IN VIVO CHARACTERIZATION OF CELLULAR XENOIMMUNITY

Abstract

47 Introduction: Rejection of xenogeneic tissue is mediated by a unique collaboration of both humoral and cellular arms of the immune system. The humoral response has been the topic of considerable investigation while the cellular response remains largely unstudied. In the present work, we characterized the cellular xenogeneic response by following lymphocytes labeled with carboxyflourescein (CFSE), a fluorescent dye that covalently binds intracellular proteins. Because CFSE fluorescence intensity is halved with each cell division, calculation of the rate, number of divisions, and precursor frequency of responding cells is possible. Methods: CFSE labeled splenocytes or lymph node cells were utilized in vivo and in vitro to compare allogeneic and xenogeneic responses. In the in vitro experiments, rat lymphoid cells were stimulated with irradiated xenogeneic cells (Lewis anti-B6). Control responders were stimulated by either Con A or allogeneic mouse cells (B6 anti-BALB/c). For in vivo experiments, Lewis responders were injected into supra-lethally irradiated (2500R) xenogeneic (B6) recipients. Responding cells were collected at designated time points, stained for T cell subset markers (CD4 and CD8) and analyzed by flow cytometry. Results: Rat cells stimulated with Con A had not divided by 48 hrs, but evidenced vigorous proliferation by 72 hrs. The proliferation kinetics showed sequential increases (to a maximum of six divisions), and the calculated precursor frequency of CD4 T cells was constant at all intervals (approx. 70%). At 72 hours, the in vitro xenoresponse yielded a precursor frequency of 0.7% with six cell divisions as compared to 4% and six divisions in allogeneic mouse controls. For in vivo experiments, the xenogeneic precursor frequency was 1.8% with six divisions at 60 hours versus 15% precursor frequency and six divisions allogeneic controls. (Figure)FigureConclusions: The use of CFSE labeling provides a unique tool to monitor the responses of xeno-reactive cells in vivo. We found that compared with allogeneic responses, xenogeneic responses have reduced division kinetics and precursor frequency. The relatively greater estimate of precursor frequencies in the in vivo experiments may result from a relative enrichment of reactive cells in the recipient spleen.