Figure 1.

Distribution of CD4pos PD-1pos ICOSpos cells in normal germinal centers and follicular lymphoma. (A1) Low power view of germinal centers (GC) in a reactive lymph node (LN) showing CD4^pos (red) T-cells mainly outside the GC. A population of cells within the GC co-express PD-1 (white) and ICOS (green). (A2) The area highlighted by the white rectangle has been enlarged showing the distribution of CD4/PD1/ICOS^pos cells in a normal GC where they are mainly polarized to the light zone (A3) Intersecting binary layer of image (A1) showing CD4/PD-1/ICOS^pos cells (magenta) in GC. DAPI staining (blue) highlights cell nuclei. (A4) High power view of the intersecting CD4/PD-1/ICOS^pos binary layer. (B1) Representative image of follicular lymphoma (FL) lymph node (LN) showing CD4^pos (red) T cells mainly outside the follicles but a population within the follicles co-express PD-1 (white) and ICOS (green). (B2) Same image as (B1) showing only DAPI (blue) and the intersecting binary layer of CD4/PD-1/ICOS^pos cells (magenta) which are restricted to the follicles where they are located predominantly in a perifollicular pattern. Scale bars represent 100 μm. (C) There was no significant difference in the proportion of CD4pos cells co-expressing PD-1 and ICOS in normal and neoplastic follicles. There was a small but significant increase in CD4/PD-1/ICOS^pos cells in the inter-follicular compartment of FL com-pared to the same area zone of reactive LN. Horizontal lines represent median, boxes represent interquartile range, ‘whiskers’ represent range. (D) Representative, magnified image showing BLC6 expression in T cells in neoplastic follicles. Four CD3^pos (blue) cells are shown, three are positive for the transcription factor BCL6 (red) and these are also PD-1^pos (white) and ICOSpos (green), indicated with white arrows. One CD3^pos cell is negative for BCL6 (green arrow), and this cell does not express PD-1 or ICOS. Overall 89.6% (88.3-91.8) of CD3^posPD1^posICOS^pos cells express BCL6.