ABSTRACT
Although graft-versus-host (GVH) disease (GVHD) is usually associated with graft versus leukemia (GVL), GVL can occur in the absence of clinical GVHD. There is evidence to suggest that GVL and GVH are mediated by different clones of T cells. The objective of this study was to identify the two types of T cells based on their receptor sequences. To this end we used irradiated nonleukemic cells from recipients as stimulator cells in a primary mixed leukocyte reaction (MLR). The activated CD4(+) donor T cells that expressed CD25 were purified by cell sorting. To prepare GVL-specific T cells, alloreactive T cells in the primary MLR were first depleted with an anti-CD25 immunotoxin. The remaining T cells had negligible alloreactivity in a secondary MLR. The allodepleted cells were then stimulated by using purified leukemia cells from the same individual as stimulator cells, and the CD25(+)-activated cells were purified by cell sorting. The GVL- and GVH-specific T cells were analyzed for their T cell receptor (TCR) clonality by using anchored RT-PCR of all the TCRbeta locus complementarity-determining region 3 (CDR3) sequences. By comparing TCRbeta CDR3 sequences from transformed bacterial colonies, we were able to demonstrate that T cells mediating GVH were different from those mediating GVL in each of the eight HLA-mismatched and one HLA-matched donor/recipient pairs. By using the appropriate TCRbeta CDR3-specific primers and probes, the GVH- and GVL-specific clones were monitored in a recipient undergoing an allogeneic stem cell transplant from her HLA-matched related donor.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Separation/methods , Clone Cells/immunology , Graft vs Host Disease/immunology , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Transfusion Reaction , Flow Cytometry , HLA Antigens , Humans , Immunophenotyping/methods , Receptors, Interleukin-2/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes , Time FactorsABSTRACT
Ovarian carcinoma multicellular spheroids are an in vitro model of micrometastasis whose adhesive abilities have not been elucidated. In this study, we identified adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to extracellular matrix proteins. The NIH:OVCAR5, but not the SKOV3, ovarian carcinoma cell line formed spheroids similar to multicellular aggregates isolated from patient ascitic fluid. NIH:OVCAR5 spheroid formation was augmented by a beta 1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the alpha 5- or beta 1-integrin subunits. By immunohistochemical staining, alpha 2-, alpha 3-, alpha 5-, alpha 6-, and beta 1-integrin subunits, CD44, and fibronectin were detected in NIH:OVCAR5 spheroids. NIH:OVCAR5 spheroids adhered to fibronectin, laminin, and type IV collagen, and this adhesion was partially inhibited by blocking antibodies against the alpha 5-, alpha 6-, and alpha 2-integrin subunits, respectively. A blocking monoclonal antibody against the beta 1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the alpha 5 beta 1-integrin and fibronectin mediate the formation of ovarian carcinoma spheroids and that their adhesion to extracellular matrix proteins at sites of secondary tumor growth may be mediated by a complex interaction between multiple integrins and their ligands.