T lymphocytic subpopulations of normal human peripheral blood defined by monoclonal antibodies: an ultrastructural study using immunogold staining method

Submicroscopic findings of T lymphocytic subpopulations of normal human peripheral blood defined by OKT monoclonal antibodies were studied using ultrastructural immunocytochemistry (immunogold staining). The results show that OKT4-and OKT8-positive lymphocytic subpopulations have a distinct morphological pattern, although some variations in the ultrastructural details of cells in each subset are evident. The most representative features of OKT8-positive cells are regular or slightly indented nucleus with condensed chromatin, abundant cytoplasm, prominent Golgi apparatus, several granules frequently localized in the Golgi area, and numerous mitochondria, often in cluster disposition. OKT4-positive cells show generally less condensed chromatin, scanty cytoplasm and poor organule pattern. Furthermore, positive lymphocytes with prominent nuclear irregularities are found in both lymphocytic subpopulations. Ultrastructural similarities between the T cell subsets phenotypically characterized by monoclonal antibodies and other immunological criteria are also discussed.     

Determination of the T-cell subset producing gamma-interferon in tuberculous pleural effusion

The percentage of cells of different T-cell subsets and their functions in tuberculous pleural effusion were investigated. The percentage of OKT4-positive cells was 65 +/- 2% (mean +/- SEM, n = 8) and that of OKT8-positive cells was 19 +/- 3% (n = 8). Pleural T lymphocytes of patients with tuberculous pleurisy responded well to stimulation with purified protein derivative of tuberculin, and deoxyribonucleic acid synthesis was observed along with gamma interferon (IFN-gamma) production. When pleural T lymphocytes of patients with tuberculous pleurisy were treated with OKT4 monoclonal antibody and complement, a significant decrease in IFN-gamma production was observed in all eight patients (P less than 0.005), whereas no definite decrease in IFN-gamma production was found after treatment with OKT8 monoclonal antibody and complement. These results suggest that at least the OKT4+/OKT8- T-cell subset is responsible for the antigen-specific IFN-gamma production in pleural T lymphocytes of patients with tuberculous pleurisy.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.     

Lymphocyte subpopulations in the neonate: identification of an immature subset of OKT8-positive, OKT3-negative cells

T cell subpopulations of lymphocytes from cord blood (CBL) of 24 newborns and from peripheral blood (a-PBL) of 24 healthy adult volunteers were assessed in T cell-enriched, T cell depleted and unseparated lymphocyte fractions by using OKT3, 4, 6, and 8 monoclonal antibodies. The results show that T cell-enriched CBL include adult numbers of OKT3+, OKT4+, OKT6+ and OKT8+ lymphocytes whereas the T cell-depleted fraction consists of a high percentage of OKT8+, OKT3-, non-E rosette-forming cells bearing a PNA receptor. The presence of the PNA receptor and the lack of the OKT3+ antigen strongly support the hypothesis that the subset of OKT8+ cells in cord blood includes immature T lymphocytes that may represent an intermediate stage between thymocytes and mature peripheral T cells.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

Identification and estimation of the proportion of limiting cell types involved in the phytohemagglutinin response by limiting cell dose-response analysis

Fluoresceinated peanut agglutinin (PNA-FITC) and a monoclonal anti-T-cell antibodies were used to identify human thymocyte subpopulations. The phenotypes of PNA+ and PNA? thymocytes were studied using two different techniques: agglutination with PNA and double-labeling immunofluorescence. The mitogen-responsive PNA- subset was shown to include early thymocytes bearing the OKT9 antigen as well as lymphocytes with the OKT3 phenotype. Nearly all PNA+ thymocytes were found to bind simultaneously OKT4, OKT6, and OKT8 antibodies, whereas about 30% of them weakly bind the OKT3 antibody. These data suggest the existence of several intermediate stages of intrathymic differentiation, including a subset simultaneously bearing the OKT6- and OKT3-defined antigens.     

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.     

Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV

T-Cell subsets identified by polyclonal and monoclonal antibodies to dipeptidyl peptidase IV (DP IV) were investigated. Analysis in a cytofluorograf revealed 63 +/- 7% positive scatter-gated T lymphocytes. DP IV-positive cells were found to be T11+, 74-81% OKT4+, and 12-19% OKT8+. DP IV-negative cells were T11+ and comprise 16-40% OKT8+, and 10-30% OKT4+ T cells. Treatment of T lymphocytes with rabbit anti-DP IV and complement as well as the presence of rabbit anti-DP IV during culture resulted in a reduction of interleukin 2 (IL-2) production. This reduction was not observed with the mouse monoclonal anti-DP IV antibody II-19-4-7. Positive enrichment of DP IV-positive lymphocytes by cell sorting revealed excellent IL-2 production of DP IV-positive cells and very poor IL-2 activity in supernatants obtained from DP IV-negative lymphocytes. Thus, DP IV may serve as cell surface marker for IL-2-producing T lymphocytes.     

Hematology and enzyme histochemistry of peripheral blood lymphocytes in domestic pigeon (Columba livia f. domestica)

The purpose of our study was to determine the percentages of alpha-naphthyl acetate esterase (ANAE)-positive and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL) in both sexes of one-year-old domestic pigeons (Columba livia f. domestica). Blood samples obtained from 12 healthy domestic pigeons were used. The mean percentages of ANAE-positive PBL for females and males were 47.8% and 48.8%, respectively, whereas the mean percentages of ACP-positive PBL were 67.5% and 68.6%, respectively. The proportions of PBL were 49.3% and 48.6% in males and females, respectively.     

Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin's disease

To better understand the immunologic effects of lymphoid irradiation (LI), blood levels of T cell subsets were sequentially monitored in 15 patients before, during, and after irradiation treatment for Hodgkin's disease. Blood levels of all lymphocytes, T cells, and T cell subsets (defined by OKT4 and OKT8) fell dramatically and in similar proportions during early therapy, reaching levels less than 20 to 25% of control by the completion of mantle irradiation, and continuing at very depressed levels through the completion of therapy. Blood levels of OKT8-reactive (OKT8+) cells returned to pretreatment levels (402 +/- 38/mm3 vs 360 +/- 32/mm3 pretreatment) between 6 to 8 mo after LI, whereas blood levels of OKT4-reactive (OKT4+) cells returned to only 42% of previous values (242 +/- 22/mm3 vs 584 +/- 34/mm3 pretreatment) over the same period. The pre-LI ratio of OKT4+ to OKT8+ cells was 1.85 +/- 0.24 and fell to 0.65 +/- 0.05 between 6 to 8 mo after LI. During the recovery period, discrepancies of 208 +/- 32 cells/mm3 (3 to 5 months post LI) and 198 +/- 32 cells/mm3 (6 to 8 mo post LI) developed between the blood levels of OKT3+ cells and the sum of OKT4+ and OKT8+ cells. This suggests the emergence of OKT4+/OKT3-, OKT8+/OKT3-, and/or OKT4+/OKT8+ cells. In five patients, the sum of OKT4+ and OKT8+ cells was compared with the number of cells simultaneously co-stained by OKT4 and OKT8. It appeared that a significant proportion of the cells were OKT4+/OKT3- and OKT8+/OKT3- lymphocytes. We concluded that LI is similarly cytotoxic to peripheral blood T cell subpopulations. The reversed ratio after LI is a result of a slower repopulation of the peripheral blood by OKT4+ cells relative to OKT8+ cells. T cells after LI show a high degree of antigenic immaturity. It is postulated that the bone marrow that lies outside the fields of treatment and contains predominantly immature OKT8+/OKT3- cells is a major source of T cells repopulating the peripheral blood after LI.     

Human autologous rosette-forming cells. I. Expression of cell surface antigens in relation to age and lymphoid organ distribution

Autologous rosette-forming cells (auto-RFC) were characterized with monoclonal antibodies to various cell surface antigens using a technique combining immunofluorescence and rosette formation. In peripheral blood, auto-RFC were T cells (Leu 1+/OKT3+) the majority being derived from the helper/inducer subset (Leu 3a+/OKT4+). A small proportion of the circulating auto-RFC were Leu 2a+/OKT8+ and virtually none of them bore T10, T6, and DR antigens or peanut agglutinin (PNA) receptors. In the elderly, the percentages of Leu 3a+ auto-RFC increased significantly along with the augmentation of the Leu 3a+ circulating pool. After Con A stimulation of peripheral blood lymphocytes the autorosette population was expanded and therefore their phenotype was again that of helper cells. In the thymus, high levels of autorosettes are found (30 to 50%). Simple or double labeling of the rosetting cells with various monoclonal antibodies permitted the confirmation of the existence of distinct thymocyte subpopulations and moreover to identify the location of the auto-RFC in the intrathymic differentiation scheme. Nearly 70% of the rosetting cells were derived from common thymocytes, those cells defined by the coexpression of T10, T6, T4, and T8 antigens whether or not they were also stained by OKT3 antibodies. The remaining auto-RFC were found with similar frequency among the T4+ and T8+ mature thymocytes. In the spleen low percentages of auto-RFC were found and the majority resided in the Leu 3a+/OKT4+ population, similarly to peripheral blood autorosettes. Taken together, these data suggest that the expression of autologous erythrocyte receptors is acquired in the thymus and is gradually lost during T-cell maturation.     

Modulation of T lymphocyte differentiation antigens: influence of aging

In vitro modulation of human T lymphocyte surface differentiation antigens T3, T8, and T4, by their respective monoclonal antibodies, was studied as a function of donor age. Kinetic studies performed on lymphocytes from young adults indicated that modulation is dependent on concentration of antibody used and duration of culture. OKT3 modulates T3 rapidly (maximum at less than 24 hr) and relatively completely (79% at the highest concentration of antibody used). By 48 hr, regeneration of the T3 antigen is apparent. T8 modulation by OKT8 is slower (continued modulation at 48 hr) and less complete than is T3 modulation by OKT3. OKT4 does not modulate the T4 antigen. In elderly individuals modulation of T3 by OKT3 is preserved whereas modulation of T8 by OKT8 is significantly reduced (24 +/- 8% at 48 hr vs 53 +/- 4% for young controls). These observations document further age-related changes in properties of human T suppressor cells.     

Interferon-alpha induction of lymphocytes containing parallel tubular structures

The induction by IFN-alpha in peripheral blood lymphocytes of parallel tubular structures (PTS) and/or electron-dense granules occurring in a minority of peripheral blood lymphocytes was examined. IFN reportedly augments natural killer (NK) cell activity of large granular lymphocytes (LGL); these cells contain PTS and/or electron-dense granules. Normal peripheral blood mononuclear cells were incubated with IFN-alpha and surface antigen expression was measured by means of indirect immunofluorescence and, at the ultrastructural level, using gold labelled monoclonal antibodies. Surface antigen reactivity with the monoclonal antibodies OKT 3, 4, 8 and Anti-Leu-7 (HNK-1) showed no difference between the IFN-alpha incubation and non-IFN-alpha groups. However, electron microscope investigation revealed significant absolute increases in the percentage of OKT 8+ and Anti-Leu-7+ cells which were PTS-positive after IFN-alpha treatment compared with the control groups. The cytotoxicity assay using the K562 cell line showed enhanced lytic activity. Our results suggest that cells coexpressing the OKT 8 and Leu-7 antigens may be responsible for a minor proportion of the increase in PTS but that IFN-alpha mainly induces PTS and/or associated structures in cells which express the OKT 8+ antigen. These PTS+/OKT 8+ cells may contribute to enhanced cell cytotoxicity.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.     

Changes in purine nucleoside phosphorylase activity during thymosin-induced human null cell differentiation

Purine nucleoside phosphorylase (PNP) is a purine salvage pathway enzyme which we have found to be 8-10 times more active (per cell) in human peripheral blood null lymphocytes than in T lymphocytes. To test the hypothesis that null cells are, in part, pre-T lymphocytes we have defined an in vitro system for null cell differentiation into T cells and examined PNP activity during this differentiation process. We found that about 10% of human null cells could be driven to differentiate into T cells using thymosin fraction 5 (TF5) an extract of bovine thymus glands. The response to TF5 was dose related to up to 250 micrograms/ml with a maximum response occurring by 42-46 hr incubation. Exposure to TF5 was necessary for more than 4 hr but no more than 8 hr in order to obtain a maximum response. Both OKT4 and OKT8 positive cells were present in the newly differentiated T cell population but OKT8 positive cells appeared to predominate (OKT4/OKT8 = 0.698 +/- 0.30, mean +/- 1 SD). The differentiation process did not involve DNA synthesis but was inhibited at 4 degrees C. In the newly differentiated T cells PNP activity per cell was 8- to 10-fold lower (36 +/- 23 nm/hr/106 cells) than in null cells (311 +/- 136), and was at a level similar to mature T cells (56 +/- 7). Thus, human peripheral blood null cells can be induced to differentiate into T lymphocytes which can be characterized by both surface markers and biochemical parameters. Future studies will look at the function of TF5-induced T cells and the regulation of PNP activity during the differentiation process.     

A study of lymphocyte subsets in human normal tonsil and lymph node

A series of T and B lymphocyte specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulation in frozen and paraffin tissue sections of human normal tonsil and lymph node by means of immunocytochemical technique. In the paracortical and interfollicular area of tonsil and lymph node, most lymphocytes reacted with Leu 1, Leu 3 a, Leu 4 and OKT4. The numbers of Leu 2 a and OKT8 positive cells were rare in tissue. These cells were not only limited in paracortical area, they also appeared in considerable numbers in medullary cords of lymph nodes. Leu 2 a and OKT 8 positive cells decreased with prominent follicular hyperplasia of tonsils. In addition, substantial leu 3 a and Leu 4 cells were found in the germinal centers. This finding supports the importance of these lymphocyte subsets in regulation of human immune response. In the mantle zone of secondary follicles, the majority of lymphocytes were positive for OKB 2 and BA 1, whereas, the IgM positive cells were predominately observed in the cytoplasma and extracellular substance of B lymphocytes in the germinal centers, but the lymphocytes bearing sIgM were rarely observed. In the mantle zone, the IgM were frequently found on the surface of membrane of small lymphocytes, however, the staining intensity was much than that in the germinal centers.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.