Abstract
Providing a method to detect avian lymphocytes by immunohistochemistry (IHC) would be helpful for analyzing immune function and diagnosing diseases in birds. In this study, we comprehensively examined the immunohistochemical identification of avian T and B lymphocytes in formalin-fixed, paraffin-embedded tissues from 53 avian species across 15 orders, using eight commercially available lymphocyte markers. T lymphocytes from all 53 avian species tested were specifically detected by IHC using the anti-CD3 antibody (clone F7.2.38). The appropriate antibody for detecting avian B lymphocytes in IHC varied depending on the avian species. B lymphocytes were specifically labeled by IHC in 46 of 53 avian species (86.8%) using any of seven B cell markers. The anti-PAX5 antibody (clone SP34) immunohistochemically detected B lymphocytes from the majority of avian species (41 out of 53 species), excluding those in the orders Falconiformes (falcons) and Passeriformes (oscines). The anti-BAFF-R antibody (clone 2C4) proved suitable for detecting B lymphocytes in the orders Galliformes (landfowls) and Anseriformes (waterfowls) in IHC. Caution is advised when using the anti-BLA36 (clone A27-42) and two anti-CD20 (clone L26 and product No. PA5-16701) antibodies, which are commonly used as B cell markers in mammals, for detecting avian B lymphocytes. These antibodies reacted with cells located in both T and B cell areas in certain avian species. The anti-Bu-1a/b (clone AV20) and anti-CD79a (clone HM57) antibodies were found not to bind to B lymphocytes in various avian species in IHC.
Keywords: avian species, immunohistochemistry, lymphocyte, poultry, wild bird
Immunohistochemistry (IHC) is an analytical method that utilizes antigen-antibody reactions to unveil the localization of target molecules and/or proteins in tissues and cells. This method has found application in both basic and clinical research, as well as disease diagnosis. The identification of lymphocytes through IHC is essential for immunological studies and tumor diagnosis.
The immunohistochemical identification of lymphocytes has been established in several mammals, and optimal lymphocyte markers vary among species [10, 19, 52, 53, 55]. However, a standardized method for the identification of lymphocytes by IHC has not been established in birds, except for chickens [28]. There are sporadic papers reporting the detection of lymphocytes from wild birds by IHC using antibodies against human lymphocytic antigens [14, 16, 18, 26, 30, 31, 36, 40, 50]. Nevertheless, the specificity of the antibodies used to identify T or B lymphocytes remains unclear in some of these papers. Confirming the specificity of the antibodies is crucial in IHC, considering the potential cross-reactivity of antibodies against other animal antigens. It is highly likely that, similar to mammals, suitable lymphocyte markers may vary among avian species. Therefore, it is important to validate and clarify appropriate lymphocyte markers for each avian species.
To date, no reports have been found that attempt to comprehensively detect lymphocytes in birds from various orders using IHC. IHC utilizing antibodies that are suitable for each avian species, with confirmed specificity, will enable more precise research and diagnosis. In this study, we examined formalin-fixed, paraffin-embedded (FFPE) sections of 53 avian species across 15 orders to identify T and B lymphocytes by IHC. We utilized commercially available antibodies raised against chicken or human antigens (CD3, PAX5, BAFF-R, BLA36, CD20, Bu-1a/b, and CD79a). The specificity of each antibody for T or B lymphocytes was determined by matching positive reactions to the corresponding T or B cell areas in lymphoid tissues.
MATERIALS AND METHODS
Birds and tissue specimens
FFPE samples of 82 birds from 53 species, prepared between 2002–2021, were collected for this study (Table 1). The classification and names of each bird were referenced from the Handbook of the Birds of the World and BirdLife International Digital Checklist of the Birds of the World [6]. We tested one to three FFPE blocks per avian species, prioritizing those with minimal postmortem changes. The fixation time of the tissues were unknown. The spleen was primarily utilized for the IHC analysis. In cases where the spleen was unavailable, another organ containing lymphoid tissue was selected on observation of hematoxylin and eosin-stained section.
Table 1. Summary of the tested specimens.
| Order | Scientific name | Species (Common name) | Tested lymphoid tissues | No. of tested birds | Case condition |
|---|---|---|---|---|---|
| Struthioniformes (Ostriches) | Struthio camelus | Common ostrich | S | 3 | A, U |
| Galliformes (Landfowls) | Gallus gallus var. domesticus | Chicken | S | 3 | A |
| Chrysolophus pictus | Golden pheasant | S | 1 | D | |
| Pavo sp. | Peafowl | S | 1 | D | |
| Meleagris gallopavo | Turkey | S | 1 | A | |
| Coturnix japonica | Japanese quail | S | 1 | U | |
| Lagopus muta hyperborea | Svalbard rock ptarmigan | S | 1 | D | |
| Numida meleagris | Helmeted guineafowl | S | 3 | U | |
| Anseriformes (Waterfowls) | ―a | Hybrid duck | B, GALT, S | 2 | A |
| Anas platyrhynchos | Mallard | S | 1 | D | |
| Anas crecca | Common teal | C | 1 | D | |
| Anas acuta | Northern pintail | S | 1 | D | |
| Sibirionetta formosa | Baikal teal | E | 1 | D | |
| Aythya ferina | Common pochard | C, S | 2 | D | |
| Aythya fuligula | Tufted duck | GALT | 1 | D | |
| Tadorna ferruginea | Ruddy shelduck | S | 1 | D | |
| Anser anser var. domesticus | Goose | B, S | 2 | A | |
| Cygnus cygnus | Whooper swan | S | 2 | D | |
| Cygnus olor | Mute swan | S | 2 | D, U | |
| Cygnus atratus | Black swan | S | 2 | U | |
| Podicipediformes (Grebes) | Podiceps cristatus | Great crested grebe | S | 1 | D |
| Columbiformes (Doves) | Columba livia | Rock dove | C, S | 2 | A |
| Streptopelia orientalis | Oriental turtle-dove | S | 2 | D | |
| Sphenisciformes (Penguins) | Spheniscus humboldti | Humboldt penguin | BALT | 1 | D |
| Spheniscus magellanicus | Magellanic penguin | E, S | 3 | D | |
| Suliformes (Cormorants) | Phalacrocorax carbo | Great cormorant | S | 2 | D |
| Phalacrocorax capillatus | Japanese cormorant | S | 2 | D | |
| Pelecaniformes (Pelicans) | Nipponia nippon | Asian crested ibis | S | 1 | D |
| Ardea intermedia | Intermediate egret | BALT | 1 | D | |
| Pelecanus sp. | Pelican | S | 1 | U | |
| Gruiformes (Cranes) | Fulica atra | Common coot | S | 2 | U |
| Charadriiformes (Plovers) | Himantopus himantopus mexicanus | Black-necked stilt | S | 2 | U |
| Larus ridibundus | Black-headed gull | B, C, GALT | 2 | D | |
| Fratercula cirrhata | Tufted puffin | S | 1 | D | |
| Synthliboramphus antiquus | Ancient murrelet | S | 2 | D | |
| Accipitriformes (Eagles and New world vultures) | ―b | Hybrid hawk | S | 1 | D |
| Haliaeetus albicilla | White-tailed sea-eagle | S | 1 | D | |
| Buteo japonicus | Japanese buzzard | S | 1 | D | |
| Parabuteo unicinctus | Harris’s hawk | S | 1 | D | |
| Strigiformes (Owls) | Bubo scandiacus | Snowy owl | S | 1 | D |
| ―c | Owl | S | 2 | D | |
| Falconiformes (Falcons) | Falco tinnunculus | Common kestrel | E | 1 | D |
| Psittaciformes (Parrots) | Ara ararauna | Blue-and-yellow macaw | S | 1 | D |
| Melopsittacus undulatus | Budgerigar | S | 2 | D | |
| Agapornis lilianae | Nyasa lovebird | S | 1 | D | |
| Alisterus scapularis | Australian king-parrot | S | 1 | D | |
| Psittacus erithacus | Grey parrot | E | 1 | D | |
| Cacatua alba | White cockatoo | S | 1 | D | |
| Cacatua goffiniana | Tanimbar corella | S | 1 | D | |
| Passeriformes (Oscines) | Corvus sp.d | Crow | S | 3 | D |
| Passer montanus | Eurasian tree sparrow | C, S | 2 | A | |
| Hypsipetes amaurotis | Brown-eared bulbul | S | 3 | D | |
| Spodiopsar cineraceus | White-cheeked starling | S | 1 | D | |
A, autopsy case; B, bursa of Fabricius; BALT, bronchus-associated lymphoid tissue; C, cecal tonsil; D, death case; E, ectopic lymphoid aggregate in the liver or kidney; GALT, gut-associated lymphoid tissue; S, spleen; U, case of unknown autopsy or death. aHybrid offspring of a male hybrid duck (Anas zonorhyncha × Anas platyrhynchos) and a female duck (Anas platyrhynchos var. domesticus). bNorthern goshawk (Accipiter gentilis) × Black sparrowhawk (Accipiter melanoleucus). cDetails are unknown. dCarrion crow (Corvus corone) or Large-billed crow (Corvus macrorhynchos).
IHC protocol
Eight commercially available primary antibodies were utilized to identify T or B lymphocytes in various avian species through IHC (Table 2). The selection of certain antibodies was based on successful outcomes observed in our previous study, which focused on detecting chicken T or B lymphocytes [28]. While the consistent detection of avian T cell antigens was achieved using a single anti-T cell antibody, the detection of B cell antigens proved to be more challenging in the present study. Hence, seven antibodies were assessed for their efficacy in detecting B cell antigens.
Table 2. Tested commercial primary antibodies for immunohistochemistry.
| Marker | Primary antibody | Host | Clonality | Clone or product No. | Source | Dilution | Antigen retrieval |
|---|---|---|---|---|---|---|---|
| T lymphocyte marker | Anti-human CD3 | Mo | Mono | F7.2.38 | Abcam | 1:400 | HIAR (pH 6.0) |
| B lymphocyte marker | Anti-human PAX5 | Rb | Mono | SP34 | Spring Bioscience | 1:100 | HIAR (pH 6.0) |
| Anti-chicken BAFF-R | Mo | Mono | 2C4 | Bio-Rad Laboratories | 1:3,200 | HIAR (pH 6.0) | |
| Anti-human BLA36 | Mo | Mono | A27-42 | Biogenex | 1:800 | HIAR (pH 6.0) | |
| Anti-human CD20 | Mo | Mono | L26 | Dako | 1:400 | HIAR (pH 6.0) | |
| Anti-human CD20 | Rb | Poly | PA5-16701 | Thermo Scientific | 1:200 | HIAR (pH 6.0) | |
| Anti-chicken Bu-1a/b | Mo | Mono | AV20 | Southern Biotechnology | 1:3,200 | HIAR (pH 6.0) | |
| Anti-human CD79a | Mo | Mono | HM57 | Novus Biologicals | 1:400 | HIAR (pH 6.0) |
BAFF-R, B cell-activating factor belonging to the TNF family receptor; BLA36, B lymphocyte antigen 36; CD, cluster of differentiation; HIAR (pH 6.0), heat-induced antigen retrieval with 10 mM citrate buffer (pH 6.0); Mo, mouse; Mono, monoclonal; PAX5, paired box protein 5; Poly, polyclonal; Rb, rabbit.
FFPE blocks were cut into 2 µm-thick sections. The histological sections were deparaffinized using xylene. Subsequently, the sections were immersed in 99% ethanol, and the endogenous peroxidase activity was suppressed by incubating them in 0.3% H2O2 methanol for 20 min at room temperature (RT). Antigen retrieval was carried out by heating the sections in a microwave oven (500 W) with citrate buffer (pH 6.0) for 15 min. Enzyme-induced antigen retrieval was not applied to the eight antibodies due to their unsuitability in the previous study [28]. Following a slow cooling period of 30 min at RT, the sections were incubated in a 5% skim milk solution (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) in phosphate-buffered saline (PBS) for 20 min at RT to inhibit nonspecific antibody binding. Each appropriately diluted primary antibody was incubated with the sections for 20–24 hr at 4°C. The sections were then rinsed with PBS and treated with Histofine Simple Stain MAX PO (M; mouse) or (R; rabbit) (Nichirei Bioscience Inc., Tokyo, Japan) as secondary antibodies for 45 min at RT. After rinsing with PBS, a DAB Substrate kit (Nichirei Bioscience Inc.) was applied to visualize the immunostaining reactions. Prior to dehydration and cover slipping, the slides were counterstained with Mayer’s hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan).
To validate the IHC, the spleens of chickens and Japanese macaques were utilized as positive control. Nonspecific binding of the secondary antibody was assessed by omitting the primary antibody.
Evaluation of IHC reaction
The IHC reactions of the antibodies were evaluated under the microscope, focusing on the positive reactions observed in the lymphocytes located in the T and B cell areas of the lymphoid tissues for each bird. Specifically, we defined periarteriolar lymphoid sheath (PALS) and interfollicular area as T cell areas, while the periellipsoidal white pulp (PWP) and follicle were considered as B cell areas [28].
The pattern of immunostaining for each antibody was classified into five categories as follows: (T) staining in the T cell area and not in the B cell area, (B) staining in the B cell area and not in the T cell area, (TB) staining in both T and B cell areas, (P) staining in plasma cells and not in both T and B cell areas, and (−) indicating no staining in either the T or B cell areas, as well as in plasma cells. Additionally, very faint or indistinct staining was considered negative (−). Lymphocytes and plasma cells were differentiated based on their morphological characteristics, with cells exhibiting an eccentric nucleus and perinuclear halo being classified as plasma cells.
Immunoreactions observed throughout the entire sections, irrespective of cell type, were considered background staining. Reactions limited to specific cells other than lymphocytes or plasma cells were regarded as nonspecific reactions.
RESULTS
The IHC, utilizing the anti-human CD3 antibody (clone F7.2.38), consistently detected lymphocytes within the T cell area on FFPE sections from all 53 avian species across 15 orders (Table 3, Fig. 1a–d, Supplementary Fig. 1a–f). The positive signal of CD3 was observed along the cellular membrane of the lymphocytes (Fig. 1c, Supplementary Fig. 1d).
Table 3. Results of immunohistochemistry in each avian species.
| Order | Species (Common name) | T lymphocyte | B lymphocyte | ||||||
|---|---|---|---|---|---|---|---|---|---|
| CD3 | PAX5 | BAFF-R | BLA36 | CD20 | CD20 | Bu-1a/b | CD79a | ||
| F7.2.38 | SP34 | 2C4 | A27-42 | L26 | PA5-16701 | AV20 | HM57 | ||
| Struthioniformes (Ostriches) | Common ostrich | T | B | - | B | - | B | - | - |
| Galliformes (Landfowls) | Chicken | T | B | B | TB | TB | - | B | - |
| Golden pheasant | T | B | B | - | TB | - | - | - | |
| Peafowl | T | B | B | B | B | - | - | - | |
| Turkey | T | B | B | B | - | B | - | - | |
| Japanese quail | T | B | B | B | TB | - | - | - | |
| Svalbard rock ptarmigan | T | - | B | TB | TB | - | - | - | |
| Helmeted guineafowl | T | - | B | - | - | - | - | - | |
| Anseriformes (Waterfowls) | Hybrid duck | T | B | B | B | TB | - | - | - |
| Mallard | T | B | B | P | B | - | - | - | |
| Common teal | T | B | B | - | - | - | - | - | |
| Northern pintail | T | B | B | P | TB | - | - | - | |
| Baikal teal | T | B | - | - | TB | TB | - | - | |
| Common pochard | T | B | B | - | TB | - | - | - | |
| Tufted duck | T | B | B | TB | TB | - | - | - | |
| Ruddy shelduck | T | B | B | P | TB | - | - | - | |
| Goose | T | B | B | TB | TB | - | - | - | |
| Whooper swan | T | B | - | - | TB | TB | - | - | |
| Mute swan | T | B | B | TB | TB | - | - | - | |
| Black swan | T | - | B | TB | TB | B | - | - | |
| Podicipediformes (Grebes) | Great crested grebe | T | B | - | - | TB | - | - | - |
| Columbiformes (Doves) | Rock dove | T | B | - | TB | TB | - | - | - |
| Oriental turtle-dove | T | B | - | - | TB | - | - | - | |
| Sphenisciformes (Penguins) | Humboldt penguin | T | B | - | - | TB | - | - | - |
| Magellanic penguin | T | B | - | TB | TB | - | - | - | |
| Suliformes (Cormorants) | Great cormorant | T | B | B | TB | TB | TB | - | - |
| Japanese cormorant | T | B | - | TB | TB | - | - | - | |
| Pelecaniformes (Pelicans) | Asian crested ibis | T | B | - | B | - | - | - | - |
| Intermediate egret | T | B | - | TB | TB | - | - | - | |
| Pelican | T | B | - | P | TB | - | - | - | |
| Gruiformes (Cranes) | Common coot | T | B | - | TB | TB | B | - | - |
| Charadriiformes (Plovers) | Black-necked stilt | T | B | - | - | TB | TB | - | - |
| Black-headed gull | T | B | - | - | TB | - | - | - | |
| Tufted puffin | T | B | - | TB | TB | B | - | - | |
| Ancient murrelet | T | - | - | TB | TB | TB | - | - | |
| Accipitriformes (Eagles and New world vultures) | Hybrid hawk | T | B | - | - | - | - | - | - |
| White-tailed sea-eagle | T | - | - | - | B | - | - | - | |
| Japanese buzzard | T | B | - | - | - | - | - | - | |
| Harris’s hawk | T | B | - | B | B | B | - | - | |
| Strigiformes (Owls) | Snowy owl | T | - | - | TB | TB | - | - | - |
| Owl | T | B | - | B | B | - | - | - | |
| Falconiformes (Falcons) | Common kestrel | T | - | - | - | TB | - | - | - |
| Psittaciformes (Parrots) | Blue-and-yellow macaw | T | B | - | - | - | - | - | - |
| Budgerigar | T | B | - | P | TB | B | - | - | |
| Nyasa lovebird | T | B | - | - | TB | - | - | - | |
| Australian king-parrot | T | - | - | - | TB | - | - | - | |
| Grey parrot | T | B | - | TB | TB | - | - | - | |
| White cockatoo | T | B | - | - | TB | - | - | - | |
| Tanimbar corella | T | B | - | - | TB | - | - | - | |
| Passeriformes (Oscines) | Crow | T | - | - | TB | - | - | - | - |
| Eurasian tree sparrow | T | - | - | - | TB | - | - | - | |
| Brown-eared bulbul | T | - | - | B | B | - | - | - | |
| White-cheeked starling | T | - | - | - | - | - | - | - | |
B, staining in the B cell area and not in the T cell area; P, staining in plasma cells and not in both T and B cell areas; T, staining in the T cell area and not in the B cell area; TB, staining in both T and B cell areas; -, no staining in both T and B cell areas, and plasma cells.
Fig. 1.
Immunohistochemistry results of using the following antibodies: anti-CD3 (clone F7.2.38), anti-PAX5 (clone SP34), anti-BAFF-R (clone 2C4), and anti-BLA36 (clone A27-42) on formalin-fixed, paraffin-embedded spleen tissues from a turkey, common pochard, Harris’s hawk, and Nyasa lovebird. Scale bar=50 μm. All pictures are at the same magnification. (a) CD3 (clone F7.2.38). Turkey. CD3-positive cells are localized around the periellipsoidal white pulp (PWP). The majority of cells in the PWP and follicle (F) are negative. (b) CD3 (clone F7.2.38). Common pochard. Immunolabeled cells are mainly distributed in the red pulp (RP). (c) CD3 (clone F7.2.38). Harris’s hawk. The periarteriolar lymphoid sheaths (PALS) contain abundant immunolabeled cells. (d) CD3 (clone F7.2.38). Nyasa lovebird. Immunolabeled cells are predominantly found in the PALS, while the F exhibits few immunolabeled cells. (e) PAX5 (clone SP34). Turkey. Nuclear immunolabeled cells are concentrated in the F and PWP. (f) PAX5 (clone SP34). Common pochard. PAX5-positive cells are mostly observed in the F and PWP. (g) PAX5 (clone SP34). Harris’s hawk. Immunolabeled cells in the PWP. (h) PAX5 (clone SP34). Nyasa lovebird. Immunolabeled cells are localized in the F and PWP. (i) BAFF-R (clone 2C4). Turkey. BAFF-R-positive cells are predominantly found in the F and PWP. (j) BAFF-R (clone 2C4). Common pochard. Immunolabeled cells are mainly distributed in the F and PWP. A fewer number of BAFF-R-positive cells are scattered in the RP. (k) BAFF-R (clone 2C4). Harris’s hawk. No immunolabeled cells are found in the PALS and PWP. (l) BAFF-R (clone 2C4). Nyasa lovebird. No immunolabeled cells are found in the PALS and F. (m) BLA36 (clone A27-42). Turkey. BLA36-positive cells are distributed in the F and PWP. (n) BLA36 (clone A27-42). Common pochard. The F and PWP show no obvious positive reactions. (o) BLA36 (clone A27-42). Harris’s hawk. The PWP comprises immunolabeled cells. (p) BLA36 (clone A27-42). Nyasa lovebird. The F and PWP show no obvious positive reactions.
Lymphocytes within the B cell area were specifically labeled using IHC in 46 avian species (86.8%) (Table 3). However, the optimal markers for B lymphocyte detection varied among the avian species, with PAX5 and BAFF-R demonstrating relatively superior efficacy in detecting avian B cells. The storage duration of the FFPE blocks did not have any discernible impact on the IHC results when comparing the same avian species. Notably, B lymphocyte detection was unsuccessful in seven avian species (ancient murrelet, snowy owl, common kestrel, Australian king-parrot, crow, Eurasian tree sparrow, and white-cheeked starling).
The anti-human PAX5 antibody (clone SP34) proved to be highly effective in detecting avian B lymphocytes in the current study (Fig. 1e–h, Supplementary Fig. 1g–m). IHC using the anti-PAX5 antibody successfully identified lymphocytes within the B cell area in 41 avian species (77.4%). However, the anti-PAX5 antibody did not bind to the B cell area of birds belonging to the orders Falconiformes (falcons) and Passeriformes (oscines). Immunoreactivity for PAX5 was observed in the nuclei of the positive cells (Supplementary Fig. 1h and 1i). The lymphocytes forming the follicles often displayed strong positive staining for PAX5 (Fig. 1e and 1f, Supplementary Fig. 1k and 1l).
The anti-chicken BAFF-R antibody (clone 2C4) exhibited successful binding to the B cell area in 18 avian species (34.0%), primarily among birds belonging to the orders Galliformes (landfowls) and Anseriformes (waterfowls) (Fig. 1i–l, Supplementary Fig. 1n–r). Positive reactions with the anti-BAFF-R antibody were observed at the cellular membranes (Fig. 1i). Although the B cell areas of common ostriches displayed very faint staining with the anti-BAFF-R antibody, we considered it as negative.
The IHC results obtained using the anti-human BLA36 (clone A27-42) and anti-human CD20 (clone L26 and product No. PA5-16701) antibodies exhibited heterogeneity (Table 3, Figs. 1m–p, 2a–h, Supplementary Fig. 1s–bb). Despite these antibodies being intended for B lymphocyte detection, positive reactions were observed not only in the B cell area but also in the T cell area in several avian species, regardless of avian genera.
Fig. 2.
Immunohistochemistry results of using the following antibodies: anti-CD20 (clone L26 and product No. PA5-16701), anti-Bu-1a/b (clone AV20), and anti-CD79a (clone HM57) on formalin-fixed, paraffin-embedded spleen tissues from a turkey, common pochard, Harris’s hawk, and Nyasa lovebird. Scale bar=50 μm. All pictures are at the same magnification. (a) CD20 (clone L26). Turkey. No immunolabeled cells are found in the periarteriolar lymphoid sheaths (PALS), follicle (F), and periellipsoidal white pulp (PWP). (b) CD20 (clone L26). Common pochard. Immunolabeled cells are distributed in both the T cell area (PALS) and B cell area (F and PWP). (c) CD20 (clone L26). Harris’s hawk. The PWP contain immunolabeled cells. (d) CD20 (clone L26). Nyasa lovebird. Immunolabeled cells are observed in the red pulp (RP), F, and PWP. (e) CD20 (product No. PA5-16701). Turkey. Immunolabeled cells in the F and PWP. (f) CD20 (product No. PA5-16701). Common pochard. No immunolabeled cells are found in the RP, F, and PWP. (g) CD20 (product No. PA5-16701). Harris’s hawk. Immunolabeled cells in the PWP. (h) CD20 (product No. PA5-16701). Nyasa lovebird. The RP, F, and PWP show no obvious positive reactions. (i) Bu-1a/b (clone AV20). Turkey. No immunolabeled cells are found in the F and PWP. (j) Bu-1a/b (clone AV20). Common pochard. No immunolabeled cells are found in the RP, F, and PWP. (k) Bu-1a/b (clone AV20). Harris’s hawk. No immunolabeled cells are found in the PWP. (l) Bu-1a/b (clone AV20). Nyasa lovebird. No immunolabeled cells are found in the PWP. (m) CD79a (clone HM57). Turkey. No significant positive reactions are observed. Nonspecific reactions are observed in nuclei, regardless of cell type. (n) CD79a (clone HM57). Common pochard. No immunolabeled cells are found in the F and PWP. (o) CD79a (clone HM57). Harris’s hawk. The RP and PWP show no obvious positive reactions. (p) CD79a (clone HM57). Nyasa lovebird. No obvious immunolabeled cells are observed in the RP and F. Nonspecific reactions in vascular endothelial cells.
The staining patterns observed in IHC using the anti-BLA36 antibody were categorized as follows: (B) exclusive to the B cell area (nine avian species; 17.0%) (Fig. 1m and 1o, Supplementary Fig. 1s and 1t), (TB) present in both T and B cell areas (17 avian species; 32.1%) (Supplementary Fig. 1u and 1v), and (P) limited to plasma cells (five avian species; 9.4%). In cases where both T and B cell areas showed positive staining, plasma cells were occasionally more intensely stained compared to lymphocytes (Supplementary Fig. 1u). Reactivity for BLA36 was observed at the cellular membrane and within the cytoplasm (Fig. 1m, Supplementary Fig. 1v). In the spleen, the anti-BLA36 antibody tended to exhibit moderate to strong staining intensity in the PWP and weak staining in the follicles across most avian species (Supplementary Fig. 1u).
The anti-CD20 antibodies (clone L26 and product No. PA5-16701) demonstrated different reactivity patterns depending on the avian species, specifically targeting either the B cell area alone or both the T and B cell areas. The anti-CD20 antibody (clone L26) exclusively reacted with the B cell area in six avian species (11.3%) (Fig. 2c), while it targeted both the T and B cell areas in 37 avian species (69.8%) (Fig. 2b and 2d, Supplementary Fig. 1w–z). Conversely, the anti-CD20 antibody (product No. PA5-16701) specifically reacted with the B cell area in seven avian species (13.2%) (Fig. 2e and 2g, Supplementary Fig. 1aa and 1bb), and with both the T and B cell areas in five avian species (9.4%). Only Harris’s hawk showed positive results for the B cell area when tested with both anti-CD20 antibodies (Fig. 2c and 2g). IHC reactions using the anti-CD20 antibodies exhibited staining on the cellular membrane and within the cytoplasm (Fig. 2b, Supplementary Fig. 1w and 1aa). The staining intensity of the anti-CD20 antibody (clone L26) was occasionally moderate to strong in the PWP and weak in the follicles of the spleen (Supplementary Fig. 1z).
The anti-chicken Bu-1a/b antibody (clone AV20), which specifically binds to chicken B lymphocytes and macrophages, exhibited reactivity exclusively in the chicken B cell area and not in the T cell area. Cells from avian species other than chicken showed no reactivity towards the anti-Bu-1a/b antibody (Fig. 2i–l). The anti-human CD79a antibody (clone HM57) did not show any reactivity towards B lymphocytes in any of the tested avian species (Fig. 2m–p). The anti-CD79 antibody exhibited nonspecific immunolabeling in the nuclei, vascular endothelial cells, and smooth muscles of several avian species (Fig. 2m and 2p).
DISCUSSION
The anti-human CD3 antibody (clone F7.2.38) is a commonly used antibody in research for detecting mammalian T lymphocytes on FFPE sections through IHC [2, 3, 17, 33, 34, 49]. In the present study, we successfully detected T lymphocytes of all analyzed avian species using this antibody under heat-induced antigen retrieval condition. This finding shows the usefulness of the antibody (clone F7.2.38) for identifying T lymphocytes in various avian species. It suggests that the epitope to which the antibody (clone F7.2.38) binds on the T lymphocytes is highly conserved among avian species and remains unaffected by formalin fixation or other histological slide preparation procedures, thereby enabling successful T lymphocyte detection through IHC. To the best of our knowledge, there are no reports of immunohistochemical detection of avian T lymphocytes on FFPE sections using the anti-CD3 antibody (clone F7.2.38) apart from chicken [28]. Other commercial antibodies targeting avian T lymphocytes have been reported for IHC, but they differ from the clone F7.2.38 of the anti-CD3 antibody [11, 26, 31, 36, 50].
In contrast, the detection of B lymphocytes in various avian species through IHC was more challenging compared to T lymphocyte detection. None of the seven anti-B lymphocyte antibodies consistently bound to B lymphocytes in all avian species tested. This observation indicates a high diversity among avian species in the amino acid sequence and structure of the B lymphocyte epitopes recognized by the antibodies tested.
In this study, the rabbit anti-human PAX5 antibody (clone SP34) exhibited the highest detection rate of avian B lymphocytes (77.4%; 41/53 avian species). This suggests that the antibody is the most effective for immunohistochemical detection of avian B lymphocytes. Different anti-human PAX5 antibodies have been employed to detect B lymphocytes in mammals and reptiles through IHC [4, 12, 19, 47, 59]. Furthermore, in avian species, the mouse anti-human PAX5 antibody (clone 24) has been reported to detect B lymphocytes in 14 species belonging to the order Psittaciformes (parrots) and domestic pigeons [14, 40]. PAX5 is a protein expressed throughout the B lymphocyte lineage and plays a role in B lymphocyte development and differentiation [1, 5, 8, 13, 20, 39]. The expression level of PAX5 in mice varies depending on the differentiation stage of B lymphocytes [1]. In our study, PAX5 was strongly expressed in the lymphocytes constituting follicles in several avian species. This observation suggests that, similar to mice, the expression level of PAX5 in avian B lymphocytes may be influenced by B lymphocyte development.
There are certain limitations in identifying avian B lymphocytes through IHC using the anti-PAX5 antibody (clone SP34). In this study, the anti-PAX5 antibody did not consistently detect B lymphocytes in all birds. The reason for this outcome is currently unknown, but it could be attributed to sample conditions or potential differences in epitopes among birds. In addition, although PAX5 is predominantly expressed in the B lymphoid lineage of the hematopoietic system, it can also be detected in non-lymphocytic cells in humans and mice [1, 32, 39, 54]. Therefore, accurate identification of B lymphocytes may also require histological typing of PAX5-positive cells.
The IHC results have demonstrated that the anti-chicken BAFF-R antibody (clone 2C4) is highly suitable for detecting B lymphocytes in birds belonging to the orders Galliformes (landfowls) and Anseriformes (waterfowls). Unlike in mammals, where BAFF-R is expressed in both T and B lymphocytes, in chickens, BAFF-R is expressed exclusively on B lymphocytes [37, 46, 51]. Consequently, IHC methods that detect BAFF-R are expected to be more specific for avian B lymphocytes compared to PAX5 based IHC, which is also expressed in non-B lymphocytes. Additionally, the immunoreactivity observed with the anti-BAFF-R antibody suggests that the epitope region of the BAFF-R antigen is shared among orders Galliformes and Anseriformes birds, exhibiting high amino acid homology. Out of the 18 species that showed a positive reaction in IHC for detecting BAFF-R, the BAFF-R gene is currently not recorded in the GenBank database for eight species, including the golden pheasant, peafowl, common teal, northern pintail, ruddy shelduck, goose, common pochard, and Japanese cormorant [35]. However, our results suggest that these eight avian species are likely to possess the BAFF-R gene. The anti-BAFF-R antibody (clone 2C4) is derived from mouse and shows positive on the plasma membrane, while the anti-PAX5 antibody (clone SP34) is derived from rabbit and exhibits positive staining in the nucleus. These differences can be useful for identifying and analyzing cells through double IHC staining.
Anti-human CD20 antibodies were successful in binding to B lymphocytes in a limited number of avian species. Although an equivalent of the mammalian CD20 (MS4A1) gene has not been identified in chickens [61], our results indicate that CD20 may be expressed by B lymphocytes in some other avian species. Interestingly, positive reactions for IHC using anti-CD20 antibodies, typically considered as a B cell marker, were also observed in lymphocytes located in the T cell area in several avian species. Similar results were obtained with IHC using the anti-BLA36 antibody. One possibility is the presence of avian lymphocytes that express both the T cell antigen CD3 and some B cell antigens, including CD20 and BLA36. For example, CD20-positive T lymphocytes exist as a subset of T lymphocytes in humans, constituting less than approximately 6% of T lymphocytes in the peripheral blood [9, 23]. Lymphoma cells that co-express T and B cell antigens have been reported in humans, dogs, cats, and birds in the genera Amazona sp. and cockatiel [7, 14, 22, 29, 38, 41, 43,44,45, 57, 58, 60]. However, the possibility of unexplained nonspecific binding of anti-CD20 and anti-BLA36 antibodies to the T cell area cannot be ruled out. The potential presence of cells co-expressing T and B cell antigens in birds needs to be further analyzed in future studies.
The anti-chicken Bu-1a/b antibody (clone AV20) and the anti-human CD79a antibody (clone HM57) were found to be unsuitable for the immunohistochemical detection of avian B lymphocytes. Bu-1 (chB6), one of the transmembrane glycoproteins, is expressed on B lymphocytes and a subset of macrophage lineage cells in chickens [15, 21]. Various antibodies targeting Bu-1 have been developed [24, 25, 42, 48, 56]. In our study using FFPE sections, the anti-Bu-1a/b antibody (clone AV20) did not show cross-reactivity with B lymphocytes from bird species other than chickens. Similar results were observed in a related study using frozen sections, where the anti-Bu-1a/b antibody (clone AV20) did not react with B cells in turkey, Japanese quail, and helmeted guineafowl samples [24]. However, different anti-Bu-1 antibodies were able to bind to B lymphocytes on frozen sections from several avian species belonging to the order Galliformes [24, 25]. As for CD79a, it was found that only five out of the 53 avian species (helmeted guineafowl, hybrid hawk, budgerigar, crow, and Eurasian tree sparrow) possess the CD79a gene [35]. The anti-CD79a antibody (clone HM57) bound exclusively to plasma cells on frozen sections of chickens [27]. However, in our study, this anti-CD79a antibody failed to bind to B lymphocytes and plasma cells on FFPE sections from the 53 avian species, including those belonging to the order Galliformes.
In conclusion, our findings suggest that the anti-CD3 antibody (clone F7.2.38) consistently demonstrated suitability for the immunohistochemical detection of avian T lymphocytes across different species. However, the detection of avian B lymphocytes requires the use of specific antibodies tailored to each species. Among the B cell markers evaluated, PAX5 showed the broadest reactivity in various avian species, making it the most effective B cell marker. The detection methods employed in this study, using commercially available antibodies and FFPE sections, can be easily implemented by various laboratories and diagnostic companies. It is important to note that the samples used in this study were obtained under varying conditions, and only a single IHC condition was employed for each antibody. Standardizing the postmortem-to-specimen preparation time and fixation conditions would contribute to obtaining more reliable and accurate results. It should also be noted that different IHC results may be obtained if the antibody concentration or antigen retrieval process deviate from those utilized in the current study.
CONFLICT OF INTEREST
The authors declare no conflicts of interest associated with this study.
Supplementary Material
Acknowledgments
We thank Ms. Megumi Shimada for her technical assistance with preparing sections. We are also grateful to Dr. Aihara Naoyuki (Azabu University), Ms. Eiko Koike (Saitama prefecture), Dr. Junichi Kamiie (Azabu University), Mr. Syun Ishizuka (Ibaraki prefecture), Mr.Yuta Hamada (Shimane prefecture), Dr. Yuta Kuribayashi (Omachi Alpine Museum), Ishikawa prefecture, and Yamaguchi prefecture for providing the samples.
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