人体正常扁桃体和淋巴结T、B淋巴细胞亚群的研究

本文用了一系列T、B淋巴细胞单抗,用免疫细胞化学技术观察了人淋巴结和扁桃体内T,B淋巴细胞及其亚群。T细胞及其亚群主要位于滤泡间的副皮质。此外在淋巴结髓质也有一定数量的T细胞亚群的分布;次级滤泡生发中心也常出现辅助T或Leu 4阳性T细胞。B淋巴细胞及其亚群多集中在初级和次级滤泡,如OKB_2和BA 1阳性细胞多集中于次级滤泡的帽状区,而IgM主要位于次级滤泡的生发中心。LN-2抗体选择性地与生发中心??和帽状区的B细胞的核膜起反应,是研究B细胞表型的一种重要试剂。     

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.     

Characterization of human large granular lymphocyte subpopulations: comparison of the phenotype of NK cells and of interleukin 2-dependent progenitors of cytolytic effector cells

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.     

Analysis of lymphoid and dendritic cells in human lymph node, tonsil and spleen. A study using monoclonal and heterologous antibodies

The distribution of lymphoid and dendritic cells in human reactive lymph nodes, tonsils and spleens was examined by means of an indirect immunoperoxidase technique, using a panel of monoclonal and heterologous antibodies. The antibodies used were directed against antigens present on T cell subsets (Leu1, leu2a, Leu3a, TA1, OKT6), various types of B cells (BA1, BA2, HLA-DR, CR1) and cells of the mononuclear phagocyte system (alpha HM1, TA1, CR1, OKM1, NA 1/34). In the lymph node and tonsil Leu3a-positive cells (T-helper/inducer phenotype) and Leu2a-positive cells (T-suppressor/cytotoxic phenotype) are found in the thymus-dependent or T-cell area; in the spleen Leu3a-positive cells are found mostly in the periarteriolar lymphocyte sheath (PALS), while Leu2a-positive T-suppressor/cytotoxic cells are almost completely restricted to the cords of Billroth in the red pulp. The cells in the mantle zone of germinal centres and in the primary follicles in lymph nodes, tonsils and spleens have B-cell properties (BA1-, HLA-DR-, and CR1-positive). The cells in the germinal centres show a similar staining pattern (HLA-DR-, and partly CR1-positive). Follicles and T-cell-dependent areas have specific dendritic cells, each with a specific staining pattern: the dendritic reticulum cell (DRC) of the follicle stain with CR1, HLA-DR, BA2 and alpha HM1; the interdigitating cell of the T-cell areas in the lymph node, tonsil and spleen stain with HLA-DR and BA1. Moreover, large dendritic OKT6-positive cells are found in the T-cell areas of some of the peripheral lymph nodes, and are probably Langerhans cells. It is concluded that human lymph nodes and tonsils have an identical compartimentalisation, clearly differing from the spleen in cellular organization.     

Human interferon-gamma (IFN-gamma) containing cells bear various surface phenotypic markers

We explored the population of Interferon-gamma (IFN-gamma) containing cells in order to clarify their cell surface phenotypic markers. Here we define gamma-IFN containing cells as gamma-IFN plaque forming cells (PFC). By this method, it was found that IFN-gamma containing cells consist of two cell fractions, i.e., OKT3+, OKT4+, and OKT8- cells and OKM1+ cells. Effective IFN-gamma production seems to require participation of plastic-adherent cells (presumably monocytes), while the addition of cyclosporin A (CyA) almost completely blocked generation of human IFN-gamma. To characterize Con A-stimulated IFN-gamma containing cells, we performed two-color flow cytometry using FACS IV. Most of the IFN-gamma containing cells have surface phenotypic markers for Leu3, Leu8, Leu15, HLA-DR, and IL-2 receptors, but most lack markers for Leu2 and Leu7. Interestingly, most of Leu3+ and IL-2 receptor+ cells belong to the dimly illuminating cell fractions of the IFN-gamma containing cell population. Our results indicate that IFN-gamma containing cells are heterogeneous with respect to surface phenotypic markers but the predominant IFN-gamma containing cell type is the helper T cell (OKT4+). Lastly, OK432, glycyrrhizin, and CCA (lobenzarit disodium) increase the number of IFN-gamma containing cells and are thought to be immunomodulators.     

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.     

Homologous human macrophage hybridomas that produce a novel cytotoxic factor in their culture supernatants

Homologous human macrophage hybridoma cell lines were obtained by somatic cell fusion between peripheral blood monocyte-derived macrophages and a subclone of the myelomonocytic cell line, U937-F9. The hybridoma cell lines grown in vitro for more than a year were confirmed by manifestations of phagocytosis, adherence, nonspecific esterase, acid phosphatase, chromosome numbers and other cell surface antigens. Cell surface antigens on hybridomas were detected by flow cytometry analysis with monoclonal antibodies. With interclonal differences, a typical phenotype of hybridoma cells was CDw14+, OKM5+, Mac-1+ (equivalent to OKM1 and Mol), OKT9+, HLA-DR- and CD20+. After stimulation with lipopolysaccharide and calcium ionophore A23187, culture supernatants of clones c18A and c29A showed cytotoxic activity against human melanoma A375 Met-Mix and other cell lines which were resistant to the tumor necrosis factor, lymphotoxin and interleukin 1. This cytotoxic factor was found to be distinct from the tumor necrosis factor, lymphotoxin and interleukin 1 using the anti-tumor necrosis factor, anti-lymphotoxin and anti-interleukin 1 antisera.     

Abnormal expansions of granular lymphocytes: reactive lymphocytosis or chronic leukemia? Case report and literature review

A case of chronic lymphoproliferative disorder is presented, wherein a morphologically homogeneous population of lymphoid cells displayed properties similar to those described for large granular lymphocytes (LGL). Besides their LGL-like phenotype (VEP 13+, OKM 1+, OKT 10+ Fc-IgG-receptor+, OKT 3-), the proliferating cells were cytotoxic to NK targets as well as to antibody-coated target cells. Clinically, our patient presented low-grade lymphocytosis, splenomegaly, neutropenia, hyperimmunoglobulinemia and recurrent infections. Based upon this and 32 similar cases reported in the literature, we conclude that lympho-proliferative disorders involving GL encompass a variety of clinical entities, ranging from reactive GL lymphocytoses to overt lymphocytic malignancies.     

Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

Study of the cellular origin of histamine release inhibitory factor using highly purified subsets of mononuclear cells

We have recently described a specific antagonist of histamine-releasing factors that inhibits histamine release from basophils and mast cells. This histamine release inhibitory factor (HRIF) is produced by PBMC upon stimulation with histamine as well as mitogens such as Con A. The objective of this study was to investigate the cellular origin of HRIF produced by PBMC. Monocytes, T cells, and B cells were isolated to 96 to 99% purity by a combination of plastic adherence, E rosetting, and negative selection with mAb (OKM1, OKT11, OKB7, OKT4, and OKT8) and C. Purified subpopulations were cultured with histamine or Con A and then the processed supernatants were assayed for the inhibition of HRF-induced histamine release from basophils. The results of this study suggest that the highest amount of HRIF is synthesized by B cells followed by T cells and monocytes. The B cell origin of HRIF was confirmed by abolishing the activity after incubation of the cells with OKB7 mAb and C. Both CD4- and CD8- T cells are capable of producing HRIF. In mixing experiments, the synthesis of HRIF by two different subpopulations has been less than additive. T + B cells produced most of the HRIF activity. Monocytes tended to suppress the synthesis of HRIF by B cells. The synthesis of HRIF by so many cell types suggests that a fine balance between HRIF and HRF may regulate the mediator release from basophils and mast cells.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Cellular origin of histamine-releasing factor produced by peripheral blood mononuclear cells

Histamine releasing factors (HRF) are a group of cytokines that release histamine and other mediators from mast cells and basophils. It has been speculated that HRF might play a major role in the pathogenesis of allergic diseases. Most investigators have studied PBMC as a source of HRF. This study was undertaken to investigate the cellular origin of HRF. Peripheral blood was processed to isolate and purify monocytes, T cells, CD4- T cells, CD8- T cells and B cells by using plastic adherence, 2-aminoethylisothiomonium-treated SRBC rosetting and negative selection with the use of mAb OKM1, OKT11, OKT8, OKT4, and OKB7 plus C. Highly purified subpopulations of PBMC were cultured alone or in the presence of Con A for 24 h. Supernatants were harvested, dialyzed, and assayed for HRF activity in the basophil histamine release test. We found that all subpopulations of PBMC including T cells, CD4- T cells, CD8- T cells, B cells, and monocytes produce variable quantities of HRF. The spontaneous production is very high in B cells but only barely measurable in T cells and monocytes. The synthesis of HRF by B cells was confirmed by abolishing the release of the activity after treatment of B cells with OKB7 mAb and C. Stimulation of cell populations by Con A significantly enhances HRF production by PBMC and T cells but not by B cells and monocytes. In mixing experiments, unstimulated monocytes + B cells showed synergism, but other combinations demonstrated an additive effect. This is the first demonstration of HRF production by human peripheral blood B cells. The results of this study also suggest that histamine releasing cytokines are of multiple cellular origin. This perhaps contributes to their molecular heterogeneity.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

Natural anti-bacterial activity against Salmonella typhi by human T4+ lymphocytes armed with IgA antibodies

Peripheral blood mononuclear cells from normal volunteers possess natural anti-bacterial (NA) activity against S. typhi that can be measured in a 2-hr in vitro assay. Employing fractionation on nylon wool columns, Percoll gradients, plastic adherence, and E rosetting, the effector cell of NA activity appeared to be a lymphocyte of the T lineage rather than a macrophage, a B lymphocyte, or a large granular cell. Moreover, complement-dependent killing with monoclonal antibodies such as OKM1, OKB7, OKT8, 5.9 and the anti-natural killer cells AB8.28 did not reduce NA activity. On the contrary, this was completely inhibited when OKT3, OKT11, or OKT4 antibodies and complement were used to pretreat the effector lymphocytes. Indeed, T4+ cells sorted with a FACS displayed an extremely high NA activity against S. typhi. By pretreatment of peripheral lymphocytes with F(ab')2 fragments against human IgA, the NA activity was blocked. It is therefore suggested that NA activity by human cells might be a mechanism of defense against infections, acting as antibody-dependent cellular cytotoxicity expressed by T4+ lymphocytes coated with preexisting anti-Salmonella IgA antibodies.     

Value of lymphocyte marker studies in diagnostic cytopathology

The separation of lymphoreticular neoplasms from poorly differentiated epithelial and mesenchymal tumors and reactive processes can be a difficult problem in cytopathology. Immunodiagnostic techniques can be applied to cytologic specimens to detect cellular antigens, which may aid in their proper identification. We have reviewed 67 cytologic specimens in which immunoperoxidase techniques were employed using antibodies to common leukocyte antigen (HLE1), B-cell markers (B1, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, Leu 12 and kappa and lambda light chains), T-cell markers (Leu 1, OKT11, OKT4 and OKT8) and monocytes (OKM1 and LeuM1). These specimens included 33 body cavity fluids (21 pleural, 8 ascitic and 4 pericardial), 22 cerebrospinal fluids (CSF) and 12 fine needle aspirates (4 brain, 1 adrenal, 2 liver, 1 kidney, 3 retroperitoneal masses and 1 lymph node). The marker studies confirmed the initial cytomorphologic diagnoses in 31 specimens and modified the final diagnoses in 16 specimens. Markers in 20 specimens were noncontributory due to low cellularity or technical difficulties. Two problems may limit the usefulness of these procedures. First, many of the CSF specimens contained too few cells for adequate processing. Second, the mesothelial cells from pleural specimens often stained with HLE1. Our findings indicate that marker studies are of value in the diagnosis of problematic cases presenting as undifferentiated tumors in cytopathology.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Effect of tissue processing on the immunostaining of inflammatory cell surface epitopes identified by monoclonal antibodies

Optimal tissue processing conditions were defined for the immunohistochemical detection of inflammatory cell surface epitopes identified by OKT3, OKT4, OKT6, OKT8, OKIa1, OKM1, Leu7 and pan B cell antibodies. Snap-freezing in isopentan was superior to liquid nitrogen in preservation of morphology. Embedding of the tissues in Tissue-Tek II O.C.T. Compound diminished the intensity of immunostaining with all antibodies studied; however, the embedded tissues tolerated longer storage without drying. Optimal fixation with satisfactory preservation of morphology and immunogenicity was achieved with fixation of the frozen sections in acetone at 4 degrees C for 5 min. Blocking of the endogenous peroxidase with methanol-H2O2 treatment destroyed all epitopes studied except those identified with OKIa1, OKT6 and Leu7.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

Improved Immunohistochemical Visualization of Helper/Inducer T-Cells by the Simultaneous Application of two Noncrossblocking Monoclonal Antibodies

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.