Defective in vitro T cell colony formation in the acquired immunodeficiency syndrome

Depressed T cell immunity is a universal characteristic of the acquired immunodeficiency syndrome (AIDS). In the present study, 25 patients with AIDS and opportunistic infections, 22 individuals with AIDS-related complex (ARC, or chronic lymphadenopathy syndrome), and 20 healthy homosexuals were evaluated by means of the T cell colony assay. Forty-seven healthy heterosexual controls showed an average of 3964 +/- 319 colonies/7.5 X 10(5) cells, with a range of 880 to 9340. The mean in the 20 healthy homosexuals (3173 +/- 483) did not differ significantly from the controls; in this group, only three patients had values less than 1000 colonies/plate. By contrast, all AIDS patients and 14 ARC patients had colony counts less than 1000. The mean value for the AIDS patients was only 24 +/- 15 (p less than 0.0005 compared with either controls or healthy homosexuals); values in the ARC group were intermediate (1180 +/- 360). The addition of interleukin 2 to the plates promoted correction of the proliferative abnormality in ARC patients. This interleukin increased colony scores in the AIDS group, but the mean value was still significantly less than controls. Comparison indicated that the colony assay is a more sensitive indicator for detecting proliferative abnormalities than responses to PHA, Con A, or pokeweed mitogen in suspension cultures.     

Growth at limiting dilution of human T cell colonies from T cell-depleted peripheral blood leukocytes

Using a new culture system, we found that up to one-fourth of nylon-wool nonadherent human peripheral blood lymphocytes (NAd) could give rise to a colony containing T cells. Even after NAd cells were depleted of T cells (rosette-forming and/or OKT3+ cells and/or OKT11+ cells), up to one-ninth of cells could still give rise to a colony containing T cells. Colonies were grown in microwell liquid cultures in a vol of 0.02 ml/culture seeded with from 2 to 40 cells. Each cell concentration was set up with 60 or more replicates, and the results were analyzed using limiting dilution theory. Growth had an absolute requirement both for lymphokine(s) present in the supernatant of PHA-stimulated peripheral blood leukocytes and for PHA. A fit to limiting dilution theory could be obtained only when heavily irradiated (3000 rad) peripheral blood leukocytes or NAd cells were also included, 300 cells/well being optimum. The system supported the development of OKT3+ and OKT11+ cells from OKT3-, OKT11-, OKM1-, and OKla1- precursors. All colonies contained some OKT3+ and OKT11+ cells, usually more than half the colony cells carrying the markers. Some colonies also contained OKTM1+ cells and OKla1+ cells at a frequency such that some colony cells must carry more than one marker.     

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.     

Agar human T cell colony growth promoted by a B + null cell-derived lymphokine distinct from IL 2

Human T cell agar colonies can be grown under PHA stimulation from either mature T cells or their E rosette-negative (E-), OKT3- peripheral blood and bone marrow precursors. Colonies comprise a majority of mature E+, OKT3+ cells and a minor (5 to 10%) population of immature E-, T3-, T8-, T4-, DR+, T10+, RFB1+ cells, which upon replating in subculture, can generate secondary colonies of OKT3+, E+, OKT4+, OKT8+ cells. Secondary colony formation can serve as a test for growth requirement of colony precursors, because it depends on the presence of both PHA and a colony-promoting activity (CPA) recovered in PHA-stimulated B + null or T + adherent cell supernatants. CPA production by B + null cells was not affected by their treatment with OKT3 or D66 (T11-like) monoclonal antibodies (MAB) + complement but was abolished by an anti-HLA-DR MAB + complement. However, B cells sorted by panning with the same anti-HLA-DR MAB did not release CPA, demonstrating the requirement of both B cells and null cells for CPA production. Neither IL 2 nor IL 1 could account for B + null cell-derived CPA.     

Identification of the B cell derived T cell colony promoting activity to soluble CD23.

We previously reported that supernatants of phytohemagglutinin (PHA)-stimulated normal human B cells (NBCsup) contain a T cell colony promoting activity. NBCsup were able to (a) increase the number of secondary colonies generated under PHA and interleukin-2 (IL-2) stimulation by peripheral blood-derived primary T colony cells, (b) enhance the ability of CD4+ but not CD8+ peripheral blood T cells to form agar colonies in the presence of PHA and IL-2 and (c) support in vitro differentiation of CD2-3-4-8- prothymocytes into CD2+3+4+ T cells. This activity was therefore refered to as Prothymocyte Differentiating Activity (PTDA). Subsequent studies pointed to striking biochemical and cell source homologies between this B cell derived factor and the 25-kDa soluble CD23 (sCD23). sCD23 has been recently found to display prothymocyte differentiating activity.     

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.     

Regulation of self-renewal of human T lymphocyte colony-forming units (TL-CFUs)

Formation of human T lymphocyte colonies in semisolid medium from T lymphocyte colony-forming units (TL-CFUs) under stimulation of phytohemagglutinin (PHA) has been reported by several authors. These TL-CFUs were present in unsensitized lymphocyte populations. We report here that such TL-CFUs are capable of renewing themselves. This was observed when colony cells from primary T cell colonies that developed in the presence of PHA were replated in methylcellulose medium containing irradiated autologous leukocytes and PHA. We have also been able to demonstrate serial transfer of TL-CFU for up to six passages. At each passage, colony-forming frequency was determined from the proportional relationship between the number of new colonies obtained and the number of colony cells plated. Examination of the number of new colonies derived from each individual T cell colony ("burst size of TL-CFU") showed that most colonies contained very few new TL-CFU and only a very small number of colonies contained many new TL-CFU. The distribution of burst sizes could be well fitted to a gamma distribution, in agreement with prediction from a stochastic model. We have identified an activity that enhanced the mean TL-CFU burst size three to ten times. This work provides the first evidence in vitro that self-renewal of human T lymphocyte progenitor cells can be stimulated by specific regulatory proteins.     

Stimulation of human T cell colony growth by a lymphocyte colony enhancement factor derived from lymphocyte subpopulations

Blood mononuclear cells (MNC) develop into T cell colonies when the cells are sensitized with PHA and seeded in a two-layer soft agar system. Conditioned medium (CM) derived from MNC enhanced lymphocyte colony formation when it was added to the culture system. CFU-TL appear to be stimulated into colony formation by molecules secreted by lymphocyte subpopulations contained in the seeded cells. In this study, human peripheral blood MNC were fractionated by a battery of techniques into adherent, E+, CD4+, CD8+, B and null cells. CM was prepared from each of the subpopulations and its effects on T cell colony growth assayed. All the lymphocyte subpopulations were found to generate lymphocyte colony enhancement factor (LCEF). After several purification procedures, CM prepared from CD4 and CD8+, displayed LCEF activity corresponding to proteins of molecular weight 30-40 and 100-140 kD.     

Lack of interaction of circulating T cells with phytohemagglutinin in bacillary positive untreated lepromatous leprosy patients--identification of subpopulation of lymphocytes by shifts in electrophoretic mobility.

Incubation of human peripheral blood lymphocytes from normal healthy subjects with phytohamagglutinin (PHA), causes the reduction of the surface charge of a subpopulation of T cells by 1363 +/- 242 e.s.u./cm2. The affected subpopulation was predominantly the high charge-bearing cells identifiable with early (10 min) rosette-forming cells with sheep erythrocytes. Purified lymphocytes obtained from untreated bacillary-positive, lepromatous leprosy patients contained high charge-bearing T lymphocyte subpopulation. However, incubation with PHA did not result in the shift of electrophoretic mobility of these cells, suggesting the absence of interacting sites for the mitogen on the surface of these cells. The absence of mitogen-interacting sites is not an inherent trait of leprosy patients; the surface charge of lymphocytes from Dapsone-treated bacillary-negative subjects was reduced upon incubation with PHA. A close correlation was found between the number of cells whose charge alters on incubation with PHA and the transformation index obtained with this mitogen.     

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

Helper (OKT4) T cells from human T cell colonies produce potent colony-stimulating activity

Confluent T cell colonies were grown by culturing blood mononuclear cells in double agar layers containing autologous plasma and phytohemagglutinin (PHA) for one week (37 degrees C, 5% CO2). The plates were then overlaid with serum-free alpha medium which was harvested after 24 h. This medium was demonstrated to have colony-stimulating activity (CSA) of greater potency than conventionally prepared PHA-leukocyte conditioned medium, which was prepared by incubating cells from the same donors. Removal of OKT4-positive cells using a monoclonal antibody and complement abolished CSA production by cells from T cell colonies while the removal of OKT8-positive cells had no effect.     

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.     

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.     

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.     

Frequent T4-positive cells with cytolytic activity in spleens of patients with Hodgkin's disease (a clonal analysis)

Purified T lymphocytes isolated from spleens of untreated patients with Hodgkin's disease (HD) were cloned by using a microculture system previously shown to allow clonal expansion of virtually all peripheral blood T lymphocytes. Cells were plated under limiting conditions with irradiated feeder cells and PHA. Interleukin 2 (IL 2)-containing supernatants were added 48 hr later. The phenotypic and functional characteristics of a total number of 221 clones derived from six different HD spleens were investigated and compared with those of 133 clones obtained from three spleens of otherwise healthy individuals who underwent posttraumatic splenectomy. The majority of T cell clones derived from HD spleens expressed the T4+ (helper/inducer) phenotype. However, further functional characterization showed that as much as 50% of these T4+ clones displayed cytolytic activity in a lectin-dependent lytic assay allowing detection of cytolytic cells of any specificity. In contrast, less than 10% T4+ clones derived from control spleens were cytolytic, as assessed by the same lectin-dependent lytic assay. The cytolytic potential of T4+ and T8+ clones established from spleens of patients with HD did not reflect the induction of lymphokine-activated killer cells, because only a minority of them displayed natural killer (NK) activity against NK-sensitive K562 and MOLT-4 cell lines. These findings indicate that T lymphocytes found in the spleens of patients with HD may represent, at least in part, the expansion of a subset present in small percentages among normal peripheral blood or spleen T lymphocytes, which is involved in a cytotoxic reaction.     

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 microg/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.     

Spontaneous growth of colonies with mature T lineage phenotype from T-cell-depleted bone marrow precursors in a patient with a lymphoproliferative disorder of the large granular lymphocytes

Bone marrow cells from a patient with pancytopenia and a lymphoproliferative disorder of large granular lymphocytes (LGL) were cultured and tested for their hemopoietic colony-forming potential. Neither erythroid nor granulocyte-macrophage colony formation could be obtained from unfractionated, or LGL-depleted bone marrow cell preparations. However, a spontaneous growth of lymphoid colonies was observed after culturing LGL-depleted (T3-) bone marrow cell suspensions for 25 days. Pooled colonies expanded with recombinant interleukin-2 yielded a population composed predominantly of mature T cells (T3+, Leu 6-). These findings suggest that some (T3-) T cell precursors may mature in the bone marrow and that, in our patient, LGL may have exerted a suppressor effect on this maturational process.     

Select growth of human T lymphocytes in single phase semisolid culture.

Selective growth and clonal proliferation of human T lymphocytes can be achieved by using a single-phase semi-solid methylcellulose system without the requirement of preincubation with lectins. Significant proliferation, however, depends upon the continued presence of Con A or PHA, but not pokeweed mitogen or lipopolysaccharide within the methylcellulose. This procedure eliminates nonspecific agglutination by lectins and allows for direct visualization of colonies and their specific removal and subsequent cloning in liquid phase. Optimal growth and production of colonies greater than 40-cell size require 3 to 9 days. Individual cells can be identified on the basis of E rosette formation and absence of surface immunoglobulin or ability to phagocytize latex particles. Moreover, proliferation is inhibited by antithymocyte but not anti-B cell sera and can be demonstrated with peripheral blood T and MOLT-4 cells, but not with B or Raji cells. Finally, colony formation is not enhanced by the presence of 2-mercaptoethanol. The clonal proliferation of human T lymphocytes has wide application in the study of both antigen recognition and lymphocyte alterations in specific diseases.     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.