Figure 6
Figure 6. T-cell infiltrate and HLA class II expression in colonic biopsies. (A) Multicolor immunofluorescent stainings for CD3 (clone PS1, IgG2a + IgG2a ALEXA 546) (red), CD8 (clone 4B11, IgG2b + IgGb ALEXA 647) (blue), and HLA class II (clone CR3/43, IgG1 + IgG1 ALEXA 488) (green) were performed on colonic biopsies from patient 1 (1) and patient 2 (2) obtained at the time of GVHD diagnosis after CD4+ DLI, a patient diagnosed with colonic GVHD after CD3+ DLI (3), and a normal colon from a healthy individual (4). Double CD3+CD8+ cells (purple) were defined as CD8+ T cells, whereas CD3+CD8-expressing cells (red) were defined as CD4+ T cells. Images were captured with a confocal laser scanning microscope (LSM510; Zeiss, Sliedrecht, The Netherlands) (original magnification ×200) (supplemental Methods). Numbers of CD4+ and CD8+ T cells were enumerated by a third party in a blinded fashion, and tissue sections showed CD4/CD8 ratios of 0.9, 1.5, 0.4, and 0.8 for images 1, 2, 3, and 4, respectively. (B) Combined immunofluorescent stainings for CD3 (clone PS1, IgG2a + IgG2a ALEXA 546) (red) and CD8 (clone 4B11, IgG2b + IgGb ALEXA 647) (blue) (1-2), and FISH analysis for detection of X (green) and Y (red) chromosomes (XX/XY) (3-4) were performed on serial sections of the colonic biopsy from patient 2 (male; URD = female). Immunofluorescent staining: original magnification ×200 (1) and framed area, original magnification ×400 (2). Corresponding fields in X/Y FISH analysis: original magnification ×200 (3) and framed area, original magnification ×400 (4). Interstitium (I) and colonic epithelial cells (E) are indicated in the stainings. Colonic epithelial cells show recipient-specific XY karyotype, whereas interstitial T-cell infiltrate predominantly shows donor-specific XX karyotype. Control normal male colonic biopsy was also examined by X/Y FISH (data not shown). All slides were digitized using a Mirax slide scanner (3DHISTEC, Budapest, Hungary) and analyzed using Panoramic Viewer (3DHistec, version 1.14.50 RTM) (supplemental Methods). (C) Immunofluorescent stainings (original magnification ×200) for CD3 (red), CD8 (blue), and HLA class II (green) on colonic biopsies from 2 kidney transplantation patients (1-2) with CMV colitis. Enumeration of CD4+ and CD8+ T cells showed CD4/CD8 ratios of 2.3 and 0.5 for images 1 and 2, respectively.

T-cell infiltrate and HLA class II expression in colonic biopsies. (A) Multicolor immunofluorescent stainings for CD3 (clone PS1, IgG2a + IgG2a ALEXA 546) (red), CD8 (clone 4B11, IgG2b + IgGb ALEXA 647) (blue), and HLA class II (clone CR3/43, IgG1 + IgG1 ALEXA 488) (green) were performed on colonic biopsies from patient 1 (1) and patient 2 (2) obtained at the time of GVHD diagnosis after CD4+ DLI, a patient diagnosed with colonic GVHD after CD3+ DLI (3), and a normal colon from a healthy individual (4). Double CD3+CD8+ cells (purple) were defined as CD8+ T cells, whereas CD3+CD8-expressing cells (red) were defined as CD4+ T cells. Images were captured with a confocal laser scanning microscope (LSM510; Zeiss, Sliedrecht, The Netherlands) (original magnification ×200) (supplemental Methods). Numbers of CD4+ and CD8+ T cells were enumerated by a third party in a blinded fashion, and tissue sections showed CD4/CD8 ratios of 0.9, 1.5, 0.4, and 0.8 for images 1, 2, 3, and 4, respectively. (B) Combined immunofluorescent stainings for CD3 (clone PS1, IgG2a + IgG2a ALEXA 546) (red) and CD8 (clone 4B11, IgG2b + IgGb ALEXA 647) (blue) (1-2), and FISH analysis for detection of X (green) and Y (red) chromosomes (XX/XY) (3-4) were performed on serial sections of the colonic biopsy from patient 2 (male; URD = female). Immunofluorescent staining: original magnification ×200 (1) and framed area, original magnification ×400 (2). Corresponding fields in X/Y FISH analysis: original magnification ×200 (3) and framed area, original magnification ×400 (4). Interstitium (I) and colonic epithelial cells (E) are indicated in the stainings. Colonic epithelial cells show recipient-specific XY karyotype, whereas interstitial T-cell infiltrate predominantly shows donor-specific XX karyotype. Control normal male colonic biopsy was also examined by X/Y FISH (data not shown). All slides were digitized using a Mirax slide scanner (3DHISTEC, Budapest, Hungary) and analyzed using Panoramic Viewer (3DHistec, version 1.14.50 RTM) (supplemental Methods). (C) Immunofluorescent stainings (original magnification ×200) for CD3 (red), CD8 (blue), and HLA class II (green) on colonic biopsies from 2 kidney transplantation patients (1-2) with CMV colitis. Enumeration of CD4+ and CD8+ T cells showed CD4/CD8 ratios of 2.3 and 0.5 for images 1 and 2, respectively.

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