The Conspicuousness of High Endothelial Venules in Angioimmunoblastic T-cell Lymphoma Is Due to Increased Cross-sectional Area, Not Increased Distribution Density

Abstract

Angioimmunoblastic T-cell lymphoma (AITL) is a T-cell lymphoma of follicular helper T-cell origin. Histologically, neoplastic T-cells proliferate to form clusters adjacent to or between arborizing high endothelial venules (HEVs). HEVs in normal lymph nodes express sulfated glycans called peripheral lymph node addressin (PNAd); however, it remains unclear whether PNAd is also expressed on HEVs in AITL. Furthermore, although it is widely accepted that HEVs are conspicuous in AITL due to their proliferation, quantitative histological support for this concept is lacking. To investigate these issues, we employed monoclonal antibodies recognizing PNAd, namely, MECA-79, HECA-452, and 297-11A, and performed quantitative immunohistochemical analysis of HEVs in 36 AITL-affected and 67 normal lymph nodes. Staining with all three antibodies confirmed that AITL HEVs express PNAd. Moreover, AITL HEVs were bound calcium-dependently by L-selectin-IgM fusion proteins, indicating that they function in the recruitment of L-selectin-expressing lymphocytes. Unexpectedly, HEV distribution density was not increased but rather decreased in AITL compared with normal lymph nodes, but HEV cross-sectional area in AITL was significantly greater than that seen in normal lymph nodes. Overall, these results indicate that the prominence of AITL HEVs is likely due to increased cross-sectional area rather than increased distribution density.

Keywords: CD34; N-acetyllactosamine; sialyl Lewis x (sLex).

Figure 1.

Glycan structure of 6,6′-disulfo sLe^x attached to both extended core 1 and core 2-branched O-glycans, one of the most fully glycosylated and sulfated forms of putative L-selectin ligands. Epitopes are shown for MECA-79, HECA-452, and 297-11A monoclonal antibodies.^–,–

Figure 2.

Immunohistochemical profiles of HEVs formed in AITL. (A, B) Histology of AITL. Normal lymph node architecture is effaced due to proliferation of lymphoma cells throughout the lymph node (A). Lymphoma cells with “clear cell” features proliferate to form clusters adjacent to or between arborizing HEVs (B). H&E staining. (C–F) HEVs formed in AITL stain positively for CD34 (C), MECA-79 (D), HECA-452 (E), and 297-11A (F). Signals were visualized with 3,3’-diaminobenzidine (DAB) (brown), and tissues were counterstained with hematoxylin. Bar = 400 µm for panel A and 40 µm for the rest. Abbreviations: HEVs, high endothelial venules; AITL, angioimmunoblastic T-cell lymphoma; H&E, hematoxylin and eosin.

Figure 3.

Dual immunofluorescence of HEVs formed in AITL for 297-11A (red) and MECA-79 (green). Yellow signals in Merged indicate colocalization of both carbohydrate antigens. Bar = 40 µm. Abbreviations: HEVs, high endothelial venules; AITL, angioimmunoblastic T-cell lymphoma.

Figure 4.

Gal-6-O-sulfation of LacNAc in the MECA-79 epitope does not inhibit reactivity to MECA-79. (A) Stable expression of the MECA-79 minimum epitope in Lec2/β3GlcNAcT3/GlcNAc6ST-2 cells (filled histogram in right panel). Filled histogram in the left panel and open histogram in the right panel represent negative controls in which the primary antibody was replaced with isotype-matched immunoglobulin. X- and Y-axes indicate fluorescence intensity and number of events, respectively. Note that cells expressing the MECA-79 epitope are distributed normally, indicating that they are of a single clone. (B) Lec2/β3GlcNAcT-3/GlcNAc6ST-2 cells were transiently transfected with empty vector (mock; upper panels) or KSGal6ST cDNA (lower panels), and then doubly immunostained with MECA-79 and 297-11A (right panels). Left panels represent negative controls replacing primary antibodies with isotype-matched immunoglobulins. X- and Y-axes indicate fluorescence intensity originating from MECA-79 and 297-11A, respectively. Abbreviations: LacNAc, N-acetyllactosamine; cDNA, complementary DNA.

Figure 5.

L- and E-selectin-IgM chimera binding assays. (A) Flow cytometric analysis of L-selectin-IgM (blue histograms) and E-selectin-IgM (red histograms) chimera binding to CHO cells expressing non-sulfated sLe^x (upper panel) or 6-sulfo sLe^x (lower panel). Black histograms represent negative controls performed using conditioned medium from untransfected COS-1 cells. X- and Y-axes indicate fluorescence intensity and number of events, respectively. (B, C) L-selectin-IgM (B) and E-selectin-IgM (C) in situ binding assays on FFPE tissue sections of normal (upper panels) and AITL-affected (lower panels) lymph nodes. Both L- and E-selectin-IgM chimeras bind to HEVs in the presence of calcium ions (without EDTA), but binding is completely abrogated in the absence of calcium ions (with EDTA). Note very weak L-selectin-IgM binding to HEVs formed in AITL (arrows). E-selectin-IgM chimera binding to HEVs is maintained even after incubating sections with CSLEX1 antibody, which binds exclusively to non-sulfated sLe^x (with CSLEX1). Signals were visualized with DAB (brown), and tissues were counterstained with hematoxylin. Bar = 40 µm. Abbreviations: FFPE, formalin-fixed, paraffin-embedded; AITL, angioimmunoblastic T-cell lymphoma; HEVs, high endothelial venules; EDTA, ethylenediaminetetraacetic acid.

Figure 6.

Distribution density of HEVs. (A) The size (area) of normal (n=67; open boxes) and AITL-affected (n=36; filled boxes) lymph nodes. (B–E) Distribution density of vessels immunoreactive for CD34 (B), MECA-79 (C), HECA-452 (D), or 297-11A (E) in normal and AITL-affected lymph nodes. Data are presented as means with SD. Abbreviations: HEVs, high endothelial venules; AITL, angioimmunoblastic T-cell lymphoma. *p < 0.05; ***p < 0.001.

Figure 7.

The proportion of HEVs immunoreactive for MECA-79 (A), HECA-452 (B), or 297-11A (C) among CD34-positive total vessels in normal (n=67; open boxes) and AITL-affected (n=36; filled boxes) lymph nodes. Data are presented as means with SD. Abbreviations: HEVs, high endothelial venules; AITL, angioimmunoblastic T-cell lymphoma; NS, not significant. **p<0.01.

Figure 8.

Increased cross-sectional area of HEVs in AITL-affected lymph nodes. (A–C) Photomicrographs of HEVs in normal (A) and AITL-affected (B and C) lymph nodes. Panel C is an enlarged view of the region indicated by asterisk in panel B. Note that in AITL, neoplastic T-cells (arrows) and reactive immunoblasts infiltrate the space between endothelial cells (arrowheads) and the basement membrane (dotted lines) likely pushing the basement membrane outward and resulting in increased cross-sectional area of HEVs. H&E staining. Bar = 40 µm for panels A and B and 20 µm for panel C. (D) The mean cross-sectional area of one HEV in normal (n=5; open box) and AITL-affected (n=5; filled box) lymph nodes. (E) The total cross-sectional area of HEVs present in a unit area (1 mm²) of normal (n=5) and AITL-affected (n=5) lymph nodes. (F) The number of HEVs per unit area in the region assessed in (E) in normal (n=5) and AITL-affected (n=5) lymph nodes. Data are presented as means with SD. Abbreviations: HEVs, high endothelial venules; AITL, angioimmunoblastic T-cell lymphoma; NS, not significant. *p<0.05, ***p<0.001.