Meeting report on recent advances on T-lymphocytes in normal and leukemia patients.

We report on the main topics discussed at the "Human T-Lymphocytes in Normal and Leukemia Patients" meeting, including the "Second International Workshop on T-Cell Colonies" (Reggio Calabria, September 16-19, 1984). Improved knowledge in the field of T-lymphocyte biology has contributed greatly to a better clinical definition of T-cell-derived lymphoproliferative disorders. The T-cell colony assay seems to represent a useful tool for studying the early steps of T-lymphocyte maturation and for characterizing accessory cells and soluble mediators that regulate T-cell proliferation.

Increase of hybridoma formation by human lymphocytes after stimulation in vitro; effect of antigen, endothelial cells, and PWM.

Peripheral blood lymphocytes from antibody-producing Rh- donors were fused with mouse myeloma cells, and the hybrids were screened for anti-Rh antibody production. Although the resulting hybrids were not stable in long-term culture, they provided a useful test system for an investigation of ways to stimulate human lymphocytes in vitro before fusion in order to maximize the recovery of hybrids producing antibodies with a desired specificity. Although 6-day culture with PWM or with antigen (Rh+ erythrocytes) increased slightly the yield of desired hybrids, exposure of the PBL to antigen in the presence of human endothelial cells was dramatically more effective. This effect was not produced with endothelial cell culture supernatants (HECS) that had been shown previously to enhance hybridoma growth. Culture of PBL before fusion with both PWM and antigen also improved the proportion of active hybridomas, and incubation with antigen plus endothelial cells plus PWM resulted in a very high number of hybridomas, with a very high proportion producing antibody to the immunizing antigen. There are several alternatives to explain the mechanisms involved, but in any case, the technique should be particularly useful when the possibility of fusion of human PBL with a human myeloma line becomes more widely available.

"In vitro" colony growth of blood T-lymphocytes from B-cell chronic lymphocytic leukemia.

T-lymphocyte colony formation in agar culture was investigated in 20 untreated B-cell Chronic Lymphocytic Leukemia (CLL) patients in stage O. As compared to the controls, colony growth was very poor when unseparated peripheral-blood lymphocytes from CLL-patients were seeded. The number of colonies was greatly higher when T-cell enriched fractions from CLL were plated; however, they failed to reach the normal range even in this experimental condition. The role of adherent cells in the colony growth was also investigated. Depletion of these cells resulted in impaired colony generation either in normals or in CLL-patients, which was subsequently restored by the addition of the same adherent cells. In the patients investigated in this study, an imbalance of T-subsets was found with increase of OKT8-positive cells (T-suppressors). When the number of colonies and the percentage of OKT8-positive cells were plotted, an inverse correlation was found. Conversely, a linear relationship was detected between the percentage of OKT4-positive cells (T-helpers) and the values of colonies. On the basis of the present experiments, it is suggested that the defective colony growth of T-cell fractions in B-cell CLL may be related to the low number of OKT4-positive cells plated, which are known to be mainly responsible for the colony generation in agar culture.

Detection of HLA antigens on blood platelets and lymphocytes by means of monoclonal antibodies in an ELISA technique.

The expression of HLA-A and -B antigens on peripheral blood lymphocytes and blood platelets was measured using monoclonal antibodies in a semi-quantitative ELISA technique. Reactively of monoclonal anti-HLA-A2 and anti-HLA-B7, with lymphocytes as well as platelets, was in agreement with the presence of these antigens as detected by conventional HLA typing of lymphocytes. When the actual amount of HLA antigens was measured, a gene-dose effect was seen: cells from HLA-B7-homozygous individuals bound more monoclonal anti-HLA-B7 antibodies compared to their HLA-B7-heterozygous siblings. At the same time, cells of different donors showed only very small differences in binding of monoclonal antibody against an HLA-"backbone" determinant. Relative to total HLA-A, -B and -C expression, the amounts of HLA-A2 on lymphocytes and platelets were similar. On the other hand, the expression of HLA-B7 on platelets was diminished compared to that on peripheral blood lymphocytes.

Reduced natural killer cell activity in non-hodgkin's lymphoma.

Natural Killer (NK) activity is investigated in 20 patients with non-Hodgkin's Lymphoma (NHL) and in 20 healthy subjects. Peripheral blood lymphocytes are used as effector cells, and 51Cr labelled K562 myeloid cells as targets. Experiments are carried out either at effector/target ratio of 20:1 or 40:1, in 4 hrs Cr released assay. As compared to controls, a reduced NK activity is found in NHL patients, but that difference is statistically significant only for the patients in active disease. Thus, patients in clinical remission display a better preservation of NK function. It is suggested that the study of NK function might offer a helpful tool in following the clinical course and the efficacy of therapy in NHL.

Human endothelial culture supernatant (HECS): evidence for a growth-promoting factor binding to hybridoma and myeloma cells.

Human endothelial culture supernatant (HECS) contains a factor(s) that enhances the proliferation of hybridoma cells as we reported previously. In the present paper, we found that, in comparison with endothelial cells, their supernatant exerts a much stronger growth-promoting effect on hybridoma cells, which can also be detected earlier. In a confluent culture of endothelial cells, detectable amounts of this factor(s) are produced after 2 hr of incubation, and the production is linear in time for 3 days. The enhancing effect of HECS on the proliferation of hybridoma cells appears to be independent of the concentration of cells in the culture. The activity of HECS is adsorbed to hybridoma or myeloma cells, but not to thymocytes. HECS acts on hybridoma cells by delivering a growth-promoting signal that induces the hybridoma cells to proliferate.

In vitro culture and ultrastructure analysis of peripheral blood cells separated by discontinuous albumin density gradient during blast crisis of chronic granulocyte leukemia.

Peripheral blood from 4 patients with chronic granulocytic leukemia during blast crisis has been investigated. One case has been studied before and after treatment with Arabinosyl-Cytosine and 6-Thioguanine. The nucleated blood cells have been separated by a discontinuous density gradient. Cells were obtained from six different gradient fractions (F1-F6: density ranging from 1.052 to 1.078). 0.5 X 10(5) cells from each density fraction have been cultured in agar culture system to evaluate the granulocyte-monocyte committed stem cells (Colony Forming Units-granulocyte monocyte: CFU-GM). Cells recovered from the same density fractions have been studied by electron microscopy to evaluate the number of less differentiated cells. A quantitative correlation between plating efficiency and blast cells number was carried out. The results indicate that the highest recovery of both CFU-GM and blast cells is present in light density fractions (specific density below 1.063). However a discrepancy between blast cell frequency and granulocyte-monocyte colony formation in the same density fractions appears to be evident. In the patient studied before and after treatment it appears that only one out of two light density fractions (F1) responds to the antiblastic treatment.