Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells

In mice, donor leukocyte infusion (DLI) given to established mixed allogeneic chimeras can mediate powerful graft-versus-host (GVH) reactions confined to the lymphohematopoietic system without inducing graft-versus-host disease (GVHD). In a clinical trial attempting to capture this approach to achieve graft-versus-leukemia/lymphoma (GVL) effects without GVHD, we have observed surprisingly powerful antitumor effects of DLI in patients achieving mixed chimerism after nonmyeloablative bone marrow transplantation. This observation led us to hypothesize that host antigen-presenting cells in mixed chimeras might be required to optimally present recipient antigens to the donor lymphocytes, leading to maximal graft-versus-tumor effects. To test this hypothesis, we established mixed and fully allogeneic hematopoietic chimeras in B6 mice and evaluated the effect of DLI on EL4 T-cell lymphoma. DLI administration to mixed chimeras produced dramatically improved leukemia-free survival compared to administration of DLI to full donor chimeras. DLI also converted mixed chimeras to full chimeras without causing GVHD. The magnitude of the GVL effect was dependent on the level of major histocompatibility complex class I expression on recipient hematopoietic cells in mixed chimeras. Thus, the induction of mixed chimerism followed by delayed DLI provides an approach to inhibiting GVHD that optimizes GVL effects.     

Primary structural studies of an H-2L molecule confirm that it is a unique gene product with homology to H-2K and H-2D antigens.

Radiochemical methodology has been used in the isolation and preliminary biochemical characterization of the murine H-2Ld major histocompatibility complex gene product. The radiolabeled molecule was isolated by immunoprecipitation from the glycoprotein fraction of detergent-solubilized H-2d tumor cells. Six major CNBr fragments were isolated from a papain fragment of this molecule; three of the fragments are connected by disulfide bonds. Due to the high degree of homology between major transplantation antigens, it was possible to align the fragments by comparison of their amino acid sequences to that of the H-2Kb gene product. Of the positions available for comparison between H-2Ld and H-2Kb, H-2Dd, and H-2Kd gene products, 61 out of 80 (78%), 45 out of 55 (82%), and 12 out of 15 (80%), respectively, are identical. Differences between the Ld and Kb and Dd molecules are distributed throughout the amino acid sequence. These data indicate that the H-2Ld gene product is a molecular species distinct from, but homologous to, the H-2K and H-2D gene products.     

Receptor Mobility and the Mechanism of Cell-Cell Binding Induced by Concanavalin A

The cell-cell binding induced by concanavalin A between single cells has been analyzed by use of cells attached to nylon fibers. Binding of a concanavalin A-coated cell to an untreated cell was found to a high degree between two lymphoma tumor cells, less frequently between a lymphoma cell and a normal lymphocyte, and only rarely between two normal lymphocytes. The binding was inhibited by the presence of a saccharide inhibitor of concanavalin A, but could not be reversed by addition of the inhibitor after the cells had bound to each other. Although no binding was obtained when both cells were coated with lectin or fixed with glutaraldehyde, fixation of a cell before coating with concanavalin A enhanced its ability to bind an untreated cell. The results indicate that cell-cell binding induced by concanavalin A requires short-range lateral movement of cell receptors for the lectin, that only one cell has to have mobile receptors, and that some receptors must be unoccupied by lectin molecules before cell-cell contact. Clustering of the receptors is not necessary and seems to hinder cell-cell binding. It is suggested that the short-range movement is required for alignment of individual receptors so as to form multi-point bridges between two cells by lectin molecules. The bridging is then followed by the formation of irreversible bonds between the cells. The receptors on tumor cells appear to have a greater ability than receptors on normal cells to align themselves for cell-cell binding.     

Polymer models for interphase chromosomes.

The overall geometry of chromosomes in mammalian cells during interphase is analyzed. On scales larger than approximately 10(5) bp, a chromosome is modeled as a Gaussian polymer subjected to additional forces that confine it to a subvolume of the cell nucleus. An appropriate partial differential equation for the polymer Green's function leads to predictions for the average geometric length between two points on the chromosome. The model reproduces several of the experimental observations: (i) a square root dependence of average geometric distance between two marked chromosome locations on their genomic separation over genomic length scales from approximately 10(5) to approximately 10(6) bp; (ii) an approach of the geometric distance to a maximum value for still larger genomic separations of the two points; (iii) overall chromosome localization in subdomains of the cell nucleus, as suggested by fluorescent labeling of whole chromosomes and by radiobiological evidence. The model is also consistent with known properties of the 30-nm chromatin fiber. It makes a testable prediction: that for two markers a given number of base pairs apart on a given chromosome, the average geometric separation is larger if the configuration is near one end of the chromosome than if it is near the center of the chromosome.     

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.     

Characterization of the Inducer Required for the Development of Macrophage and Granulocyte Colonies

A substance produced by various types of cells can induce from single hematopoietic cells the formation of colonies of normal macrophages and granulocytes. This inducer has been purified 600-fold from serum-free conditioned medium from a tissue culture line of mouse cells. It is shown that the inducer is a protein, with a molecular weight of 65-70,000, whose inducing activity was about 1 ng per colony. Acrylamide gel electrophoresis of this material gave four bands, only one of which contained inducing activity. The purified protein was inactive. Activity was regained by the addition of a low molecular weight cofactor that is present in conditioned medium. Under certain conditions, activity was also regained by the addition of adenosine 3':5'-cyclic monophosphate (cyclic AMP).     

Pharmacokinetic and pharmacodynamic study of a clinically effective anti-CD2 monoclonal antibody

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2⁺ cells in circulation and tissue. Siplizumab had a longer t_1/2 and fewer AEs compared to BTI-322.