The CD45 tyrosine phosphatase regulates Campath-1H (CD52)-induced TCR-dependent signal transduction in human T cells

Abstract

Campath-1H, a humanized mAb undergoing clinical trials for treatment of leukemia, transplantation and autoimmune diseases, produces substantial lymphocyte depletion in vivo. The antibody binds to CD52, a highly glycosylated molecule attached to the membrane by a glycosylphosphatidylinositol anchor. Cross-linked Campath-1H is known to activate T cells in vitro. We have investigated the molecular basis for these effects by comparing the protein tyrosine phosphorylation signals induced by Campath-1H and the CD3 mAb OKT3 in primary T cells, and in CD45(+)TCR(+), CD45(-)TCR(+) and CD45(+)TCR(-) Jurkat subclones transfected with CD52. Our results show that Campath-1H triggers similar tyrosine phosphorylation events as OKT3 in both primary T cells and in the CD45(+)TCR(+) Jurkat sub-clone, albeit at quantitatively lower levels. However, no phospholipase C gamma 1 activation nor calcium signals were detected in response to CD52 ligation. The CD52-mediated induction of protein tyrosine phosphorylation was absolutely dependent upon the expression of both the TCR and the CD45 phosphotyrosine phosphatase at the cell surface. Cross-linking of Campath-1H was essential for signal transduction in all cells investigated. Fluorescence resonance energy transfer was used to demonstrate CD52 homo-association at the cell surface in Jurkat T cells in a TCR- and CD45-independent manner, and CD52-TCR association in CD45(+)TCR(+) cells. We propose a model to explain the activating effects of Campath-1H in which CD52 mAb cross-linking causes the trapping of TCR polypeptides within molecular complexes at the cell surface, thereby inducing signals via the TCR by a process which depends on the CD45-mediated regulation of the p56(lck) and p59(fyn) tyrosine kinases.

Significance of circulating T-cell clones in Sézary syndrome

“…Thus, we may also ask whether treatment in itself may generate T-cell clones through the exertion of negative selection on the entire T-cell repertoire. The role of alemtuzumab in generating such a clonal expansion in our patient remains speculative, but, in agreement with this hypothesis, it has been shown that alemtuzumab cross-linking may result in T-cell activation in vitro 38,39 and that alemtuzumab may promote the expansion of CD52 Ϫ T-cell clones in vivo. [40][41][42] On the other hand, the expansion of nonmalignant T-cell clones may be unrelated to the disease or the treatment because expanded T-cell clones can be detected by molecular studies in the blood of patients, especially elderly patients, with benign dermatoses or non-CTCL malignant diseases.…”

Differential Association of CD45 Isoforms with CD4 and CD8 Regulates the Actions of Specific Pools of p56lck Tyrosine Kinase in T Cell Antigen Receptor Signal Transduction

“…The reaction mixture was passed through a Sephadex G-100 fine column and the collected F(abЈ) fractions were further separated from intact IgG on a protein A-Sepharose column. Purified mAbs and F(abЈ) fragments were conjugated with 6-(fluorescein-5-carboxamido)-hexanoic acid succinimidyl ester and 6-(tetramethylrhodamine-5-carboxamido)-hexanoic acid succinimidyl ester (Molecular Probes, Eugene, OR) or with sulfoindo-cyanin succinimidyl bifunctional ester derivative of Cy-3 and Cy-5 (Amersham Biosciences, Inc.) as described (34). The dye:protein ratios were separately determined for each labeled aliquot by spectrophotometric measurements and in the case of F(abЈ) fragments were ϳ1.…”

Effective Treatment of a Refractory Hairy Cell Leukemia Variant with Splenic Pre-Irradiation and Alemtuzumab

“…Alemtuzumab (MabCampath; Bayer Schering) was used at a concentration of 10 µg/ml, which is within the range of serum concentrations achieved with clinical dosing [14] and similar to the range of concentrations used in previous studies [15, 16]. …”