T3 Antigen Research Articles

Induction of cytotoxicity by interleukin‐2 in tγ‐lymphoproliferative disorders

We have studied 7 patients with T gamma-lymphoproliferative disorders, in whom 78-88% of circulating nonadherent lymphocytes had the morphology of large granular lymphocytes (LGL) as assessed by light and transmission electron microscopy. The main common features of the membrane phenotype of these LGL expansions included expression of T3, HNK-1 and AB8.28. Other monoclonal antibody-defined surface markers of LGL (OKM1, B73.1, N901) were variably expressed or absent in these patients. Patients' LGL had little or no natural killer (NK) activity but mediated antibody-dependent cellular cytotoxicity (ADCC). Exposure to interferons (type B or gamma) for 20-72 hr resulted in no appreciable induction of cytolytic activity. In contrast, culture in the presence of interleukin-2 (IL-2) for 3 days resulted in the expression of strong cytolytic activity in all the patients tested against an NK-susceptible (K562) and an NK-resistant (Daudi) target. The expression of T3 antigen, the low levels or lack of native NK activity and the induction of consistent cytotoxicity by prolonged exposure to IL-2 led us to suggest that the cells expanding in these subjects are related to the effectors involved in lymphokine-activated killer (LAK) activity.

Evidence for a regulatory role of the T8 (CD8) antigen in antigen‐specific and anti‐T3‐(CD3)‐induced lytic activity of allospecific cytotoxic T lymphocyte clones

The functional role of the T8 antigen of human T cells was studied by inhibition with anti-T8 monoclonal antibodies (mAb) of the cytotoxic action of T8+ cytotoxic T lymphocyte clones (CTL). All clones were allospecific and directed against HLA-B7. The ability of seven different anti-T8 mAb to inhibit the cytotoxicity of these alloreactive CTL clones corresponded with their avidity for a particular target cell. The lysis of cross-reactive antigen-bearing target cells was more readily blocked by anti-T8 mAb than lysis of the specific B7 target cell against which a clone was raised. The seven anti-T8 mAb showed a spectrum of CTL blocking ability ranging from strong blocking with all five CTL clones tested to weak inhibition of only two out of five clones. mAb inhibition of CTL reactivity and cold target inhibition studies with one of the five CTL clones indicate a post-binding role of the T8 molecule. Functional epitope mapping based on CTL blocking with the anti-T8 mAb resulted in the definition of one nonfunctional epitope on the T8 molecule which is only expressed on mature T lymphocytes and a cluster of closely related functional epitopes expressed on both thymocytes and mature T lymphocytes. Not only allospecific cytotoxicity, but also nonspecific cytotoxicity induced anti-T3 mAb in these allospecific clones was inhibited by anti-T8 mAb in the absence of HLA class I expression on the target cell (Daudi cell line). The hierarchy of blocking with anti-T8 mAb and the classification of functional epitopes on T8 in anti-T3-induced nonspecific cytotoxicity were similar to those obtained in blocking of allospecific reactivity of the CTL clones. This analogy points to an identical function of the T8 antigen in both allospecific and anti-T3-induced nonspecific cytotoxicity. If HLA class I molecules are the counter structures of the T8 antigen, then these results argue against an adhesion-like function of the T8 structure. The combined results show that the T8 molecule has a regulatory role in CTL activation. It is postulated that the T8 antigen might serve as a receptor that transduces a negative feedback signal for T cell activation which prevents T cell triggering by nonspecific interaction.

Modulation of surface T11 molecules induced by monoclonal antibodies: analysis of the functional relationship between antigen‐dependent and antigen‐independent pathways of human T cell activation

Previous data indicated that T lymphocyte activation can be achieved by using a combination of anti-T11 monoclonal antibodies (mAb) directed to the "T11(2)" and the "T11(3)" epitopes, respectively. Unlike the T cell activation induced by antibodies directed to the T3-T cell receptor (Ti) complex or to T44 molecules, the anti-T11 mAb-induced cell activation was not accompanied by surface modulation of the T11 antigen. In the present study we show that appropriate stimulatory combinations of anti-T11 mAb are able to induce T11 antigen modulation in a variety of T cells including polyclonal peripheral blood populations, normal as well as leukemic (JA3) T cell clones. The first anti-T11 mAb combination leading to both cell activation and T11 antigen modulation was given by a mAb directed to the T11(2) epitope and by another mAb recognizing an epitope belonging to the T11(1) group. The second combination was given by two mAb directed against two different determinants of the T11(1) group. The ability to induce T11 antigen modulation allowed a more precise analysis of the pathway of T cell activation initiated by T11 molecules and its physical and functional relationship with the other known pathways of T cell activation. T cells following T11 antigen modulation failed to respond to subsequent stimulation with anti-T11 mAb. The refractory period lasted for 48-72 h and the restoration of the responsiveness to anti-T11 mAb coincided with the re-expression of T11 molecules at the cell surface. Modulation of T11 antigen did not affect the surface expression of T3, Ti or T44 molecules, in addition, "modulated" cells maintained their ability to respond to mAb directed against T3, Ti or T44 molecules. On the contrary, antibody-induced modulation of the T3-Ti receptor complex abrogated both T11- and T44-dependent T cell activation. Finally, antibody-induced modulation of T44 antigen did not inhibit either the T11- or the T3-Ti-dependent pathway of T cell activation. These data indicate that down-regulation of the pathway of T cell activation initiated by T11 molecules can be induced not only by modulation of the antigen receptor complex but also by appropriate mAb to T11 molecules and, presumably, by the natural ligand binding to T11 molecules.

Three distinct subpopulations of sheep T lymphocytes.

Monoclonal antibodies reactive with distinct T lymphocyte subpopulations have been described in man, mouse and rat and structural analyses of these antigens have demonstrated a high degree of evolutionary conservation. This report describes the reactivity of three monoclonal antibodies (mAb), 19-19, alpha SBU-T4 and alpha SBU-T8, which define T cell subpopulations in the sheep. The mAb alpha SBU-T4 and alpha SBU-T8 define the sheep CD4 and CD8 molecules, respectively. These two antigens show similar tissue distributions, molecular weights and fluorescence-activated cell sorter profiles to human, mouse and rat CD4 and CD8 molecules. The mAb 19-19 is reactive with a subpopulation of T lymphocytes which displays a tissue distribution unlike that reported for a T cell subset in any other species. 19-19 stains 7% of efferent lymph lymphocytes, 15% of peripheral blood lymphocytes but only 1-3% of lymph node lymphocytes. Two-color immunofluorescence demonstrates that the 19-19+ T cell subset is SBU-T4- and SBU-T8-, and thus defines a third T cell subpopulation in sheep. Immunohistology on frozen lymph node tissue sections localizes 19-19 mAb-reactive cells to the subcapsular region of the lymph node and lymph node trabeculae. Only 1% of thymocytes are 19-19+ and these cells are located mainly in the medulla and often arranged as foci around blood vessels. The 19-19 mAb immunoprecipitates from sheep lymphocytes an antigen with an apparent molecular mass of 215 kDa under both reducing and nonreducing conditions. It is concluded that alpha SBU-T4 and alpha SBU-T8 recognize the sheep homologues of the human T4 and T8 antigens, respectively, whereas 19-19 recognizes an antigen (termed SBU-T19) which has not been reported in any other species.

Heterogeneity of Human Natural Killer Cells

The cell surface antigens and specificity of cultures of human natural killer (NK) cells, propagated in the presence of interleukin 2, were analyzed at the single-cell level. With the use of a micropipette isolation procedure, clones were initiated from cells of defined phenotype. The cell surface markers on the resultant clones were not stable and often diverged from those on the cells selected for initiation. Clones manifesting NK activity had low expression of T4 and T8 and most had some expression of T10, whereas clones without detectable cytotoxic activity had a considerable proportion of cells expressing T4 and/or T8 antigens. Within a cytotoxic clone, single cells shown to have killer activity were shown to express often T10 and infrequently T8; T4 was not detected on any of the reactive cells examined. The NK, reactive cells were found to express also antibody-dependent cell-mediated cytotoxicity and lectin-dependent cell-mediated cytotoxicity (LDCC), and some of the individual NK cells were shown to have the ability to lyse more than one type of target cell. However, there appeared to be considerable heterogeneity among the cells in the clones, with only 50-60% of cells displaying lytic activity against K562 cells at a given time. In addition, one cytotoxic clone appeared to have specific immune alloreactivity as well as NK and LDCC activities. The results of this study indicated that the cell surface phenotype and specificity of cytotoxic reactivity of cultured human NK cells were heterogeneous and suggested that such heterogeneity may be due to variations in the levels of differentiation and/or activation.

Phenotypic characterization of individual interferon-gamma-producing cells after OKT3 antibody activation.

This is the first study using a direct approach to determine the phenotype of interferon gamma (IFN-gamma)-producing cells after polyclonal T cell activation. Blood mononuclear cells (MNC) were cultured and stimulated by OKT3 antibody to produce IFN-gamma. They were then stained with a panel of monoclonal antibodies with specificity for cell surface antigens, fixed and permeabilized in suspension and stained for cytoplasmic IFN-gamma using monoclonal antibodies and indirect immunofluorescence technique. IFN-gamma appeared mainly as a local, polar accumulation in the cytoplasm in a juxtanuclear position, probably identical to the Golgi apparatus. After a culture period of 20 h, 6-11% and 0.2-0.6% of the OKT3-activated MNC from adults or newborns, respectively, produced IFN-gamma in cultures. Unstimulated cultures contained maximally 0.1% IFN-gamma-positive MNC. The majority of the IFN-gamma-producing MNC were T cells and expressed the T11 antigen and receptors for interleukin 2. To our surprise most of these T cells also carried receptors for complement 3bi (OKM1) but did not express class II molecules. Less than 50% displayed T4 or T8 antigens in three out of the four experiments performed. Natural killer cells, as recognized by Leu 7 or B73.1 antibodies, contributed only marginally to the IFN-gamma synthesis whereas B cells (OKB7) and monocytes (Leu M3) showed no production of IFN-gamma.

Rearrangement of immunoglobulin heavy chain genes and t3 expression in the absence of rearrangement of t-cell receptor β-chain gene in a patient with t-cell malignant lymphoma

We describe the rearrangement of immunoglobulin genes and T3 expression in the absence of rearrangement of T-cell receptor β-chain genes in a patient wih T-cell malignant lymphoma. He had a mediastinal mass and his lymphoma cells expressed T-cell antigens (OKT3+, OKT9+, and OKT10+). When we examined genomic DNA from the lymphoma cells, we detected the rearrangement of immunoglobulin heavy chain genes with a germ-line configuration of light chain genes and no rearrangement of T-cell receptor β-chain gene. These results indicated that the rearrangement of immunoglobulin genes could occur in T-cell malignant lymphoma, and that T3 antigen could be expressed prior to the rearrangement of T-cell receptor β-chain genes under certain circumstances.

Differentiation antigens on rhesus monkey lymphocytes II. Characterization of RhT3, a CD3‐like antigen on T cells

A monoclonal antibody FN18 is described, which is specific for mature rhesus monkey T lymphocytes. It defines a cell surface antigen, composed of two polypeptide chains with a molecular mass of 22 and 27 kDa. In view of these and other similarities with the human T3 or CD3 antigen, it was designated as RhT3. Expression, distribution and certain functions of RhT3 were compared with those of its human counterpart. Analogous to anti-CD3 antibodies in man, FN18 is able to modulate its target antigen, has mitogenic properties and is able to block the pokeweed mitogen and concanavalin A-driven cell proliferation, but not that caused by phytohemagglutinin. In spite of such minor dissimilarities the available data strongly suggest that the RhT3 antigen is the rhesus monkey's homologue of the human CD3 antigen.

NK‐9, a distinct sialylated antigen of the T200 family

The nature and properties of antigens detected by a novel monoclonal antibody, NK-9, were studied. The NK-9 antigens had apparent molecular masses of 190, 200 and 220 kDa and were sensitive to neuraminidase and sodium metaperiodate treatments, which destroy the sialic acid residues of the cell surface glycoproteins. Trypsin treatment also removed the NK-9 reactivity, but the antigens were restored within a few hours thereafter. Tunicamycin, which inhibits the N-linked glycosylation after neuraminidase treatment, had no effect on the reappearance of the NK-9 positivity. Neither did endoglycosidase F, which removes the N-linked sugars, abolish the NK-9 antigenicity. Monensin, which blocks the cellular secretion, inhibited the restoration of the antigens, and monensin block also without preceding treatment with neuraminidase led to the disappearance of NK-9 reactivity, suggesting possible recycling molecules as carriers of the NK-9 detected epitopes. The NK-9 antigens appear to belong to the T200 antigen family, but are distinct from the antigens reactive with the available anti-T200 antibodies anti-LC, T29/33 and HLe-1, based on their different cell type distribution and absence of cross-reactivity in sequential immunoprecipitations.

Discrete stages of human thymocyte activation and maturation in vitro: correlation between phenotype and function.

Using light scatter, flow cytometric and functional analyses, three major stages in the in vitro activation of human thymocytes were defined. The earliest stage (day 0-2) was characterized by the induction of the interleukin 2 (IL2) and transferrin receptors, the T cell lineage specific Ta1 antigen, and increased reactivity with anti-T8 antibody. At this time, major changes in nuclear morphology but not cell size were observed. Early-stage thymocytes were immature with regard to T3 and cytotoxic capacities. The second stage (day 3-4) of in vitro culture was distinguished by loss of T6, maximal activation (both in cell size and nuclear morphology) and maximal expression of both transferrin and IL2 receptors. At this stage, nearly all thymocytes expressed T3, 30-70% of thymocytes were T4+T8+, and functionally, only small increases in cytotoxic capacities were observed. The third stage of maturation (day 5-7) represented thymocytes with reduced levels of activation as measured by forward and right angle light scatter analysis and declining IL2 and transferrin receptor expression. However, these thymocytes exhibited high levels of T3 antigen density, loss of T4, T8 coexpression and pronounced cytotoxic and detectable inducer function capabilities.

Immunological and genotypic analysis of human T gamma-lymphoproliferative disorders.

T gamma-lymphoproliferative disorders (T gamma-LPD) are rare diseases characterized by expansion of circulating elements with resemblance to large granular lymphocytes (LGL). We have studied 12 patients with T gamma-LPD. Morphological evaluation revealed 79-88% of LGL in non-adherent peripheral blood lymphocytes as assessed by light and electron microscopy. The most common features of the membrane phenotype included expression of T3, HNK-1 and AB8.28 (anti-Fc gamma); other surface markers of LGL (OKM1, B73.1, N901) were variably expressed or absent. Patients' LGL usually had little or no NK activity, with the exception of two patients who had values comparable to those of normal donors; in addition, cell preparations from all patients mediated antibody-dependent cellular cytotoxicity. The recent availability of the T cell receptor beta chain probes allowed us to investigate the lineage and the clonality of T gamma-LPD. Of the 12 patients analyzed, 10 displayed clonal rearrangements of T beta locus and expression of the T3 antigen, whereas the two remaining cases displayed a germ-line configuration of the T beta gene and no expression of the T3 antigen. We suggest that individual T gamma-LPD cases represent the clonal expansion of cells frozen at different stages of differentiation/activation within an individual hematopoietic LGL/NK lineage. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage.

T cell activation by anti-T3 antibodies: comparison of IgG1 and IgG2b switch variants and direct evidence for accessory function of macrophage Fc receptors.

The human T3 antigen is closely associated with the T cell receptor. Some anti-T3 antibodies cause T cell proliferation in the presence of monocytes which have Fc receptors (FcR) that bind particular antibody subclasses. Such an interaction is thought to determine whether or not an anti-T3 antibody is mitogenic. We examined the mitogenicity of an IgG1 antibody, UCHT1, and an IgG2b switch variant of identical specificity, UCHT1B. With autologous monocytes, 76% of individuals responded to UCHT1 and 9% to UCHT1B, falling into three patterns of responsiveness. Both antibodies in the absence of monocytes induced responsiveness to recombinant interleukin 2, even for UCHT1B nonresponder T cells. The proliferation induced by UCHT1B, however, was always less than that induced by UCHT1. These findings demonstrate the critical role played by the Fc region for mitogenesis, and suggest a possible role for the hinge region. We then obtained direct evidence that mitogenicity can be mediated exclusively via FcR. Mouse macrophages have distinct FcR: FcRI binds IgG2a but FcRII binds IgG1 and IgG2b and its function can be inhibited by the specific antibody 2.4G2. Because UCHT1 and UCHT1B were of the correct subclass to interact with FcRII we examined the accessory function of mouse peritoneal macrophages. Without exception, human T cells now responded to both antibodies. Proliferation was drastically inhibited by 2.4G2 but not by an irrelevant anti-macrophage antibody, F4/80, nor by an anti-human neutrophil FcR antibody, 3G8. Furthermore, 2.4G2 did not inhibit the accessory function of mouse macrophages for OKT3, an IgG2a antibody that presumably interacts with FcRI, and did not inhibit the function of human monocytes for UCHT1 and UCHT1B. Mouse B cells, in contrast to macrophages, have an FcR which binds all three subclasses, but which can be inhibited by 2.4G2. B cells, however, were not accessory cells for mitogenesis with UCHT1, UCHT1B or OKT3. These findings are discussed in relation to other requirements for T cell activation by anti-T3 antibodies.

Immunotoxins show rapid entry of diphtheria toxin but not ricin via the T3 antigen.

We compared immunotoxins made with ricin and diphtheria toxin (DT) and with two monoclonal antibodies against different T cell-specific antigens, CD5 (T101) and CD3 (UCHT1). Only one reagent, UCHT1 linked to DT (UCHT1-DT), had exceptional properties. UCHT1-DT killed human peripheral T cells and T leukemia cells (Jurkat) at 2 to 10 pM, a concentration 10- to 100-fold lower than UCHT1-ricin and 10 to 500 times lower than native DT. The toxicity was blocked 50- to 100-fold by excess UCHT1 antibody. Human multipotent stem cells were not killed at up to 2000 pM UCHT1-DT. UCHT1-DT shows greater selectivity between T cells and stem cells than UCHT1-ricin, and may better prevent graft-vs-host disease in allogeneic bone marrow transplantation. The kinetics of UCHT1-DT were extremely rapid. UCHT1-DT inhibited Jurkat cell protein synthesis faster than DT and had a different ratio of lag time to inactivation rate. UCHT1-DT killed 90% of Jurkat cells within 2 hr at concentrations nontoxic to human stem cells. In contrast, UCHT1-ricin required more than 18 hr to kill one log of Jurkat cells. Another monoclonal antibody, T101, against the 65 kD CD5 antigen on Jurkat cells was linked to DT and ricin, and was compared with the UCHT1 immunotoxins. UCHT1-DT was 100 times more potent and five to 10 times faster than T101-DT and T101-ricin. Standardization to other antibodies with regard to the number of molecules bound per cell shows that UCHT1-DT is 10 to 100 times faster than previously reported immunotoxins. The role of the T3 antigen in transporting DT to the cytosol is discussed.

Differentiation of human mature thymocytes: existence of a T3+4-8- intermediate stage.

A T3 complex-bearing subpopulation was characterized within an in vivo cycling T4-8- early thymocyte compartment which contains cells constitutively expressing interleukin 2 and transferrin receptors. We show differentiation in vitro of both mature subsets of thymocytes (T3+4+8- and T3+4-8+) from the above T4-8- compartment, their appearance being preceded by cells in a T3+4-8- intermediate stage. Furthermore, those mature thymocytes generated in vitro contain functionally competent cells which use T3, T4 and T8 structures for their cytolytic activity. The finding of T3+4-8- thymocytes in vivo, together with the observation that T3 antigen expression precedes that of T4 or T8 molecules in vitro, shows that T3 (and presumably Ti) is present early in ontogeny, and suggests that T3+4-8- cells constitute an "intermediate" stage relevant to the connection between early precursors and mature thymocytes during T lymphocyte ontogeny.

Functional surface structures on human natural killer cells

This manuscript reviews recent studies on the characterization of functional surface antigens on human NK cells. A series of cloned NK cell lines has been utilized for examination of these structures. These clones provide a relatively large number of cells with a stable phenotype and consistent specific cytotoxicity, which reflect the diversity of uncultured NK cells in normal peripheral blood. Almost all clones express the T11 antigen, some have a mature T-cell phenotype (T3+, T11+), and only one (JT1) does not reveal any T-cell antigen at all (T3-, T11+). Using NK clones to generate monoclonal antibodies specific for NK-associated antigens, two structures have been identified, NKH1 and NKH2. NKH1 appears to be exclusively expressed on large granular lymphocytes (LGL) of peripheral blood and was found to be a pan-NK cell antigen. NKH2 is also expressed primarily by LGL, but NKH2-positive LGL do not display a high level of NK activity. Another surface structure that has been found to play an important role in NK cell function is the T11 antigen/E rosette receptor complex, which is expressed in 80% of peripheral blood NK cells. The T11 antigen complex has been described as possessing the T11(1), T11(2), and T11(3) antigens and is an important alternate pathway for antigen-independent T-cell activation. Using anti-T11(2) and anti-T11(3) monoclonal antibodies, IL-2 receptor expression could be induced on various NK clones if they expressed the correct T11 antigenic epitope. As anti-T11 2/3 antibodies had a direct proliferative effect on NK cells with mature T-cell phenotype (T3+), it is proposed that the production of IL-2 by NK clones is largely dependent on the T-cell phenotype of NK cells. All NK clones expressed IL-2 receptor at low density and therefore needed a ten fold higher concentration for maximal proliferation than T-cell clones. For some T-cell-like NK clones, the T3 antigen complex and a T-cell receptor-like structure, NKTa or NKTb, have been shown to define the target cell specificity. The activation antigen, TNKTAR, was characterized as the recognition structure on the target cell for these NK cells. For both T3- and T3+ NK clones, the LFA-1 antigen has been shown to play an important role in effector/target cell interaction. As previously described for CTL, the LFA-1 molecule is involved in NK cytotoxicity as a nonspecific adhesion-strengthening molecule at the effector cell level.(ABSTRACT TRUNCATED AT 400 WORDS)

Failure of Histiocytosis X Cells to Express i Blood Group Antigen

Expression of HLADR, I, i blood group antigen and T6 antigen were studied in Histiocytosis X cells and pulmonary alveolar macrophages using double labelling immunofluorescence technique or immuno-peroxidase procedure. Alveolar macrophages express simultaneously HLADR and i blood group antigen. Histiocytosis X cells, characterized by HLADR and T6 antigens, and by their ultra-structural marker do not express i antigen. These results confirm the hypothesis that histiocytosis X cells constitute a specialized sub-population of the mononuclear phagocyte system.

Cell surface molecular changes on the activation of human thymocytes.

The changes in the expression of antigen molecules on the cell surface membranes of uncultured (nonactivated) and activated human thymocytes have been studied by flow cytometry and immunoprecipitation techniques. Nonactivated thymocytes do not have the phenotypic profile of a resting population because they express cell proliferation molecules such as the transferrin receptor and the 4F2 antigens (mainly the 100,000 dalton subunit). After activation with IL 2-containing supernatants, mature T3+, T6- thymocytes proliferate and are able to nonspecifically kill different target cells. The activated thymocytes are T3+, T11+, T6-, OKM1- and bear T4 or T8 antigens in mutually exclusive cell subpopulations. They also "de novo" express the IL 2 receptor, and the 210.000/130.000 molecular complex defined by the TS2/7 MAb. Activated human thymocytes express higher amounts of class I and class II MHC antigens, equal T3 and LFA-1, and lower quantities of T11 and T4 molecules than nonactivated thymocytes. Furthermore, activated thymocytes only express the T8 34,000 dalton polypeptide subunit, whereas the nonactivated thymocyte population expressed the T8 34,000 dalton associated with a 46,000 glycoprotein. We have demonstrated that this structural change in the T8 molecule from a complex of two polypeptide subunits of 46,000 and 32,000 does not indeed occur in the activation process but rather in the maturation from T6+ to T6- thymocytes.

T cell receptor beta chain gene rearrangements in lymphoproliferative disorders of large granular lymphocytes/natural killer cells.

Twelve cases of T gamma LPD (lymphoproliferative disorders of Fc gamma receptor-bearing T cells) involving an expansion of large granular lymphocyte/natural killer (LGL/NK) cells were investigated for the expression of LGL/NK-associated markers and for T beta gene rearrangement. All the cases selected were classified as T gamma LPD on the basis of morphology, function, and phenotype of the circulating cells. 10 to 12 cases displayed clonal rearrangements of the T beta locus and expression of the T3 antigen, whereas the 2 remaining cases displayed the germline configuration of the T beta gene and no expression of the T3 antigen. T8, Mol, B73.1, and N901 antigens were variably expressed among both T beta+T3+ and T beta-T3- T gamma LPD cases. We suggest that individual T gamma LPD cases represent the clonal expansion of cells frozen at different stages of differentiation/activation within an individual hematopoietic LGL/NK lineage.

Analysis of human bone marrow with monoclonal antibodies.

In order to study the antigenic phenotype of different hemopoietic cells, we used a series of monoclonal antibodies to investigate normal bone marrow in a standard immunofluorescence assay. The antibodies detected the following antigens: HLA-ABC, beta 2-microglobulin (beta 2m), HLA-DR (Ia), a lymphocyte subset and specific antigen (T and B) HuLy-m2, m3, T lymphocyte antigen (HuLy-m1), lymphocyte T200 antigen (HuLy-m4), a viral-associated antigen (HuLy-m5), and platelet-specific glycoproteins IIb-IIIa (HuPl-m1). The following results were obtained: (a) normoblasts were weakly HLA-ABC+, beta 2m+ and Ia-; all other lymphocyte and platelet antigens were not detected. (b) Myeloid cells at all stages of differentiation (promyelocytes, myelocytes, metamyelocytes, and neutrophils) were HLA-ABC+; beta 2m+; HuLy-m1-, m2-, m3+/- (20%), m4+, m5+/- (20%); HuPl-m1-; in addition, promyelocytes and myelocytes were Ia+ but neutrophils and metamyelocytes were Ia-. (c) Lymphocytes were HLA-ABC+, beta 2m+, Ia+/- (20-30%), HuLy-m1+/- (40-50%), m2+/- (60-70%), m3+, m4+, m5+; Pl-m1-. (d) Platelets and megakaryocytes were HLA-ABC+; beta 2m+; Ia-; HuLy-m1+-, m2-, m3-, m4-, m5-, HuPl-m1+, and the putative "megakaryocyte precursors" were HuPl-m1+, Ia-, HuLy-m1-. The different cell types in bone marrow could readily be distinguished, particularly cells of the myeloid series (Ia and HuLy-m4, m5), lymphocytes (Ia and HuLy-m1, m2, m3), and platelets and their precursor cells (HuPl-m1). This simple method of defining cellular phenotypes in bone marrow has demonstrated the practicality of using monoclonal antibodies to identify marrow cells and should be of diagnostic value.

A late-differentiation antigen associated with the helper inducer function of human T cells.

T lymphocytes possessing helper function produce soluble factors that greatly augment B-cell proliferation and differentiation into antibody-secreting cells. In humans the subset of T lymphocytes bearing the T4 surface antigen comprises most of the cells that display helper activity and recognize class II antigens of the major histocompatibility complex (MHC), while the subset bearing the T8 antigen comprises T cells recognizing class I MHC antigens and exhibiting cytotoxic or suppressor function. Monoclonal antibodies to T4 or T8 greatly inhibit the cognitive and effector function of cells with the corresponding phenotype. This function/phenotype correlation is not absolute, however, for there are many examples of T8-positive clones that recognize MHC class II antigens and have helper activity, as well as of T4-positive clones with suppressor or cytotoxic function. Recently a family of cell-surface neoantigens, which might be relevant to T-cell function and which are present on activated but not on resting T lymphocytes, has been identified in mouse and humans using monoclonal antibodies. Some of these antibodies block the cytolytic activity of alloreactive T-cell clones, suggesting the possible involvement of such molecules in the activation of cytotoxic T-cell clones or in the lytic process itself. We now describe a similar late-differentiation antigen (LDA1) that is expressed by human T lymphocytes only following activation and is recognized by a monoclonal antibody that inhibits the antibody-inducing helper function of T lymphocytes.