OK T-positivity in stimulated T-lymphocytes

T-lymphocytes express different antigenic determinants which can be recognized using specific anti-T monoclonal antibodies. OK T3 (Ortho Diagnostics, Raritan, N.J.) detects 95% circulating T-lymphocytes, while OK T4 reacts with helper/inducer T-lymphocytes and OK T8 with suppressor/cytotoxic T-lymphocytes. In normal peripheral blood the proportion of mononuclear cells (after "Lymphoprep' separation) positive with the various anti-T monoclonal antibodies is, according to our standards, as follows: OK T3 77 +/- 9.4%, OK T4 51 +/- 7.8%, OK T8 27+/- 6.4%. In this study we have evaluated the positivity with OK T MoAb after activation and proliferation of the T-cell population in a double layer T-lymphocyte colony assay. After 4-5 days of incubation, the proportion of OK T3 + cells had increased to 94 +/- 4.6%, while that of OK T4+ and OK T8+ had raised to 62 +/- 14.1% and 65 +/- 7.1% respectively. These data suggest that T-colony formation gives rise to an increased expression of OK T8 positivity, possibly through a mechanism of T-cell activation (shown also by the 'Ia' positivity), and/or of proliferation of T-cells with a double antigenic phenotype.     

In vitro immune response of human peripheral lymphocytes. VI. Distribution and characterization of precursors for PHA- and protein A-induced colony-forming B cells

B cells from peripheral blood or cord blood formed colonies by stimulation with either PHA or protein A. On the other hand, tonsillar B cells did not form protein A-induced colonies, although PHA-induced colony formation was comparable to that observed in peripheral B cells. Lack of protein A-induced colony formation in tonsillar B cells was not due to the defect of helper T cells in preculture or to the presence of suppressor cells but was due to the absence of precursors for colony formation. The result showed that PHA- and protein A-induced colony-forming cells belonged to distinct subsets of B cells. Depletion of mu-bearing cells from peripheral B cells abrogated both PHA- and protein A-induced colony formation. Depletion of delta-bearing cells did not affect PHA- and protein A-induced colony formation and the population enriched with delta-bearing cells also showed colony formation. Depletion of complement receptor (CR)-positive cells removed precursors for both PHA- and protein A-induced colony formation. These results showed that precursor cells for PHA- and protein A-induced colony formation were IgM+, IgD+ and CR+ or IgM+, IgD- and CR+.     

T-lymphocyte colony formation by lymphocytes from patients with aplastic anaemia

T-lymphocyte colonies were cultured using lymphocytes from patients with aplastic anaemia and normal donors to assess their respective proliferative activities. Colony numbers from aplastic patient's cells were lower than from normal donors', though this was not significant. When lymphocytes from patients were co-cultured with normal lymphocytes, inhibition of T-colony formation was observed in 8 out of 12 experiments. As the degree of inhibition was greater than if patient cells grew no colonies, then, clearly, normal T-colony formation was inhibited. This ability of patients' lymphocytes to suppress lymphopoiesis might account for the low levels of patient T-colony formation, as well as low in vivo numbers of lymphocytes found in patients with aplastic anaemia. The role of patients' lymphocytes in causing marrow aplasia was investigated. Although the incorporation of patients' lymphocytes in normal granulocyte-macrophage (GM) colony-forming systems inhibited colony growth, in only 1 out of 8 patients was this inhibition significantly greater than that caused by the addition of normal lymphocytes to GM colony systems. Therefore, lymphocytes may not be the primary cause of aplastic anaemia, except for a few rare cases.     

Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

Approximately 80% of human peripheral blood T-lymphocytes could be cloned in the presence of crude interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80% of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20% had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets in relation to colony type.     

Reaginic antibody formation in the mouse. VII. Depression of the ongoing IgE antibody formation by suppressor T cells.

The ongoing IgE antibody formation against ovalbumin (OA) in high responder mice was depressed by i.v. injections of either native or urea-denatured ovalbumin (UD-OA). Adoptive transfer experiments to determine the helper function of spleen cells from the treated animals showed that helper function for both IgE and IgG antibody responses diminished after treatment. Evidence was obtained that treatment suppressed the expansion of IgE-G memory cells. When the same treatment with OA or UD-OA was given to OA-primed mice before the appearance of IgE antibody in their serum, OA-specific splenic suppressor T cells were demonstrable. Thus, the transfer of splenic T cells from treated mice into normal mice suppressed the primary IgE and IgG antibody responses of the recipeints to DNP-OA. It was also found that the transfer of the splenic T cells from UD-OA-treated mice into OA-primed mice depressed ongoing IgE antibody formation in the recipients. The results suggested strongly that the decrease of helper function and the depression of ongoing IgE antibody formation by repeated injections of UD-OA was caused by generation of antigen (OA)-specific suppressor T cells.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

The mechanisms of lymphoid regulation and the radiosensitivity of the hematopoietic system]

On the basis of the authors' own data and the literature it has been inferred that the key principles of the haemopoietic system regulation are similar to those of the immune system. The cells of a lymphoid origin are found, which implement helper and suppressor functions with respect to early haemopoiesis precursors; the influence of lymphokines on this compartment under the effect of radiation is described. Disturbances in the haemopoiesis system regulation, that result from various damaging effects, might be corrected by T-lymphocytes and lymphokines. The data obtained suggest that the formation of splenic colonies is the result of the interaction of some cell populations. That is why many radiobiological characteristics of CFUs may be attributed to partner cells (for instance, T-lymphocytes).     

Lymphocyte subset responses to repeated submaximal exercise in men

The effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1, 3, and 5 of a 5-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P less than 0.001), a decrease in the percentage of T-helper/inducer cells before vs. after exercise on day 3 (P less than 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

The role of regulatory components from resident T lymphocytes in polyclonal B cell activation

Resident T lymphocytes have been found to exert helper and suppressor regulatory influences with regard to polyclonal activation of murine splenic B lymphocytes elicited by lipopolysaccharide. In the normal adult spleen, only T cell helper influences are exercised over polyclonal B cell activation. This activity is a property of Lyt 1+2- T cells and does not appear to be subject to MHC restriction. Suppressive influence evidently is either latent or it exists at such a low level that its effects are difficult to detect. No regulatory activity can be recovered from the supernatants of T cells, cultured either with or without LPS. However, suppressor T cell function may be evoked by activating splenic T cells with Concanavalin A or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these unstimulated cells when intact. The soluble fraction of resident splenic T cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt l+2- T cells, whereas suppressor activity is generated from Lyt 1-2+ T cells. The suppressive activity of T cell sonicates is not restricted by the MHC gene complex. Helper and suppressor activities contained in splenic T cell sonicates were separated by gel chromatography; the suppressive activity was found to elute with a molecular weight between 68,000 and 84,000 daltons, and the helper activity eluted with a molecular weight between 15,000 and 23,000 daltons. The data indicate that helper and suppressor activities are distinct molecular entities derived from distinct splenic T lymphocyte subpopulations. The possibility that these molecules are precursors to or components of antigen-specific or nonspecific helper and suppressor factors described in the literature is discussed.     

Functional characterization of T lymphocytes grown in semisolid agar

T lymphocyte colony forming cells (TL-CFC) grown in agar in the presence of PHA were assayed for their capacity to induce or suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells. This was measured by identifying cells containing intracytoplasmatic immunoglobulins by direct immunofluorescence. To validate the helper and suppressor system used in this paper, the inductive capacity of unfractionated T lymphocytes and their subpopulations bearing Fc-receptors for IgM (TM) and for IgG (TG) was measured. The unfractionated T cells and the TM fraction showed helper activity, whereas the TG cells expressed suppressor activity. The TL-CFC grown in agar in the presence of PHA manifested helper activity at low cell concentration. However, increasing the TL-CFC concentration finally caused suppression of B cell differentiation. The suppressor effect could be abolished by prior irradiation of the TL-CFC before seeding them in agar. These results indicate that T cells grown in agar have the functional capacity of T helper and T suppressor cells to induce and suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells.     

The helper and suppressor functions of primate T cells elicited by a 185K streptococcal antigen, as compared with the helper function elicited by a 4K streptococcal antigen

Helper and suppressor functions of human T lymphocytes that act on antibody-forming B cells were elicited by a large 185K streptococcal cell wall antigen. However, a small 4K streptococcal peptide elicited helper but no suppressor function. These differences in the functional activities of the large and small m.w. streptococcal antigens (SA) were confirmed by direct immunisation of rhesus monkeys with the 185K-SA and 4K-SA. Sequential studies have shown that whereas the 185K-SA elicits dose-dependent helper and suppressor activities, the 4K-SA elicits only helper function. Cell-depletion studies with human cells suggest that removal of T8+ cells by killing with OK.T8 and complement leads to a loss of suppressor and a broadening in the concentration of 185K-SA, which elicits helper activity. Because the 4K-SA does not elicit suppression, removal of T8+ cells does not affect this function. However, similar depletion of T4+ cells results in loss of the helper activities, both with the 185K-SA and 4K-SA, and again a broadening in the concentration of the 185K-SA, which elicits suppression. Direct comparison by autoradiography between 125I-labeled 185K-SA and 4K-SA suggests that both antigens can bind directly to monocytes or T8+ VV+ cells. Furthermore, both antigens can induce helper function if T4+ cells are reconstituted with either monocytes or T8+ VV+ cells. Attempts will now be made to sequence the amino acid determinants of the 185K-SA, so as to define the epitopes responsible for the two major regulating functions elicited by this antigen.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

T-cell subsets in peripheral blood of patients with newly diagnosed and posttreatment Hodgkin's and non-Hodgkin's lymphomas. Analysis by monoclonal antibody staining and flow cytometry

T-cell subsets in the peripheral blood of patients with Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHLs) were determined using anti T-cell monoclonal antibodies and flow cytometry. Forty HD patients and 30 NHL patients were evaluated; 76 normal blood donors served as controls. Newly diagnosed (untreated) HD and NHL patients had relatively normal values for percentages of total T-cells, helper cells and suppressor cells; their helper/suppressor ratios were also normal. The total lymphocyte count was normal for pretreatment HD, but lower than normal for NHL. Following treatment, both HD and NHL patients showed significantly decreased helper/suppressor ratios, caused by a significant decrease in the percentage of helper cells in HD patients and a significant increase in the percentage of suppressor cells in the small number of NHL patients studied. A small number of NHL patients, followed without specific treatment (passive follow-up), had relatively normal values for percentages of helper and suppressor cells and total T-cells. For both groups of patients off treatment, it is concluded that the lower helper/suppressor ratios are due to the prolonged effects of treatment (predominantly irradiation).     

Interferon is the suppressor of hematopoiesis generated by stimulated lymphocytes in vitro

Lymphocytes that inhibit hematopoiesis may have a pathogenic role in some forms of bone marrow failure, and lymphocyte-mediated suppression may also be important in the normal regulation of bone marrow function. We have investigated the mechanism of in vitro suppression of hematopoiesis by T cells by using the methylcellulose colony culture system. Total peripheral blood T cells and separated subpopulations of helper (OKT4+) and suppressor (OKT8+) cells that have been stimulated by exposure to lectin suppress autologous colony formation by bone marrow myeloid (CFU-C) and erythroid (BFU-E) progenitor cells. Medium conditioned by these cells is also inhibitory, indicating that the suppressor activity is a soluble factor. A strong correlation existed for the concentration of interferon and the degree of hematopoietic suppressor activity in these supernatants; both activities peaked at days 3 to 5 of incubation and had sharply declined by day 7. Interferon production was enhanced by exposure of lymphocytes to sheep red blood cells during the rosetting procedure. Specific antiserum and a monoclonal antibody directed against gamma-(immune) interferon abrogated the inhibitory activity for hematopoiesis produced by lectin-stimulated T cells; an antiserum to alpha-interferon was generally much less effective in neutralizing activity. We infer from these results that gamma-interferon is the mediator of hematopoietic suppression generated by lectin-treated T-cells.     

Long-term expression of gene introduction into normal human T-lymphocytes by retroviral-mediated gene transfer

Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.     

In vitro immune response of human peripheral lymphocytes. II. Properties and functions of concanavalin A-induced suppressor T cells.

Con A-stimulation of human peripheral T lymphocytes induced both suppressor and helper T cells. ConA-generated suppressor T cells inhibited PWM-induced IgG and IgM production in PBL. Lower concentrations of Con A (0.5 micrograms/ml) or shorter incubation periods (6 to 24 hr) induced mainly helper T cells, while higher concentrations of Con A (10 micrograms/ml) or longer incubation periods (at least 48 hr) induced suppressor T cells. Con A-generated suppressor T cells were sensitive to mitomycin treatment and exerted their suppressor function on the early phase of differentiation and/or proliferation of B cells but not on the final differentiation of B cells to Ig-producing cells. The identity of the MHC was not required for the expression of suppressor function. Suppressor T cells competed with helper T cells in PWM-induced Ig-production of PBL. This experimental system can be applied to estimate the regulatory function of T cells in several disease states.     

Altered levels of mononuclear leukocytes in tumor-bearing rats: decrease of helper T lymphocytes and increase of suppressor cells

In the spleen and peripheral blood of BN rats with progressive tumors, W3/25+ T helper cells were significantly reduced and OX8+ T suppressor/cytotoxic cells were significantly increased. The ratio of helper to suppressor elements was decreased to 1.6 from a ratio of 3 in normal BN rats without tumors, and this decreased ratio correlated with tumor growth. When tumors were eliminated in vivo by infusion of effector cells (W3/25+ T lymphocytes), the levels of W3/25+ and OX8+ T cells returned to normal and the ratio of helper to suppressor/cytotoxic cells in the spleen and peripheral blood reverted to 3.0 or higher. Macrophages and null cells, T-sIg-, were also elevated in the spleen and peripheral blood of rats bearing expanding tumors and returned to normal levels after cure. Assays of spleen cells for cell-mediated cytotoxicity in rats with large tumors revealed little or no specific cytotoxicity. Cytotoxic activity was high in spleen of rats cured of their neoplasms by infusion of helper cells.     

Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.     

Surface markers on the T cells that regulate cytotoxic T-cell responses I. The Ly phenotype of suppressor T cells changes as a function of time, and is distinct from that of helper or cytotoxic T cells

Regulatory T cells can be obtained from primary mixed lymphocyte cultures of CBA spleen cells responding to BALB/c stimulators. At day 3 of culture, T cells are generated which can either help or suppress the generation of cytotoxic T cells in a second primary MLC culture. The regulatory activity observed depends on the conditions employed in the assay system allowing independent assay of different functional cell types which coexist in the cultures. Both the helper activity and the suppressor activity are mediated by differentiated antigen-specific T cells whose function is radioresistant. The Ly phenotype of these regulatory cells was tested. At day 3 of the first-step culture, the phenotype of the helper cells is Ly 1.1+ Ly 2.1-, whereas the inhibitory cells are Ly 1.1 Ly 2.1+. At day 5 of M LC culture, suppressor activity and helper activity are also observed. However, at this point, a suppressor cell which is Ly 1.1-Ly 2.1+ represents the major inhibitory activity. It is not clear whether this change in suppressor cell phenotype as a function of time in culture represents one differentiation pathway or cells derived from two different precursor cells. The Ly phenotype of helper or cytotoxic T cells did not change as a function of time in culture. In day 5 first-step cells, the cytotoxic cells were typed as Ly 1.1+ 2.1+, whereas the inhibitory cells present in aliquots of the same treated cell population expressed the Ly 1.1- Ly 2.1 phenotype. Taken together, these observations show that the antigen-specific suppressor cells and helper cells which regulate the generation of cytotoxicity, and the cytotoxic cells themselves represent physically distinct subclasses of T cells.