Michael J. Yellin

A single amino acid substitution in a common African allele of the CD4 molecule ablates binding of the monoclonal antibody, OKT4

The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G----A transition at bp 867 encoding an arginine----tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine----tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4- protein.

LIGATION OF CD40 ON FIBROBLASTS INDUCES CD54 (ICAM-1) AND CD106 (VCAM-1) UP-REGULATION AND IL-6 PRODUCTION AND PROLIFERATION

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon‐γ (rINF‐γ) induces fibroblast CD40 up‐regulation. This effect of rINF‐γ is augmented by recombinant interleukinlα or recombinant tumor necrosis factor‐α. CD40 expression on fibroblasts is functionally significant because CD40L‐CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule‐1) and CD106 (vascular cell adhesion molecule‐1) up‐regulation. Moreover, ligation of CD40 augments IL‐6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF‐γ enhances the effect of CD40L‐CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation. J. Leukoc. Biol. 58: 209–216; 1995.

CD40L‐CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression

During immune responses, activated endothelial cells down‐regulate thrombomodulin and up‐regulate tissue factor expression leading to the development of a procoagulant surface. CD4+ T cells are known to promote endothelial cell procoagulant activity, however, the molecular interactions that mediate this effect are not completely known. CD40L is an activation‐induced CD4+ T cell surface molecule that functionally interacts with CD40 expressed on endothelial cells. In this study we ask if CD40L‐CD40 interactions modulate endothelial cell surface tissue factor or thrombomodulin expression in vitro. Human umbilical vein endothelial cells (HUVEC) were cocultured with control cells or CD40L+ Jurkat T cells in the presence or absence of anti‐CD40L mAb. By two‐color FACS analysis we demonstrated that CD40 ligation induces HUVEC tissue factor expression and thrombomodulin down‐regulation. Utilizing neutralizing antibodies, we show that CD40L‐mediated tissue factor and thrombomodulin modulation, as well as E‐selectin and VCAM‐1 up‐regulation, is independent of tumor necrosis factor α, interleukin‐1α, or interleukin‐1β production. Together these data suggest that CD40L‐CD40 interactions may directly regulate endothelial cell procoagulant activity during inflammatory responses. J. Leukoc. Biol. 63: 373–379; 1998.

Suppression of Murine Cardiac Allograft Arteriopathy by Long-Term Blockade of CD40-CD154 Interactions

Background—The interaction between CD40 on antigen-presenting cells and CD40L on T cells is critical in allograft rejection. CD154 blockade suppresses allograft rejection, but the role of this pathway in allograft vasculopathy remains obscure. Methods and Results—A vascularized murine heterotopic cardiac transplant model was used to test whether perioperative CD154 blockade suppresses allograft vasculopathy or whether long-term CD154 blockade is required to suppress allograft vasculopathy. Perioperative CD154 blockade consisted of MR1 given on days −1, 1, and 3; long-term blockade consisted of MR1 given on days −1, 1, and 3 and continued twice weekly for 8 weeks. Allografts treated with perioperative or long-term CD154 blockade survived indefinitely. Perioperative and long-term treatment with control antibody (Ha4/8) resulted in uniform early rejection. Perioperative CD154 blockade transiently reduced early T-cell and macrophage infiltration in parallel with a transient reduction in endothelial adhesion receptor expression. Although perioperative CD154 blockade prevented allograft failure, it did not reduce allograft vasculopathy; mean neointimal cross-sectional area in perioperative MR1-treated and Ha4/8-treated recipients was 43±7% and 50±12%, respectively (P =NS). In contrast, mean neointimal cross-sectional area in long-term, MR1-treated recipients was 19±3% (P <0.001 versus perioperative MR1). Long-term CD154 blockade also suppressed endothelial E-selectin, P-selectin, and intracellular adhesion molecule-1 expression and improved graft function 3.5-fold versus control (P <0.05). Conclusions—These data show that perioperative CD154 blockade mitigates acute rejection but long-term CD154 blockade may result in decreased allograft endothelial activation and is required to suppress allograft arteriopathy.

Analysis of CD154 and CD40 expression in native coronary atherosclerosis and transplant associated coronary artery disease.

Abstract T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4+ T cell surface molecule that interacts with CD40+ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40+ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154+ and CD40+ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40+ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases.

The central role of the CD40-ligand and CD40 pathway in T-lymphocyte-mediated differentiation of B lymphocytes.

This review summarizes recent findings concerning the role of CD40‐ligand and CD40 interactions in B‐cell differentiation. CD40‐ligand on helper CD4+ T lymphocytes interacts with CD40 on B cells and directs the selection and differentiation of clones of B lymphocytes to generate specific antibodydependent immune responses. CD40‐ligand is necessary for normal B‐cell differentiation and plays several distinctive roles in this multistage process. The CD40 signaling pathway that normally regulates B‐cell death appears to be usurped by the Epstein‐Barr virus to mediate B‐cell transformation.

Non-antigen signals for B-cell growth and differentiation to antibody secretion.

Recently, significant progress had been made in understanding the T-B lymphocyte interactions that control humoral immunity. This review highlights experiments that demonstrate a central role for interactions between T-cell-B-cell-activating molecule (CD40 ligand) expressed on T cells and CD40 on B cells in B-cell activation and immunoglobulin isotype switching, both in vitro and in vivo.