Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations

We report a new technology for in vivo tracking of hematopoietic cells, using fluorescent lipophilic probes. Because the probe is irreversibly bound in the lipids of the cell membrane; substantial numbers of dye molecules can be incorporated per cell and thus substantial signal to noise can be achieved. Although this technology can be used for all hematopoietic cells, these first findings are reported on red blood cells (RBCs) owing to the importance of the membrane to RBC function and integrity. We demonstrated that labeling 10% of the RBCs of a rabbit and reinjecting them into the animal makes possible the tracking of these cells at various times after injection. Furthermore, the labeling appears not to affect in vivo cell lifetime or cellular volume changes in response to hypotonic shock. The single cell fluorescence intensity of the labeled RBCs remains relatively constant for 60 days, and an immune response appears not to be generated against labeled cells. That labeled RBCs have lifetime kinetics in vivo, as shown in other studies, indicates that the membranes are functioning normally and are unaltered by the labeling technology. The technology we present is also applicable to white blood cells, bone marrow, and platelets.     

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Transforming growth factor-beta trans-modulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines

Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor-dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF- beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM- CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF-beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF- beta involves reducing the cell surface expression on growth factor receptors.     

Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen

Even if neoplastic cells express tumor associated antigens they still may fail to function as antigen presenting cells (APC) if they lack expression of one or more molecules critical for the induction of productive immunity. These cellular defects can be repaired by physiologic activation, transfection, or fusion of tumor cells with professional APC. Although such defects can be repaired, antitumor specific T cells may still fail to respond in vivo if they may have been tolerized. Here, human pre-B cell acute lymphoblastic leukemia (pre-B ALL) was used as a model to determine if primary human tumor cells can function as alloantigen presenting cells (alloAPC) or alternatively whether they induce anergy. In the present report, we show that pre-B cell ALL express alloantigen and adhesion molecules but uniformly lack B7-1 (CD80) and only a subset express B7-2 (CD86). Pre-B ALL cells are inefficient or ineffective alloAPC and those cases that lack expression of B7-1 and B7-2 also induce alloantigen specific T- cell unresponsiveness. Under these circumstances, T-cell unresponsiveness could be prevented by physiologic activation of tumor cells via CD40, cross-linking CD28, or signaling through the common gamma chain of the interleukin-2 receptor on T cells. Taken together, these results suggest that pre-B ALL may be incapable of inducing clinically significant T-cell-mediated antileukemia responses. This defect may be not only due to their inability to function as APC, but also due to their potential to induce tolerance. Attempts to induce clinically significant antitumor immune responses may then require not only mechanisms to repair the antigen presenting capacity of the tumor cells, but also reversal of tolerance.