Surface phenotype of Japanese adult T-cell leukemia cells characterized by monoclonal antibodies

We studies the surface phenotype and the functional activities of leukemic cells from three patients with Japanese adult T-cell leukemic (ATL) using the panel of OK and anti-Tac monoclonal antibodies, which react with differentiation antigens and define functionally distinct T- cell subsets or activated and terminally differentiated T cells. The phenotype of ATL cells were determined to be OKT1+T3+T4+T10+T5-T8-Okla1- , although cells from two patients suppressed pokeweed mitogen (PWM) induced normal B-cell differentiation, and cells from all patients lacked helper activity in this system. In addition, after cultivation with PWM, ATL cells from all patients were reactive with anti-Tac monoclonal antibody, and cells from one patient were reactive with OKlal. These findings suggest that ATL cells arise from peripheral mature T-cell subsets and also suggest that the transition of surface phenotype of ATL cells to functionally mature and activated T cells occurs in culture.     

T-cell subpopulations in patients with monoclonal gammopathies: essential monoclonal gammopathy, multiple myeloma, and Waldenstrom macroglobulinemia

T-cell subsets defined by monoclonal antibodies (OKT3, OKT4, and OKT8) were analyzed in 117 patients with monoclonal gammopathies--69 multiple myeloma (MM) (30 untreated and 39 treated), 14 Waldenstr?m's macroglobulinaemia (WM), and 34 essential monoclonal gammopathy (EMG) patients. The percentage and absolute numbers of total T-lymphocytes (E+, OKT3+ cells) were within the normal range in all groups except for the treated MM patients, in which a decrease in the absolute number could be observed. The percentages of OKT4+ cells were significantly lower in MM (35 +/- 1.7) than in EMG patients (43 +/- 2) and controls (50 +/- 2). In contrast, OKT8 cells correspondingly increased in MM (38 +/- 1.6) compared with EMG patients (29 +/- 1) and controls (27 +/- 1). The OKT4/OKT8 ratio was lower in MM than that in EMG patients and controls (p less than 0.01) and was shown to be one of the four most significant variables in a linear discriminant analysis used to distinguish between MM and EMG groups. The MM patients in clinical stage III as well as Bence-Jones myeloma patients showed a more pronounced OKT4/OKT8 imbalance. The treatment did not influence the percent distribution of T-cell subpopulations. The patients with WM exhibit an alteration in the distribution of the T-cell subsets similar to the MM patients with a T4/T8 ratio of 1.1 +/- 0.1. This imbalance was more pronounced in WM patients with monoclonal B-lymphocytes in peripheral blood (leukaemic phase of WM). The functional significance of the altered T-cell subsets in MM and WM patients remains to be established, though it is probable that such an imbalance plays an important role in regulating these B-cell proliferations.     

Dipeptidylaminopeptidase IV (DAP IV) activity in normal and malignant T-cell subsets as defined by monoclonal antibodies

The activity of dipeptidylaminopeptidase IV (DAP IV) was investigated by a cytochemical method in isolated fractions of normal peripheral blood mononuclear cells and malignant cells from 9 patients with chronic T-cell leukaemia. A positive DAP IV reaction was observed in 52% of normal T cells, a consistent negative reaction in normal B cells and monocytes. 2 distinct T-cell subsets, helper/inducer cells (T4+/T8-) and cytotoxic/suppressor cells (T4-/T8+), were negatively selected by complement-mediated cytolysis utilizing the monoclonal antibodies OKT4 and OKT8. On examination of these T-cell subsets a positive DAP IV reaction was restricted to the majority of normal T4+/T8- cells. The malignant cells from 3 patients with T-chronic lymphocytic leukaemia, expressing the cytotoxic/suppressor phenotype (T4-/T8+) lacked DAP IV activities. In contrast, almost all leukaemic cells from 3 other cases, expressing the helper/inducer phenotype (T4+/T8-), were strongly positive. Despite their T4+/T8- phenotype, the malignant cells from 3 patients with Sézary syndrome were completely DAP IV negative. These findings suggest that the DAP IV reaction may be helpful in the further characterization of normal and malignant T-cell subsets.     

Morphological and immunological change in the predominant type of leukaemic cells in a patient with T-cell chronic lymphocytic leukaemia

A case of T-cell chronic lymphocytic leukaemia is described. Intracerebellar tumour was demonstrated by the characteristic feature of contrast-enhanced computerized tomography and was evidenced by surgical procedure. Histological examination revealed lymphocyte infiltration in the cerebellum. Initially, the majority of leukaemic cells were mature, medium-sized lymphocytes with surface marker phenotype of E+, OKT3+, OKT4+, OKT6-, OKT8-. In the terminal stage, large atypical lymphocytes which were morphologically distinct from the original lymphocytes and had different surface marker phenotype of E+, OKT3+, OKT4-, OKT6-, OKT8+, Leu7+ became increasingly prominent. The remaining medium-sized lymphocytes were morphologically and immunologically unchanged. Subsequently, the disease developed to a more aggressive pattern and the patient died in spite of chemotherapy.     

Phenotypic heterogeneity of human T-cell malignancies: demonstration by monoclonal antibodies and cytochemical markers

The present study sought to delineate the phenotypic heterogeneity of the human T-cell malignancies. Twenty T-cell neoplasms were investigated for reactivity with the OKT hybridoma monoclonal antibodies and expression of acid alpha-naphthyl acetate esterase (ANAE), beta-glucuronidase (BG), and acid phosphatase (AP) activity. Twelve cases (Mycosis fungoides, Sezary syndrome, cutaneous T-cell lymphoma, chronic lymphocytic leukemia) were OKT3'T4', ie, expressed the phenotype commonly associated with mature T-helper cells. These cases were further divisible into ANAE+BG+ (6 cases), ANAE-BG+ (5 cases), and ANAE-BG- (1 case) phenotypes. In contrast to the 12 OKT3+T4+ cases, the remaining 8 cases showed considerable inter- and intratumor heterogeneity with respect to reactivity with the OKT antibodies. Six of these cases (acute lymphoblastic leukemia, lymphoblastic lymphoma) expressed phenotypes consistent with various intrathymic stages of T-cell differentiation. Five of the latter 6 cases were AP+BG+ANAE-, analogous to the majority of normal cortical thymocytes; an OKT3+T4-T8+T10+ neoplasm was ANAE+, analogous to normal medullary thymocytes. Two cases expressed the previously undescribed OKT3+T4-T8-T10+ phenotype. These studies demonstrate that the T-cell malignancies are divisible into phenotypes which correspond to normal maturational stages of T-cell differentiation and functionally distinct T-cell subsets. Phenotypic analysis of the human T-cell malignancies may provide a basis for understanding their biological heterogeneity and may aid in the identification of transitional stages of T-cell differentiation and minor T-cell subsets.     

Analysis of T lymphocytes after bone marrow transplantation using monoclonal antibodies

Using monoclonal antibodies to cell surface antigens and fluorescent cell sorter analysis, we studied peripheral blood lymphocyte subsets after bone marrow transplantation (BMT). In 13 patients studied 3 mo or more after BMT, the ratio of T-cell subsets defined by antibodies OKT4 and OKT8 was reversed (OKT4/OK%8 = 0.7 +/- 0.3) in comparison to normal volunteers or bone marrow donors (ratio OKT4/OKT8 = 1.7 +/- 0.4) (p less than 0.001). This reversed ratio persisted for up to 3 yr after BMT. In contrast to a previous report, presence of an abnormal ratio of T-cell subsets did not correlate with clinically significant graft- versus-host disease (GVHD). In agreement with a previous study, (26% +/- 8%; less than 4% in normals (p less than 0.001) and antibody OKT10 reactive cells (39% +/- 20% versus 10% +/- 4%) (p less than 0.01), suggesting in vivo activation. However, their PBL did not react with antibody B3/25 (antitransferrin receptor), a marker found on normal PBL after in vitro activation by mitogens (BMT patients less than 5%; normal PBL T cells plus PHA 45% +/- 11%). These results demonstrate that BMT patients have: (A) an abnormal ratio of T-cell subsets in the presence or absence of clinically significant GVDH disease so that these measurements were not useful in monitoring patients; (B) an increased number of T cells with cell surface phenotype (OKT8+, Ia+, OKT10+, B3/25-) that is distinct from normals but similar to patients with infectious mononucleosis or acquired hypogammaglobulinemia.     

Functional and multimarker analysis of T-cell chronic lymphocytic leukaemia

The morphology, immunophenotype, cytoenzymatic and functional activities of T lymphocytes from 4 patients with chronic lymphoproliferative disease of T-cell origin were studied. Clonal proliferation was demonstrated by distinctive chromosomal abnormalities involving chromosomes 2 and 14. Patients 1 and 2 were classifiable as OKT4+ T-cell chronic lymphocytic leukaemia (T-CLL) and patient 3 as OKT4+/OKT8+ T-CLL, with helper function in vitro only in patient 1. Patient 4 has low-grade lymphocytosis with benign clinical course, with cells showing morphology of large granular lymphocytes (LGL), and immunophenotype HNK-1+, ER+, Fc gamma receptor+, OKT3+, OKT11+ and OKT8+, as well as natural killer activity, radiosensitive suppressor activity on Ig secretion and responsiveness to PHA; this case was interpreted as LGL leukaemia. This study indicates that a large proportion of cases of true T-CLL may belong to the OKT4 subset, and that extensive investigations should be made of the lymphocytic OKT8+/T gamma forms to characterize them precisely.     

Phenotypic characterization of skin-infiltrating T cells in cutaneous T- cell lymphoma: comparison with benign cutaneous T-cell infiltrates

Using a panel of monoclonal antibodies, we have studied cell surface antigens of infiltrating mononuclear cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and compared them with the T-cell surface phenotype seen in benign cutaneous T-cell infiltrations (e.g., contact dermatitis, delayed hypersensitivity skin tests, granuloma annulare) and in dermal infiltrates of lymphomatoid granulomatosis patients. We found that unlike circulating CTCL (Sezary) cells, CTCL cells infiltrating skin epidermis frequently expressed the T-cell antigen 3A1. Cutaneous infiltrates in 10 patients with mycosis fungoides (MF) and 1 patient with Sezary syndrome were OKT4 (inducer T cell), OKT8 (suppressor/cytotoxic T cell); 2 patients with MF were OKT4- , OKT8; and one MF patients's skin T cell were OKT4-, OKT8+. Similar to CTCL infiltrating cells, most of the benign skin T-cell infiltrates were usually 3A1+. OKT4+, and OKT8-. Our study shows the complex nature of T-cell antigen patterns in inflammatory and malignant skin T-cell infiltrations. We demonstrated that the CTCL the skin epidermal infiltrating T-cell phenotype is not invariate, and in many cases, is similar to the phenotype of clinically benign cutaneous T-cell infiltrations.     

Intestinal lymphocyte subpopulations in inflammatory bowel disease: an analysis by immunohistological and cell isolation techniques.

Lymphocyte subpopulations in the intestinal mucosa of patients with ulcerative colitis or Crohn's disease have been studied using a double marker immunofluorescence technique. Analysis of tissue sections revealed that the majority of intraepithelial lymphocytes (IEL) were T cells (Hle-1+ HuTLA+ UCHT1+). Of these, over 80% were of suppressor-cytotoxic phenotype (OKT8+:83 +/- 10.2%) with a small population of helper type IEL (OKT4+). Only one third of OKT8+ IEL reacted with the T cell antibody, anti-Leu-1. IEL were also Tac-, C3b-receptor- (C3RT05-), and Ig-. Within the lamina propria, OKT4+ T cells predominated (ulcerative colitis 64 +/- 6.0%; Crohn's disease 63 +/- 6.0%). Less than half of the smaller OKT8+ population in the lamina propria was Leu-1+. These finding did not differ from those seen in histologically normal tissues from controls, and are similar to those reported in the small intestine. Mononuclear cells were also isolated from the intestinal lamina propria using an enzymatic technique. The majority of lymphocytes obtained were T cells (OKT3+), with populations of OKT4+ and OKT8+ cells. Comparison of the ratio of OKT4+ to OKT8+ lymphocytes determined by immunohistological analysis with that obtained in mucosal isolates, however, suggested that the isolation procedure may deplete OKT8+ cells. These findings indicate that an imbalance of mucosal immunoregulatory T cells, as defined by monoclonal antibodies, does not occur in inflammatory bowel disease. They also emphasize that functional studies of isolated intestinal mucosal cells should be combined with morphological studies of cell populations in situ.     

Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.     

Stimulation and inhibition of human granulopoiesis in vitro by normal and malignant T4- or T8-lymphocyte subpopulations

We studied the role of total peripheral blood T-lymphocytes and separated subpopulations of OKT4+ and OKT8+ cells stimulated with concanavalin A in the regulation of human granulopoiesis. Unfractionated total T cells depleted of monocytes are capable of producing colony-stimulating activity (CSA) and colony-forming unit-clone (CFU-C) suppressor activity simultaneously. After positive selection of cell subsets using a fluorescence-activated cell sorter, these two activities were produced by OKT4+ lymphocytes, whereas OKT8+ cells displayed small amounts of CSA and were incapable of releasing suppressor activity. On the other hand, total human thymocytes and subsets defined by monoclonal antibodies Leu2a/Leu3a failed to express any detectable CSA or CFU-C suppressor activity. A total of 14 cases of phenotyped lymphoid malignancies were also studied: the results showed that the production of stimulating and/or inhibiting factors is neither clearly related to a discrete stage of differentiation nor to the OKT4/OKT8 phenotype. Moreover, three monoclonal T leukemias, OKT4+OKT8-, OKT4-OKT8-, and OKT4-OKT8+ have been able in each case to produce simultaneously CSA and CFU-C suppressor activity. Finally, these studies strongly suggest that the activities of T-lymphocytes involved in the regulation of granulopoiesis are not closely linked with OKT4/OKT8 phenotype.     

Established IL-2-dependent double-negative (CD4- CD8-) TCRαβ/CD3+ ATL cells: induction of CD4 expression

Summary. We established IL-2-dependent T cells from an adult T-cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single-p-positive in the initial phase to double-negative (CD4^- CD8^-) at the time of exacerbation. The cells termed SO-4 were of ATL cell origin and showed the double-negative TCRαβ/CD3⁺ T-cell phenotype. SO-4 cells acquired CD4 antigen expression following stimulation with concanavlin A (ConA) or immobilized anti-CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO-4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO-4 cells return to their original phenotype (CD4 single-positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single-positive and double-negative T cells.     

Abnormal monoclonal antibody-defined helper/suppressor T-cell subpopulations in multiple myeloma: relationship to treatment and clinical stage

Among peripheral blood T cells from 25 patients with myeloma, % OKT8+ suppressor cells were increased in all but two patients (mean 63 +/- 12%; normal 34 +/- 8%), but were present in normal absolute numbers (mean 0.5 +/- 0.3 x 10(9)/l; normal range 0.2-0.6 x 10(9)/l). OKT4+ helper cells were reduced in relative and absolute numbers (mean 40 +/- 12%, 0.3 +/- 0.2 x 10(9)/l; normal 64 +/- 8%, 0.6-1.4 x 10(9)/l). Similar results were obtained in treated and untreated cases, and in patients of different clinical stage. In the 12 patients tested, gamma Fc-rosetting T cells were present in normal numbers (mean 28 +/- 10%; normal 34 +/- 10%). In four patients with IgM disease, similar, but less marked, changes in monoclonal antibody-defined T-cell subsets were observed. The findings are discussed in relation to those in other B-cell malignancies, and it is suggested that imbalances of OKT4+ helper/OKT8+ suppressor cells may be important in the immune suppression of many of these disorders.     

Blood and lymph node T-lymphocyte subsets in non-Hodgkin lymphomas

Blood and lymph node T-lymphocyte subpopulations have been studied in untreated non-Hodgkin lymphoma (NHL) patients and healthy controls. T-lymphocytes were determined by the E-rosette technique and by OKT3/LEU4 monoclonal antibodies; OKT4/LEU3 and OKT8/LEU2 monoclonal antibodies were used to identify T-cell subsets with helper/inducer and suppressor/cytotoxic activity, respectively. OKT4+ T-cells were low in patients, while OKT8+ T-cell numbers were normal. The OKT4+/OKT8+ blood lymphocyte ratio was below the normal range in about 50% of the patients. The ratio was higher in lymph nodes than in blood of patients and controls. The results may suggest that untreated NHL patients have a reduced pool of T-cells with phenotypic markers of OKT4/LEU3. Monoclonal blood B-lymphocytes were found in 45% of the cases. The presence of such cells in blood was frequently associated with a low OKT4+/OKT8+ ratio.     

Leu 11+ T gamma cell chronic lymphocytic leukemia with partially activated natural killer function and its further activation by recombinant IL2 in vitro

A patient with T gamma cell chronic lymphocytic leukemia with the Leu 11+ phenotype and novel function of activated natural killer cells is reported. The peripheral blood mononuclear cells of this patient showed large granular lymphocytes by May-Giemsa staining and lamellipodia by scanning electron micrography. Tests on reactivity with monoclonal antibodies showed that most cells were Leu 11+, OKT3-/Leu 1-, OKT4-, OKT8-, Leu 7-, OKM1-, and Tac-. Freshly collected cells lysed not only K562, which is highly sensitive to natural killer cells, but also Raji cells and Daudi cells, which are not. Leu 11+ cells were triggered by recombinant interleukin 2 (rIL2) to proliferate, produce gamma- interferon (gamma IFN), and show enhanced HLA-DR antigen expression, and 30% of the Leu 11+ cells became positive for IL2 receptor antigen (Tac). The spectrum of cytotoxic activity of these cells against target cells was extended by rIL2; after treatment with rIL2, the cells also lysed HeLa cells and even fresh cancer cells. This stimulation also increased the activities of acid phosphatase and tartrate-resistant acid phosphatase of the cells and resulted in the appearance of nonspecific esterase activity. The expanded cell population may represent a neoplasm, but these findings provide information on a novel differentiation stage of activated NK cells.     

Imbalance among T-cell subsets in patients with coronary arterial aneurysms in Kawasaki disease

The populations of T cells were studied in 46 patients with Kawasaki disease, separated into 2 groups: group I--11 patients with coronary aneurysms; and group II--35 patients with normal coronary arteries. Patients from both groups with early acute illness, before day 5, had a significant reduction in the population of OKT3+ (p less than 0.001), OKT4+ (p less than 0.02) and OKT8+ cells (p less than 0.002), but normal OKT4/OKT8 ratios compared with age-matched control subjects. These abnormal values quickly returned to normal levels during week 2 in patients with normal coronary arteries. In contrast, patients in whom coronary aneurysms developed within 3 weeks of the onset had an imbalance between OKT4 and OKT8 during week 2, characterized by a decrease in the number of OKT8+ cells and an increase in the number of OKT4+ cells, resulting in a high OKT4/OKT8 ratio (p less than 0.01). Three patients in whom large coronary aneurysms developed had ratios higher than 4.50. Follow-up analysis of T-cell subsets from individual patients with coronary aneurysms showed that the OKT4/OKT8 ratio during the acute stage was reduced during the convalescent stage (p less than 0.005). In contrast, the ratio in patients with normal coronary arteries was normal during the course of the illness. These observations suggest that an immune regulatory process operating in coronary aneurysm formation is present.     

Modulation of T-lymphocyte differentiation antigens: potential relevance for multiple sclerosis.

Effects of the anti-T-cell monoclonal antibodies OKT3, OKT5, and OKT8 on T-cell surface properties and cell functions were evaluated. Incubation of mononuclear cells isolated from peripheral blood for 48 hr with each monoclonal antibody in the absence of complement resulted in modulation of their respective surface antigens; i.e., the number of cells detected by immunofluorescence as positive for the T3, T5, and T8 surface antigens was reduced. T3, T5, and T8 antigens modulated independently. A radiolabeled second antibody technique confirmed modulation by OKT3 and OKT8 and indicated that T-cell differentiation antigens can regenerate in culture. Incubation of mononuclear cells with OKT3 increased the number of sheep erythrocyte-binding lymphocytes (E+-rosetting cells) and markedly increased the number of avidly E+-rosetting cells. Incubation with OKT8 reduced the number of E+- and of avidly E+-rosetting cells. OKT3 induced both mitogenic reactivity and suppressor cell activity; cells modulated by OKT8 exhibited reduced mitogenic reactivity and reduced suppressor cell function. The decreases in total T cells, in avid T cells, in suppressor cell number, and in suppressor cell function that follow modulation by OKT8 mimic changes observed in multiple sclerosis patients.     

In vitro and in vivo effects of 2'-deoxycoformycin (Pentostatin) on tumour cells from human gammadelta+ T-cell malignancies

Hepatosplenic gammadelta+ T-cell lymphoma represents a rare neoplasm of post-thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2'-deoxycoformycin (dCF) on cultured mononuclear cells and purified gammadelta+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic gammadelta+ T-cell lymphoma. At a concentration of 10 microM, dCF, in the presence of 2'-deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on gammadelta+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/gammadelta+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/alphabeta+ lymphocytes being reduced only by 6-40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/alphabeta+ T cells, which accounted, however, for only 20-25% of the initial cell input. Accordingly, the combination of dCF (10-100 microM) plus dAdo was able to induce a dose-dependent inhibition of clonogenic growth and [3H]-thymidine incorporation in purified CD3+/CD4-/CD8- gammadelta+ tumour cells. We also report that one patient with hepatosplenic gammadelta+ T-cell lymphoma in terminal leukaemic phase showed a striking haematological response to single-agent dCF given as fourth-line treatment. In particular, the selective clearance of gammadelta+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T-cell lymphoma.     

Existence of a pool of T-lymphocyte colony-forming cells (T-CFC) in human bone marrow and their place in the differentiation of the T- lymphocyte lineage

The existence and characteristics of bone marrow T-cell progenitors have not yet been established in man. Several pieces of evidence such as the reconstitution of certain immunodeficiencies by bone marrow graft suggest that T-cell precursors are present in the bone marrow. We report the growth of T-cell colonies from bone marrow populations using PHA-stimulated lymphocyte-conditioned medium containing T-cell growth factor (TCGF). Rosetting experiments and complement-dependent cytotoxicity assays with monoclonal antibodies indicate that the bone marrow T colony-forming cells (T-CFC) are E- OKT 3- and la+, i.e., immature progenitors. The colonies derived from these cells have the phenotype of mature T cells: E + OKT 3 + la- with either helper (OKT 4+) and suppressor (OKT 8 +) antigens. These results suggest that a thymic microenvironment may not be necessary for the in vitro proliferation and differentiation of the T-cell lineage in adult humans. These methodologies may permit direct investigation of early phenomena concerning the T-cell lineage, such as the acquisition of self-tolerance, the formation of a repertoire of specificities, and the HLA restriction phenomena that we believe takes place before the thymic maturation.