Showing papers on "Cytotoxic T cell published in 1984"

T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV

Abstract: Many viruses, including retroviruses, are characterized by their specific cell tropism. Lymphadenopathy-associated virus (LAV) is a human lymphotropic retrovirus isolated from patients with acquired immune deficiency syndrome (AIDS) or related syndromes, that displays selective tropism for a subset of T lymphocytes defined by the expression of a surface glycoprotein of relative molecular mass 62,000 (62K) termed T4 (refs 6-8). This glycoprotein delineates a subset of T lymphocytes with mainly helper/inducer functions, while T lymphocytes of the reciprocal subset express a glycoprotein termed T8, have mainly cytotoxic/suppressor activities, and are unable to replicate LAV. Such a tropism may be controlled at the genomic level by regulatory sequences, as described for the human T-cell leukaemia viruses HTLV-I and -II (refs 2, 3). Alternatively or concomitantly, productive cell infection may be controlled at the membrane level, requiring the interaction of a specific cellular receptor with the virus envelope, as demonstrated recently for Epstein-Barr virus (EBV). Therefore, we have investigated whether the T4 molecule itself is related to the receptor for LAV. We report here that preincubation of T4+ lymphocytes with three individual monoclonal antibodies directed at the T4 glycoprotein blocked cell infection by LAV. This blocking effect was specific, as other monoclonal antibodies--such as antibody to histocompatibility locus antigen (HLA) class II or anti-T-cell natural killer (TNK) target--directed at other surface structures strongly expressed on activated cultured T4+ cells, did not prevent LAV infection. Direct virus neutralization by monoclonal antibodies was also ruled out. These results strongly support the view that a surface molecule directly involved in cellular functions acts as, or is related to, the receptor for a human retrovirus.

Allogeneic Stimulation of Cytotoxic T Cells by Supported Planar Membranes

Abstract: Phospholipid vesicles containing the transmembrane protein H-2Kk spontaneously fuse to form planar membranes when incubated on treated glass surfaces. Pattern photobleaching of fluorescent lipid probes indicates that these planar membranes are continuous and that the lipids are as mobile as they are in conventional fluid bilayers or monolayers. H-2Kk molecules in these planar membranes are immobile. These membranes stimulate cytotoxic T lymphocytes when cultured with immune spleen cells. The response to H-2Kk in planar membranes is greatly enhanced by the addition of supernatant from concanavalin A-stimulated spleen cells, indicating that relatively little antigen processing or presentation by accessory cells occurs. Cytotoxic T cells induced by purified alloantigen are found to be as susceptible to antibody blockade as are effectors from conventional mixed lymphocyte culture, where the antibody is directed against a T-cell surface antigen reputed to strengthen target cell adhesion through an interaction independent of major histocompatibility antigens.

Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence.

Abstract: We studied the mechanism of lymphocytic choriomeningitis virus (LCMV) persistence and the suppression of cytotoxic T lymphocyte (CTL) responses in BALB/c WEHI mice infected at birth with LCMV Armstrong strain. Using adoptive transfer experiments we found that spleen cells from persistently infected (carrier) mice actively suppressed the expected LCMV-specific CTL response of spleen cells from normal adult mice. The suppression was specific for the CTL response and LCMV -specific antibody responses were not affected. Associated with the specific CTL suppression was the establishment of persistent LCMV infection. The transfer of spleen or lymph node cells containing LCMV -specific CTL resulted in virus clearance and prevented establishment of the carrier state. The suppression of LCMV -specific CTL responses by carrier spleen cells is not mediated by a suppressor cell, but is due to the presence of genetic variants of LCMV in spleens of carrier mice. Such virus variants selectively suppress LCMV-specific CTL responses and cause persistent infections in immunocompetent mice. In striking contrast, wild-type LCMV Armstrong, from which these variants were generated, induces a potent CTL response in immunocompetent mice and the LCMV infection is rapidly cleared. Our results show that LCMV variants that emerge during infection in vivo play a crucial role in the suppression of virus-specific CTL responses and in the maintenance of virus persistence.

Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences.

Abstract: Two related, but distinct, cDNA clones have been isolated and sequenced from a functional murine cytotoxic T-lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and both have similarities to immunoglobulin variable and constant region genes. It is concluded that these genes code for the two subunits of the heterodimeric antigen receptor on the surface of the T cell; its complete deduced primary structure is presented.

Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2.

Abstract: By using as an experimental system the induction of growth of a cloned, antigen:Ia-reactive helper T cell line by an antigen receptor-specific monoclonal antibody, we demonstrated that growth requires two essential co-factors, exogenously produced IL 1 and endogenously produced IL 2. The primary role of the IL 1 is in the expression of receptors on the T cell surface for IL 2, rather than for promoting the synthesis of IL 2. The use of a clone-specific activating monoclonal antibody at nanogram amounts to activate a cloned helper T cell should allow a detailed characterization of T cell activation via antigen receptor cross-linking.

1 alpha,25-dihydroxyvitamin D3 suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells.

Abstract: Activated B and T lymphocytes from normal human subjects are known to have the specific high-affinity receptor for 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). In an attempt to determine a functional role for the sterol in such cells, we studied the effect of 1,25-(OH)2-D3 on DNA synthesis and Ig production by normal human peripheral blood mononuclear (PBM) cells activated in vitro by the polyclonal lymphocyte activators pokeweed mitogen and phytohemagglutinin, and the specific antigen dermatophyton O. A dose-dependent inhibition of [3H]thymidine incorporation was observed in cells incubated with 1,25-(OH)2-D3 in concentrations ranging from 10(-10) to 10(-7) M. Production of IgG and IgM, determined by enzyme-linked immunosorbent assay, was similarly inhibited by increasing concentrations of 1,25-(OH)2-D3. Half-maximal inhibition of DNA and Ig synthesis was found at 10(-10) to 10(-9) M 1,25-(OH)2-D3. This suppressive effect was specific for 1,25-(OH)2-D3; of the other vitamin D metabolites examined, only 10(-7) M 24R,25 dihydroxyvitamin D3 (24,25-(OH)2-D3) had a similar inhibitory effect. 1,25-(OH)2-D3 was not cytotoxic and did not affect unactivated PBMs. These data demonstrate that 1,25-(OH)2-D3 is a potent inhibitor of human PBM Ig production in vitro and suggest that this action is mediated through the hormone's antiproliferative effect on Ig-producing B cells and/or helper T cells.

Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.

Abstract: A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.

Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells

Abstract: The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histo-compatibility complex (MHC)1,2. These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments3,4. Interestingly, in certain pathological conditions5–8, for example in autoimmune diseases such as thyroiditis5 and diabetic insulitis6, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity9. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.

In vivo effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific.

Abstract: Cloned lines of murine cytotoxic T lymphocytes (CTL) directed to type A influenza virus confer complete protection upon adoptive transfer to syngeneic mice lethally infected by influenza virus. The exquisite specificity exhibited by a subtype-specific cloned CTL in culture is reflected in its capacity to eliminate pulmonary virus and mediate recovery only in those mice infected by the virus subtype recognized by this cloned line in vitro. A cross-reactive CTL cloned line protects mice infected by either of two influenza virus subtypes. In mice dually infected with two virus subtypes, the subtype-specific CTL clone only reduces lung virus levels of the recognized virus subtype and cannot prevent these mice from dying. In contrast, adoptive transfer of the cross-reactive CTL clone into mice simultaneously infected with two virus subtypes results in reduction of pulmonary titers of both subtypes and promotes complete recovery. These results directly implicate CTL as an important antiviral defense mechanism in experimental influenza infection. In addition, these results indicate that both the induction and expression of antiviral effector activity by CTL in vivo is highly specific and therefore favor the concept that CTL express their antiviral effect in vivo by direct cytolysis of infected cells.

Preparation and characterization of monoclonal antibodies reactive with porcine PBL.

Abstract: Partially inbred miniature swine have been developed in this laboratory as a large animal model for immunologic studies. For many of these studies, a source of large quantities of well standardized antibodies to lymphocyte cell surface markers would be extremely helpful. Screening of a large panel of murine anti-human monoclonal antibodies (MAb) for anti-pig cross-reactions was nonproductive. We have therefore prepared a panel of MAb from mice immunized with pig thymocytes. Twenty-six stable MAb were produced that reacted with Ficoll/Hypaque-purified porcine lymphocytes. Reactivity was detected by complement-mediated cytotoxicity and/or cell surface binding ELISA. FMF analysis was performed on unseparated, glass-adherent, nylon wool-adherent, or nylon wool-nonadherent PBL and on cell preparations from lymphoid organs. In addition, the m.w. of the antigens detected by certain of these MAb were determined by immunoprecipitation of extracts of 125I surface-labeled cells. The majority of the MAb were not subpopulation specific. Included in these were 74-11-10 (IgG2b), which was specific for a polymorphic class I MHC determinant, and 76-3-2 (IgG1), which reacted with either a monomorphic class I MHC determinant or beta 2-microglobulin. Another MAb, 74-9-3 (IgM), precipitated a high m.w. complex of three polypeptide chains that appeared similar to that of the common leukocyte antigen. Of special interest were six MAb that were specific for various subpopulations of PBL: 76-7-4 (IgG2a) reacts with peripheral B cells, and 60% of thymocytes but not peripheral T cells, a pattern consistent with the murine ThB antigen; 74-22-15 (IgG1) and 76-5-28 (IgM) react with macrophages and granulocytes; 76-6-7 (IgM) reacts with T cells, macrophages, and granulocytes but not B cells, a combination of reactivities not previously reported in other species; 74-12-4 (IgG2b) and 76-2-11 (IgG2a) react specifically with T cells. 74-12-4 precipitated an antigen of 55,000 m.w., and 76-2-11 precipitated an antigen of 35,000 m.w. The m.w. and tissue distributions suggest that 74-12-4 recognizes porcine T helper cells, whereas 76-2-11 recognizes cytotoxic and/or suppressor T cells.

Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects.

Abstract: Studies were designed to investigate whether the cellular immunodeficiency state observed in human glioblastoma patients could be due to inhibitory factors released by the tumor cells. Cultured human glioblastoma cells were found to secrete an interleukin 1-like factor (m.w. 22,000) and a factor (m.w. 97,000) that inhibits interleukin 2 (IL 2)-dependent T cell mechanisms. This is demonstrated by its inhibitory effect on the IL 2-induced proliferation of T cell clones and on the induction of alloreactive cytotoxic T cells in mixed lymphocyte cultures. Additionally the glioblastoma cell-derived 97,000-m.w. factor inhibited growth of neuroblasts but not of fibroblasts and thus shares the characteristics of the neuroblast growth inhibition factor (NGIF) previously detected in the supernatant of fetal rat glia cell cultures. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.

Antigen recognition by H-2-restricted T cells. II. A tryptic ovalbumin peptide that substitutes for processed antigen.

Abstract: A 17-amino acid tryptic peptide of chicken ovalbumin, designated P323-339, that substituted for processed antigen when presented by glutaraldehyde prefixed accessory cells to specific I-restricted T hybridomas was characterized. The peptide antigen could not be demonstrated to have any specific or stable interactions with accessory cell Ia antigens by either direct binding or functional assays for inhibition of specific T cell activation. In addition, the T cell receptor for I-restricted antigen had no affinity for free antigen alone. A rabbit antibody specific for the antigenic peptide inhibited presentation when introduced before but not after binding of the peptide to accessory cells. These results extend our earlier finding that accessory cell-mediated processing of chicken ovalbumin can be completely explained by the fragmentation of the native molecule into smaller m.w. peptides, and suggests that if an antigen/Ia complex is important in T cell activation, it forms significantly only in the presence of the T cell receptor for I-restricted antigen.

Rejection of transplantable AKR leukaemia cells following MHC DNA-mediated cell transformation.

Abstract: Major histocompatibility complex (MHC) class I molecules can function as specific target antigens in T-cell-mediated cytotoxity. In addition, T cells can kill target cells through non-MHC antigens, for example, virally infected cells, if the target and effector cells express the same MHC class I antigens. Consequently, quantitative and/or qualitative variations in the expression of the H-2/HLA antigens on the target cells could interfere with MHC-restricted immune reactions. We have reported that the AKR leukaemia cell line K36.16, a subline of K36 (ref. 3), on which the H-2Kk antigen cannot be detected, is resistant to T-cell lysis and grows very easily in AKR mice. Other AKR tumour cell lines, like 369, which have a relatively large amount of H-2Kk on their surface, are easily killed by T cells in vitro and require a much larger inoculum to grow in vivo. Monoclonal antibodies against H-2Kk, but not against H-2Dk, prevented the killing by T cells. This suggests that some tumour cells grow in vivo because tumour-associated antigen(s) cannot be recognized efficiently by the host's immune system, due to the absence of MHC molecules which would function as restriction elements for T-cell cytotoxicity. We have tested this hypothesis by introducing the H-2Kk gene into the H-2Kk-deficient AKR tumour cell line K36.16 and have now demonstrated directly the biological relevance of H-2Kk antigen expression in the regulation of the in vivo growth of this tumour cell line.

Heterogeneity of natural killer cells

Abstract: Natural killer cells were discovered about ten years ago (1-5) during studies of cell-mediated cytotoxicity. Although investigators expected to find specific cytotoxic activity of tumor-bearing individuals against autologous tumor cells or against allogeneic tumors of similar or the same histologic type, appreciable cytotoxic activity was observed with lymphocytes from normal individuals. Since this time, the studies of natural killer cells have expanded into a broad and multifaceted research area, stimulated by the increasing indications that these cells may play important roles in natural host resistance against cancer and infectious diseases (6-24). With the wide array of recent studies related to natural cell-mediated cytotoxicity, there has been considerable diversity in the terminology related to the effector cells and consequently some confusion in the literature. However, at a recent workshop devoted to the study of natural killer cells, a consensus definition for these effector cells was developed (25). Natural killer (NK) cells were defined as effector cells with spontaneous cyto­ toxicity against various target cells; these effector �ells lack the properties of classical macrophages, granulocytes, or cytotoxic T lymphocytes (CTL); and the observed cytotoxicity does not show restriction related to the major his­ tocompatibility complex (MHC). This definition is sufficiently broad to include not only "classical" NK cells but also other natural effector cells such as natural cytotoxic (NC) cells. The workshop participants agreed that the obser­ vations relating to the development of cytotoxic cells in culture [e.g. lectin­ activated killers (LAK), anomalous killers (AK)] remain difficult to interpret and that, for the moment, it is best to categorize separately cultured or activated cells with cytotoxic reactivity that cannot be classified as CTL. Such culture-

The antigen-specific, major histocompatibility complex-restricted receptor on T cells. VI. An antibody to a receptor allotype.

Abstract: We have prepared a monoclonal antibody, KJ16-133, from the cells of a rat immunized with the purified receptor for antigen plus I-A of a BALB/c T cell hybridoma, DO-11.10. Unlike most other monoclonal anti-receptor antibodies that have been described before, KJ16-133 is not clone specific. It reacts with approximately 20% of the receptors on T cells of normal BALB/c mice. It also reacts with about the same percentage of antigen-specific, major histocompatibility complex (MHC)-restricted or allogeneic I-region specific T cell hybridomas. Reaction of KJ16-133 with a given T cell hybridoma does not seem to depend on the antigen specificity or MHC-restricting element of the T cell in question. The determinant recognized by KJ16-133 has some unexpected properties. It is absent in several strains of mice including SJL/J and SJA/20, but present on the T cells of most other commonly used strains. The determinant recognized therefore does not map to Igh. Our experiments suggest that a clone-specific "antiidiotypic" antibody and KJ16-133 recognize determinants on different parts of the receptor. For example, the binding of a clone-specific antibody to target T cells is relatively temperature insensitive, whereas KJ16-133 binds well to cells at 37 degrees C but poorly to cells at 4 degrees C. The determinant recognized by a clone-specific antibody is sensitive to reduction and alkylation of the receptor, whereas KJ16-133 reactivity is not. Finally, binding of KJ16-133 at saturating concentrations to target T cells does not block the binding of a clone-specific antibody. Similarly, binding of a clone-specific antibody only marginally inhibits binding of KJ16-133. Taken together, these results suggest that KJ16-133 is directed against an allelic determinant on T cells that may be close to the membrane, and not in the receptor binding site for antigen plus MHC. The antibody may recognize an allele of a constant region isotype, or an allele of a J region.

Resting and sensitized T lymphocytes exhibit distinct stimulatory (antigen-presenting cell) requirements for growth and lymphokine release.

Abstract: Previous studies have shown that unprimed or resting T lymphocytes will grow and release lymphokines when stimulated by dendritic cells (DC). We now have examined the stimulatory requirements for antigen-primed or blast-transformed T cells. The latter were derived from dendritic/T cell clusters that developed during the primary mixed leukocyte reaction (MLR). The specificity of the blasts was established by a binding assay in which most T cells aggregated small B lymphocytes of the appropriate haplotype within 2 h at 4 or 37 degrees C. Since unprimed T cells did not aggregate allogeneic B cells, we suggest that DC induce T lymphocytes to express additional functioning receptors for antigen. Lyt-2-T blasts did not grow or release interleukin 2 or B cell helper factors unless rechallenged with specific alloantigen, whereupon growth (generation time of 14-18 h) and lymphokine release rapidly resumed. The blasts could be stimulated by allogeneic macrophages, B cells, and B lymphoblasts, whereas the primary MLR was initiated primarily by DC. responsiveness appeared restricted to the I region of the major histocompatibility complex, and varied directly with the level of Ia antigens on the stimulator cells. The interaction of B cells and T blasts was bidirectional. The T blasts would grow and form B cell helper factors, while the B cells grew and secreted antibody. However, the efficacy of T cell-mediated antibody formation was enhanced some 10-fold by the addition of specific antigen. Therefore, responses of resting helper T cells, then, are initiated by antigen plus DC. Once sensitized, T blasts interact independently with antigen presented by other leukocytes.

Activated B cells express receptors for, and proliferate in response to, pure interleukin 2.

Abstract: In this study we investigated whether interleukin 2 (IL-2) acts on B cell proliferation and whether activated B cells express IL-2 receptors. First, the functional activity of immunoaffinity-purified or recombinant human IL-2 was studied in a B blast assay using positively selected murine surface Ig-positive cells that had been activated by lipopolysaccharide (LPS) plus anti-Ig antibodies (anti-Ig). In this assay, T cells were not detected by fluorescence-activated cell sorter analysis. It was found that both IL-2 preparations led to optimal B cell proliferation compared with supernatants obtained from murine or human spleen cells or murine cloned T helper cells. Second, we observed that the IL-2 requirement in this assay was about the same as in a proliferation assay using lectin-activated polyclonal murine Lyt-2-positive T cells. Third, analysis of the binding of radiolabeled immunoaffinity-purified IL-2 to B cells indicated that LPS plus anti-Ig-activated B cells expressed a mean of 3,500 IL-2 receptors per cell with an apparent dissociation constant of 150 pM. However, neither nonactivated B cells nor B cells activated by LPS alone exhibited significant specific IL-2 binding. The functional and the receptor data are consistent with the conclusion that IL-2 is a growth factor not only for T cells but also for B cells.

The Fc receptor for IgG on human natural killer cells: phenotypic, functional, and comparative studies with monoclonal antibodies.

Abstract: We compare five monoclonal antibodies ( B73 .1, 3G8 , Leu- 11a , Leu- 11b , and VEP13 ) that react with natural killer (NK) cells and polymorphonuclear cells (PMN). We show that all of these antibodies are directed against and inhibit the functional properties of the receptor for the Fc portion of IgG (FcR). Modulation of the FcR on NK cells after reaction with immune complexes induces the disappearance of the antigen(s) recognized by each of the five antibodies. Conversely, the antibodies block binding of IgG-sensitized erythrocytes to the NK cells and PMN and inhibit their ability to mediate cytotoxicity against antibody-sensitized tumor target cells. By using two-color immunofluorescence techniques, we characterize directly the lymphocyte population recognized by these antibodies and show that it is a homogeneous subset that does not bear markers of either B or T cells, with the exception of the 33,000 dalton antigen characteristic of suppressor/cytotoxic T cells present in 20 to 50% of the cells, and the 45,000 dalton receptor for sheep erythrocytes present on 80 to 90% of the cells. The phenotype of the cells reacting with the monoclonal antibodies corresponds to that of NK cells. Cross-competition experiments indicate that these antibodies detect at least two distinct epitopes on FcR, one ( B73 .1) preferentially expressed on NK cells and one or more ( 3G8 /Leu- 11a /Leu- 11b / VEP13 ) preferentially expressed on PMN. The lack of reactivity of these antibodies with B cells suggests that human B cells bear a different FcR from that on NK cells and PMN.

Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity.

Abstract: Mice previously infected with an aerosol of A/Rec 31 influenza virus were strongly protected against an aerosol challenge with A/Vic influenza as judged by lung virus titers recovered 2 days after the challenge infection. Such complete homotypic immunity was not achieved by priming with live Rec 31 virus injected i.v. or UV-inactivated Rec 31 virus administered s.c. together with Al(OH)3 and saponin. The reason for the superior protective effect of the natural infection was investigated. The protection induced by respiratory infection with Rec 31 virus was specific for influenza A viruses. It was not correlated with specific serum hemagglutination inhibition antibody titer or cross-reactive cytotoxic T (Tc) cell reactivity. Moreover, the transfer of splenic and lymphoid T cell populations with strong secondary Tc activity did not significantly reduce lung virus titers in recipient mice 3 days after infection. The protection however occurred in parallel with the presence of cross-reactive IgA antibody in the lung washings. It thus appears that local secretory IgA plays a causal role in the prevention of cross-infection by influenza A virus. Serum antibody and Tc cells, on the other hand, may be crucial for recovery from such infection. All mice primed with live Rec 31 virus, administered i.v. or by aerosol and expressing equally high levels of Tc reactivity, survived a lethal challenge with A/PR8 virus. The same challenge, however, killed half of the mice immunized s.c. with inactivated Rec 31 virus which induced only a low level of Tc reactivity.

1,25-dihydroxyvitamin D3 inhibits antigen-induced T cell activation

Abstract: The proliferative response of murine spleen and thymus cells to antigen but not to lectin was inhibited by the active metabolite of vitamin D3, 1,25-(OH)2D3. To directly examine the effect of 1,25-(OH)2D3 on T cell activation in the absence of other complicating interactions, we utilized a panel of cloned Ia-restricted T cell hybridomas that secrete IL 2 on activation by cloned Ia-bearing stimulator cells (TA3) or when stimulated by mitogen. Physiologic concentrations of 1,25-(OH)2D3 (0.01 to 0.1 nm) inhibited the antigen-induced secretion of IL 2 by several of these T cell hybridomas. This inhibition was dependent on the concentration of the free hormone and could be overcome by increasing the number of Ia-bearing stimulator cells used. Pretreatment of the T hybridoma but not the TA3 stimulator cell with 1,25-(OH)2D3 resulted in inhibition of activation. These results are consistent with the finding that specific 1,25-(OH)2D3 receptors are present on the T cell hybridomas but are lacking in TA3 cells. 1,25-(OH)2D3 failed, however, to inhibit the activation of the T cell hybridomas by lectin or by an anti-Thy-1 antibody. These findings suggest that 1,25-(OH)2D3 may be interfering with early events of antigen-induced T cell activation, perhaps by hindering T cell recognition of the relevant antigen on stimulator cell surfaces. This system should prove useful in studying the molecular mechanisms by which 1,25-(OH)2D3 acts to inhibit T cell activation and subsequent IL 2 production.

Monoclonal antibody analysis of mononuclear cells in myopathies. II: Phenotypes of autoinvasive cells in polymyositis and inclusion body myositis.

Abstract: In 6 cases of polymyositis and 6 of inclusion body myositis, phenotypes of mononuclear cells focally surrounding and invading muscle fibers were analyzed. By localizing the T8, T4, and Ia markers with direct immunofluorescence and acid phosphatase enzyme cytochemically in the same sections, five different phenotypes were simultaneously identified in a given section: T8+ and T4+ cells that were either activated (Ia+) or not activated (Ia-), and acid phosphatase--reactive and Ia+ macrophages. This approach permitted the separate and quantitative assessment of the distributions of the different phenotypes among the invading versus the surrounding cells. In both polymyositis and inclusion body myositis, the invading cells were selectively enriched in the T8+ phenotype. One-third of all invading cells and one-half of the invading T8+ cells were activated. T4+ cells were more abundant among the surrounding than the invading cells, and only a small proportion of the T4+ cells were activated. These findings are especially significant in view of the cytotoxic capability of the T8+ cells and because histocompatibility factors permit T8+ but not T4+ cells to recognize an antigen on muscle fibers. Macrophages accounted for 21 to 31% of the cells invading or surrounding nonnecrotic fibers. For purposes of comparison, we also analyzed mononuclear cells in necrotic fibers: 80% of these cells were macrophages, and only 20% were T cells. The findings indicate that in polymyositis and inclusion body myositis, nonnecrotic muscle fibers are injured by autoinvasive T8+ cells that act in concert with macrophages. Further, the findings strongly imply previous sensitization of clones of T cells to muscle fiber-associated surface antigen(s).

Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors.

Abstract: Purified cytoplasmic granules from cytotoxic rat large granular lymphocytes (LGL) tumors were cytolytic to erythrocytes, splenocytes, and a number of different lymphoid tumor cells. Granule concentrations of approximately 1 microgram/ml granule protein were adequate to lyse 100% of the erythrocytes, while the nucleated cells required up to 100 micrograms/ml granule protein to achieve complete lysis. Cytoplasmic granules purified from noncytotoxic lymphoid cells did not contain detectable cytolytic activity; purified granules from rat mast cells and rat liver lysosomes likewise failed to display cytolytic activity. However, granules prepared from normal rat peripheral blood LGL were cytolytic. Granule-mediated lysis of erythrocytes and nucleated cells was complete within 3 min at room temperature. The lytic activity required calcium at concentrations of 10(-4)-10(-2) M; magnesium or barium failed to replace calcium, while strontium could replace calcium at 10(-3)-10(-2) M when nucleated cells were the target. Exposure of LGL tumor granules to calcium before the addition of target cells resulted in an inactivation of granule cytolytic activity over the course of 20 min at room temperature. Granule cytolytic activity was heat and Pronase sensitive, and could be solubilized by 2 M salt. Examination of granules exposed to calcium in the electron microscope using negative staining showed that calcium treatment of granules results in the formation of ring-shaped structures previously described to be associated with LGL-mediated cytotoxicity. These results provide support for the hypothesis that the cytotoxic processes mediated by LGL are a secretory event characterized by the release of cytolytic material from the cytoplasmic granules after triggering by a surface receptor. The results further suggest that the ring structures visible in the electron microscope are associated with the lytic event.

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo.

Abstract: Our data show that 1 X 10(7) to 1.5 X 10(7) lymphocytic choriomeningitis virus-specific, H-2-restricted cloned cytotoxic T lymphocytes (CTL) administered intravenously into acutely infected mice totally cleared virus from the spleens (10(4) to 10(5) PFU per spleen reduced to less than 50 PFU per spleen) by 24 h. This activity was genetically restricted in that cloned CTL could reduce titers of infectious virus in syngeneic C57BL/6 mice but not allogeneic BALB/c mice. Dose-response analysis indicated that at least 3 X 10(6) to 5 X 10(6) cloned CTL injected intravenously were needed to reduce significant amounts of infectious virus in the spleens. No infectious virus could be recovered from the spleens for at least 4 days after injection of cloned CTL. Hence, CTL play a major role in elimination of infectious virus from spleens during lymphocytic choriomeningitis virus infection. Our results also indicate that cloned CTL propagated in vitro for long periods of time can mediate a biologically relevant effect in vivo. These cells should be of considerable value in defining the precise manner in which CTL bring about control of viral infection, analyzing lymphocyte trafficking, and the potential use of cloned CTL in immunotherapy against viral disease.

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones.

Abstract: Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.

T cell-activating properties of an anti-Thy-1 monoclonal antibody. Possible analogy to OKT3/Leu-4.

Abstract: We have identified a single rat monoclonal antibody, G7, that is a potent inducer of interleukin (IL-2) production from all functioning T cell hybridomas as well as from normal T cells. G7 is also mitogenic for normal T cells and is a very effective inducer of IL-2 receptor expression. On fluorescence-activated cell sorter analysis, G7 recognized a pan-T cell antigen. Immunoprecipitation studies demonstrated that G7 recognized a cell surface molecule of 28-32 kD that appeared to be identical to Thy-1 in coprecipitation studies. In addition, G7 precipitated a protein of 50 kD. The possible relationship of the putative molecular complex identified by G7 on murine cells to the molecular complex identified on human T cells with anti-T3 reagents is discussed. In addition, G7 should prove to be a very useful reagent for studying the early events of lymphocyte activation as well as an inducer of lymphokine-rich supernatants.

Effects of natural and recombinant IL 2 on regulation of IFN gamma production and natural killer activity: lack of involvement of the Tac antigen for these immunoregulatory effects.

Abstract: Highly enriched populations of human large granular lymphocytes (LGL), natural killer (NK) cells, and T cells were obtained from low and high density fractions, respectively, of discontinuous Percoll gradients. The NK cells were composed of 75 to 90% LGL, with the majority of the contaminating cells being monocytes. The T cells were greater than 95% OKT3+. The proliferative and cytotoxic progenitors in both fractions were examined by using a limiting dilution assay with interleukin 2 (IL 2) from four sources: 1) crude supernatant of a gibbon lymphoma (MLA-144), 2) purified (150,000-fold) MLA-144 IL 2, 3) partially purified human IL 2, and 4) purified recombinant human IL 2. The proliferative capacity was measured at day 7 by [3H]thymidine incorporation, whereas the progenitors of cells with NK-like activity were evaluated by assessing cytotoxic activity against K562 cells at day 8 in a 4-hr 51Cr-release assay. The frequency of proliferative progenitors among T cells was approximately 1/5 and was approximately 1/60 with LGL. Titration of the highly purified IL 2 preparation demonstrated that LGL proliferated with as little as 2 U of IL 2. The frequency of detectable cytotoxic progenitors in the LGL population, however, fell sharply when less than 40 U of IL 2 were employed. The T cells failed to demonstrate cytotoxic activity against the NK-susceptible target cells at any concentration of IL 2 tested. The IL 2 preparations also were examined for their ability to directly and rapidly enhance the cytotoxic activity of highly purified NK cells. All four preparations of IL 2 enhanced the cytotoxic activity of LGL without any detectable accessory requirement after incubation for as little as 6 hr, even though the MLA-144 IL 2 preparations were devoid of detectable interferons (IFN). These data indicate that IL 2 has dual effects on NK cells, regulating their activity was well as promoting their proliferation. Collectively, these results demonstrate that highly purified IL 2, devoid of other detectable lymphokines, is capable of supporting the growth of human NK cells and augmenting their in vitro activity. In parallel experiments, these same IL 2 preparations were quite active in causing the proliferation of T lymphocytes, clearly demonstrating a role of IL 2 in promoting the proliferation of NK cells as well as T cells. The mechanism of IL 2 boosting appears to be a direct interaction with LGL, resulting in the production of IFN gamma.(ABSTRACT TRUNCATED AT 400 WORDS)