Pi. Hornick et al., OPTIMIZING A LIMITING DILUTION CULTURE SYSTEM FOR QUANTIFYING THE FREQUENCY OF INTERLEUKIN-2-PRODUCING ALLOREACTIVE T-HELPER LYMPHOCYTES, Transplantation, 64(3), 1997, pp. 472-479
Background. The development of sensitive, specific, and reproducible t
echniques to quantify T cells with direct allospecificity has potentia
l applications in the selection of bone marrow donors and in the monit
oring of the antidonor alloresponse in patients after organ transplant
ation. Such data may provide an objective basis for altering existing
immunosuppression, monitoring novel antirejection therapies, and predi
cting long-term graft outcome. We have previously published a correlat
ion between donor antirecipient T helper frequencies (HTLf) and the se
verity of acute graft-versus-host disease after bone marrow transplant
ation. Using the same assay protocol, we have described the developmen
t of donor-specific hyporesponsiveness in a proportion of renal transp
lant recipients. However, several imperfections existed in the protoco
ls used in these studies. Cellular interactions within the stimulator
and the responder cell populations, and back stimulation of T cells wi
thin the stimulator cell population, could give rise to extraneous int
erleukin-2 and alter the validity or estimation of derived recipient a
ntidonor HTLf. Methods. Using peripheral blood mononuclear cells as th
e responding population and splenic mononuclear cells as the stimulati
ng population, we have examined the possible effects of these cellular
interactions on the results of limiting dilution analysis assays for
HTLf measurement, Results. These interactions have the ability to alte
r the validity or estimation of HTLf. We show that by depleting the re
sponder population of HLA class II+ cells and depleting T cells from t
he stimulating population, these interactions are effectively abrogate
d. Conclusions. On the basis of the findings reported here, we describ
e an optimized HTLf assay which is sensitive, specific, and reproducib
le, This has obvious applications in the analysis of alloimmune respon
ses in transplantation.