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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.


Spectral imaging analysis of human spleen stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). a Representative original multispectral image. Scale bar = 100 μm. Composite RGB image (b) of unmixed CD3 (c in red), CD37 (d in blue) and Nuclear Red (e in green in composite RGB image) signal using the spectral library (Fig. 4a). f Tissue segmentation; B cell follicle (B, yellow), T cell zone (T, red), red pulp (RP, green) and other tissue (blood vessels, collagen; blue). g Segmentation of individual cells (green) based on Nuclear Red staining. h Thresholds for Warp Red and True Blue staining were set to score CD3−CD37dim (blue), CD3+CD37dim (red), CD3+CD37bright (yellow) or CD3−CD37bright (green) cells. i–l Scatter plots showing optical densities for CD3 (Y-axis) and CD37 (X-axis) of individual cells in T cell zones (i, lgreen), B cell follicles (j, lblue) and red pulp regions (k–lred) and thresholds used for scoring (dotted lines). A representative of 2000 cells per tissue region is plotted
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Fig5: Spectral imaging analysis of human spleen stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). a Representative original multispectral image. Scale bar = 100 μm. Composite RGB image (b) of unmixed CD3 (c in red), CD37 (d in blue) and Nuclear Red (e in green in composite RGB image) signal using the spectral library (Fig. 4a). f Tissue segmentation; B cell follicle (B, yellow), T cell zone (T, red), red pulp (RP, green) and other tissue (blood vessels, collagen; blue). g Segmentation of individual cells (green) based on Nuclear Red staining. h Thresholds for Warp Red and True Blue staining were set to score CD3−CD37dim (blue), CD3+CD37dim (red), CD3+CD37bright (yellow) or CD3−CD37bright (green) cells. i–l Scatter plots showing optical densities for CD3 (Y-axis) and CD37 (X-axis) of individual cells in T cell zones (i, lgreen), B cell follicles (j, lblue) and red pulp regions (k–lred) and thresholds used for scoring (dotted lines). A representative of 2000 cells per tissue region is plotted

Mentions: We investigated the tissue distribution of CD37 and CD53 in human lymphoid organs by multispectral imaging. In contrast to classical immunohistochemistry, multispectral imaging directly provides quantitative information into the differential tissue distribution of individual cell subsets. First, we investigated localization of CD37 and CD53 in human spleen. We observed that CD37 was more locally expressed in follicle-like structures when compared to CD53 which showed a more dispersed expression profile (Fig. 3a–e). To explore this in more detail, we performed double staining of either the T cell marker CD3 or the B cell marker CD20 combined with CD37 or CD53 on primary (bone marrow) and secondary (spleen and appendix) lymphoid tissues. Figure 4 illustrates the technology of multispectral imaging and analysis of lymphoid tissue stained for the B cell marker CD20 (Warp Red), tetraspanin CD53 (True Blue) and cell nuclei (Nuclear Red). Single-stained tissues for each chromogen (Warp Red, True Blue and Nuclear Red) were used to create a spectral library containing the specific spectra of each used chromogen (Fig. 4a) allowing to unmix the original multispectral images (Fig. 4b). This resulted in separate images for each marker (Fig. 4d–f) that were used to generate the composite RGB image (Fig. 4c). We made use of two red chromogens (Warp Red and Nuclear Red) with highly similar spectra of which correct unmixing has been described before (Van Der Loos 2010). Next, analysis software was trained using ten representative original multispectral images to segment the different tissue regions (B cell follicle and stromal tissue (red pulp in spleen or lamina propria in appendix)) based on a combination of parameters including cell morphology and specific staining (Fig. 4g) and individual cells based on nuclear characteristics (Fig. 4h). For each cell, CD20 positivity and CD53 expression were determined in relation to tissue localization (Fig. 4i–l). These settings were saved within an algorithm allowing batch analysis of multiple original multispectral images of the same tissue and stainings. Figure 5 shows similar analysis for lymphoid tissue stained for the T cell marker CD3 (Warp Red), tetraspanin CD37 (True Blue) and cell nuclei (Nuclear Red). Original multispectral images were unmixed using the spectral library showed in Fig. 4a (Fig. 5a–e). Next, tissue segmentation was performed for T cell zones, B cell follicles and red pulp regions (Fig. 5f), followed by cell segmentation (Fig. 5g) and analysis of CD3 and CD37 expression within the different tissue regions (Fig. 5h–l). As expected, B cell follicles mainly consisted of CD20-positive cells, and T cell zones contained mainly CD3-positive cells. The stromal tissue consisted of both CD20- or CD3-negative and CD20- or CD3-positive cells. Altogether, we established multispectral imaging analysis to combine quantitative tetraspanin expression data with specific tissue localization in human lymphoid tissues.Fig. 3


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)

Spectral imaging analysis of human spleen stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). a Representative original multispectral image. Scale bar = 100 μm. Composite RGB image (b) of unmixed CD3 (c in red), CD37 (d in blue) and Nuclear Red (e in green in composite RGB image) signal using the spectral library (Fig. 4a). f Tissue segmentation; B cell follicle (B, yellow), T cell zone (T, red), red pulp (RP, green) and other tissue (blood vessels, collagen; blue). g Segmentation of individual cells (green) based on Nuclear Red staining. h Thresholds for Warp Red and True Blue staining were set to score CD3−CD37dim (blue), CD3+CD37dim (red), CD3+CD37bright (yellow) or CD3−CD37bright (green) cells. i–l Scatter plots showing optical densities for CD3 (Y-axis) and CD37 (X-axis) of individual cells in T cell zones (i, lgreen), B cell follicles (j, lblue) and red pulp regions (k–lred) and thresholds used for scoring (dotted lines). A representative of 2000 cells per tissue region is plotted
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Related In: Results  -  Collection

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Fig5: Spectral imaging analysis of human spleen stained for CD3 (Warp Red), CD37 (True Blue) and cell nuclei (Nuclear Red). a Representative original multispectral image. Scale bar = 100 μm. Composite RGB image (b) of unmixed CD3 (c in red), CD37 (d in blue) and Nuclear Red (e in green in composite RGB image) signal using the spectral library (Fig. 4a). f Tissue segmentation; B cell follicle (B, yellow), T cell zone (T, red), red pulp (RP, green) and other tissue (blood vessels, collagen; blue). g Segmentation of individual cells (green) based on Nuclear Red staining. h Thresholds for Warp Red and True Blue staining were set to score CD3−CD37dim (blue), CD3+CD37dim (red), CD3+CD37bright (yellow) or CD3−CD37bright (green) cells. i–l Scatter plots showing optical densities for CD3 (Y-axis) and CD37 (X-axis) of individual cells in T cell zones (i, lgreen), B cell follicles (j, lblue) and red pulp regions (k–lred) and thresholds used for scoring (dotted lines). A representative of 2000 cells per tissue region is plotted
Mentions: We investigated the tissue distribution of CD37 and CD53 in human lymphoid organs by multispectral imaging. In contrast to classical immunohistochemistry, multispectral imaging directly provides quantitative information into the differential tissue distribution of individual cell subsets. First, we investigated localization of CD37 and CD53 in human spleen. We observed that CD37 was more locally expressed in follicle-like structures when compared to CD53 which showed a more dispersed expression profile (Fig. 3a–e). To explore this in more detail, we performed double staining of either the T cell marker CD3 or the B cell marker CD20 combined with CD37 or CD53 on primary (bone marrow) and secondary (spleen and appendix) lymphoid tissues. Figure 4 illustrates the technology of multispectral imaging and analysis of lymphoid tissue stained for the B cell marker CD20 (Warp Red), tetraspanin CD53 (True Blue) and cell nuclei (Nuclear Red). Single-stained tissues for each chromogen (Warp Red, True Blue and Nuclear Red) were used to create a spectral library containing the specific spectra of each used chromogen (Fig. 4a) allowing to unmix the original multispectral images (Fig. 4b). This resulted in separate images for each marker (Fig. 4d–f) that were used to generate the composite RGB image (Fig. 4c). We made use of two red chromogens (Warp Red and Nuclear Red) with highly similar spectra of which correct unmixing has been described before (Van Der Loos 2010). Next, analysis software was trained using ten representative original multispectral images to segment the different tissue regions (B cell follicle and stromal tissue (red pulp in spleen or lamina propria in appendix)) based on a combination of parameters including cell morphology and specific staining (Fig. 4g) and individual cells based on nuclear characteristics (Fig. 4h). For each cell, CD20 positivity and CD53 expression were determined in relation to tissue localization (Fig. 4i–l). These settings were saved within an algorithm allowing batch analysis of multiple original multispectral images of the same tissue and stainings. Figure 5 shows similar analysis for lymphoid tissue stained for the T cell marker CD3 (Warp Red), tetraspanin CD37 (True Blue) and cell nuclei (Nuclear Red). Original multispectral images were unmixed using the spectral library showed in Fig. 4a (Fig. 5a–e). Next, tissue segmentation was performed for T cell zones, B cell follicles and red pulp regions (Fig. 5f), followed by cell segmentation (Fig. 5g) and analysis of CD3 and CD37 expression within the different tissue regions (Fig. 5h–l). As expected, B cell follicles mainly consisted of CD20-positive cells, and T cell zones contained mainly CD3-positive cells. The stromal tissue consisted of both CD20- or CD3-negative and CD20- or CD3-positive cells. Altogether, we established multispectral imaging analysis to combine quantitative tetraspanin expression data with specific tissue localization in human lymphoid tissues.Fig. 3

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.