Showing papers on "Cytotoxic T cell published in 1983"

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule.

Abstract: Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.

Cytotoxic T-cell immunity to influenza.

Abstract: In a study designed to determine whether cytotoxic T lymphocytes contribute to immunity against influenza virus infection, we inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/1/79 virus. Over the next seven days clinical observations were made, and the amount of virus shed was measured. The protective effects of preinfection serum antibody and of cytotoxic T-cell immunity against influenza A virus were assessed for each participant. All subjects with demonstrable T-cell responses cleared virus effectively. This response was observed in volunteers in all age groups, including those born after 1956, who did not have specific antibody and hence had probably not been exposed to this subtype of influenza A virus before. Cytotoxic T cells show cross-reactivity in their recognition of the different subtypes of influenza A virus, in contrast to the antibody response that is specific for each virus subtype. We conclude that cytotoxic T cells play a part in recovery from influenza virus infection.

The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody.

Abstract: An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1-26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties.

Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens.

Abstract: The functional and phenotypic characteristics of cells in human peripheral blood that mediate "natural killer" (NK) cytolysis have been examined with the use of multiparameter flow cytometry analysis and cell sorting. Essentially, all lymphocytes expressing NK and ADCC activity reacted with the anti-Leu-11a monoclonal antibody. The Leu-11a antigen was expressed on cytotoxic large granular lymphocytes (LGL), neutrophils, and basophils, but was not present on B cells, mitogen-activated T lymphoblasts, or Leu-1+ and Leu 4+ resting T cells. Anti-Leu-11a antibody selectively inhibited the binding of FITC heat-aggregated IgG complexes to granulocytes and LGL, and it may recognize a type of Fc receptor on these cells. Two-color FACS cell sorting indicated the existence of four lymphocyte subsets defined by the expression of Leu-11a and Leu-7 antigens. The Leu-11a+, -7- cells were highly active in 4-hr NK assays with the use of 51Cr-labeled K562 as the target. In contrast, the Leu-11a-, -7+ cells demonstrated weak activity and the Leu-11a-, -7- cells demonstrated no activity. The function of the Leu 11a+, -7+ cells varied considerably among several individuals examined. Multiparameter analysis with the use of two-color flow cytometry was used to determine the relationship between the expression of these NK-associated antigens and T and B cell-associated markers. These data indicate that considerable heterogeneity exists within human peripheral lymphocytes with regard to cell phenotype and function, but that several defined cellular subsets can be clearly revealed by using multiparameter FACS analysis and sorting.

Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex.

Abstract: Monoclonal antibodies were produced against a human cytotoxic T cell clone, CT8III (specificity: HLA-A3), with the view of defining clonally restricted (clonotypic) surface molecules involved in its antigen recognition function. Two individual antibodies, termed anti-Ti1A and anti-Ti1B, reacted exclusively with the CT8III clone when tested on a panel of 80 additional clones from the same donor, resting or activated T cells, B cells, macrophages, thymocytes, or other hematopoietic cells. More importantly, the two antibodies inhibited cell-mediated killing and antigen-specific proliferation of the CT8III clone but did not affect the functions of any other clone tested. This inhibition was not secondary to generalized abrogation of the CT8III clone's function, because interleukin 2 responsiveness was enhanced. To examine the relationship of the structures defined by anti-clonotypic antibodies with known T cell surface molecules, antibody-induced modulation studies and competitive binding assays were performed. The results indicated that the clonotypic structures were associated with, but distinct from, the 20,000-mol wt T3 molecule expressed on all mature T lymphocytes. Moreover, in contrast to anti-T3, anti-Ti1A and anti-Ti1B each immunoprecipitated two molecules of 49,000 and 43,000-mol wt from 131I-labeled CT8III cells under reducing conditions. The development of monoclonal antibodies to such polymorphic T cell surface structures should provide important probes to further define the surface receptor for antigen.

Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells.

Abstract: Two antisera and a monoclonal antibody raised in BALB.K mice against cloned, major histocompatibility complex (MHC)-restricted, antigen-specific helper T cell lines are described. These antibodies are specific for individual cloned T cell lines and are potent inducers of T cell proliferation. The induction of T cell proliferation by these antibodies requires the presence of an adherent accessory cell. There is no H-2 restriction between this accessory cell and the cloned T cell, nor is this antibody-induced proliferation blocked by a monoclonal anti-Fc receptor antibody. The requirement for an accessory cell, however, is eliminated in the presence of an IL-1- or IL-2-rich supernatant. Thus this system allows the analysis of helper T cell activation with only a single cell type present. Anti-T cell sera also induce T cell-dependent B cell proliferation and immunoglobulin secretion. The induction of T cell-dependent B cell activation by these sera does not require H-2-matched T cells and B cells. The specificity of these antibodies and their ability to stimulate cloned helper T cells in the absence of antigen and antigen-presenting cells strongly suggest that these antibodies are directed against antigen and/or Ia recognition sites on the T cell.

Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis.

Abstract: Within minutes of exposure of target cells to cytotoxic T lymphocytes, their nuclear DNA begins to be fragmented. This phenomenon precedes 51Cr release by at least an hour. DNA fragmentation occurs only when appropriately sensitized cytotoxic T cells are used and is not merely a result of cell death because killing of target cells by heating, freeze/thawing, or lysing with antibody and complement did not yield DNA fragments. Agarose gel electrophoresis of target cell DNA showed discrete multiples of an approximately 200-base-pair subunit, suggesting that fragmentation was the result of activation of a specific endonuclease. A similar pattern of DNA fragments is observed during glucocorticoid-induced killing of mouse thymocytes. The endonuclease in that case is inhibited by zinc ions, and we find that Zn2+ also inhibits DNA fragmentation and 51Cr release induced by cytotoxic T cells, suggesting a final common biochemical pathway for both types of cell death.

Transient expression of interleukin 2 receptors. Consequences for T cell growth.

Abstract: T lymphocyte mitosis results from the interaction of interleukin 2 (IL-2) with specific receptors that appear only after appropriate immune stimulation. To assess the potential role of IL-2 receptor levels in determining the rate and magnitude of T cell proliferation, the expression of IL-2 receptors by lectin-stimulated human peripheral blood T cells was examined and correlated with T cell growth. Using biosynthetically radiolabeled IL-2 and anti-Tac, a monoclonal antibody that blocks IL-2 receptor binding, IL-2 receptors were found to accumulate slowly and asynchronously among lectin-stimulated T cells and to precede the onset of DNA synthesis. Moreover, a critical threshold of IL-2 receptor density appeared to be required before the commitment to cell cycle progression, as analyzed quantitatively by tritiated thymidine incorporation and flow cytometric analysis of cellular DNA content. Once maximal IL-2 receptor expression occurred, continued proliferation was IL-2 concentration dependent as assessed using homogenous immunoaffinity-purified IL-2. Upon removal of the activating lectin, IL-2 receptor levels progressively declined, and, in parallel, the rate of proliferation diminished. The decay of IL-2 receptors could not be attributed to IL-2-mediated down-regulation. Instead, renewed IL-2 receptor expression was dependent upon the reintroduction of the initial activating signal. Repetitive exposure to lectin resulted in a more rapid reexpression of maximal IL-2 receptor levels, which was then followed by an accelerated resumption of proliferation. Thus, the extent of T cell proliferation after immune stimulation depends upon the interplay of the IL-2 concentration available and the density of IL-2 receptors expressed, both of which are ultimately determined by antigen/lectin stimulation. The awareness of the transience and the antigen/lectin dependence of IL-2 receptor expression, together with the capacity to monitor T cell cultures for IL-2 receptor levels, should facilitate the initiation and maintenance of cloned, antigen-specific T cells in long-term culture. In addition, these findings suggest that, in vivo, the rapidity of acquisition of maximum IL-2 receptor levels by activated T cells and the duration of IL-2 receptor expression may well direct the magnitude of T cell clonal expansion and resultant immune responses.

Lymphokine-activated killer cell phenomenon. II. Precursor phenotype is serologically distinct from peripheral T lymphocytes, memory cytotoxic thymus-derived lymphocytes, and natural killer cells

Abstract: Culture of human peripheral blood lymphocytes (PBL) in partially purified and lectin-free interleukin 2 results in the generation of cytotoxic effector cells which have the unique property of lysing natural killer (NK)-resistant fresh human tumor cells. We have termed these effector cells "lymphokine- activated killer" cells (LAK). LAK are generated from both normal and cancer patients' PBL and are able to lyse both autologous and allogeneic tumor cells from all histologic tumor types tested. Our previous studies suggested that the LAK phenomenon was distinct from either the cytotoxic thymus-derived lymphocyte (CTL) or NK systems based on a variety of criteria. This study reports that the cell type involved is also distinct, as determined by phenotypic characteristics. The LAK effector cell phenotype was analyzed in parallel with alloimmune CTL, and LAK were found to be similarly susceptible to the monoclonal anti-T cell antibodies OKT-3 or OKT-8 plus complement. In contrast the LAK precursor was not susceptible to the OKT-3 or Leu-1 antibodies plus complement, while the ability to generate alloimmune CTL was totally obliterated when tested using the same PBL responder population; in fact, generation of LAK was found to be augmented five- to sixfold, clearly suggesting that LAK precursor cells are not T lymphocytes as defined by these antibodies. LAK precursors were found to be abundant in NK cell-enriched Percoll gradient fractions, which had been depleted of the 29 degrees C E- rosetting "high affinity" T cells. However, LAK precursors were found to be distinct from the majority of NK cells since lysis of fresh PBL with the monoclonal antibodies OKM-1, Leu-7, or OKT-11 significantly depleted or totally eliminated NK activity, while subsequent activation of the remaining cells generated high levels of LAK and in some cases augmented levels of LAK. LAK precursors were found to be distributed in the thymus, bone marrow, spleen, lymph node, and thoracic duct in addition to the PBL. Therefore, while the cell(s) responsible for activation and expression of LAK activity have some common features with the classic T cell-mediated CTL and NK cell systems, the LAK precursor cells are clearly distinct as determined by phenotype analysis using monoclonal antibodies and complement, and at present must be classified as a "null" cell.

Human tumor necrosis factor produced by human B-cell lines: synergistic cytotoxic interaction with human interferon

Abstract: Human cell lines of hematopoietic origin were tested for production of tumor necrosis factor (TNF). B-cell lines transformed by Epstein-Barr virus release a factor (referred to as hTNF) that is cytotoxic for mouse L cells sensitive to mouse TNF but not for L cells resistant to mouse TNF. Exposure to 4 beta-phorbol 12 beta-myristate 13 alpha-acetate augmented production of hTNF. hTNF activity was not found in supernatants of cell lines of T-cell, monocytic, or promyelocytic origin. Partially purified hTNF has a molecular weight of approximately 70,000, has no interferon activity, is acid labile, is destroyed by heating at 70 degrees C for 1 hr, induces cross-resistance to mouse TNF in vitro, and causes hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay. Tests with a panel of 23 human cancer cell lines showed that hTNF is cytotoxic for 7 cell lines, cytostatic for 5, and has no effect on 11. Comparative studies with human alpha, beta, and gamma interferons indicated that sensitivity to hTNF and interferon can be distinguished. Combined treatment with hTNF and alpha or gamma interferon resulted in a synergistic cytotoxic effect.

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets.

Abstract: Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.

Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin.

Abstract: Antigen-specific human T cell clones specific for defined peptides of influenza A hemagglutinin were found to be rendered unresponsive by incubation with moderately high concentrations of antigen. This was the case whether the synthetic peptide antigen was present for the duration of the culture or the cloned T cells were preincubated with antigen for 3-18 h at 37 degrees C, before stimulation with T-depleted irradiated sheep erythrocyte non-rosette-forming lymphocytes (E-) pulsed with the optimal dose of peptide. Tolerance could not be overcome by culture with various numbers of E- cells and antigen. The induction of unresponsiveness was antigen specific, since it depended upon incubation with the appropriate peptide recognized by that clone. In addition, the tolerant T cells remained unresponsive to stimulation with the specific peptide for at least 7 d after induction even though maintained in culture in the presence of T cell growth factor. This state of antigen-specific unresponsiveness is akin to immunological tolerance. Furthermore, the experiments reported here demonstrate that the helper T cell clone can be inhibited by the relevant peptide in the absence of any suppressor cells or their precursors. This suggests that antigen-induced unresponsiveness need not always depend on the presence of suppressor T cells. The induction of tolerance in T cell clones does not result in early T cell death, since cells that no longer proliferate in response to the specific antigen and accessory cells still proliferate in response to T cell growth factor.

Evidence for the T3-associated 90K heterodimer as the T-cell antigen receptor

Abstract: Several surface molecules appear to be involved in antigen recognition by human T lymphocytes including the monomorphic 20/25K T3 structure present on all mature T lymphocytes and the subset-specific associative recognition elements, T4 and T8 (refs 1–8). More recently, Ti1, a clonally unique antigen recognition structure comprised of a 49,000 molecular weight (49K) α-chain and a 43K β-chain, linked to T3 was identified on a major histocompatibility complex (MHC) class I specific T8+ T-cell clone, CT8III (ref. 9). To determine whether analogous receptor molecules could be found on other T-cell clones of differing specificity, we produced monoclonal antibodies against a clonal structure (Ti2) on an MHC class II specific T4+ lymphocyte, CT4II, derived from the same donor as CT8III. The Ti2 structure on CT4II is shown here to be a disulphide-linked heterodimer like Ti1 on CT8III and is composed of summits of similar molecular weight. Monoclonal antibodies against Ti2 or Ti1 block antigen specific functions of the respective clone without showing any cross-reactivity. These findings suggest that each T lymphocyte, regardless of subset derivation or specificity, uses an analogous Ti heterodimer for antigen specific function. The latter is linked to T3 and expressed on the cell surface at an identical density (30,000–40,000 sites per cell).

Multiple sclerosis: distribution of T cell subsets within active chronic lesions

Abstract: The distribution of T cells and T cell subsets was examined within the human central nervous system in active lesions from seven patients with chronic multiple sclerosis. The monoclonal antibodies anti-T11, anti-T4, and anti-T8 were used to detect total (whole) T cells, helper T cells, and suppressor-cytotoxic T cells, respectively, and a monoclonal antibody against human Ia was used for macrophages and B cells. Lesion progression was associated with large numbers of T4+ cells at the lesion margin and these extended great distances into the adjacent normal-appearing white matter. T8+ cells were most commonly concentrated around the lesion margin and displayed a preferential perivascular distribution. Within the lesion center, only a few T cells were found. Ia+ macrophages were most numerous within the centers of active lesions and were always present in the adjacent normal white matter. The monoclonal antibodies to T cells did not cross-react with glial cells including oligodendrocytes. These results indicate that T4+ cells are actively involved in lesion extension and Ia+ cells, in demyelination.

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1

Abstract: We describe the production of the monoclonal antibody B731, reacting with a subset of human lymphocytes and, in about one-half of the donors, with neutrophilic polymorphonuclear leukocytes In the peripheral blood from normal adult donors, 146 +/- 85% of the lymphocytes react with B731 antibody The B731(+) lymphocyte subset does not bear markers of typical T or B cells and corresponds to the lymphocyte subset containing antibody-dependent killer (K) and natural killer (NK) cells We demonstrate that: a) virtually all lymphocytes with K/NK cytotoxic activity are found in the lymphocyte subpopulation bearing the B731-defined antigen; b) the B731(+) lymphocyte subset bears the combination of antigens known to be present on K/NK cells; and c) there is a positive correlation between the level of cytotoxicity and the actual number of B731(+) lymphocytes in individual donors We also report the distribution of B731(+) lymphocytes according to donor age and tissue types The use of the B731 antibody in quantitating the actual number of K/NK cells and in performing functional studies on spontaneous cytotoxicity is discussed

Interleukin-2 enhances the depressed natural killer and cytomegalovirus-specific cytotoxic activities of lymphocytes from patients with the acquired immune deficiency syndrome.

Abstract: The recently described acquired immune deficiency syndrome (AIDS) is characterized by the occurrence of severe opportunistic infections and an aggressive form of Kaposi's sarcoma A variety of profound defects in cell-mediated immunity have been reported in association with the AIDS, including deficiencies in natural killer (NK) cell activity and cytomegalovirus (CMV)-specific cytotoxicity In the present study, the in vitro effects of interleukin-2 (IL-2) and interferon beta (IFN Beta) on these abnormalities were examined to assess the potential use of these lymphokines in the immunotherapeutic treatment of this syndrome The peripheral blood lymphocytes (PBL) from six male homosexuals with AIDS and an active CMV infection exhibited markedly depressed NK cell and CMV-specific cytotoxic lymphocyte responses compared with uninfected, heterosexual control subjects Incubation of PBL with IFN Beta enhanced the NK cell activity and the CMV-specific cytotoxicity of only one of six and neither of two AIDS patients, respectively, while enhancing the NK cell activity of all six control subjects In contrast, IL-2 dramatically enhanced both the NK cell and the CMV-specific cytotoxic lymphocyte activities of all of the patients These results indicate that IL-2 can substantially potentiate the depressed cytotoxic effector functions of PBL from AIDS patients, while IFN Beta has little effect

Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GK1.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation, release of lymphokines, and binding by cloned murine helper T lymphocyte lines.

Abstract: Monoclonal antibody GK1.5 recognizes a determinant, designated L3T4a, on the murine T cell surface molecule L3T4. The expression of L3T4a by functional murine T cell clones appears to correlate primarily with class II MHC antigen reactivity rather than with functional phenotype. In previous studies, antigen-specific cytolysis by a cloned class II MHC antigen(I-Ak)-reactive CTL line was found to be blocked entirely by monoclonal antibody (mAb) GK1.5, at a step before the lethal hit. In the present studies, we demonstrate that mAb GK1.5 profoundly blocks antigen-specific proliferation and release of lymphokines by cloned murine class II MHC antigen-reactive helper T lymphocyte (HTL) lines. Analysis of cloned T cell hybridomas, however, suggests that there exists clonal heterogeneity in the degree of inhibition of class II MHC antigen-specific function by mAb GK 1.5. Finally, we present evidence that mAb GK1.5 blocks class II MHC antigen-specific function by blocking class II MHC antigen-specific binding. The data presented here lend considerable support to the concept both that L3T4 and the human Leu-3/T4 molecules are similar and that L3T4 plays a role in class II MHC antigen-reactivity by murine T cells.

Transformation of human umbilical cord blood T cells by human T-cell leukemia/lymphoma virus.

Abstract: Several isolates of human T-cell leukemia/lymphoma virus (HTLV) were transmitted to normal human T cells obtained from the umbilical cord blood of newborns. T cells from seven specimens were immortalized by infection with different HTLV isolates and their properties were compared with those of activated uninfected normal T cells grown in the presence of T-cell growth factor (TCGF) and with those of HTLV-positive neoplastic T-cell lines derived from patients with T-cell malignancies. The HTLV-infected cells generally belonged to a class of mature T cells (OKT4+ and Leu 3A+) and differed from the normal uninfected cells in that they could be propagated in culture indefinitely; possessed altered morphology, including convoluted nuclei and some bi- and multinucleated giant cells; formed large clumps in culture; demonstrated a diminished requirement for TCGF; had an increased density of TCGF receptors; often became completely independent of exogenous TCGF; and expressed HLA-DR determinants. These properties of the HTLV-infected cord blood T cells contrasted to those of uncultured cord blood T cells and of cord blood cells stimulated with mitogen and grown with TCGF but resembled the characteristics of T-cell lines established previously from patients with HTLV-associated T-cell malignancies. This in vitro system offers a unique opportunity to study the basic mechanism involved in abnormal growth and neoplastic transformation of a specific class of human T cells.

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

Abstract: In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

A human lymphocyte‐associated antigen involved in cell‐mediated lympholysis

Abstract: Human lymphocyte function antigen (HLFA) is a cell surface protein defined by two monoclonal antibodies MHM23 and MHM24. It is present on both B and T lymphocytes but in greater amounts on the latter. Both antibodies precipitated antigen, from radiolabeled HSB-2 cells, which ran as two chains on sodium dodecyl sulfate polyacrylamide gel electrophoresis at 180 and 94 kDa. Neither antibody inhibited binding of the other, indicating that distinct epitopes were recognized. Both antibodies were shown to inhibit HLA-restricted lysis of influenza virus-infected and Epstein-Barr virus-transformed target cells by cytotoxic T lymphocytes. Blocking occurred at the level of the effector cells and in the presence of subsaturating concentrations of antibody. Both reagents also inhibited lysis of K562 cells, mediated by natural killer cells. These blocking effects differ from the inhibitory effects of monoclonal anti-HLA ABC and anti-suppressor cytotoxic T cell antibodies which inhibit only HLA-restricted lysis when present in saturating amounts. It is concluded therefore that HLFA is likely to be involved in the nonspecific adherence or lytic functions of killer cells rather than specific antigen recognition.

Simultaneous flow cytometric analysis of human t cell activation antigen expression and dna content

Abstract: Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice

Abstract: Clone 33D1 is a mouse-rat hybridoma that secretes a specific anti-dendritic cell (DC) monoclonal antibody (14). Because the antibody kills DC in the presence of rabbit complement, it can be used to study the functional consequences of selective DC depletion. Previous data on the cell specificity of 33D1 were first extended. By cytotoxicity (rabbit complement) and indirect immunofluorescence (biotin-avidin technique), 33D1 reacted with DC but not with macrophages nor other splenocytes. In contrast, the monoclonal antibody, F4/80 (15), reacted with macrophages but not DC. The functional assay evaluated in this paper was stimulation of the primary mixed leukocyte reaction (MLR). 33D1 antibody itself did not inhibit stimulation by enriched populations of DC. In the presence of complement, 33D1 killed DC and ablated stimulatory function. The effect of 33D1 and complement on MLR stimulation by heterogenous cell mixtures was then evaluated. Removal of DC from unfractionated spleen suspensions reduced stimulatory capacity 75-90 percent, comparable to that produced with specific anti-Ia antibody and complement. Stimulation of both proliferative and cytotoxic responses was reduced. DC depletion had similar effects on MLR generated across full strain differences, or across selected subregions (H2I, H-2K/D) of the major histocompatibility complex. To further compare the functional properties of spleen DC and macrophages, MLR stimulation by adherent and nonadherent fractions of spleen were tested separately. 62 +/- 8 percent of the total stimulatory capacity of spleen was in the plastic adherent population. Activity was ablated greater than 90 percent after elimination of DC. MLR stimulation by 24-h cultures of spleen adherent cells, which contained a three- to sixfold excess of Ia(+) macrophages, was also ablated when DC were removed. Stimulation by nonadherent spleen was more resistant, but was reduced 50-75 percent by 33D1 and complement. The function of spleen cells treated with 33D1 or anti-Ia antibody and complement was restored with a small inoculum of purified DC. The latter corresponded to 0.5 percent of total stimulator cells and were enriched by previously described techniques that did not require the 33D1 antibody. We conclude that the DC, a trace component of mouse spleen, is the principal cell type required for stimulation of the primary MLR. Because other cells are not immunogenic, but do express Ia and H-2 alloantigens, DC likely represent the critical accessory cell required for the induction of lymphocyte responses.

Sex steroid receptors in peripheral T cells: absence of androgen receptors and restriction of estrogen receptors to OKT8-positive cells.

Abstract: The immune response has been reported to be modulated by sex hormones in several models, and estrogen receptors have been demonstrated in the human thymus. We therefore investigated the presence of estrogen and androgen receptors among human peripheral T cells; thoracic duct lymph provided large amounts of circulating lymphocytes. Pure T cells were obtained by negative selection by using complement-dependent cytotoxicity with a monoclonal antibody against a monomorphic determinant of class II histocompatibility antigen (HLA-DR). Furthermore, subsets of OKT8-positive and OKT8-negative lymphocytes were selected by using an OKT8-like monoclonal antibody. Sex steroid binding was determined on purified nuclei; no androgen receptors could be demonstrated on peripheral T cells. The cytoplasmic [3H] 17-beta-estradiol-receptor complex was always translocated to the nucleus in vitro within 1 hr at 37 degrees C; no estrogen receptors were demonstrable on purified OKT4-positive subsets. Assuming that estrogen receptors were evenly distributed among OKT8-positive cells, their level could be estimated to be about 40 fmol/mg DNA. The restriction of estrogen receptors to T cells bearing the "suppressor-cytotoxic" phenotype suggests a possible pathway for the modulation of T cell suppressive activities by estrogens.

Definition of a common leukocyte cell-surface antigen (Lp95-150) associated with diverse cell-mediated immune functions.

Abstract: We have described a monoclonal antibody, designated 603, which reacts with a cell surface antigen expressed by most peripheral blood and bone marrow leukocytes Immunoprecipitation showed at least three major components with relative mw of 95,000, 130,000, and 150,000 under reducing conditions Antibody 603 inhibited several cell-mediated immune functions The lytic activity of both cytotoxic T cells and natural killer cells was blocked in the presence of the antibody The proliferative responses of T cells stimulated by soluble antigens, mitogens, or allogeneic cells were inhibited when antibody 603 was added at the initiation of culture, but not when added after 48 hr Antibody 603 also blocked the migration of neutrophilic granulocytes The antigen identified by antibody 603 thus appears to be involved in a membrane-dependent cell activation pathway that is common to diverse functional systems and is shared by both lymphoid and myeloid cells

A new function for platelets: IgE-dependent killing of schistosomes.

Abstract: Several killing mechanisms against schistosomes have been described in vitro, involving cellular and humoral factors. Neutrophils, eosinophils—with an accessory role for mast cells—monocytes and macrophages have been shown to exhibit cytotoxic properties against Schistosoma mansoni larvae, in association with antibodies of various isotypes or with complement (reviewed in ref. 1). Lymphocyte participation in effector functions is mediated mainly through lymphokines inducing cytotoxic macrophages2, and, in certain cases, directly by T cells3. The experiments reported here show that platelets, taken from rats after specific periods of infection with S. mansoni, were able to kill schistosomula, and that normal human or rat platelets acquired toxic properties towards the same target in the presence of serum from infected individuals. The humoral factor involved in this process was shown to be IgE, and evidence was obtained of a Fc receptor for IgE on human and rat platelets. The passive transfer of immune platelets to normal rats conferred a high degree of protection towards a challenge infection by the parasite.

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication.

Abstract: Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.

Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I. T cells derived from Peyer's patches that switch sIgM B cells to sIgA B cells in vitro.

Abstract: To explore mechanisms of T cell regulation governing mucosal IgA immune response, concanavalin A-induced cloned T cell lines from Peyer's patches (PP) as well as spleen were established. The cloned cell lines expressed Thy- 1.2(+), Lyt-l(+)2(-) and were radioresistant (1,500 rad). The capacity of the cloned T cells to regulate Ig synthesis was determined by measuring their effect on lipopolysaccharide (LPS)-induced polyclonal Ig synthesis by PP B cells. In initial studies Ig secreted by B cells was determined by double antibody radioimmunoassay. LPS in the absence of cloned T cells induced abundant amounts of IgM (average 8,860 ng/2 × 10(5) B cells) and IgG (average 1,190 ng/2 × 10(5) B cells), but little or no IgA. The addition of PP cloned T cells markedly suppressed production of IgM (88 percent at the highest T/B cell ratio, 4:1), but the addition of spleen cloned T cells suppressed only a little or not at all. IgG production was inhibited by both PP and spleen T clone cells (70 percent at the 4:1 T/B ratio), wheras IgA synthesis was enhanced by both clones, but only to a limited degree. In subsequent studies the expression of class-specific surface Ig (sIg) and cytoplasmic Ig (cIg) on/in unseparated PP B cells as well as Ig class- specific PP B cells and spleen B cells during culture with or without the cloned T cells was determined by immunofluorescence. The major findings were as follows: (a) Compared with unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS alone, cultures containing LPS and PP cloned T cells showed a marked decrease in cIgM-, sIgG-, and cIgG-expressing cells that was accompanied by a striking increase in sIgA-bearing, but not cIgA-containing, cells. In contrast, unseparated B cell cultures and cultures of purified sIgM B cells derived from PP containing LPS and spleen cloned T cells did not show any increase in sIgA- bearing cells. (b) Compared with purified sIgG-bearing PP B cell cultures containing LPS alone, purified sIgG-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no substantial change in sIgG- or cIgG- expressing cells, and no sIgA- or cIgA- expressing cells appeared. (c) Compared with sIgA-bearing PP B cell cultures containing LPS alone, purified sIgA-bearing PP B cell cultures containing both LPS and PP cloned T cells showed no increased proliferation, and cIgA cells did not occur. Cultures of purified sIgM B cells derived from spleen containing LPS and PP cloned T cells showed qualitatively similar changes. From these results we conclude that PP cloned T cells induced class-specific switching from sIgM- to sIgA- bearing B cells, whereas spleen cloned T cells lacked this property, although they may have induced an IgM {arrow} IgG or intersubclass IgG switch. These processes seem to be in part tissue dependent. Furthermore, the PP switch T cells appear to operate as true switch cells, which govern the pathway of DNA recombination events, rather than as classical helper cells, which act to expand already differentiated cells. Finally, these switch T cells probably account for the fact that PP are an important source of IgA B cells and also a major site of IgA heavy chain class switching during gut-associated mucosal B cell proliferation and differentation.

Striking paucity of HLA-A, B, C and beta 2-microglobulin on human neuroblastoma cell lines.

Abstract: Monoclonal antibodies to beta 2-microglobulin (beta 2m), and to the native two-chain molecule, were used to assess the expression of the HLA-A, B, C molecules on human neuroblastoma-derived cell lines. In radioimmuno-, cytotoxic, and microscopic assays, employing fresh and fixed cells, neuroblastoma cells show at best weak activity as compared to glial or lymphoid cells. In binding inhibition assays, neuroblastoma extracts were 200- to 1800-fold less efficient in inhibiting the antibodies than were glial or lymphoid extracts. Immunoprecipitation and SDS-PAGE analysis confirmed that a beta m-like chain is synthesized by the neuroblastoma cells, but the HLA chain could not be visualized by this technique. HLA-A, B, C and beta 2m levels are known to vary among tissues and cell lines. Yet the magnitude of the differences between the neuroblastoma and lymphoid lines is much greater than the reported differences in expression between some of these same lymphoid lines and many other nonlymphoid malignant or nonmalignant cell types. Metastatic neuroblastoma tumor in bone marrow also showed weak HLA-A, B, C activity, with the cells appearing negative in microscopic assays. Possible clinical implications are discussed.

Epinephrine-induced changes in the distribution of lymphocyte subsets in peripheral blood of humans.

Abstract: We have previously demonstrated that mitogen responsiveness of mononuclear cells (MNC) from peripheral blood is reduced after a single injection of epinephrine to human subjects. The purpose of the present study was to characterize the relative distributions of MNC subsets after epinephrine administration using monoclonal antibodies and conventional cell markers. The absolute number of circulating MNC increased 64% within 30 min after injection of epinephrine, and returned to baseline by 2 hr. Analysis of MNC subsets revealed that there were no changes in the relative percentages of total T lymphocytes [T3+ cells, or neuraminidase-treated sheep red blood cell rosettes (EN-rosettes)], B lymphocytes (B1+, or cells with surface-bound immunoglobulin), or monocytes (by morphologic criteria) after epinephrine administration. The percentage of inducer T cells (T4+) declined at 30 and 60 min postinjection. Overall, the percentage of suppressor/cytotoxic T cells (T8+) did not change after injection of epinephrine; however, analysis of individual subjects revealed opposing responses of this subset. The T4:T8 ratio was 2.19 before injection, declined to 1.56 at 60 min, then increased to 3.10 2 hr postinjection. The percentage of natural killer/killer cells (HNK-1+) increased from a baseline of 15.5% before epinephrine injection to 29.6% at 30 min postinjection, then declined to 11.4% at 2 hr. Therefore, the administration of physiologic doses of epinephrine results in changes in the relative proportions of lymphocyte subsets in peripheral blood, in addition to reduced mitogen responsiveness as reported previously.