CTLA-4 is not restricted to the lymphoid cell lineage and can function as a target molecule for apoptosis induction of leukemic cells.

The expression of cytotoxic T-lymphocyte antigen-4 (CTLA-4) molecule in human normal and neoplastic hematopoietic cells, both on the cell membrane and in the intracellular compartment, was evaluated. Flow cytometric analysis carried out with a panel of anti-CTLA-4 human single-chain fragment of variable domain (scFv) antibodies revealed that CTLA-4 was not expressed on the surface, whereas it was highly expressed within the cytoplasm, in freshly isolated peripheral blood mononuclear cells (PBMCs), T cells, B cells, CD34(+) stem cells, and granulocytes. Various treatments with agents able to specifically activate each cell type induced CTLA-4 expression on the surface of these cells. Similarly, increased CTLA-4 expression was observed in different hematopoietic cell lines although they also expressed surface CTLA-4, at different degrees of intensity, before activation. Surprisingly, CTLA-4 RNA transcripts were detectable in such cell lines only after nested polymerase chain reaction (PCR) specific for CTLA-4 extracellular domain, suggesting a very fast CTLA-4 RNA processing accompanied by prolonged CTLA-4 protein accumulation. We further demonstrated surface expression of CTLA-4 in a variety of acute and chronic myeloid leukemias (AMLs and CMLs) and B- and T-lymphoid leukemias, either adult or pediatric. CTLA-4 was expressed in 25% to 85% of AMLs and CMLs depending on the leukemia subtype and the epitope analyzed, whereas in acute B- and T-leukemias CTLA-4 expression was mainly cytoplasmic. Chronic B leukemias appeared to express CTLA-4, both on the surface and in cytoplasm, whereas few cases tested of chronic T leukemias were negative. Two anti-CTLA-4 immunotoxins (scFvs-saporin) induced in vitro apoptosis of neoplastic cells from a representative AML, suggesting a novel immunotherapeutic approach to AML based on CTLA-4 targeting.

Analysis of HLA-G expression in breast cancer tissues.

Among the different mechanisms by which cancer can elude the immune system, alterations in the expression of human leukocyte antigen (HLA) class I molecules on tumor cells may play a crucial role by impairing the HLA molecules interaction with T and natural killer (NK) cells specific receptors. More recently, aberrant expression of HLA-G has been described in different tumor tissues in addition to HLA class I downregulation. The HLA-G molecule is a nonclassical HLA class I antigen selectively expressed by trophoblast and thymic epithelial cells. Several studies reported that the HLA-G function might represent an additional mechanism of tumor immune escape, mainly inhibiting NK and cytotoxic T-cell activity. Here we report the analysis of HLA-G expression both at RNA level by reverse transcriptase-polymerase chain reaction and at protein level by Western blot and immunohistochemistry in 25 breast cancer patient tissues. The aim of this study was to elucidate the HLA-G gene expression pattern in breast tumor tissues and correlate it with HLA class I alterations. Our results demonstrated that HLA-G molecules expression was never found even in a group of patients revealing HLA class I total loss, and that HLA-G is not expressed in breast cancer tissue with a low-tumor grade (G1-G2) and minimal stromal contamination.