Defective in vitro production of influenza virus-specific cytotoxic T lymphocytes in ataxia-telangiectasia

Peripheral blood mononuclear cells (PBMC) from patients with ataxia-telangiectasia (A-T) were studied for their capacity to proliferate and to generate influenza virus-specific cytotoxic T lymphocytes (CTL) after in vitro stimulation with influenza A/Hong Kong (A/HK (H3N2)) virus. PBMC from 11 patients proliferated poorly to A/HK and 10 of the 11 patients failed to exhibit significant CTL effector activity when tested on influenza A/HK virus-infected autologous target cells. In contrast, PBMC from each of 18 simultaneously studied, unrelated normal individuals proliferated to A/HK and generated influenza-immune CTL. In each of the 10 A-T patients, deficient CTL activity was shown to be due to a lack of generation of CTL and not to target cell resistance to lysis, because the virtually infected target cells of the patients were lysed by parental influenza-immune CTL. Determinations of T cell numbers and existing serum antibody titers to H3N2 influenza virus suggest this nonresponsiveness cannot be simply explained by a lack of T cells or the absence of exposure to type A (H3N2) influenza virus. Studies in which CTL were generated in A-T plasmas and during co-culture of PBMC from an A-T patient and an MHC-matched sibling failed to demonstrate either plasma or cellular suppression as a mechanism for the lack of CTL production in A-T patients. This immune defect in the production of cytotoxic effector T cells may be a cause of the increased frequency of infections and neoplasms observed in A-T patients.     

Augmentation of auto-anti-idiotypic antibody production by hapten-reactive helper T lymphocytes

Augmented auto-anti-idiotypic antibody production was effectively achieved by immunization of mice with haptenated myeloma protein in the presence of hapten-reactive helper T lymphocytes. Hapten-reactive helper T-lymphocyte activities were raised in BALB/c mice by immunization with para-azobenzoate (PAB)-derived mouse gamma globulin (MGG) prepared by amidination reaction (PAB_im-MGG). Helper T cell activity was effectively enhanced by pretreatment of mice with a PAB-derived nonimmunogenic copolymer of D-glutamic acid and D-lysine (D-GL) (PAB-D-GL) 3 days before priming with PAB_im-MGG; PAB-D-GL is a potent tolerogen of both PAB-specific suppressor T lymphocytes and PAB-specific B cells. After induction of these enhanced PAB-reactive helper T lymphocytes, mice were immunized with PAB-coupled TEPC-15 myeloma protein (PAB_im-T-15), which was also prepared by amidination reaction. Mice immunized in this way manifested strikingly enhanced titers of auto-anti-idiotypic antibodies, specific for the T-15 idiotype, as compared to control mice which had not been preimmunized with PAB_im-MGG. The ability of PAB_im-MGG preimmunization to facilitate auto-anti-idiotypic antibody production was due to the activity of PAB-reactive helper T cells since PAB-specific B cells had been abolished by prior treatment with PAB-D-GL. The implications of this model for future studies on immunological engineering the analysis of idiotype network phenomena are discussed.     

Stable expression of cytolytic activity by influenza virus-specific cytotoxic T lymphocytes

Influenza-specific immune cytotoxic T lymphocyte (CTL) populations maintain a constant level of in vitro cytolytic activity. This is demonstrable with both heterogeneous populations of anti-viral CTL from immune donors and long-term CTL clones derived from primed CTL precursors. Cytolytic machinery is stably expressed by these CTL populations under a variety of in vitro cultivation conditions. This finding is in contrast to results with alloreactive CTL generated by stimulation of primed CTL precursors that lose cytolytic activity on a per cell basis with time after stimulation. The results indicate that virus-specific, cloned CTL that stably express cytolytic activity are representative of the heterogeneous populations from which they are derived and further suggest a qualitative difference in the regulation and expression of cytolytic machinery between heterogeneous populations of influenza-specific CTL and alloreactive CTL.     

Tolerance induction in antigen-specific helper T cell clones and lines in vitro

The induction of T cell tolerance in vitro was investigated by using HGG-specific murine helper T cell (Th) clones and cell lines. It was found that exposure of Th to monomeric HGG (tolerogen) for 18 hr rendered the Th unable to reconstitute the PFC response of HGG-primed B cells. The tolerant state was not a result of Th cell death, as up to 100% of Th could be recovered after exposure to the monomer, and in addition, the recovered cells proliferated in response to IL2. B cells were shown not to be significantly affected by the presence of monomeric HGG in amounts calculated to be carried over from the tolerization cultures into the assay cultures. Consequently, it was concluded that interaction between Th and monomeric HGG induced unresponsiveness at the T cell level. A comparison of the tolerogenic potential of monomeric, soluble, and aggregated HGG revealed that only the monomer could induce tolerance in Th. Monomeric HGG was also shown to induce tolerance in an antigen-specific manner. Th reactive to HGG could be tolerized by monomeric HGG, but not Th reactive to FGG or OVA. Helper function of Th was also shown to be antigen specific in that HGG-reactive Th helped only HGG-primed B cells. Certain HGG-specific Th clones were found to be refractory to tolerization with monomeric HGG, whereas other clones derived from the same uncloned cell line were tolerizable.     

Target-cell recognition by cloned lines of influenza virus-specific cytotoxic T lymphocytes. Selective inhibition by a monoclonal H-2-specific antibody

A monoclonal H-2^d-specific antibody markedly inhibits target-cell lysis mediated by two influenza virus A/JAP/57-specific, H-2K ^d -restricted cloned CTL lines. Three other A/JAP/57-specific, H-2 ^d -restricted CTL clones (two of which are also restricted to H-2K ^d in target-cell recognition) are only minimally inhibited by this monoclonal antibody. The inhibitory effect of the antibody is not due to selective binding to certain cloned CTL lines but rather is due to blocking of a determinant on the target cell. The monoclonal antibody produces partial inhibition of lysis mediated by a heterogeneous population of A/JAP/57-specific, H-2 ^d -restricted CTL. Likewise the profound, selective inhibition of cytolysis produced by the H-2^d-specific monoclonal antibody could not be reproduced with a conventional H-2^d alloantiserum. These observations suggest that more than one site on a particular H-2K or H-2D molecule can serve as a determinant for H-2-restricted CTL recognition. They furthermore imply that there is more than one recognition structure (receptor) for self MHC products clonally distributed among a population of H-2-restricted CTL directed to a particular antigen.     

MIs locus recognition by a cloned line of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

This report demonstrates the expression of strong MIs locus MIsd) recognition by a cloned line of H-2-restricted influenza virus-specific CTL. This clone of F1 (H-2b/d; MIsb) origin was found to specifically proliferate in response to uninfected cells of CBA/J (H-2k, MIsd) origin but not to uninfected B10.BR or CBA/CaJ cells (H-2k, MIsb). In addition, proliferation by this cTL line was observed in response to histocompatible cells expressing cross-reactive MIsa determinants (DBA/2, NZB; H-2d, MIsa). This recognition was observed only at the level of CTL proliferation. The CTL line exhibited no cytotoxic activity for target cells of these MIs types. These observations are contrasted with the response of another cloned H-2-restricted influenza-specific CTL line that simultaneously exhibits alloreactivity for H-2k. The significance of these results for T lymphocyte recognition is discussed.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

HIV promoter activity in primary antigen-specific human T lymphocytes

Human retroviruses, such as the HIV, infects human T cells, and efficient HIV replication occurs primarily in activated T cells rather than resting cells. Increased HIV production is likely caused by the activation of the retroviral promoter, and the HIV promoter may be regulated by intracellular signals induced during immune stimulation. To examine the regulation of retroviral promoter activity in normal, Ag-specific primary T lymphoblasts, a heterogeneous population of primary human T cells was transfected with either the HIV promoter or a promoter from a different retrovirus, Rous sarcoma virus (RSV) by protoplast fusion technique. Transfected T cells responded normally to Ag or mitogen stimulation, and activation of these T cells increased both the HIV and RSV promoter activity. Promoter activity was assessed by using transient expression assays after the T cells were restimulated with Ag, mitogen, or IL-2. In situ hybridization of transfected human T cells showed that 68 to 95% of activated lymphocytes expressed CAT mRNA directed by HIV or RSV. Thus, protoplast transfection of primary T cells was efficient in that the majority of cells expressed CAT message. By deletion of different regions of the HIV promoter, the enhancer region was identified as necessary for effective HIV promoter activity. In addition these deletion studies identified a region that negatively affects HIV promoter activity in primary T cells. Cotransfection of the HIV promoter with the HIV transactivator protein, tat, increases HIV promoter activity in both resting and activated primary human T cells only when the tat target sequences were present.     

Efficiency and effectiveness of cloned virus-specific cytotoxic T lymphocytes in vivo

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.     

Enhancement of a human antibody response in vitro mediated by interaction of interferon-alpha with T lymphocytes

This study describes the effects of human recombinant IFN-alpha 2 on antibody production in vitro. Whereas the inclusion of IFN-alpha 2 in cultures for 7 days had a relatively minor effect on pokeweed mitogen (PWM)-induced antibody production, it resulted in a dose-related enhancement of a hapten-specific primary antibody response. Comparison of PWM and IFN-induced [3H]thymidine uptake indicated that the observed IFN activation was not polyclonal. Pretreatment of T cells with IFN for 1 hr before recombination with untreated autologous B lymphocytes increased the anti-TNP response four-fold, whereas similar pretreatment of B lymphocytes had no effect. Furthermore, 2000 R x-irradiation of T cells before coculture with autologous B lymphocytes and IFN abrogated the TNP-specific response. These results indicate that IFN modulates TNP-specific antibody production via a radiosensitive T-helper function. Further subfractionation by panning suggests that the enhancement is mediated by the Leu-3a+ helper/inducer T cell subset. Evidence that a 1-hr exposure to IFN was sufficient to modulate antibody production prompted the examination of T cells for possible receptor mechanisms. Scatchard analysis of 125I-IFN-alpha 2 binding revealed approximately 65 high affinity IFN receptors per cell with an apparent dissociation constant (Kd) of 4.4 X 10(-10) M. This paper is the first demonstration of the role of T cells in mediating the effects of recombinant IFN-alpha 2 on human primary antibody responses in vitro. These data further suggest that the observed modulation of hapten-specific antibody production in vitro by IFN may involve the binding of IFN to specific cellular receptors expressed by T lymphocytes.     

Clonal analysis of HLA-restricted virus-specific cytotoxic T lymphocytes from cerebrospinal fluid in mumps meningitis

T lymphocytes were cloned directly from the cerebrospinal fluid of a patient with mumps meningitis by limiting dilution in the presence of irradiated feeder cells and T cell growth factor. Of 84 colonies analyzed, 41 were cytotoxic, as shown by their ability to exert phytohemagglutinin-dependent killing. Of these, 39 showed specificity for the autologous mumps-virus infected target cells. The cytotoxic T cell colonies showed the same pattern of HLA restriction as bulk cultures of CSF lymphocytes. This study shows that it is possible to perform functional assays on inflammatory exudate cells at the clonal level. The data also suggest that recruitment of effector cells from the peripheral compartment into the meningeal spaces in mumps meningitis is highly antigen-specific.     

In vitro induction of chromosome damage by sulphasalazine in human lymphocytes

Two different endpoints, sister-chromatid exchange and micronucleus induction, were measured in human peripheral blood lymphocytes stimulated to divide in short-term in vitro cultures. The cultures were exposed to sulphasalazine and 6 of its metabolites for 72 h in the absence of any exogenous metabolic activation system. Analysis of the sister-chromatid exchange and micronuclei frequencies clearly indicates that sulphasalazine itself is capable of inducing both sister-chromatid exchange and micronuclei while sulphapyridine and its acetylated metabolites only induce sister-chromatid exchange. 5-Aminosalicylic acid, the therapeutic moiety of sulphasalazine, and its acetylated metabolite did not induce either sister-chromatid exchange or micronuclei at the concentrations tested. The data from these in vitro experiments are discussed in relation to the previously reported elevations in sister-chromatid exchange and micronucleus frequencies in inflammatory bowel disease patients receiving sulphasalazine therapy.     

In vitro antibody response to influenza virus. II. Specificity of helper T cell recognizing hemagglutinin

Intraperitoneal immunization of mice with liver influenza virus was shown to induce helper T (TH) cells with specificity for the hemagglutinin (HA). The interaction of virus-primed TH cells with purified HA was studied independently of B cell reactivity to the same antigen by using the generation of nonspecific help as an index of activation of HA-specific TH cells. TH cells from mice primed with any of the H3 viruses A/Aichi/68 X A/Bel/42 (H3N1), A/Memphis/102/72 X A/Bel/42 (H3N1) or A/Port Chalmers/73 (H3N2) were strongly cross-reactive towards HA of other strains within the H3 subtype. In addition, several examples of cross-reactivity towards HA of a different subtype were observed, usually of a lower magnitude. TH cells from mice primed to any of the H3 viruses above or to A/Bel/42 (H1N1) virus cross-reacted with the HA of A/Japan/305/57 (H2); furthermore, priming with A/Bel/42 or with A/Jap/305/57 X A/Bel/42 (h2N1) virus yielded TH cells that cross-reacted with certain of the H3 HA preparations. The cross-reactivity observed between subtypes was not due to the common chicken host carbohydrate component of HA, since no response to the purified type A HA preparations was obtained with T cells from mice primed with egg-grown influenza B/Hong-Kong/8/73 virus. The results indicate that HA of different subtypes may share cross-reactive antigenic determinants recognized by TH cells. Within a subtype, HA are highly cross-reactive with respect to tH cell recognition.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Propagation of antigen-specific T cell helper function in vitro.

Antigen-induced proliferation of primed lymph node cells was abrogated by treatment with anti-Ly 1 serum and complement (C) but not with anti-Ly 2 serum and C. Lymph node cells from animals primed to ovalbumin were activated with antigen in vitro, followed by propagation in an antigen-free supernatant fluid obtained from lectin-induced normal spleen cells. T cells processed in this manner displayed a stepwise enrichment of helper activity for antibody production as measured in a secondary hapten-carrier response. The sequential increase in antigen-specific help was paralleled by a rise in the antigen-induced proliferative response, a phenomenon whose expression was dependent on the presence of syngeneic or semi-syngeneic irradiated filler cells.     

Antigen-specific helper factor production by immortalized clone of OVA-specific T cells. I. In vitro activity

Immortalized clones of virally transformed OVA-specific T cells produce antigen-specific helper factor upon stimulation in vitro. The helper factor activate DNP-primed B cells to multiply and synthesize IgG anti-DNP antibodies. The trigger of the helper clone is antigen specific and the B cell-stimulating hapten must be coupled to the specific T cell carrier in order to transfer the help signal from the activated T clone to the B lymphocytes. Activation of the helper clone is performed by antigen-pulsed macrophages and cannot be achieved by the free soluble antigen. However, cell-free supernatant of the antigen-pulsed macrophages can stimulate the helper cells. Thus the antigenic determinant must be presented to the helper cell in the form of macrophage-processed antigen. These requirements for antigenic stimulation and the activity of the secreted helper factor demonstrate that the immortalized helper clone preserved the cellular components which control the antigen-specific immune function of the normal T lymphocyte.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

Regulation of influenza virus-specific cytotoxic T cell responses by monoclonal antibody to a human T cell differentiation antigen

The results in this report indicate that the OKT3 monoclonal antibody, which is specific for a human T cell differentiation antigen present on 90 to 95% of peripheral T cells, can exert several effects that regulate the generation and expression of human influenza virus-immune cytotoxic T lymphocytes (CTL). The OKT3 antibody, but not OKT1 or OKT11 (which bind to all peripheral T cells), is able to inhibit anti-influenza CTL effector cell activity. An F(ab')2 preparation of OKT3 IgG were as effective as whole IgG for the inhibition of CTL effectors, indicating that the inhibitory activity of the antibody was not a function of the Fc portion of the molecule. OKT3 IgG and OKT3 F(ab')2 fragments (but not OKT4, OKT8, or OKI were able to inhibit the generation of anti-influenza CTL. The culture of human lymphoid cells with OKT3 in the presence or absence of influenza virus induced radioresistant cells that could suppress the CTL response of fresh autologous lymphocytes to influenza. These results suggest that T cell functions can be regulated by signals that are initiated by the binding of antibody to cell surface molecules that may not be related to the T cell antigen-specific receptor(s).     

人淋巴细胞的体外抗体诱发及其在杂交瘤中的应用

Sources of immunized lymphocytes constitute one of the main obstacles in the production of human monoclonal antibodies. We tried to get them through in vitro immunization. Cells from excised tonsils or trauma spleens were used for the induction of antibody responses in vitro. Antibodies to different antigens including sheep red blood cells, ovalbumin, tetanus toxoid, and hepatitis B surface antigens were induced in 7-14 days' cultures. Taking tetanus toxoid as antigen, we analysed the various factors required for antibody induction with statistics analysis, which included cell separation method, T cell conditioned medium, antigen dosage, serum content, and concentration of mitogen PWM and LPS. The results showed: (1) The cell separation method influenced the antibody production significantly in comparison with other factors. It signified that immune cells' combination was the most influential factor. (2) Serum also constituted quite important influencing factor especially in the later period of culture. However, it did not make much differences if it attended 10% or so. The antigens and mitogens tended to be used at low concentration. (3) Due to the significant variation among individuals and among different antigens, it is suggested to set up the culture system with some flexibility so as to adapt to the variation in cells and antigens from different sources. The present culture system we use includes nylon wall column separation of cells, suitable range of antigens (three doses instead of one), and either 10% T cell conditioned medium or a mixture of 1 microgram PWM/ ml + 0.1 microgram LPS/ml. The human B lymphocytes stimulated in vitro with tetanus toxoid were used for the construction of human hybridomas.