CD40 ligandŽCD40L.is important for TB lymphocyte interaction. To understand the cellular basis of humoral allosensitization we, therefore: Ž1. measured CD40L protein and gene expression in sensitized and non-sensitized uremic unactivated peripheral CD4 T lymphocytes; Ž2. studied the impact of blocking the CD40CD40L pathway on alloreactive antibodyŽallo-Ab. production by engrafted sensitized PBLs into severe combined immunodeficientŽSCID.mice after in vitro preactivation with IL2LPsHLA class II allopeptides and adjuvants as a potent stimulus to produce allo-Ab ŽShoker et al. Transplantation 1999;68;1188.; andŽ3.studied the modifying effect of CD40 CD40L blockade on T helper type I and II cytokine gene expression in the respective mice spleen. The CD40L protein was measured by flow cytometry and the gene expression was measured by quantitative RT-PCR. Alloreactive antibodiesŽalo-Abs.produced by sensitized PBLs engrafted into SCID mice with and without blockade of the CD40 receptor were measured by the PRA-STAT ELISA method. The modifying effects of CD40 blocking on allo-Ab production and cytokine gene expression by the engrafted cells measured by RT-PCR were then compared. There was no detectable CD40L protein expression in either the uremic or the control groups. The CD40L gene expression of 0.04 0.02 attomolesŽaM.in the sensitized group was significantly higher than in the non-sensitized patientsŽ0.009 0.007 aM, P 0.0001. or the control CD4 T cellsŽ0.016 0.004 aM, P0.001.. Blockade of the CD40 receptor abrogated the production of allo-Ab antibodies by the engrafted sensitized cells in 60% of the tested miceŽn 10.; decreased the mean S.D. optic density of allo-Ab to 0.1 0.13 and the meanS.D. PRA to 1216.. In the presence of the control Ab, allo-Ab production in SCID sera was present in 100% of the 10 SCID mice tested; the meanS.D. PRA was 7520, and the meanS.D. OD activity was 0.4120.17. All cytokine genes were, otherwise, expressed in the presence or absence of CD40 blockade. The results suggest a potential role of an enhanced CD40 CD40L interaction in the sustenance of alloreactive antibody production without significant deviation to T helper-like I or II responses. Blocking the CD40 CD40L pathway may have a potential therapeutic benefit to treat sensitized uremic patients. 2001 Elsevier Science B.V. All rights reserved.