T3 Antigen Research Articles

Relationship Between T200 Antigen Expression and Stages of B Cell Differentiation in Resurgent Hyperplasia of Bone Marrow

AbstractUsing monoclonal antibodies (MoAbs) and dual-parameter flow cytometric techniques, bone marrow mononuclear cells (MMC) from patients with resurgent hyperplasia were analyzed for their coexpression of HLe-1 (T200) and antigens normally associated with particular stages of B cell differentiation. The marrow from those with resurgent hyperplasia contained increased numbers of B cell precursors in multiple stages of differentiation compared to controls, thus providing a useful model system for studies of B cell differentiation. These studies indicate that the quantitative expression of T200 is differentiation-related on normal and malignant B cells and B cell precursors. Immature cells express low amounts of T200, while increasing levels of maturity correlated with increasing amounts of the antigen. This study increases the understanding of relationships between B cell surface antigens and T200 and further demonstrates that B cell hyperplasia occurs commonly in association with bone marrow reactive or resurgent processes. The quantitative, rather than only the qualitative, expression of T200 is therefore a useful marker of B cell differentiation in reactive hyperplasia and in further investigation of B cell malignancy.

Induction of an Immature T-Cell Phenotype in Malignant Helper T Cells by Cocultivation With Epidermal Cell Cultures

The possible inductive effect of epidermal cells on T-cell maturation has been examined employing an in vitro cocultivation technique. Mononuclear cells from 6 patients with cutaneous T-cell lymphoma (CTCL) and from 12 healthy volunteers were studied. In the 6 CTCL patients, all showed an expansion of the helper T-cell subpopulation and in one patient with leukemic CTCL, there was almost complete replacement of peripheral blood mononuclear cells by malignant cells with a helper T-cell phenotype. Epidermal cells derived from normal human skin were cultured to confluent monolayers, and were cocultivated with the mononuclear cells from CTCL patients or normal controls for 48 h at a density of 10(6)/ml. Following cocultivation, the surface phenotype of the cells from the 12 healthy volunteers and 5 of the patients with CTCL showed no significant phenotypic change. In the patient with leukemic CTCL, however, the surface phenotype of the malignant T cells had changed, with the acquisition of the T6 antigen by the majority of the cells. Cells cocultivated in medium alone and with human fibroblast monolayers showed no change in surface phenotype. The malignant T cells from the leukemic CTCL patient failed to react in a mixed lymphocyte culture to lymphocytes from 2 different healthy donors, and showed no phenotypic change following culture with these lymphocytes, indicating that the phenotypic change seen was not due to allogeneic stimulation.

Recognition of a Purified <italic>M</italic><sub>r</sub>-40,000 Organ-Specific Immunogen of Human Lung Cancer by Class II-Restricted T-Cells<xref ref-type="fn" rid="FN1">2</xref>

An Mr-40,000 polypeptide (p40) was purified from a lung cancer cell line on the basis of its antigenicity with leukocytes from lung cancer patients in an in vitro immunological assay of leukocyte adherence inhibition. Here, the cells and mechanism responsible for recognizing the purified p40 organ-specific cancer neoantigen (OSN) were studied. Buffy coat leukocytes from lung cancer patients showed a bell-shaped dose response with a peak response at 0.75 micrograms/assay. Mononuclear cells showed a similar response pattern, whereas pure T-cells were unreactive. Monoclonal antibodies (MAbs) to T-cell differentiation antigens T3 and T4 inhibited the response in a dose-dependent fashion, whereas anti-T8 had no effect, indicating T helper cells and their T3-antigen receptor complex recognized the antigen. MAbs to class II major histocompatibility complex (MHC) antigens also inhibited the response, whereas MAbs to class I MHC had no effect, indicating an important role for class II MHC antigens of monocytes. None of the MAbs inhibited the response to OSN on membrane fragments, which is mediated by antibody-dependent monocytes. Trypsin- or cyanogen bromide-cleaved p40 OSN triggered a response at the same concentrations as the intact molecule. The p40 OSN incubated with live leukocytes showed less than 30% proteolytic digestion. The results indicate that class II-restricted T-cells recognize, via their antigen-specific cell surface receptors, contiguous sequences within the immunogenic tumor molecule in the context of the molecule or peptides binding to class II transplantation antigens.

Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

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 CrCl 3. 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.

Keratinocyte T6 antigen expression after PUVA therapy

We report T6 antigen expression on keratinocytes in 11 cutaneous T lymphomas treated by PUVA therapy. This staining was absent before treatment. T6 reactivity was strictly limited to cell membrane. The nature of this expression is discussed, and it is suggested that it could be attributed to a passive diffusion from Langerhans´s cells.

Anti-clonotypic autoantibodies in pregnancy

Idiotypic T-cell receptors for antigen have been identified in previous studies by use of anticlonotypic monoclonal antibodies. To determine whether anti-receptor autoantibodies play a role in immune regulation, we have used normal pregnancy as a model. T-cell clones were generated from the peripheral blood of a primiparous woman by priming her lymphocytes to stimulating cells from her husband. As a control, T cells primed to lymphocytes from HLA different controls were used. Purified IgG was prepared from this woman's serum and tested for its reactivity with cell surface antigens expressed by autologous T-cell clones. We have identified one anti-HLA-DR reactive clone specific for the immunizing HLA haplotype of the child that reacted with autologous F(ab′) 2 in immunofluorescence studies. When tested in functional studies, the F(ab′) 2 blocked the capacity of the clone to lyse specific target cells, while triggering clonal proliferation in the absence of stimulating cells. The determinant(s) recognized by autoantibodies in the F(ab′) 2 preparation comodulate with the T3 antigen present on the surface of the cells. These data suggest that sera obtained during pregnancy contain autoantibodies which behave similarly to heterologous anti-clonotypic antibodies. Such anti-T-cell receptor antibodies may play a role in maternal tolerance to the fetus.

Effect of immunosuppressants on OKT3 associated T cell activation: Clinical implications

The monoclonal antibody directed at the T cell differentiation antigen T3 (CD3, T.gp 20-25) appears to be superior to conventional high-dose steroids in the treatment of rejection in cadaveric renal graft recipients. Re-rejection episodes and other adverse reactions, probably secondary to T cell activating potential of anti-T3, continue to be clinical problems with anti-T3 therapy. We therefore examined the relative efficacy of cyclosporin A (CSA), methylprednisolone (MP), or 6-mercaptopurine (6-MP), at concentrations that are readily accomplished in clinical practice, on the activation of T cells by anti-T3. CSA or MP mediated marked and 6-MP mediated modest inhibition of anti-T3 induced proliferation of alloimmune memory T cells. CSA- or MP-inhibited anti-T3 elicited specific secondary cytolytic activity and natural killer (NK) cell activity, and 6-MP failed to prevent the augmentation of NK cell activity mediated by anti-T3. The immunosuppressants also exhibited differential effects on anti-T3-associated lymphokine production by peripheral blood mononuclear cells. Interleukin 2 production was completely inhibited by CSA, modestly inhibited by MP and not inhibited by 6-MP. Interferon gamma production was completely inhibited by CSA or MP and not inhibited by 6-MP. Our findings, in addition to providing a plausible immunological basis for some of the complications of anti-T3 therapy, provide experimental support for therapeutic strategies that include the use of CSA and/or MP along with anti-T3.

Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes.

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.

Identification and isolation of a T4+T8+ cell with high T3 expression in human thymus: a possible late intermediate in thymocyte differentiation.

Abstract By using sensitive three-color fluorescence flow cytometric techniques, we were able to identify a T4+T8+ thymocyte with high T3 surface density (T3H) representing 4 to 9% of thymocytes. To characterize the T3HT4+T8+ cell, thymic subpopulations with high T3 surface density (T3H) and lower T3 density (T3L/T3-) were compared with regard to T6 expression. The T3H subpopulation was characterized by lower numbers of T6+ cells and reduced levels of T6 antigen density, whereas the T3L/T3- population was greater than 90% T6+ and expressed this antigen at high cell surface density. In addition, T3H fractions appeared to possess higher levels of nuclear activation with respect to the T3L/T3- population as indicated by increased log 90 degrees scatter profiles. These results suggest that thymocytes with high T3 surface expression are not only more differentiated, but also more activated than the majority of the thymic population. The T3HT4+T8+ fraction could be distinguished from T4+T8+ thymocytes with lower T3 density not only by an increased log 90 degrees scatter profile, but also by the presence of T4+T8+ cells with reduced levels of T8 surface antigen. Our results indicate that T4+T8+ thymocytes with high T3 surface density are a distinct subpopulation and may represent the immediate precursors of the phenotypically more mature T3HT4+T8- and T3HT8+T4- subpopulations found in human thymus.

Human Epidermal Langerhans Cells Internalize by Receptor-Mediated Endocytosis T6 (CD1 “NA1/34”) Surface Antigen. Birbeck Granules Are Involved in the Intracellular Traffic of the T6 Antigen

Using immunogold staining of a suspension of living human epidermal cells to identify the Langerhans cell membrane-associated antigen T6 (revealed by the monoclonal antibody BL6), we have observed internalization of T6 antigen in Langerhans cells. This phenomenon is at least partly due to receptor-mediated endocytosis involving coated pits, coated vesicles, endosomes, the smooth endoplasmic reticulum, and lysosomes. These ultrastructural results suggest that T6 antigen may be part of a receptor site. Following receptor-mediated endocytosis, the appearance in the cell center of the first labeled Birbeck granules suggests that Birbeck granules could represent T6 intracellular transport organelles carrying T6 from the central part of the cell to an unknown destination.

An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations.

The inhibitory effect of phenylhydrazine and azide combined with either pre-formed or nascent hydrogen peroxide H2O2 upon endogenous peroxidatic activity, expressed by tissue eosinophils in different disease states, was investigated. It was found that whilst endogenous peroxidatic activity due to eosinophils in a Hodgkin's disease and a histiocytosis X case were adequately inhibited by phenylhydrazine combines with pre-formed or nascent H2O2, the eosinophils in the Onchocerca volvulus nodule were either not at all or only partly inhibited by the two regimens. On the other hand, a combination of azide with nascent H2O2 proved consistently effective against this resistant form of endogenous peroxidatic activity. Using human tonsil sections this protocol was shown to be non-deleterious to T4('CD4'), T6('CD1') and T8('CD8') lymphocyte surface antigens as evidenced by the application of a standard indirect immunoperoxidase technique and the relevant monoclonal antibodies.

Absence of Langerhans Cells in Oral Hairy Leukoplakia, an AIDS-Associated Lesion

Oral hairy leukoplakia (HL) is a recently described manifestation of human immunodeficiency virus (HIV) infection in which Epstein-Barr virus (EBV) has been shown to replicate. To seek evidence for a local defect in mucosal immunity, we assessed the presence of epithelial Langerhans cells (LC) in these lesions and in autologous nonlesional mucosa. We used monoclonal antibodies against HLA-DR, HLA-DQ, and T6 antigens to identify LC in biopsy specimens of HL from 23 homosexual men. In all lesion specimens, LC either were not detected or were present only in greatly reduced numbers with at least 1 of the antibodies. In nonlesional oral mucosa from the same patients, LC were detected with all 3 antibodies in 11/12 specimens (92%) and were found in approximately normal numbers with at least 1 antibody. There was close correlation between the absence of LC and positive staining for EBV, human papillomavirus antigens, and candidal hyphae in the epithelium. We conclude that LC are absent or greatly reduced in the lesions of HL. Absence of normal LC function may be important in the pathogenesis of HL and may reflect an event in the pathogenesis of other features of the acquired immune deficiency syndrome.

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P. vulgaris phytohemagglutinin or when protein kinase C was directly activated by phorbol 12,13-dibutyrate. Immune activation of T cells via the protein kinase C pathway thus induces phosphorylation of a single site on the T3 gamma chain, namely serine 126.

T6 Monoclonal Antibody Reacts with Blasts from Cases of Common Antigen Acute Lymphocytic Leukemia

The expression of the T6 antigen on malignant lymphoid cells has been considered strong evidence in support of T-cell lineage and thymic stage of differentiation of the neoplastic cells. Thus, the authors have used the T6 monoclonal antibody for the last three years in the immunophenotyping of blasts from 60 consecutive cases of acute lymphocytic leukemia (ALL) and 8 cases of T-cell lymphoma. Blasts from 12 of 46 (26%) cases of common type ALL, 4 of 7 (57%) cases of T-cell ALL, 2 of 3 (66%) cases of lymphoblastic lymphoma, and 1 of 5 (20%) cases of peripheral (postthymic) T-cell lymphoma were positive for the T6 antigen. The authors conclude that the expression of T6 antigen on malignant lymphoid cells may not always indicate T-cell lineage.

Clinical significance of low levels of myeloperoxidase positivity in childhood acute nonlymphoblastic leukemia

The clinical significance of a low percentage of myeloperoxidase- positive blast cells in childhood acute nonlymphoblastic leukemia was determined. Of 155 consecutive cases studied by cytochemical staining methods, 14 were characterized by 4% to 15% (median 6%) myeloperoxidase- positive blasts. All 14 cases showed reactivity to Sudan black B stain, and 7 had Auer rods. The morphological subtypes of leukemia were M1 (8 cases), M2 (3), M4 (1), and M5 (2). Immunological marker studies disclosed the lymphoid-associated T11 antigen on cells from 8 of the 11 cases tested. Other lymphoid-related findings in these 8 cases included the T3 antigen and E rosette formation in 1 case each. Among cases that were prospectively studied for the expression of lymphoid-associated markers, 6 of 8 with low levels of myeloperoxidase positivity compared with only 1 of 44 with higher levels (greater than 15%) possessed such features (P less than 0.001). We conclude that low levels of myeloperoxidase reactivity distinguish cases of acute leukemia in which the blast cells coexpress lymphoid (T11 antigen) and myeloid markers.

Clinical Significance of Low Levels of Myeloperoxidase Positivity in Childhood Acute Nonlymphoblastic Leukemia

The clinical significance of a low percentage of myeloperoxidase-positive blast cells in childhood acute nonlymphoblastic leukemia was determined. Of 155 consecutive cases studied by cytochemical staining methods, 14 were characterized by 4% to 15% (median 6%) myeloperoxidase-positive blasts. All 14 cases showed reactivity to Sudan black B stain, and 7 had Auer rods. The morphological subtypes of leukemia were M1 (8 cases), M2 (3), M4 (1), and M5 (2). Immunological marker studies disclosed the lymphoid-associated T11 antigen on cells from 8 of the 11 cases tested. Other lymphoid-related findings in these 8 cases included the T3 antigen and E rosette formation in 1 case each. Among cases that were prospectively studied for the expression of lymphoid-associated markers, 6 of 8 with low levels of myeloperoxidase positivity compared with only 1 of 44 with higher levels (greater than 15%) possessed such features (P less than 0.001). We conclude that low levels of myeloperoxidase reactivity distinguish cases of acute leukemia in which the blast cells coexpress lymphoid (T11 antigen) and myeloid markers.

Melanoma cell lysis by human CTL clones: differential involvement of T3, T8 and HLA antigens.

Three lymphocyte clones, derived by micromanipulation from peripheral blood lymphocytes (PBL) of a melanoma patient and expressing a broad pattern of reactivity against different target cells, were analyzed for the involvement of T-cell markers and HLA antigens in the lysis of target cells by blocking experiments with a panel of monoclonal antibodies (MAbs). The clones lysed autologous melanoma cells (Me 28) and 18 out of 22 allogeneic targets including neoplastic and normal cells of different histological origin. Anti-T3 and anti-T8 MAbs strongly inhibited the cytotoxicity of the lymphocyte clones against Me 28, 3 allogeneic melanomas and 3 carcinomas, but failed to affect the lysis of K562. Anti-HLA class-I MAb (w6/32) produced a significant enhancement of the lysis of Me 28 by the 3 clones without modifying cytotoxicity against one allogeneic melanoma or against K562 cells. Anti-HLA class-II MAb (D1.12) did not affect the lysis of the same targets by the 3 clones. These results thus indicate that some anti-melanoma CTL clones may interact with autologous tumor cells by the T3 and T8 structures in an HLA class-I unrestricted manner.

Lymphokine activation of T4+ T lymphocytes and monocytes.

The function of the T4 antigen, a marker for a differentiated T cell subset, is not well understood. Our previous observation that a chemoattractant human lymphokine, lymphocyte chemoattractant factor (LCF), which selectively induces motile responses in unactivated T4+ lymphocytes, led us to investigate whether LCF could also induce T4+ cell activation. Because LCF acts selectively on T4+ cells, we next determined whether the T4 antigen has a function in this LCF-induced cellular activation. A T4+ lymphocyte migratory response is induced by divalent anti-T4 antibody, but not by corresponding Fab fragments of the same antibody. Fab fragments of anti-T4 antibody, but not Fab fragments of anti-T3 antibody, block the migratory effect of both LCF and divalent anti-T4. Furthermore, LCF but not divalent anti-T4, evokes the expression of interleukin 2 (IL 2) receptors and HLA-DR antigen on T4+ lymphocytes in 24 hr. These effects are quantitatively similar to those observed by anti-T3 antibody activation. LCF-induced IL 2 receptor expression is blocked by co-incubation with anti-T4 antibody and anti-T4 Fab fragments, whereas anti-T3 activation is not inhibitable by anti-T4 Fab fragments. Because cultured monocytes express the T4 antigen, we investigated the action of LCF on cultured monocyte migration and HLA-DR expression. Induction of monocyte migration by LCF and anti-T4 antibody increases proportionally as T4 antigen expression increases in vitro. This enhanced migration is inhibitable by anti-T4 Fab fragments. Monocyte activation, as measured by augmented HLA-DR expression 24 hr after incubation with LCF, but not anti-T4 antibody, is quantitatively similar to the effects of interferon-gamma. Augmented HLA-DR expression is blocked by anti-T4 Fab fragments but not by antibody to interferon-gamma. These studies indicate that LCF interacts with T4+ lymphocytes and monocytes to induce migration and cellular activation.

Identification and cloning of a prethymic precursor T lymphocyte from a population of common acute lymphoblastic leukemia antigen (CALLA)-positive fetal bone marrow cells.

We have cloned common acute lymphoblastic leukemia (CALLA)-positive cells from human fetal bone marrow containing less than 1 in 10,000 E-RFC in round-bottomed microtiter wells (one cell per well) using the autocloning unit of an EPICS-V cell sorter. Expansion of such cells (with IL-2 and heavily irradiated autologous thymocytes as feeder cells) resulted in growth in 6-14% of the wells (mean, 11%) with cells with mature T lymphocyte phenotype. Two-color fluorescence analysis of outgrowing cultures furthermore ascertained that these cells had differentiated through a phase of simultaneous expression of T4 and T8 antigens and at the same time expression of the thymocyte-associated T6 antigens. Thus, given the fact that 10-20% of T cell acute lymphoblastic leukemia (T-ALLs) are CALLA+, we have been able to identify a human prethymic T lymphocyte population that might be the normal counterpart of precursor cell to the CALLA+ T-ALL cell.