George M. Gagne

Technique of leukocyte harvesting and labeling: Problems and perspectives

Mixed leukocyte suspensions obtained after gravity sedimentation of red cells and labeled with 111In lipophilic chelates are now widely used clinically for abscess localization at many medical centers. So far, labeling with 111In-oxine or tropolone has been more successful than any 99mTc method. More sophisticated approaches are available for isolation and labeling of specific leukocyte cell types, to study their migration in vivo. The most significant advances in cell harvesting include newer density gradients for isopyknic centrifugation (nonionic contrast media such as Nycodenz and Percoll, PVP-coated colloidal silica), centrifugal elutriation, and flow cytometry. Unlike current radioactive agents which label many cell types indiscriminately, more selective ligands are being developed which bind to specific cell surface receptors. These will label certain leukocyte populations or subtypes while not reacting with others, thereby avoiding laborious separation techniques. Monoclonal antibodies against leukocyte cell-surface antigens appear particularly promising as agents for selective cell labeling.

Preferential homing of tumor-infiltrating lymphocytes in tumor-bearing mice

SummaryIn view of the current interest in the use of lymphoid cells in adoptive immunotherapy of patients with advanced cancer, we have studied the homing patterns of various lymphoid effector cells in mammary-tumor-bearing mice. Single-cell suspensions of total splenocytes, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells were prepared from the spleens of C3H/OuJ mice. Tumor-infiltrating lymphocytes (TIL) were isolated from mammary adenocarcinomas excised from retired breeder females of the same substrain. Effector cells were labeled with indium-111 and injected via a tail vein into female C3H/OuJ mice bearing one or more mammary tumors. Twenty-four hours after administration, total splenocytes, NK cells, and LAK cells distributed themselves evenly between normal mammary tissue and mammary adenocarcinomas. Only TIL had a higher concentration in tumors than in corresponding normal mammary tissue. The ability of the different lymphocyte preparations to lyse YAC-1 cells was determined by means of a 4-h 51Cr-release cytotoxicity assay. Cells harvested from LAK cell cultures and further enriched by centrifugation through a discontinuous Percoll gradient and interleukin-2 (IL-2)-stimulated TIL demonstrated the highest levels of cytotoxicity, while total splenocytes and fresh TIL were characterized by the lowest levels. Since IL-2-stimulated TIL were highly cytotoxic and exhibited better tumor localization than both NK cells and LAK cells in this system, they may be the lymphoid effectors of choice for adoptive immunotherapy of advanced cancer.

Experimental and clinical trials of new 99mTc-labeled hepatobiliary agents.

Several new derivatives of lidocaine were synthesized and used to examine the intra- and extrahepatic bile ducts and gallbladder. Diisopropyl-IDA exhibited approximately twice the bile concentration of 131I-rose bengal during the first hour after injection. P-butyl-IDA also concentrates moderately well in the bile and has the added advantage of very low excretion into the urine (2%). It seems more effective than the other IDA derivatives at high bilirubin levels.

Transmission Computed Tomographic Diagnosis of Experimentally Produced Acute Pulmonary Vascular Occlusion in the Dog

Acute experimental pulmonary arterial occlusions were produced in 5 dogs. The chest was subsequently imaged with computed tomography (CT), 99mTc-MAA scintigraphy, and plain radiography. Gamma images revealed all 6 lesions, and plain radiographs were uniformly negative. Enhanced CT scans demonstrated 3 of 5 lesions, and unenhanced scans revealed 1 of 6. CT findings were variable.

Present Trends and Future Directions in “Leukocyte Labeling”

Imaging with In-111-labeled leukocytes has become an established clinical method for the detection of focal inflammatory lesions in many medical centers but has been rejected by others because of the technical complexities of the labeling procedure. Gaining knowledge of the in vivo migratory pattern of the different leukocyte populations and subtypes in health and disease remains an important goal(1), particularly in the field of immunology. Techniques for examining the in vivo distribution of lymphocytes, monocytes and the eosinophils are still under development, and differences in migratory patterns of mononuclear subtypes remain to be explored. This paper attempts to summarize recent progress in the techniques of harvesting and labeling leukocytes and suggests possible directions for future research.

Radioactive Monoclonal Antibodies Against Cell Surface Antigens for Labeling Leukocyte Subpopulations

The ability to follow the kinetics and migration of various leukocyte populations, particularly with the oxine and tropolone lipophilic chelates of In-111, has contributed greatly to our expanding knowledge of cellular immunology in recent years (1, 2, 3, 4). In the cellular immune system, lymph nodes collect and process antigen from extracellular fluid — the peripheral nodes for superficial tissues and the spleen for blood-borne antigens. The gastrointestinal tract has its own lymphoid organs for processing ingested antigens — Peyer’s patches, appendix, tonsils and adenoids (5). Labeled T cells migrate preferentially to peripheral lymph nodes and B cells to the spleen and Peyer’s patches (6). This organ-specific homing is controlled by interaction between recirculating lymphocytes and endothelial cells of post-capillary high endothelial venules (HEV) through specific surface receptors, thereby directing the cells into the lymphoid organs. A greater localization of cytotoxic T cells than helper T cells has been observed in Peyer’s patches, but their localization is equal in peripheral nodes (7).