Antigen-specific T-cell Lines Research Articles

Isolation of CD4+and CD8+T-Cell Clones from Mice Immunized with Synthetic Peptides on Splenic Dendritic Cells

Splenic dendritic cells (DC) express high levels of MHC, co-stimulator, and adhesion molecules and have been shown to be extremely potent antigen-presenting cells for both CD4+and CD8+T-cell responses. Previous studies have shown that murine DC can be loaded with exogenous antigens and used to prime CD4+, Class II-restricted T-cell responsesin vivo.This article describes protocols for immunization using DC loaded with peptides bound to Class I or Class II molecules and for the derivation and characterization of CD4+and CD8+antigen-specific T-cell lines and clones. The rationale for using DC as antigen-presenting cells is discussed, together with the advantages and disadvantages of these cells compared with more conventional methods of immunization.

Human T leukaemia virus type 1 (HTLV‐1) specific T‐helper cell response: clonal fluctuations and repertoire heterogeneity

The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vbeta gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the V beta gene usage were determined by comparing the TCR Vbeta gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro. Diverse Vbeta genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro. Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vbeta gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo, the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development.

Secretion of Nitrite by Schwann Cells and Its Effect on T-Cell Activationin Vitro

To assess a potential immunoregulatory role of Schwann cells in the peripheral nervous system we examined whether they are able to secrete nitric oxide metabolites. Schwann cells treated with IFN-γ and TNF-α upregulated iNOS-specific mRNA within 12 hr and released nitrite in a time- and dose-dependent manner, reaching a plateau of secretion after 3 days. Nitrite secretion was inhibited by NMMA, suggesting that Schwann cells are endowed with a cytokine-inducible NO synthase. TGF-β and IL-1 failed to modulate nitrite release. When assessing their role as APC, we noted that Schwann cells activated CD4+antigen-specific T-cell lines, but in contrast to professional thymic APC this ability declined markedly after Day 1. Theoretically diminished T-cell proliferation and finally death might be achieved by secretion of nitric oxide metabolites by Schwann cells. Inhibition of NO production by NMMA did not restore T-cell proliferation after Day 2 or prevent apoptosis of T-cells. However, in a coculture model Schwann cells exerted a strong suppressive effect on T-cell activation by thymic APC, which was almost completely abrogated by addition of NMMA. We suggest that Schwann cells may exert potent immunoregulatory functions beyond their role as APC. They may terminate immunoinflammatory reactions in the peripheral nervous system by releasing NO.

The Unique Amino-Terminal Domain of p56lckRegulates Interactions with Tyrosine Protein Phosphatases in T Lymphocytes

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.

Regulation of Zap-70 by Src Family Tyrosine Protein Kinases in an Antigen-specific T-cell Line

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.

The role of duplication of tumour-derived chromosome 15 carrying the rearranged pvt-1 gene in the transformed phenotype of YACUT T-cell lymphoma x G4 T-cell line somatic cell hybrids in dictating the terminal differentiation program of the parental G4 cell.

Fusion of the YACUT T-cell lymphoma with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 was previously reported to produce growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. The proliferation-suppressed hybrid lines exhibited phenotypic changes as follows: the usually high levels in YACUT of J11d antigen, IL-2 receptor, and c-myb expression, which are markers of immature T cells, were all down-regulated; the G4 T-cell function, i.e., contact helper activity for B-cell proliferation in T/B cell collaboration, was retained. Furthermore, fusion of the YACUT lymphoma with a killer T-cell line produced growth-arrested and tetraploid somatic cell hybrids having killer activity. Thus, in addition to the transformed phenotype (autonomous proliferation in vitro), the antigen-specific non-tumorigenic T-cell line genomes introduced into the YACUT lymphoma suppressed the immature phenotypes of YACUT and imposed their own programming of terminally differentiated traits on the hybrids. Prolonged growth of the proliferation-suppressed hybrid lines by repeated antigenic stimulation was previously reported to result in the appearance of transformed hybrids, which was accompanied by both a reversion of c-myc expression to the levels of YACUT and an increase in the number of chromosome 15. The present study revealed that the amplification of chromosome 15 resulted from the duplication of the tumour-derived chromosome 15 carrying the rearranged pvt-1 gene. However, the differentiated phenotypes of the hybrids remained mostly unchanged upon cell transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of porcine T lymphocytes and T-cell lines

Monoclonal antibodies (mAb) against leucocyte differentiation antigens have altered the way in which immunologists examine the immune system. These mAb allow us to identify distinct surface molecules on leucocyte populations, by which these cells can be classified, isolated and studied for their functional properties. This review summarizes the knowledge about differentiation antigens useful in the characterization of porcine T lymphocytes. Furthermore, it focuses on several properties of porcine T lymphocytes, T-lymphocyte subpopulations and the generation of porcine antigen-specific T-cell lines.

MHC restriction of T-cell proliferative responses in Xenopus

The MHC restriction of Xenopus allogeneic MHC- and antigen-specific T-cell proliferative responses was assessed. Xenopus MHC-specific monoclonal antibodies that recognize class I and class II molecules were tested for inhibitory effects on the generation of secondary T-cell proliferative responses. Antigen-specific T-cell lines were inhibited by anti-class II but not anti-class I monoclonal antibodies. Secondary alloantigen-specific proliferative responses also demonstrated MHC class II restriction. Allogeneic MHC- and antigen-specific T-cell lines demonstrated differential sensitivity to anti-class II monoclonal antibodies directed at discrete class II epitopes. These results indicate that Xenopus T cells interact with antigen-presenting cells similarly to mammals, and directly confirm previous data indicating that MHC class II restriction of proliferative responses is present in amphibians.

Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk.

Tyrosine protein phosphorylation is necessary for antigen receptor-mediated activation of T lymphocytes. This signal is generated at least in part by the Src-related tyrosine protein kinases p56lck and p59fynT (refs 2, 3). The activity of these two enzymes is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory phosphorylation may be caused by p50csk (for C-terminal Src kinase), a tyrosine protein kinase which accumulates most abundantly in thymus and spleen. To investigate the function of Csk in T lymphocytes and characterize the processes regulating T-cell receptor (TCR) signalling, we examined the effects of overexpression of Csk on the physiology of an antigen-specific mouse T-cell line. We report here that p50csk negatively regulates TCR-induced tyrosine protein phosphorylation and lymphokine production. This provides evidence for the involvement of Csk in the regulation of T-cell activation.

Effect of macrophage infection by Leishmania on the proliferation of an antigen-specific T-cell line, TPB1, to a non-parasite antigen.

The ability of Leishmania mexicana amazonensis to inhibit antigen specific T-cell proliferation against a non-parasite polypeptide antigen, poly(LTyr, LGlu)-poly(DLAla)--poly(LLys), was examined. Infection of mouse peritoneal macrophages by promastigotes blocked the proliferation of the T-cell line, TPB1. This effect was correlated with the level of parasite infection, and the timing of macrophage infection and antigen addition. Peritoneal macrophages from both BALB/b and C57BL/6 mice showed reduced ability to serve as antigen presenting cells.

CD8 T cells can protect against an intracellular bacterium in an interferon gamma-independent fashion.

Specific T-cell immunity to Listeria monocytogenes is thought to occur through the action of lymphokines which activate phagocytes to ingest and kill microorganisms. Interferon gamma (IFN-gamma) has been shown to be an effective mediator of this type of macrophage activation in vivo and in vitro. The monoclonal antibody H22.1 efficiently neutralizes endogenous IFN-gamma, exacerbates disease in a mouse model of L. monocytogenes infection, and inhibits the in vivo protective activity of a Listeria antigen-specific CD4 T-cell line. In contrast, in vivo protection by Listeria-immune CD8 T cells is not inhibited by the neutralizing anti-IFN-gamma monoclonal antibody. These results suggest that CD8 T cells can protect against an intracellular pathogen in an IFN-gamma-independent manner.

Analysis of antigenic determinants shared by two different allergens recognized by human T cells: house dust mite (Dermatophagoides pteronyssinus) and chironomid midge (Chironomus yoshimatsui).

To analyse the cross-reactivity of T-cell-mediated immunity between Dermatophagoides pteronyssinus (Dp) and Chironomus yoshimatsui (Cy), the most common allergens in Japan, we established antigen-specific human T-cell lines and clones. Some but not all of the Cy-induced T-cell lines showed a significant proliferative response not only to Cy, but also to Dp. No T-cell line responded to other unrelated antigens. When we stimulated the Dp-induced T-cell clones with Cy, 3 of the 40 clones (7.5%) showed a significant proliferation, and 2 of the 3 clones produced interleukin-4 and interferon-gamma, indicating their helper function. Cross-reactivity was diminished significantly after the absorption of Dp antigen in an anti-Cy affinity column. The cross-reactive epitopes were thought to be expressed on the Dp molecule of 45-53 kD. The presence of helper T cells reactive to both allergens suggests a possibility that this cross-reactivity might be involved in part in the high incidence of allergy to the 2 major allergens.

Immune Responses to 6 and 30‐kDa Mycobacterial Antigens in Rheumatoid Patients, and Vβ Usage by Specific Synovial T‐Cell Lines and Fresh T Cells

We have investigated both the humoral and the cellular immune responses of patients with juvenile rheumatoid arthritis (JRA) and rheumatoid arthritis (RA) to mycobacterial antigens. The JRA group was not Bacillus Calmette Guerin (BCG) vaccinated whilst the majority of the RA group was. As determined by immunoblotting, 79% of sera from patients with JRA reacted mainly with a 18.6-kDa protein (P18.6), whilst 70% of sera from patients with RA reacted mainly with a 30-kDa protein (P30) of BCG, M. tuberculosis and M. kansasii. In contrast, only a moderate proportion of the control sera (25% of adult and 20% of children) showed reactivity to P30, and none of the samples had significant reactivity with the P18.6 antigen. Furthermore, T-cell proliferation to the P18.6 and P30 antigens was detected in the majority of JRA and RA patients, and was nearly always higher in synovial fluid (SF) than in the peripheral blood (PB). We also investigated the usage of V beta family genes in P18.6 and P30 antigen-specific T-cell lines established from the SF of one patient with active RA. We showed that V beta 2, -4, -5, -6, -7, -14, -17, -18 and V beta 19 were over-represented compared with other known V beta families. We also noted that the proportion of V beta 14 was higher in freshly isolated SF mononuclear cells compared with the blood in this patient and in 2 out of 4 other RA patients examined. Other V beta families such as V beta 6, V beta 8, V beta 16, V beta 18 and V beta 19 were also over-represented in the SF compared with the blood in some patients. Taken together our results provide more information concerning the role of mycobacterial antigens in RA and suggest that there may be an in vivo clonal expansion of T lymphocytes in the synovium.

Coccidioides immitis fractions which are antigenic for immune T lymphocytes

The principal mechanism of resistance to coccidioidomycosis in experimental animals has been reported to be T-cell-mediated immunity. We have generated a Coccidioides immitis antigen-specific murine T-cell line to identify specific macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T cells were stimulated in vitro with a soluble conidial wall fraction (SCWF), which has been previously characterized by humoral and cellular immunoassays. The SCWF was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane, and the stained blot was cut into seven pieces based on the molecular size of the SCWF components. The nitrocellulose membrane strips were converted into antigen-bearing particles and tested in a T-cell proliferation assay. Antigenic components of the SCWF in the molecular size range of 43 to 66 kDa were identified as the most immunoreactive. In a parallel study, we used a cDNA expression library derived from mRNA of the mycelial phase of C. immitis, which was constructed in lambda gt11 to identify clones that encoded T-cell-reactive fusion proteins (FPs). The cDNA library was screened by using anti-SCWF rabbit serum, and the FPs expressed in Escherichia coli were isolated and tested for T-cell response in the same manner as the SCWF components. The nucleotide sequence of a 0.2-kb cDNA insert encoding a protein which elicited vigorous T-cell response was determined. The isolated cDNA insert hybridized to a single 1.9-kb mRNA band in a Northern blot of the total RNA fraction of the mycelial phase of C. immitis. Antibody with affinity for the T-cell-reactive FP was isolated from anti-SCWF rabbit serum by solid-phase immunoadsorption. The FP-specific antibody reacted with a 47-kDa polypeptide in Western blots (immunoblots) of the SCWF. The same antibody preparation was used for immunoelectron microscopy to show that the FP was localized in the walls of arthroconidia and spherules of C. immitis. Attempts to clone and sequence the entire gene which encodes the T-cell-reactive protein are under way. The results of this study should lead to the determination of the complete structure of an important T-cell-stimulating antigen of C. immitis.

Cellular and Molecular Characteristics of Transformed T Cells from an Antigen‐Specific T‐Cell Line

An antigen-specific T-cell line which transforms into T-lymphoma cells in vitro but apparently not in vivo is described. Membrane markers, tumorigenicity and T-cell receptor (TcR) V alpha and V beta-gene usage of the in vitro transformed T-cell line were analysed to investigate whether the transformation event was poly-, oligo-, or monoclonal. The results indicate that the T lymphoma has no chromosome abnormalities, contains no tumour-inducing virus, can induce clone-specific immunity, and is oligoclonal with respect to TcR V alpha and V beta expression. The nature of the transformation event and clinical application of vaccination against T lymphomas is discussed. In addition, the expressed TcR V alpha and V beta repertoire of Con A T blasts was apparently not affected by the Igh-l or the MHC haplotype, as investigated in Igh-l and MHC congeneic C57Bl mice.

The Generation of Guinea Pig T‐Cell Lines Reactive to Antigens from Mycobacterium tuberculosis. Selected Lines Induce Erythematous Skin Reactions

We describe methods for the development and partial characterization of antigen-specific T-cell lines from the guinea pig, which is the classical experimental animal in tuberculosis research. T cells were obtained from strain 2 guinea pigs immunized with BCG vaccine or with killed Mycobacterium tuberculosis in oil. T-cell lines were obtained from limited dilution cloning of antigen-stimulated, blast-enriched lymphocyte cultures. The lines were grown with weekly reseedings in the alternating absence and presence of mycobacterial antigen. Antigen reactivity of the cell lines was studied with lymphocyte stimulation tests. With these methods we have consistently obtained antigen-reactive cell lines. When injected in small numbers intradermally in the presence of antigen in syngeneic guinea pigs, some of these cell lines gave rise to antigen-specific erythematous tuberculin-like skin reactions. The skin reactions, which were usually without induration, were most pronounced after 24 h. Histological examinations of skin undergoing such reactions showed that the erythemas were not accompanied by mononuclear infiltrations. We expect that transfer experiments with guinea pig T-cell lines will prove useful tools in the analysis of the contribution of defined mycobacterial antigen preparations to tuberculosis immunity.

Influence of bone marrow-derived Ia-bearing cells on the selection of the T-cell repertoire.

We have presented the results of studies using chimeric animals to examine the role of distinct cell subsets in shaping the TCR repertoire. Examination of specificity and TCR expression in antigen-specific long-term T-cell lines derived from these chimeras suggests that bone marrow-derived APC play a role in the selection of T cells for MHC restriction during development, and also profoundly influence the expansion of the TCR repertoire following antigen priming. An understanding of the molecular mechanisms that govern TCR repertoire maturation may await the development of effective in vitro model systems of T-cell differentiation.

Establishment and Characterization of an Antigen-Specific T-Cell Line from Liver Granulomas of Schistosoma mansoni -Infected Mice

Establishment and Characterization of an Antigen-Specific T-Cell Line from Liver Granulomas of <i>Schistosoma mansoni</i> -Infected Mice

Stimulation of B-cell proliferation by membrane-associated molecules from activated T cells.

Activation of B cells to proliferation and antibody secretion is dependent on soluble lymphokines secreted by activated T cells. Activation of T cells results from physical contact between B cells and T cells through binding of the T-cell antigen receptor to a complex of antigen and class II major histocompatibility complex (MHC) molecules. To determine whether this interaction also contributes to B-cell activation by mechanisms other than those mediated by soluble T cell-derived lymphokines, I examined the ability of isolated T-cell plasma membranes to stimulate proliferation in cultures of unfractionated B cells. Membranes prepared from a cloned antigen-specific helper T-cell line induced substantial proliferation provided that the T cells had been mitogen-activated before isolation of membranes. Membranes from splenic Con A-treated blasts also stimulated B-cell proliferation, suggesting that this activity may be a common property of some subsets of activated T cells. Induction of B-cell proliferation was not found to be antigen-dependent or MHC-restricted, indicating no significant contribution by the T-cell receptor for antigen. The presence of interleukins 4 and 5 in membrane fractions was indicated by proliferation of lymphokine-sensitive cell lines, although culture supernatants from mitogen-activated T cells proved to be far more potent sources of these activities. The combined effect of membranes and lymphokine-containing culture supernatants in B-cell cultures was greater than their added effects in separate cultures. This observation suggests that lymphokines or molecules with mitogenic activity for B cells other than those found in abundance in culture supernatants may be present on activated T-cell membranes.

Establishment and characterization of an antigen-specific T-cell line from liver granulomas of Schistosoma mansoni-infected mice.

Granulomatous inflammations in schistosomiasis mansoni are the result of T-cell-mediated reactions to soluble egg antigens (SEA) secreted by parasite ova. To study TDH effector cell function, a granuloma T-cell line was established from collagenase-digested liver granulomas of acutely infected CBA/J mice. Dispersed nonadherent granuloma cells were cultured with feeder layer cells and SEA or with feeder layer cells alone in alternate cycles for 32 weeks. The granuloma T-cell line was L3T4+ Lyt-1+. In vitro, the SEA-stimulated T cells showed proliferation and interleukin 2 production. One million T cells adoptively transferred SEA-specific footpad swelling, and 7.5 X 10(6) T cells adoptively transferred granulomatous hypersensitivity to injected ova or SEA-coated beads. Anti-L3T4 monoclonal antibody blocked the SEA-specific cell proliferation. Depletion of L3T4+ cells abrogated, while that of Lyt-1+ cells diminished the adoptive transfer of SEA-specific footpad swelling. These experiments demonstrate that the granuloma T-lymphocyte population contains TDH-type effector cells. Establishment of an SEA-specific granuloma T-cell line will allow the study of the effector functions of the hitherto uncharacterized intralesional granuloma T lymphocyte.