged from 0.05 to 0.85, with a mean of 0.29 (ODmean; Fig. 6d). We observed around 80 % of all bone marrow cells to be CD37bright (Fig. 6e) and 90 % of all T cells to be highly positive for CD37 (Fig. 6f). The scatter plots with the set thresholds to annotate cells with dim and bright expression of CD37 and CD53 are shown in Figs. 4j–l, 5i–l and Supplementary Figures 2 and 3. In human spleen, B cell follicles, T cell zones and red pulp areas were efficiently distinguished (Figs. 6g–i, 7g–i). CD37 showed highest expression in the B cell follicles (ODmean = 0.22) compared to the T cell zones and red pulp areas (ODmean = 0.15; Fig. 6j). In splenic B cell follicles, twice as many cells were CD37bright, compared to the red pulp where only 45 % of the cells was CD37bright (Fig. 6k). When focusing on CD37 expression on splenic T cells, we observed that significantly more T cells were CD37bright in the red pulp as compared to T cells in T cell zones (Fig. 6l). In the appendix, B cell follicles and lamina propria regions were located immediately below the crypts (Figs. 6m–o, 7m–o). CD37 showed highest expression in B cell follicles (ODmean = 0.12) compared to the lamina propria (ODmean = 0.09) in appendix (Fig. 6p) which is in line with CD37 expression in spleen. In the B cell follicles in human appendix, almost all cells were CD37bright, which was significantly more than in the lamina propria where around 80 % of total cells expressed high levels of CD37 (Fig. 6q). Similar to the red pulp in spleen, significantly more T cells in the lamina propria were CD37bright compared to the T cells within B cell follicles of the appendix (Fig. 6r). However, we need to be careful with interpreting these data, because the frequency of T cells in the appendix is very low.Fig. 6

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.