Joseph Wybran

Thymus-Derived Rosette-Forming Cells in Various Human Disease States: Cancer, Lymphoma, Bacterial and Viral Infections, and Other Diseases

Lymphocytes that bind in vitro to sheep erythrocytes in a rosette formation are thymus-derived. A modified technique that does not detect the total number of rosette-forming cells (RFC) was used to study normal subjects and various disease states. Of 100 healthy subjects, 95 had more than 15% RFC (mean 28.4+/-6.5%). We studied 104 patients with solid tumors, who were classified according to clinical status and stage of therapy. Of 19 newly diagnosed patients, 13 had less than 15% RFC. Of 44 untreated patients undergoing relapse, 32 had less than 15% RFC. In both categories, patients with metastases had fewer RFC than patients with localized disease. 11 patients were studied 2 wk after cessation of therapy; four of them showed less than 15% RFC. Only one of 30 patients in remission had less than 15% RFC. In seven patients followed for various periods of time, the numbers of RFC correlated generally with clinical status. 11 patients with chronic lymphatic leukemia had very low percentages of RFC. 21 of 21 patients with symptoms of viral upper respiratory diseases had less than 15% RFC. RFC returned to normal values between 5 days and 7 wk after disappearance of clinical symptoms. 20 patients with bacterial infections had normal numbers of RFC. Of 25 patients with miscellaneous nonimmunologically related diseases, two had low numbers of RFC. It appears that the percentage of RFC may be valuable in evaluating not only immunological defenses but also the status of patients with solid tumors, lymphomas, viral diseases and, perhaps, bacterial infections.

The Human Rosette-Forming Cell as a Marker of a Population of Thymus-Derived Cells

Sheep red blood cells can surround, in vitro, some human peripheral blood lymphocytes in a formation called a rosette. The number of rosetteforming cells (RFC) in 50 normal persons had a wide range (4-40%). The organs of 13 human fetuses (11-19 wk conceptional age) were examined for the presence of RFC. The thymus possessed the highest percentage of RFC, the maximum being 65% of total thymocytes in two 15-16 wk fetal specimens. Blood RFC were always present and their number slightly increased in the oldest fetuses. The bone-marrow showed 0-8% in the six fetuses studied. RFC were found in the spleen around the 13th wk and in the liver around the 17th wk of gestation. These observations lead to the hypothesis that human blood RFC may be chiefly thymic derived. Studies of patients with immunological disorders support this hypothesis: one patient with Nezelof syndrome had no blood RFC and four patients with Wiskott-Aldrich syndrome had a low number of blood RFC (1 and 1.5%). Patients with acquired hypogammaglobulinemia showed a normal percentage of RFC. With the fetal thymocytes, the percentage of inhibition with anti-mu serum increased with the fetal age to become complete in the oldest fetuses studied. Incubation of the oldest fetal thymocytes or the blood lymphocytes with anti-gamma serum of anti-mu serum completely inhibited the rosette formation. These results suggest that mu-chain determinants are present on human fetal thymocytes and blood RFC. The significance of the presence of gamma-chain determinants on these cells is unclear.

ISOLATION OF NORMAL T CELLS IN CHRONIC LYMPHATIC LEUKÆMIA

Abstract Lymphocytes from patients with chronic lymphatic leukaemia (C.L.L.) show a poor response to mitogens. Lymphocytes from these patients and from healthy patients were separated into two populations. One population, very rich in T cells, showed the same type of response for both the controls and patients. In contrast, the population poor in T cells derived from C.L.L. lymphocytes did not respond at all to mitogens. Recombination experiments indicate that the response can be restored by the addition of T cells. These findings demonstrate the presence of a normally reactive T-cell population in patients with C.L.L. It seems that the lack of responsiveness in C.L.L. is due to the dilution of normally responding cells.

Osteogenic sarcoma. Immunologic parameters before and during immunotherapy with tumor-specific transfer factor.

18 patients with osteogenic sarcoma were followed by serial measurements in vitro of tumor-specific cell-mediated cytotoxicity and of active and total rosette-forming T-cells. 13 of these patients have had or are currently receiving injections of osteogenic sarcoma-specific dialyzable transfer factor derived from healthy donors. In three patients with very small lesions, cytotoxicity was high before amputation and decreased within 2 mo after removal of tumor. Cytotoxicity was low at time of diagnosis in all patients with large tumor masses. The cytotoxicity of the patients lymphocytes increased after administration of tumor-specific transfer factor in all patients so treated. Patients receiving nonspecific transfer factor showed evidence of declining cell-mediated cytotoxicity. Tumor-specific transfer factor may produce an increase in cell-mediated cytotoxicity to the tumor in patients with osteogenic sarcoma. This possibility is suggested by the pain and edema that occurred in the area of the tumor in patients who had metastatic disease when therapy was started and by lymphocytic infiltrates in the tumor, as well as by the increase in cell-mediated cytotoxicity and the increase in percentage of active rosette-forming cells from subnormal to normal. Serial measurements of cell-mediated cytotoxicity are helpful in monitoring the efficacy of transfer factor and other modes of therapy in these patients, and these measurements are the best available criteria for selection of donors of tumor-specific transfer factor.

THYMOSIN: EFFECTS ON NORMAL HUMAN BLOOD T-CELLS*

It has recently been shown that thymosin, an extract of calf thymus, can rapidly induce the appearance of specific T-cell markers on lymphoid cells of It has been postulated that thymosin produces this effect by acting on a stem T-cell to confer upon it T-cell characteristics. Thus, thymosin may be the factor responsible for the differentiation of T-cells within the thymus. In both animals and man it is assumed that the T-cells present in the peripheral blood are fully differentiated. Stem cells are probably present in the bone marrow but rarely, if at all, in normal peripheral blood. Thymosin is not species specific; indeed, most of the extracts are prepared from calf thymus and have been assayed in mice.’-:: Therefore, calf thymosin presumably could similarly affect human cells. The present data confirm this hypothesis. In view of the difficulties of obtaining normal human bone marrow, we decided to investigate the action of thymosin on human peripheral blood lymphocytes through their ability to bind to normal sheep red blood cells (SRBC). Such binding produces a morphologic configuration termed a “rosette.” The rosette-forming cells (RFC) so detected are T-cells.d-7 In the present reports two techniques were used for rosette formation. ( 1 ) One, termed the “active” rosette test, requires only a short incubation time between SRBC and lymphocytes; 8-10 it detects some subpopulation( s) of T-cells. (2) The other rosette test requires a substantially longer incubation time between SRBC and lymphocytes; it probably detects all or almost all T-cells.”

Cellular interactions: Scanning electron microscopy of human thymus-derived rosette-forming lymphocytes

Abstract Scanning electron microscopy was used to study morphologic features of the surface of human T-lymphocytes which were incubated with sheep red blood cells without antibody or complement and then separated into rosette forming cells and nonrosette forming cells. Microvilli, 1800–2300 A in diameter and of variable length, were demonstrated on the surface of all lymphocytes. Microvilli formed the attachment with sheep red blood cells suggesting that the receptor sites for this interaction are located on the microvilli. Microvilli on some rosette forming lymphocytes were unequally distributed with the higher density being adjacent to the red blood cells. Rosette forming cells had far more microvilli than did nonrosette forming cells.

Effect of serum on human rosette-forming cells in fetuses and adult blood

Abstract The action of fetal calf serum and gamma globulin-free calf serum in the rosette formation between sheep red blood cells and mononuclear cells was studied in the lymphoid tissues of 12 human fetuses and the blood of 50 adults. It was found that serum increases the number of rosette-forming cells (RFC) in the fetal thymus, spleen, and blood, and in the adult blood. In contrast, rosette formation was decreased or abolished in fetal bone marrow. In one fetus studied, the bone marrow RFC showed phagocytic activity while in the other lymphoid tissues the RFC did not possess such activity. RFC from adult blood were not able to ingest carbon particles. Some normal subjects were followed during several months for their blood RFC performed in presence of serum; no important variation was noted. Therefore, the use of serum appears to be a valuable tool for studying thymus-derived cells.

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

Abstract The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.