A comparative nuclear localization study of galectin-1 with other splicing components.

Using both conventional and laser confocal fluorescence microscopy, the intracellular distribution of galectin-1 in HeLa cells was analyzed and compared with the localization of previously documented markers of the nucleus and cytoplasm. The Sm epitopes of the small nuclear ribonucleoprotein complexes (snRNPs) and the non-snRNP splicing factor SC35 yielded only nuclear staining. On the other hand, the enzyme lactate dehydrogenase was cytoplasmic. In contrast to these patterns in which nuclear versus cytoplasmic localizations appeared to be mutually exclusive, galectin-1, as well as galectin-3, yielded simultaneous nuclear and cytoplasmic staining. Confocal microscopy showed galectin-1 fluorescence throughout most of the sections from the top of the cell to the bottom. Through the middle sections, as the plane of focus cuts through the nucleus, there was definite fluorescence staining in the nuclear compartment. This nuclear localization was critically dependent on the type of detergent used to permeabilize the cell: cells treated with saponin or digitonin yielded exclusively cytoplasmic staining while Triton X-100-treated cells showed nuclear as well as cytoplasmic labeling. Finally, double-immunofluorescence analysis showed that, within the nucleoplasm, the following pairs of nuclear antigens could be colocalized in certain speckled structures: (a) SC35 versus Sm; (b) galectin-1 versus Sm; (c) galectin-3 versus Sm; and (d) galectin-1 versus galectin-3. These results establish the presence of galectin-1 in the nuclei of HeLa cells, a conclusion consistent with the identification of the protein in nuclear extracts of the same cells and with its documentation as a factor in pre-mRNA splicing.

Evidence for a role for galectin-1 in pre-mRNA splicing.

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.

Immunization of human HIV-seronegative volunteers with recombinant p17/p24:Ty virus-like particles elicits HIV-1 p24-specific cellular and humoral immune responses.

OBJECTIVE: To evaluate the immune response to HIV-1 p24 generated in vivo by p17/p24:Ty virus-like particles (p17/p24:Ty-VLP) by examining the lymphoproliferative and antibody (Ab) responses to HIV-1 p24, as well as Gag-specific cytotoxic T lymphocytes (CTL), in HIV-seronegative volunteers immunized with hybrid p17/p24:Ty-VLP. DESIGN AND METHODS: Sixteen HIV-seronegative volunteers were immunized with p17/p24:Ty-VLP at two dose levels (100 or 500 micrograms) and monitored for the following 48 weeks for production of anti-p24 and anti-p17 Ab, in vitro lymphoproliferative responses to HIV-1 p24 and p17, and in vitro CTL responses to HIV-1 Gag. RESULTS: Twelve out of the 16 volunteers had significant p24-specific proliferative responses, with volunteers on the higher dose schedule exhibiting earlier proliferative responses than those on the lower dose schedule. Proliferative responses in both volunteer groups were similar in overall magnitude but appeared at different times during the immunization schedule. Anti-p24 Ab were detected in six out of the nine individuals in the lower dose group and in five out of the seven in the higher dose group. There was a good correlation between the presence of p24-specific Ab and the detection of lymphoproliferative responses to the p24 protein in peripheral blood mononuclear cells isolated from the same individuals. Anti-p17 Ab were detected in five volunteers. No Gag-specific CTL responses were detected. CONCLUSION: We conclude that hybrid HIV-1 p17/p24:Ty-VLP are capable of inducing both cellular and humoral immunity to HIV-1 Gag p17 and p24 components and are worthy of further study as a potential HIV immunotherapeutic.

T cell responses to peptides covering the gag p24 region of HIV-1 occur in HIV-1 seronegative individuals.

We demonstrate that peptides (16 amino acids long) covering the sequence of the HIV-1 core protein p24 induce significant proliferation in peripheral blood mononuclear cells (PBMC) of several (greater than 50%) healthy seronegative volunteers as well as seronegative homosexual men. The nature of this response was characterized and compared with those of HIV-infected patients. Several peptides induced responses; however, the most frequent responses in both seropositive and seronegative individuals were noted to the following peptides: 1 and 2 (aa 133-157); 6 and 7 (aa 183-207); 15 (aa 273-287); and 17 and 18 (aa 293-317). The response pattern was related to the disease stage of the patients; seronegative individuals as well as asymptomatic seropositive individuals (CDC II/III) responded to low concentrations of several peptides, but symptomatic patients (CDC IV) only responded to high concentrations of a few peptides. Cell separation studies of PBMC from healthy volunteers showed that the responding cells were CD4+ and expressed the CD45RO differentiation antigen. Furthermore, cord-blood mononuclear cells with less than 5% of CD45RO T cells did not proliferative to any of the peptides. Finally, CD4+ T cell lines specific for both peptides and p24 protein were successfully established from the PBMC of seronegative individuals confirming the data obtained with freshly isolated cells. These studies therefore suggest that the CD4+ cell response to p24 is not strictly disease related, instead, the response may be due to priming of the host with cross-reactive antigens.

Altered production of tumour necrosis factors alpha and beta and interferon gamma by HIV-infected individuals.

In vitro studies shows that recombinant tumour necrosis factor (TNF) alpha and beta, and interferon-gamma (IFN-gamma) can enhance HIV replication, and peripheral blood mononuclear cells (PBMC) infected with HIV in vitro secrete high levels of the same cytokines. As T cells secrete all three mediators, the capacity of T cell activation signals to trigger cytokine production in PBMC from HIV-infected individuals was investigated as such patients may be immunocompromised. We demonstrate that asymptomatic seropositives in CDC group II/III as well as patients who have progressed to CDC group IV of the disease proliferate efficiently to anti-CD3 antibody, recombinant interleukin-2 (rIL-2), phytohaemagglutinin (PHA), PHA plus phorbol 12,13 dibutyrate (PMA) but secrete significantly (P less than 0.05) higher amounts of TNF-alpha, TNF-beta and IFN-gamma compared with controls in response to the same stimulants. We also show a difference between group II/III and group IV patients with the latter secreting more TNF-alpha and IFN-gamma. The kinetics of TNF-alpha and -beta, and IFN-gamma production was stimulus dependent with overall levels varying in time for each stimulus. Furthermore, the kinetics of the response to all three stimulants were altered in seropositives; CDC group II/III and group IV patients secreted higher levels of cytokines over several time points compared to controls. The altered production of these mediators by HIV-infected patients may contribute to disease progression and to the pathogenesis of AIDS.

Tumour necrosis factors (alpha, beta) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication.

Cytokines such as tumour necrosis factor (TNF) can induce HIV-1 production in T-cell tumour lines. However, it is not known if the same occurs in freshly isolated mononuclear cells, nor is it known if the virus can itself regulate cellular cytokine production. In this paper we report that HIV-1 induces peripheral blood mononuclear cells (PBMC) and CD4+ T lymphocytes to secrete TNF alpha, TNF beta and interferon-gamma (IFN gamma), three cytokines having multifunctional activities and complex physiological roles. We also show that separate addition of exogenous recombinant (r) TNF alpha or rTNF beta or rIFN gamma increases HIV-1-induced syncytium formation in both PBMC and CD4+ cells by up to 10,000-fold, with TNF alpha being most potent in this regard. Finally, we show that syncytium formation induced by diverse HIV-1 isolates and LAV-2 is inhibited without the addition of exogenous r-cytokines by the respective anti-cytokine antibodies. Our study therefore demonstrates that efficient HIV replication in primary mononuclear cells is associated with the ability of the virus to induce TNF and IFN gamma secretion.

Cytolysis by cloned helper T cells: induction by specific antigen or by anti-CD3 hybrid antibodies.

We have explored the use of hybrid antibodies--prepared by covalently linking anti-CD3 to an antibody specific for a monomorphic major histocompatibility complex (MHC) class II determinant using N-succinimidyl 3-(2-pyridyldithio)proprionate/succinimidyl 4-(N-malcimidomethyl)cyclohexane-1-carboxylate (SPDP/SMCC) as coupling reagent--in inducing cytolysis in human tuberculin (PPD)-specific T helper (TH) clones. These clones have been shown to lyse PPD-bound Epstein-Barr virus (EBV)-transformed B-cell lines (B-EBV) in an MHC class II-restricted manner. In this paper anti-CD4-induced cytolysis is compared with antigen/MHC-induced cytolysis with the same clones. Cytolysis induced by the hybrid antibodies was highly efficient, with killing of both syngeneic and allogeneic tumour cells positive for MHC class II. Conjugate-induced cytolysis was maxima within 4 h; that of antigen-positive targets at 16 h. Killing of bystander cells was seen only when cytolysis was triggered by antigen/MHC, suggesting that the mechanism of cytolysis in the two systems may be distinct. Targets treated simultaneously with hybrid antibody and with antigen, thereby providing both activation signals to the clones, are lysed less efficiently than those treated with either PPD or hybrid antibody alone. Evidence is presented showing that this inhibition is most marked against syngeneic PPD-coated cells treated with hybrid antibody, suggesting that two signals independently capable of activating cytolytic function in the clones, when presented simultaneously, interfere with the induction of the cytolytic process.

Tumour necrosis factor as an autocrine tumour growth factor for chronic B-cell malignancies.

Recombinant tumour necrosis factor (TNF) promotes survival and induces proliferation in the tumour cells from two malignancies of B lymphocytes--hairy-cell leukaemia and B-chronic lymphocytic leukaemia. Culture with TNF also induces TNF mRNA and protein, so the cytokine may act as an autocrine tumour growth factor. These growth promoting effects are antagonised by alpha but not by gamma interferon.

The killing of tumour cell targets coupled to tuberculin (PPD) by human and murine PPD-reactive T helper clones. I. PPD specificity of killing.

This paper reports on the characteristics of killing by a human and a murine tuberculin (PPD)-specific T helper clone of targets to which PPD was attached via the lectin concanavalin A (Con A). The killing was specific for PPD from M. tuberculosis; and targets coupled to Con A alone or to PPD from M. paratuberculosis were not killed. Target cells carrying Con A-PPD were more effectively lysed than PPD-pulsed cells. This form of lymphocyte killing, though highly significant, was inefficient. Maximum killing of PPD carrying targets was 30-40% at effector to target ratios of 20:1 and at 16 h. Cells carrying 2 x 10(6) molecules of PPD and less than 1.5 x 10(6) molecules Con A per cell were killed most efficiently. A major distinction between this helper T cell killing and that mediated by cytotoxic T cells was that both TH clones displayed bystander lysis and killed PPD uncoupled targets when these were cultured with syngeneic PPD-bound targets. This suggests that the mechanism of cytotoxicity may involve soluble mediators.

The killing of tumour cell targets coupled to tuberculin (PPD) by human and murine PPD-reactive T helper clones. II. Major histocompatibility complex restriction of killing.

This paper takes up the major histocompatibility complex (MHC) restriction of killing by a murine and a human tuberculin (PPD)-specific T helper clone of PPD-Con A bound targets. In the previous paper we demonstrated that the specificity of killing of such targets was directed against PPD and not the lectin. This paper provides further evidence to suggest that the PPD-specific clones recognize PPD on PPD-Con A-bound cells though the T cell antigen receptor complex, since the killing was restricted by MHC class II products. Using a range of syngeneic, allogeneic, and semi-syngeneic targets we have shown the fine specificity of the restricting element to be one of the two alleles of the DR region (DR 2) for the human clone, and to be the I-A subregion for the murine clone. Binding studies with radiolabelled class II antibodies were performed to see whether killing efficiency was dependent on the number of class II products expressed. The findings showed that the human B-EBV targets express 2-3 x 10(6) molecules per cell, while the susceptible murine tumours, the Abelson line and the 6A tumour, only expressed 600-800 binding sites per cell. Target cell susceptibility appeared to be linked to the number of class II molecules expressed; thus the syngeneic murine MBL-2 tumour expressing 200-300 binding sites per cell was not killed and the lysis of the 6A and Abelson tumours could be enhanced by doubling the number of class II binding sites by incubating cells with Con A-conditioned medium. However, maximum lysis did not exceed 30-40%, suggesting that class II expression alone did not govern killing.

Human large granular lymphocytes induce immunoglobulin synthesis after bone marrow transplantation.

Following T cell-depleted bone marrow transplantation, helper T cell numbers remain depressed for some months. Nonetheless, functional B cells can be adoptively transferred to the recipients of such grafts, where they continue to secrete antibody. We now show that immunoglobulin production by these transferred B cells is induced by activated large granular lymphocytes (LGL) which circulate in the recipients in substantial numbers during the immediate post-transplant period. The LGL are CD3 negative and therefore provide help in an antigen-unlinked manner. Helper effects for autologous (donor) B cells are augmented by the addition of anti-LFA-2 (anti-CD2) which appears to act by blocking recruitment of LGL inhibitory to developing B cells. In contrast antibody to the beta chain of LFA-1, which effectively reduces natural killer activity of LGL, does not influence their helper function. The peripheral blood LGL fraction thus contains both helper and cytotoxic activity, which can be distinguished by appropriate monoclonal antibodies.

Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals.

The use of PPD (purified protein derivative of tuberculin) as a carrier has several significant advantages. It provides very powerful T cell help and it gives rise to virtually no antibody response against itself. This is particularly useful if it is intended to go on to make monoclonal antibodies, where the presence of a large amount of anti-carrier antibody is a nuisance! Furthermore, unlike most comparably powerful adjuvant systems, it can be used in man. PPD coupling has been used to raise antibodies to haptens and to raise T cell responses to tumour cells. It is here reported that small peptides coupled to PPD will give rise to good titres of anti-peptide antibody. For peptides that contain no cysteine, coupling has been achieved by attaching succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to the alpha-amino group of the peptide and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to the PPD and allowing an uncleavable bond to form between them. Data on immunization with the leucotactic nonapeptide of the alpha chain of the complement component C3 and with some oncogene-related peptides have been obtained.

Migration inhibition factor secreting human T-cell lines reactive to PPD: a study of their antigen specificity, MHC restriction and the use of Epstein-Barr virus-transformed B-cell lines as requirement for antigen-presenting cells.

Two PPD-reactive T-cell lines and two clones derived from them have been characterized. The lines were maintained for a period of 10-12 weeks in I1-2 containing medium. The clones were derived from the uncloned lines by the limiting dilution method and maintained in culture for 12 weeks. The cloning efficiency was 1%. Both the cloned and the uncloned lines were highly reactive to tuberculin in a proliferation assay and produced migration inhibition factors following antigenic stimulation. Both these functions were dependent on the addition of antigen-presenting cells and genetically regulated by Class II molecules of the MHC. Each uncloned line and the clones derived from them were restricted by just one of the DR alleles of the autologous host. An analysis of cell types involved in antigen presentation showed that macrophages and Epstein-Barr virus-transformed B-cell lines induced both proliferation and MIF secretion in the T-cell lines and clones cultured with PPD. Phenotypic studies indicate that the cells are Sheep E+, OKT4+, OKT8- and HLA-DR+.

The heterogenization of tumour cells with tuberculin. I. The coupling of tuberculin to cell surfaces using con-A as ligand.

The introduction of an antigenic determinant strongly recognized by T cells on to a cell enhances the immune response to weak cellular antigens. Tuberculin (PPD) is a particularly suitable antigenic determinant for this purpose since it behaves in many ways as a 'T-cell hapten'. It has been found that direct chemical coupling of PPD to cell surfaces damages their antigenicity, but that this can be circumvented by coupling PPD to the lectin Concanavalin A and using this as the ligand for binding PPD to the cell. Techniques for preparing Con-A/PPD using little glutaraldehyde or SPDP as cross-linking agents are described.

The heterogenization of tumour cells with tuberculin. II. Studies of the antigenicity of tuberculin-heterogenized murine tumour cells in syngeneic BCG positive and BCG negative mice.

The possibility of enhancing the antigenicity of tumour cells by chemically attaching purified protein derivative (PPD) of tubercle bacillus as an immunogenic carrier determinant into the tumour cell surface has been explored in these studies. It was observed that multiple immunizations with PPD-coupled tumour cells did potentiate a marked anti-rumour response even to tumours that were very weakly antigenic. Moreover, such immunizations could be used to retard the growth of tumours in previously unimmunized animals. An attempt has been made to elucidate the important factors involved when PPD heterogenized tumour cells are used for immunization.