Izutsu Y et al. (2000), Larval antigen molecules recognized by adult im...

XB-ART-11092

Dev Biol 2000 May 15;2212:365-74. doi: 10.1006/dbio.2000.9681.

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Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: two pathways for recognition by adult splenic T cells.

Izutsu Y , Tochinai S , Iwabuchi K , Onoè K .

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During anuran metamorphosis, larval cells of the tadpole are completely eliminated and replaced by adult cells in the corresponding tissues of the frog for the adaptation to terrestrial life from an aquatic life. Before the metamorphic climax, most of the cells have already transformed from larval cells into adult-type cells, but the tail cells remain as larval cells even at the climax stages of metamorphosis. In our previous works, we demonstrated that larval skin grafts are rejected by an inbred strain of adult Xenopus and that the larval cells are recognized and made apoptotic by splenocytes obtained from adults and/or metamorphosing tadpoles in vitro (Y. Izutsu and K. Yoshizato, 1993, J. Exp. Zool. 266, 163-167; Y. Izutsu et al., 1996, Differentiation 60, 277-286). In the present study, it was found that there were two types of larval epidermal cells, classified according to the presence of major histocompatibility complex (MHC); one is the apical cell expressing both MHC classes I and II, and the other is the skein cell, which expresses no MHC. By a Percoll gradient, we were able to separate these two types of cells and examined the proliferative response of adult T cells to each of them. It was revealed that the apical cells (MHC-positive) were recognized directly by adult splenic T cells, whereas the skein cells (MHC-negative) were recognized by the T cells via the antigen presentation by adult splenocytes. Both of these proliferative responses were restricted to MHC class II. This is the first report showing how the larval-specific antigens present in different forms in epidermal cells are recognized as immunological targets by syngeneic adult T lymphocytes.

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Species referenced: Xenopus laevis
Genes referenced: mhc2-dcb myh6 tbx2

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	FIG. 1. Developmental change in the expression of MHC class II molecules on epidermal cells in the skin during metamorphosis. Frozen sections were subjected to immunohistochemical staining using anti-Xenopus MHC class II monoclonal antibody (red signals) and counterstained with quinacrine (green). Larval-positive cells sometimes looked yellowish, due to the overlaid counterstaining with quinacrine. The thickness of the epidermis is shown by a white line on the left side of each photo. The images on the left show the back epidermis of stage 57 (A), stage 59 (B), and stage 64 tadpoles (C) and a 2- to 3-month-old froglet (D). The images on the right show the tail epidermis of stage 57 (E), stage 59 (F), and stage 64 tadpoles (G). Irregularly shaped large cells expressing MHC class II were seen in the tail epidermis at stage 64 (arrowheads in G). The letter G in B, C, and D indicates the developing granular gland. Arrows in C and D indicate the sites of granular gland formation. Bar represents 100 mm.
	FIG. 2. Expression of MHC molecules on the epidermal cells around the back and tail junction region at metamorphic climax. Frozen sections including the boundary of the tail and back of stage 64 larvae were immunostained as described in the legend to Fig. 1, demonstrating the expression of MHC class I (A) and MHC class II (B). A conspicuous difference was not seen between the expression patterns of MHC class I and II on the epidermal cells. Note the clear immunohistochemical difference between the back and the tail areas of the epidermis. Broken lines show the locations of the basement membrane. E and D represent the thickness of epidermis and dermis, respectively. Bar represents 100 mm.
	FIG. 3. In vitro induction of MHC expression in larval epidermal cells (A–D) and the responses of adult splenocytes to epidermal cells (E). Epidermal cells (1.2 3 105) were obtained from tadpole trunk skin at stage 55/56 and cultured in 96-well dishes for 3 days at 28°C in the presence (A, C) or absence (B, D) of 100 nM T3. The cells were immunostained with anti-Xenopus MHC class I (A, B) or class II (C, D) mAb and detected by enzyme reaction. The cells expressing MHC class I or class II were stained brown, especially strong in keratinized cells differentiated by T3. A few differentiated cells appeared even in the hormone-free culture (arrows). A significant difference was not seen between MHC class I and II expressions. Bar represents 100 mm. (E) The epidermal cells were precultured for 24 h and then cocultured with adult splenocytes for 4 days. Response of adult splenocytes cultured without stimulators (W/O), with larval tail epidermal cells (Tail-epi), and with larval trunk epidermal cells (Trunk-epi). Open columns represent the response to the epidermal cells cultured in the absence of hormone, and striped columns represent the response to those cells treated with hormone (100 nM T3). A total of 1000–1500 cells were counted in five or six randomly selected areas of individual specimens prepared from each of the responder splenocytes, which were labeled with randomized numbers to avoid possible anticipation. Proliferative responses are shown as percentages to the total number of leukocytes. Values represent means 6 SD obtained from three different experiments.
	FIG. 4. Stimulator-dependent proliferative responses of adult splenocytes. Proliferative responses are shown as percentages of the total number of leukocytes after scoring by the same methods as mentioned in Fig. 3E. (A) Proliferative responses of adult splenocytes including APC. J strain adult splenocytes were cocultured with syngeneic larval trunk epidermal cells (JJLa-epi), which are mostly MHC class II-negative as shown in Fig. 3D; with larval tail tissues at stage 55/56 (JJLa-tis) including MHC class II-positive apical cells as shown in Fig. 1E; with JB adult skin tissues obtained from F1 of the J strain of X. laevis and the B strain of X. borealis (JBAd-tis); or with adult skin of J strain (JJAd-tis). Cross-hatched columns indicate cultures to which 20 mg/ml anti-Xenopus MHC class I mAb was added, closed columns indicate cultures to which 20 mg/ml anti-Xenopus MHC class II mAb was added, and open columns indicate cultures without antibodies. (B) Proliferative responses of adult splenocytes that were completely depleted of the adhesive APC (macrophages, dendritic cells, and B cells) by nylon wool columns. Nonadherent adult splenocytes were cocultured with each stimulator as described in A. Response of adult splenocytes cultured without stimulators (W/O). Values represent means 6 SD obtained from three different experiments. The higher percentages were generally obtained in the proliferative responses of splenocytes in the absence of APC, which may be attributed to the large number of T cells in the responder population.
	FIG. 5. A schematic model depicting two pathways of T cell recognition of larval antigens derived from epidermal cells. The yellow-colored cells indicate apical epidermal cells expressing MHC class II molecules (gray-colored mushroom-like), and the blue cells are MHC-negative skein cells. Adult T cells directly recognize the larval-specific antigens (red ball) on the MHC class II molecules of apical cells or the larval-specific antigens that have been processed and presented on the MHC class II molecules on splenic APC. T, Mp, and B represent the responding T cells, the antigen-presenting macrophages, and the B cells, respectively.