Figure 1.
Selection of hens with normal ovaries or ovarian tumors using (A–C) color Doppler ultrasound and (D–F) gross morphology of the ovaries.
A normal ovary (A and D) with maturing ovarian follicles (F1, F2) and stromal blood vessels (arrows) with normal gross morphology showing maturing (F1, F2) ovarian follicles. An example of an abnormal ovary (B and E) that contains more blood vessels (arrows) than in the normal ovary, no detectable large mature follicles and a small solid tissue mass (circle) in the ovary. An example of late stage ovarian cancer (C) characterized by a large solid mass with increased blood flow (arrows) and profuse ascites (*).The gross morphology (F) confirmed the presence of multiple solid masses and tumor metastasis to other organs. Abbreviations: CT = cecal tonsil; F1–F2 = large preovulatory follicles; GI = gastrointestinal tract; OV = ovary; S = ovarian stroma; SM = solid tissue mass in the ovary; SP = spleen; UOD = upper oviduct; UT = uterus.
Figure 2.
Localization of immune cells in normal hen ovaries.
Bu1a+, CD4+ and CD8+ cells (arrows) detected by immunohistochemistry in normal ovaries in the ovarian stroma (S), adjacent to follicles (f), in the thecal layer of follicles (double arrow) and were abundant in the cell layer under the ovarian surface epithelium (SE). Original magnification, 10x; scale bar = 100 µm. Selected areas (dotted boxes) are shown in an inset at higher magnification for Bu1a and CD8 staining; inset scale bars = 10 µm.
Figure 3.
Localization of immune cells in early stage ovarian tumors.
Bu1a+, CD4+ and CD8+ cells are shown in similar regions of serial sections that contain tumor foci. More CD8+ T cells and B cells are present in tumor foci than CD4+ T cells. (A) Early-stage tumor showing CD4+ cells in a tumor with poorly differentiated (PD) structure, and CD8+ and B cells in an adjacent well differentiated (WD) area. (B) A small lesion (dotted circle) containing CD8+ T cells and Bu1a+ cells, but not CD4+ cells. (C) An area with neoplastic cells containing CD8+ and Bu1a+ cells, but not CD4+ cells. Original magnification, 10x; scale bar = 100 µm. Stained CD8+ lymphocytes in a selected area (dotted box) is shown in an inset at higher magnification; scale bar = 10 µm.
Figure 4.
Immune cell distribution in ovaries with late stage ovarian tumors.
Similar regions of three tumor types are shown in serial sections stained for Bu1a, CD4 and CD8. Top Row: An example of sections from a serous ovarian tumor showing few CD4+ cells within the tumor compared to Bu1a+ and CD8+ cells. Middle Row: An example from a mucinous ovarian tumor showing deposits of Bu1a+, CD4+ and CD8+ cells between glands and throughout the tumor. Bottom Row: An example of an endometrioid ovarian tumor showing Bu1a+, CD4+ and CD8+ cells. Original magnification, 10x; scale bar = 100 µm. Stained CD8+ and CD4+ lymphocytes in selected areas (dotted boxes) are shown in insets at higher magnification; scale bar = 10 µm.
Figure 5.
The number of ovarian CD4+, CD8+ and Bu1a+ cells estimated by flow cytometry.
(A): Representative forward vs. side scatter plot for cells from a whole ovary showing the gate (R1) used for lymphocytes. (B) Cells were first gated using the gate in (A) and then stained with either Bu1a-APC, CD4-FITC or CD8-PE. The percent of labeled cells within the gate is shown in the lower right quadrant for each stain. (C) The total number of Bu1a-APC, CD4-FITC and CD8-PE labeled cells from normal and tumor hen ovaries (n = 3/group) are shown with the median indicated by the horizontal line.
Figure 6.
Comparison of the number of immune cells in ovarian follicles determined by morphometric analysis.
(A) B (Bu1a+) cells were rarely found in follicles and therefore were not counted. The number of CD4+ T cells increased slightly (p = 0.052) and the number of CD8+ T cells decreased (p = 0.009) in late stage ovarian tumors compared to normal ovaries. At each stage there were more CD8+ compared to CD4+ T cells (normal ovary, p = 0.003; early stage tumor, p = 0.008; late stage tumor, p = 0.007). Follicles were defined as shown in (B) and cells within the designated area were counted and the average determined per 2×105 µm2 area of follicle. Three sections from each ovary for each hen were counted at a magnification of 20x; scale bar = 50 µm. Error bars represent mean ± SEM.
Figure 7.
The number of Bu1a+ B cells and CD4+ and CD8+ T cells in normal ovaries and ovarian tumors determined by morphometric analysis.
As shown in panels A-C for normal (N), early (E) and late stage (L) ovarian cancer, Bu1a+ cells and CD8+ T cells were more numerous overall than CD4+ T cells, particularly in late stage tumors. In panel D, the B and T cells counts were added. There was an overall increase in total (non-follicular) immune cells from normal ovary to late stage tumors. Cells were counted in multiple fields in three sections from each ovary for each hen at 20x magnification. The average number of B (Bu1a+) cells and CD4+ and CD8+ T cells was estimated from 11 hens with normal ovaries (number of fields counted was Bu1a = 524, CD4 = 425 and CD8 = 360), 8 hens with early stage ovarian cancer (number of fields counted was Bu1a = 228, CD4 = 190 and CD8 = 225) and 7 hens with late stage ovarian cancer (number of fields counted was Bu1a = 180, CD4 = 190 and CD8 = 200). Since the areas evaluated varied in size, all counts were normalized to 8×104 µm2.
Figure 8.
Comparison of the number of Bu1a+, CD4+ and CD8+ cells by ovarian tumor type.
Since the histological classification of tumors is clearest in late stage ovarian tumors, the morphometric analysis was confined to late stage tumors. Clear cell tumors occurred in relatively low numbers and were not included in the analysis. The number of Bu1a+, CD4+ and CD8+ cells was counted as described in the Methods for serous (66 fields in 3 hen ovaries); mucinous (53 fields in 2 hen ovaries) and endometrioid (37 fields counted in 2 hen ovaries) histology tumors. There were significantly more B (Bu1a+) cells in serous tumors than in mucinous (p = 2.6×10−14) or endometrioid (p = 7.6×10−9) tumors. In contrast, serous tumors had significantly fewer CD4+T cells compared to mucinous (p = 3.8×10−5) or endometrioid (p = 2.9×10−9). The number of CD+8 T cells did not differ significantly by tumor histology (p>0.2).