Analysis of the CD40/CD40L role in the sustenance of alloreactive antibody production.
CD40 ligand (CD40L) is important for T/B lymphocyte interaction. To understand the cellular basis of humoral allosensitization we, therefore: (1) measured CD40L protein and gene expression in sensitized and non-sensitized uremic unactivated peripheral CD4+ T lymphocytes; (2) studied the impact of blocking the CD40/CD40L pathway on alloreactive antibody (allo-Ab) production by engrafted sensitized PBLs into severe combined immunodeficient (SCID) mice after in vitro preactivation with IL2/LPs/HLA class II allopeptides and adjuvants as a potent stimulus to produce allo-Ab (Shoker et al. Transplantation 1999;68;1188); and (3) 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 antibodies (alo-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 attomoles (aM) in the sensitized group was significantly higher than in the non-sensitized patients (0.009 +/- 0.007 aM, P < 0.0001) or the control CD4+ T cells (0.016 +/- 0.004 aM, P < 0.001). Blockade of the CD40 receptor abrogated the production of allo-Ab antibodies by the engrafted sensitized cells in 60% of the tested mice (n = 10); decreased the mean +/- S.D. optic density of allo-Ab to 0.1 +/- 0.13 and the mean +/- S.D. PRA to 12 + 16). In the presence of the control Ab, allo-Ab production in SCID sera was present in 100% of the 10 SCID mice tested; the mean +/- S.D. PRA was 75 +/- 20, and the mean + S.D. OD activity was 0.412 +/- 0.17. 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.
Shoker
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Department of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. shoker@sask.usask.ca
Lun
Z R
ZR
Choudry
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Saxena
A
A
D000276:Adjuvants, Immunologic; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000637:transplantation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D019013:CD40 Antigens / Q000276:immunology*; D023201:CD40 Ligand / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006084:Graft Rejection / Q000276:immunology; D006085:Graft Survival; D006684:HLA-DR Antigens / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007156:Immunologic Memory / Q000276:immunology*; D007376:Interleukin-2 / Q000494:pharmacology; D007518:Isoantibodies / Q000096:biosynthesis* / Q000276:immunology; D016030:Kidney Transplantation / Q000276:immunology; D008213:Lymphocyte Activation; D008211:Lymphocyte Cooperation / Q000276:immunology*; D008222:Lymphokines / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D014511:Uremia / Q000276:immunology
D000276:Adjuvants, Immunologic; D019013:CD40 Antigens; D006684:HLA-DR Antigens; D007376:Interleukin-2; D007518:Isoantibodies; D008222:Lymphokines; D023201:CD40 Ligand