Table 1.
Expression of TOX in normal human tissues.
Table 2.
TOX expression in lymphomas.
Fig 1.
Boxplot on mRNA expression of TOX across low-grade B-cell lymphomas and reactive lymphoid tissues.
Relative gene expression of TOX mRNA expression across B-cell lymphomas and lymphoid reactive tissues (n = 72): 16 lymph nodes with FL (blue), 4 lymph nodes infiltrated by MALT lymphoma (yellow), 8 reactive lymph nodes (grey), 15 lymph nodes with NMZL (red), 4 lymph nodes infiltrated by SMZL (green), 14 spleens infiltrated by CLL (light blue), 7 reactive spleens and 4 spleens infiltrated by MCL (magenta). Statistical significance was obtained when TOX expression in FL was separately compared to each of the other histotypes (FDR <0.05, fold-change >2.0. S1 Table in S1 Text). Expression data from each individual case is illustrated by single dots.
Fig 2.
Validation of the reactivity and specificity of the anti-TOX mAb NAN448B.
A) Immunocytochemical staining for TOX on cytocentrifuge preparations using NAN448B mAb. Staining of antibody NAN448B was observed in HEK-TOX-Myc transfectants but not in HEK-TOX2-GFP, HEK-TOX3-GFP and HEK-TOX4-GFP. The anti-Myc and anti-GFP mAbs were used to confirm transfection efficiency. Two bands of 63 kDa and 57kDa (weaker intensity) were detected by WB in HEK-TOX cell extracts while no expression was found in HEK-TOX2, HEK-TOX3 and HEK-TOX4. Anti-Vinculin antibody was used as loading control. B) Immunocytochemical staining for TOX on cytocentrifuge preparations of MOLT4 wild type (WT) and MOLT4 cell line after TOX gene inactivation using CRISPR-Cas9 technology (TOX KO MOLT4). Strong TOX staining was observed in the WT MOLT4 cytocentrifuge preparation while no staining was observed in the TOX MOLT4 KO cells. Two bands of 63 kDa and 57kDa (weaker intensity) were detected by WB in WT MOLT4 cell extracts while no expression was found in TOX KO MOLT4. Anti-Vinculin antibody was used as loading control.
Fig 3.
TOX protein expression in lymphoid cell lines.
TOX expression in whole cell extracts of multiple tumor lymphoid cell lines were analyzed by WB using the NAN448B antibody. Anti-Vinculin antibody was used as loading control. The images represent three different gels.
Fig 4.
Expression of TOX in normal lymphoid tissues.
(A) Single immunoperoxidase labeling in tonsil showed strong nuclear staining of TOX in GC B and T cells and in T cells in the interfollicular area. (B) TOX was also highly expressed by CD4/CD8 double positive T cells in thymus cortex and in proportion of mature B and T cells in the medulla. (C) In spleen, TOX was expressed by B and T cells in the white pulp GC and in scattered cells in the red pulp. Double immunenzymatic staining showed high expression of TOX [25] in CD20+/CD10+ cells (red) (D and E) in the GC and also in a proportion of B cells in the T-cell area. (F) Double immunofluorescence staining showed that the large majority of BCL6+ cells (green) also expressed TOX (red). High numbers of CD3+ T lymphocytes (red) (G) were TOX+ [25], the majority of them being CD4+ (red) (H) and only 5–10% were CD8+ (red) (I). Double immunofluorescence staining showed that a high number of Ki67 positive cells (green) co-expressed TOX (red) (J). All the PD1+ TFH (red) (K) and CD30+ activated B cells (L) [25] were TOX+ (brown and red respectively). CD68 positive macrophages (M) [25], IgD + lymphocytes (red) (N) and CD138+ plasma cells (red) (O) lacked TOX staining.
Fig 5.
TOX expression in B and T-cell lymphomas.
Strong TOX expression was found in the nuclei of tumor cells in FL (A), DLBCL (B) and precursor B-LBL (C). In CLL (D) and NMZL (E) TOX was found in the tumor microenvironment. In NLPHL (F) TOX was expressed by L&H cells and in the PD1+ rosette surrounding the tumor cells as shown by the double immunoenzymatic staining of TOX and PD1 (red) (upper image F and G). In cHL (G) TOX was found only in the tumor microenvironment. MF and AITL and MF also expressed high level of TOX (H and I). Double immunoenzymatic staining confirmed the expression of TOX [25] in PD1+ (red) in AITL tumor cells (inset image I).