The Expression Patterns of CD44 and CD45RB on Peripheral Blood T Lymphocytes in the Rejection of Allogeneic Skin Transplantation in Mice

Yang, Jae Seok; Ahn, Curie; Jung, Hee Kyeong; Kim, Eun Kyeong; Kim, Jae Young; Kim, Ki Won; Hwang, Dae Yeon; Oh, Jieun; Oh, Yoon Kyu; Oh, Kook Hwan; Kim, Sang Joon; Kim, Joong Gon; Kim, Yon Su; Han, Jin Suk; Kim, Suhnggwon; Lee, Jung Sang

Abstract

PURPOSE: Until now, the rejection was diagnosed through a biopsy, but this method of diagnosis reflected the advanced tissue damage of the transplanted organ and contained the innate problem of being invasive. Activation of T lymphocytes, which occurs before the overt tissue damage has a pivotal role in rejection. In relation, our research attempted to evaluate the viability of analyzing the surface antigens of the peripheral blood activated T lymphocytes in mice after skin transplantation as a noninvasive and early diagnostic tool for diagnosis of rejection.
METHODS
After the mouse's skin was transplanted, the expression patterns of activated T lymphocyte markers, CD44 and CD45RB were analyzed along with T lymphocyte markers, CD3, CD4, and CD8 using flow cytometry. The skins from the tails of allogeneic Balb/c (H2(d)) mice and syngeneic C57BL/6J mice were transplanted to C57BL/6J (H2(b)) mice as test and control groups, respectively. Peripheral blood, which was sampled from the tail every other day from day 3 to day 15 was stained with anti-CD44 (or CD45RB), anti-CD4 (or CD8) and anti-CD3 monoclonal antibodies simultaneously, and analyzed by 3-color FACS. Repeated ANOVA test and Mann-Whitey test were used to analyze the differences between the expression patterns of peripheral blood T lymphocyte surface antigen in the control and test groups (SPSS 8.0).
RESULTS
Rejection occurred only in the test group from day 8 to day 13 (median: day 10). Although the proportions of CD3(+)lymphocytes (CD3(+)%), CD4(+)lymphocytes (CD4(+) %), and CD8 lymphocytes (CD8(+)%) showed no difference between the control and test groups, the total number of peripheral blood lymphocytes and the number of CD3(+)lymphocytes (CD3(+)) and CD8(+)lymphocytes (CD8(+)) decreased more sharply in the control group after day 7. The proportion and the number of CD44 CD3(+)lymphocytes, CD44 CD4(+)lymphocytes, and CD44(+) CD4(+) CD3(+)lymphocytes began to increase after day 7, to peak on day 11, and then to decrease, showing a significant difference from those of the control group. The proportion and number of CD44(+) CD3(+)lymphocytes, in particular, showed the most significant difference among these significant markers. The proportion and number of CD44(+) CD8(+) lymphocytes and CD44(+) CD8(+) CD3(+)lymphocytes showed similar trends to those of CD44(+) CD3(+) or CD44(+) CD4(+), but the differences between the subset proportions in control and test groups were statistically insignificant. No significant difference was observed in any subsets of the CD45RB antigen.
CONCLUSION
CD44(+)CD3(+) lymphocytes representing activated T lymphocytes increased significantly compared to the control group during the rejection period of skin transplantation. The analysis of the expression patterns of surface antigen CD44 on peripheral blood T lymphocytes using flow cytometry is sensitive, safe, easily repeatable, and controllable, and, therefore, can be considered a promising tool for the diagnosis of rejection. However, the clear change in CD44 occurred between day 9 and day 13, when rejection was observed grossly. Therefore, it is regarded more useful as a screening test or follow-up indicator rather than as an early diagnostic tool.