Enumeration of human lymphocyte subsets by monoclonal antibodies and flow cytometry: a comparative study using whole blood or mononuclear cells separated by density gradient centrifugation.

Enumeration of lymphocyte subpopulations by combined use of flow cytometry and monoclonal antibodies is influenced by the separation method used. T or B lymphocyte antigen frequencies do not differ in samples of whole blood or after separation on Ficoll-Paque or Percoll, but there is a significant increase of Leu+ Leu11+ lymphocytes showing strong natural killer activity at the sample-separation medium interface. At the bottom of the tubes a selective loss of OKT+, Leu7+, Leu11- cells is found; at this level cytotoxicity is very low. Our data suggest that Leu7+ cells could be subdivided into 2 subpopulations differing in reactivity with the monoclonal antibody to Leu11, natural killer activity and density. Differences observed in the percentages of Leu7+, Leu11+ lymphocytes between whole blood and separated cells could be due to an enrichment produced by centrifugation or to the presence in whole blood of a factor interfering with antibody binding.

A method for the determination of the adherence of granulocytes to microtitre plates.

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system. Using three different cellular concentrations (1 X 10(6), 5 X 10(5), 2.5 X 10(5) PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation less than 8%; inter-assay coefficient of variation less than 11%).

Expression and modulation of common acute lymphoblastic leukaemia antigen by B prolymphocytic leukaemia cells.

Leukaemic blood lymphocytes from patients with B prolymphocytic leukaemia express the common acute lymphoblastic leukaemia antigen (CALLA). The in vitro incubation of prolymphocytes with leucocyte conditioned medium produced the acquisition of plasma cell morphology, of intracytoplasmic immunoglobulins and the simultaneous reduction of CALLA expression. The presence of CALLA appears to be unrelated to different clinical courses. Our report demonstrates that the antigen is present also in relatively mature stages of B lymphocyte differentiation. The blastic appearance of prolymphocytes could suggest that CALLA may be related to active metabolism or function of these cells.

Selective depletion of the OKT 4+ 4B4+ subset in lymph nodes from HIV+ patients.

We have used 4B4 and 2H4 monoclonal antibodies in conjunction with OKT 4 to quantify T cell subsets in lymph node suspensions from human immunodeficiency virus (HIV) positive subjects with lymphadenopathy syndrome. The data indicate that the reduced OKT 4:OKT 8 ratio was due to a depletion of the OKT 4+ 4B4+ subset. In contrast, there were no differences compared to reactive controls, considering the OKT8+ subpopulation. These alterations may be related to the immunological deficiency associated with HIV infection.

Age-related changes in human lymphocyte subsets: progressive reduction of the CD4 CD45R (suppressor inducer) population.

The purpose of this study was to investigate, by double-labeling immunofluorescence, lymphocyte subsets in the peripheral blood of healthy children, adults, and aged individuals. The absolute number of T and B lymphocytes decreases with age. The decline in T cells was attributed to a decrease in CD4 lymphocytes. We also found that the composition of the CD4 subset changes with age: in children the CD45R molecule is expressed on the majority of CD4 cells, whereas in aged subjects the absolute number of these lymphocytes is greatly reduced. The reciprocal CD4 CD29 population is not modified during the life span. Aging is also associated with the appearance of CD8 Leu 7 lymphocytes. Putative contrasuppressor cells, identified by Vicia villosa binding, represent a very small population in peripheral blood and are not subject to age-dependent variations.

OKT4+ chronic lymphocytic leukaemia cells display different reactivity with selected monoclonal antibody combinations.

Four cases of chronic T lymphocytic leukaemia are characterized by selected monoclonal antibody combinations and double-fluorescence studies. We found 3 possible combinations: OKT4++ Leu8++ Leu9+, OKT4++ Leu8+ Leu9- and the OKT4++ Leu9++ Leu8- phenotypes are correlated with different biological aspects of the disease. No markers of T cell activation were found, and serological examinations did not reveal anti-HTLV-I antibodies. In considering a larger number of patients, multiparametric analysis could be useful to better classify this heterogeneous type of leukaemia.

Alteration of T cell subsets during tetanus infection treated with high doses of specific immunoglobulins.

Peripheral blood lymphocytes of 8 tetanus patients have been studied with monoclonal antibodies and flow cytometry. During the progression of the disease OKT3+ and Leu7+ cells undergo significative daily variations and OKT4+ cells become the majority of peripheral blood T cells. In two patients is present a severe T lymphopenia and one of these subjects presented a more prolonged disease compared to the other ones. B cells are greatly increased after 10-12 days. Two possibilities could be considered to explain our data: firstly a direct effect of the tetanic infection on the recirculation of lymphocytes producing also an expansion of OKT4+ cells containing a helper population; secondly the observed alterations of lymphocytes subsets are not due to the infection itself but rather to the massive administration of specific immunoglobulins to these patients.

A subset of gamma delta lymphocytes is increased during HIV-1 infection.

The gamma delta T cell receptor (TcR) lymphocytes constitute 3-10% of human peripheral blood lymphocytes. Only a very small fraction of these cells is recognized by the delta TCS1 monoclonal antibody, directed against the V delta 1 chain of the receptor. We describe the immunological, virological and clinical data of a small group of seropositive subjects having high levels of gamma delta TcR T cells in the peripheral blood. Our flow cytometric studies show that most of these cells belong to the delta TCS1+ (V delta 1+), CD8 +/- (dim staining) subset. Patients with high gamma delta TcR T cell numbers were not characterized by the presence of an acute (IgM positive) or reactivated (as defined by high IgG titres against early antigen or IgA titres against viral capsidic antigen) Epstein-Barr virus infection. Cytomegalovirus infection was excluded by serological assays, and other herpes viral infections were not found after clinical examination. HIV p24 antigenaemia was present in two out of 11 subjects. AIDS patients had very high percentages of gamma delta TcR T cells. Altogether these data show that the selective expansion of delta TCS1+ cells in HIV1 seropositive subjects is not related to some exogenous antigen stimulation, but may be related to peculiar pathologic processes involving the immune system.

Persistent generalized lymphadenopathy in Northeastern Italy: increased frequency of HLA-DR 5.

We undertake a study of Italian intravenous drug abusers and male homosexuals with the prolonged generalized lymphadenopathy syndrome (PGL). 75% of the subjects had the HLA-DR 5 antigen (relative risk 3.25). Only one patient was DR 2 positive. 14/20 subjects had anti-HTLV III antibodies and immunological malities were a rather constant finding.

Selective reduction of the OKT 4 positive 4B4 positive subset during HTLV III infection in humans.

Combinations of monoclonal antibodies defining the OKT 4 positive lymphocyte subset in the peripheral blood of intravenous drug abusers suggest that the 4B4 positive helper inducer population is greatly reduced in subjects infected with HTLV III. No differences were seen instead using different antibodies directed against various epitopes of the T4 molecule. These results suggest that the multiple marker assay could be very useful to define immunological status during HTLV III infection in humans.

Immunologic abnormalities related to antigenaemia during HIV-1 infection.

The expression of phenotypic markers on T and B lymphocytes in long-term human immunodeficiency virus type 1 (HIV-1) seropositive, antigen negative patients, in seropositive, antigen positive individuals without AIDS and in seronegative intravenous drug abusers was examined by two colour flow cytometry. Seropositive, antigen positive patients showed decreased CD4+ lymphocyte numbers, causing lower CD4/CD8 ratios when compared to seropositive, antigen negative subjects. While CD4 CDw29+ (4B4) lymphocytes are selectively reduced in seropositive, antigen negative individuals, both CD4 CDw29+ and CD4 CD45R+ (2H4) lymphocytes are decreased when antigenaemia is present. An increased percentage of CD3 HLA DR+ activated T lymphocytes and of CD20+ (B1) Leu 8 negative activated B cells was seen in HIV-1 seropositive antigen positive patients. These results demonstrate that, in long-term seropositive individuals, antigenaemia is associated with peculiar phenotypic changes of lymphocyte subsets.

Peripheral blood and bone marrow lymphocytes of B chronic lymphocytic leukaemia patients.

The percentages of lymphocytes subpopulations were assessed in the peripheral blood and bone marrow of B chronic lymphocytic leukaemia patients with monoclonal antibodies and flow cytometry. The absolute number of peripheral T cells is higher compared to healthy subjects; the imbalance of OKT 4 and OKT 8 positive subsets in peripheral blood is characterized by an inversion of the T 4/T 8 ratio. T 4 positive cells are predominant in the bone marrow; the Leu 7 positive population is increased in absolute numbers. These precocious phenomena could be important in the immunodeficiency associated with B CLL and/or in the progression of the disease.

Immunological abnormalities in intravenous drug abusers and relationship to the prolonged generalized lymphadenopathy syndrome in Italy.

The prolonged generalized lymphadenopathy syndrome (PGL) has been considered a prodromal condition to the Acquired immunodeficiency syndrome (AIDS), but the clinical, virological and immunological characteristics of patients who will develop AIDS are not known. We report on the immunological profile of intravenous drug abusers with or without PGL in Northeastern Italy. We found a reduction of lymphocyte-absolute numbers with reversal of the T4/T8 ratio and decreased Leu-11b+ cells. The response to mitogens and natural killer activity are compromised in PGL patients. Neutrophil function is reduced both in drug abusers with or without lymphadenopathy. The serological investigations revealed a high prevalence of antibodies against HTLV III and the Epstein-Barr viruses. The recognition of immune dysfunction in the intravenous drug abusers appears to be important since these patients develop AIDS and these abnormalities may precede AIDS.

Immunological characterization of renal glycosuria patients.

We have evaluated the autoantibody pattern, the production of specific immunoglobulins against bacteria in the urinary tract and lymphocytes populations in peripheral blood of primary renal glycosuria patients. All the affected members present autoantibodies against various antigens. In nine patients immunofluorescence revealed antibody coated bacteria in urine specimens. Imbalances in lymphocytes subpopulations are also present, with a reduction of OKT8+ cells and an increase of B and natural killer cells. These results support previous observations about immunological abnormalities in these patients.

Chronic T lymphocytic leukaemia: antigenic, morphological and functional properties of the neoplastic lymphocytes.

We describe 2 cases of T cell leukaemia. Both patients react with OKT 4 monoclonal antibody, while their clinical course, surface markers and in vitro immunological reactivity present opposite behaviour. The cells of 1 patient react with all pan T cell markers (OKT 3, OKT 11, Leu 1), from E and Ea rosettes and respond well to mitogens. This patient does not present liver, spleen or lymph node enlargement; white cell count is stable and no other haematological alterations are present. The cells from the 2 patient are unreactive with OKT 3 and are unable to form Ea rosettes. The response to mitogens is almost abolished. The disease has an aggressive clinical course with progressively increasing blood leucocyte count and infiltration of liver and spleen. These data suggest that the study of antigenic and functional properties of T CLL may be useful to better define the biologic characteristics and the prognostic criteria of the disease.