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Lymphotoxin (LT)-α regulates many biologic activities, yet little is known of the regulation of its gene. In this study, the contribution to LTA transcriptional regulation of the region between the transcription and translation start sites (downstream segment) was investigated. The LTA downstream segment was found to be required for, and alone to be sufficient for, maximal transcriptional activity in both T and B lymphocytes. The latter observation suggested that an alternate core promoter might be present in the downstream segment. Characterization of LTA mRNAs isolated from primary and from transformed human T cells under different stimulation conditions identified eight unique transcript variants (TVs), including one (LTA TV8) that initiated within a polypyrimidine tract near the 3' end of the downstream segment. Further investigation determined that the LTA downstream segment alternate core promoter that produces the LTA TV8 transcript most likely consists of a stimulating protein 1 binding site and an initiator element and that factors involved in transcription initiation (stimulating protein 1, TFII-I, and RNA polymerase II) bind to this LTA region in vivo. Interestingly, the LTA downstream segment alternate core promoter was active only after specific cellular stimulation and was the major promoter used when human T cells were stimulated with TGF-β1 and fibroblast growth factor-7. Most importantly, this study provides evidence of a direct link for crosstalk between T cells and epithelial/stromal cells that has implications for LT signaling by T cells in the cooperative regulation of various processes typically associated with TGF-βR and fibroblast growth factor-R2 signaling.

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.

Lymphotoxin-beta (LT- beta) is a tumor necrosis factor (TNF)-related membrane-bound cytokine that forms a heterotrimeric surface lymphotoxin (LT) complex with LT-alpha on the surface of lymphoid cells. Although knockout studies have revealed a role in lymph node biogenesis during development, the regulation and function of surface LT in mature cell types are poorly understood. The present study aims to understand the physiologic signals that regulate the components of surface LT in Jurkat T cells. We show that the previously observed upregulation of surface LT by phorbol myristate acetate (PMA) is markedly abrogated by cotreatment with ionomycin through posttranscriptional mechanisms. In addition, the observation of striking similarities between the mRNA accumulation kinetics of LT-alpha and LT-beta during these treatments indicates tight coupling of expression under certain conditions. In investigating the reported upregulation of LT-beta during inflammation, we tested the effects of various proinflammatory and anti-inflammatory cytokines on LT-beta expression. Our data demonstrate an upregulation of LT-beta mRNA by the inflammatory cytokines TNF and LT-alpha.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

Subcutaneous application of interferon-beta1b (IFN-beta1b) is an established therapy for patients with relapsing-remitting multiple sclerosis (RRMS), but early side effects are still a major concern. In vitro studies with myelin basic protein (MBP)-specific T-cell lines revealed a synergistic suppressive effect of IFN-beta1b and the phosphodiesterase inhibitor pentoxifylline (PTX) on proliferation and the production of tumor necrosis factor-alpha (TNF-alpha), lymphotoxin (LT), and interferon-gamma (IFN-gamma). In an initial, open labeled prospective trial, the cytokine messenger RNA (mRNA) expression of blood mononuclear cells from MS patients, receiving either IFN-beta1b alone or in combination with oral PTX, was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Patients treated with IFN-beta1b alone reported more side effects during the first 3 months of treatment and had upregulated TNF-alpha as well as IFN-gamma mRNA expression during the first month, which was not detected in patients receiving both drugs. A synergistic effect of both drugs was observed on the upregulation of interleukin (IL)-10 mRNA, which was accompanied by an increase in IL-10 serum levels. Both in vitro and in vivo data suggest that co-treatment of IFN-beta1b with PTX is a promising approach to correct the disturbed cytokine balance in MS patients.

Proliferating human medullary thymocytes can exhibit characteristic T helper cell type 1 cytokine responses exemplified by the immediate early expression of interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and lymphotoxin-beta. Here we report that cAMP-mediated attenuation of the transcription of T helper-1-specific cytokine genes in human medullary thymocytes correlates with the induction of the cAMP-mediated transcriptional repressor ICER (inducible cAMP early repressor). We show that ICER binds specifically to several NFAT/AP-1 (nuclear factor of activated T cells/activating protein-1) composite DNA sites essential for the activation of the interleukin (IL)-2 promoter as well as to a homologous DNA motif present in the proximal segment of the interferon-gamma promoter. In the presence of the minimal NFAT DNA-binding domain, which is sufficient for both DNA binding and AP-1 complex formation, ICER and NFAT form NFAT/ICER ternary complexes on several NFAT/AP-1 DNA composite sites previously identified as essential for the expression of the immunoregulatory cytokines such as IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. In extracts prepared from human medullary thymocytes treated with forskolin and ionomycin, these composite sites bind endogenously expressed ICER either singly or in complexes. Moreover, in Jurkat cells, ectopically expressed ICER represses transcription from NFAT-mediated, phorbol ester/ionophore-activated IL-2, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha promoters. We present evidence that ICER interactions with NFAT/AP-1 composite DNA sites correlate with its ability to repress transcription. These findings provide further insight into the mechanisms involved in cAMP-mediated transcriptional attenuation of cytokine expression.

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.

Lymphotoxin (LT) and tumor necrosis factor-alpha (TNF-alpha) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-alpha are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-alpha, and interferon-gamma (IFN-gamma) by CD4+ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2+ donors produced significantly more LT and TNF-alpha than TCLs from DR2- donors. In contrast, HLA-DR2+ and DR2- TCLs did not differ in the production of IFN-gamma, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-alpha was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-alpha alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-alpha.

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR+ or DQ+ cells, but only marginal levels of TNF production were induced in the presence of DP+ cells. Although both CD4+ T-cells and CD8+ T-cells produced TNF-alpha and TNF-beta in response to toxin stimulation in the presence of HLA class II+ cells, the former T-cell subset was the major source of producers of TNF-alpha and TNF-beta.

Murine tumorlytic factor (TF), immunologically distinct from murine tumor necrosis factor (TNF)-alpha and -beta, was purified to a homogeneity from the serum of mice injected with a T-cell mitogen of Corynebacterium kutscheri. The treated mouse serum was purified by Lentil lectin-Sepharose chromatography, DEAE-cellulose chromatography, preparative isoelectric focusing, and high-pressure liquid chromatography to the specific activity of 1.5 x 10(6) U/mg protein. TF was 42 kDa in its oligomeric form and 14 kDa in its monomeric form. TF activity was not impaired with hamster monoclonal antibody (mAb) to recombinant murine TNF-alpha and -beta and, reciprocally, rabbit antibody to TF neutralized the bioactivity of neither murine TNF-alpha nor -beta. TF was not precipitated with the mAb to murine TNF-alpha and -beta in Western blot analysis. The partial amino acid sequence of TF was at most 33% homologous to the 46-63 sequence of mouse TNF-beta. Thus, these results suggest that TF might be a novel tumorlytic factor which is immunologically distinct from mouse TNF-alpha and -beta.

Spleen cells from BALB/c mice bearing a syngeneic tumor (CSA1 M) 2 to 3 wk after inoculation with CSA1 M cells produced IL-2, IFN-gamma, and TNF upon in vitro cultures. This was previously demonstrated to be a result of collaboration between tumor-primed CD4+ T cells and APCs binding CSA1 M tumor Ags in vivo. The IL-2- and IFN-gamma-producing capacities decreased with the progress of tumor-bearing stages. This was parallel to the levels of IL-2 and IFN-gamma mRNAs expressed by cultured spleen cells. In contrast, comparable levels of TNF mRNA were expressed by all groups of cultured cells. However, large amounts of TNF were secreted by the cells from early but not from late tumor-bearing mice. TNF was produced mainly by the non-T cell fraction upon stimulation with CD4+ T cell-derived IFN-gamma. Therefore, the reduced TNF production by whole spleen cells from late tumor-bearing mice was restored by addition of rIFN-gamma to their cultures. Reciprocally to the progressive decrease in the production of IFN-gamma/TNF, the capacities of tumor-bearing mice to produce TGF-beta and IL-6 increased along with tumor growth. TGF-beta suppressed production of IL-2, IFN-gamma, and TNF, but not of IL-6. Moreover, IFN-gamma/TNF production was negatively regulated by IL-6. Taken together with the fact that the growth of CSA1 M cells is completely inhibited by the combination of TNF and IFN-gamma, these results demonstrate that the tumor-bearing state induces an abnormal cytokine network under which the production of antitumor cytokines is negatively regulated.

In the presence of IFN-gamma-stimulated vascular endothelial cells which have acquired HLA class II molecules, T cells produced tumor necrosis factors (TNF-alpha and TNF-beta) and IL-2 in response to stimulation with streptococcal pyrogenic exotoxin A.

We studied the physiological role of complement receptor type II (CR2, C3d/EBV receptor) expressed on T cells using MT-2 cells. First, we confirmed CR2 expression on MT-2 cells by flow cytometry and found that the MW of CR2 molecules on these cells and Raji B cells were the same by SDS-PAGE analysis. When MT-2 lysates were incubated with anti-CR2 mAb HB5 and thereafter with 32P-labeled ATP, 52- and 74-kDa proteins were phosphorylated, suggesting the activation of MT-2 cells through the complex of CR2 with these proteins. In this respect, we measured lymphotoxin production by MT-2 cells when incubated with C3d or EBV. The cytotoxicity of the MT-2 supernatant against L929 cells was elevated in a dose- and time-dependent manner. Next, we confirmed EBNA expression on EBV-infected MT-2 cells and attempted to establish an EBV-positive MT-2 clone by in vitro EBV infection. However, these clones disappeared during cloning. To clarify this mechanism, we examined the EBV genome in MT-2 cells. By Southern blot analysis, BamHI digestion of DNA extracts from MT-2 cells 3 days after EBV treatment gave a 3.0-kb signal which comigrated with the EBV BamHI-W probe. The 3.0-kb signal of genomic EBV-DNA was detected at 1, 2, 3, 5, and 7 days after EBV treatment, but could not be detected at 14 days. Thus, natural ligands of CR2 stimulate CR2-positive MT-2 cells through their functionally active CR2 molecules and in vitro EBV infection of MT-2 cells might be transient.

The capacity of human T cell subsets, CD4+ or CD8+ T cells, to produce tumour necrosis factors (TNF-alpha and TNF-beta) upon stimulation with toxic shock syndrome toxin-1 (TSST-1) and the requirement for MHC class II molecules on accessory cells (AC) in the response were investigated. The capacity of CD4+ T cells was much higher than that of CD8+ T cells in TSST-1-induced production of TNF-alpha and TNF-beta. The expression of MHC class II molecules on AC was required in the response.

Different activation parameters of peripheral blood mononuclear cells (PBMC) from 31 patients with oral lichen planus (OLP) were examined and compared with 23 healthy donors. Impaired spontaneous (450 +/- 241 vs 1290 +/- 480 cpm) and mitogen-induced (39580 +/- 14470 vs 67000 +/- 11810 cpm) lymphocyte blastogenesis was observed in OLP patients. Furthermore, reduced cytokine production was found after phytohemagglutinin A (PHA) stimulation for all cytokines studied-tumour necrosis factor alpha (TNF alpha, 432.2 +/- 73.4 vs 979.8 +/- 46.3 units/ml), interleukin 2 (IL-2, 156.2 +/- 14.9 vs 572.6 +/- 12.9 pg/ml), interferon gamma (IFN gamma, 48.5 +/- 11.9 vs 82.6 +/- 12.4 pg/ml) and interleukin 6 (IL-6, 253.6 +/- 57.7 vs 1,419.0 +/- 279.6 units/ml)-except for interleukin 1 beta (IL-1 beta) and lymphotoxin (LT). In contrast, unstimulated culture supernatants showed increased TNF alpha (38.2 +/- 13.1 vs 8.0 +/- 0.2 units/ml), LT (10.2 +/- 2.2 units/ml vs < 0.4) and IL-6 (18.5 +/- 5.6 units/ml vs < 0.5) activity. Similarly, elevated concentrations of TNF alpha (19.6 +/- 6.3 units/ml) and IL-6 (22.9 +/- 4.7 units/ml) were detected in the sera of OLP patients. Combination of PHA and phorbol myristate acetate (PMA) could restore OLP proliferative T cell response and cytokine production to the level of healthy donors, whereas exogenous recombinant human IL-2 (rhuIL-2) plus PMA did not seem to be an effective stimulant for OLP T cells. These results indicate an alteration in the immune condition of OLP patients and an impairment in T lymphocyte function.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to be a potent inhibitor of lymphocyte proliferation in vitro. The present study was undertaken to determine if this is caused by a direct effect on the lymphocytes, and to evaluate to what degree this suppression may be restored by the addition of cytokines. 1,25-(OH)2D3, > or = 10(-10) M, significantly inhibited the proliferation of pokeweed mitogen (PWM)-driven human peripheral blood mononuclear cells (MNC). Depletion of monocytes did not alter the response to 1,25-(OH)2D3. The antiproliferative effect was preceded by decreased production of interleukin (IL)-1 alpha and lymphotoxin (LT), both of which are crucially involved in T cell activation. However, the suppressive effect of 1,25-(OH)2D3, seen in MNC cultures stimulated with PWM alone, was of the same magnitude as the effect seen in MNC cultures stimulated with a combination of PWM and recombinant (r)IL-1 alpha, rIL-6, recombinant tumour necrosis factor (rTNF) alpha, rIL-2 or rLT, as well as PWM plus conditioned medium. Although pretreatment of monocytes for 2 h with 1,25-(OH)2D3 caused significant reduction in the release of IL-1 alpha and TNF alpha, reconstitution of monocyte-depleted cultures with similarly treated monocytes had no inhibitory effect on the proliferative response. In conclusion, even though it cannot be excluded that a low but critical number of monocytes are essential for the suppressive effect of 1,25-(OH)2D3-mediated inhibition of MNC proliferation, the inhibition is most likely the result of a direct effect on the lymphocytes and independent of monocytes and exogenously added cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)

(TGF)-beta is a pluripotent cytokine exerting differential effects on distinct components of the immune response. The present report, based on lymphokine determination in culture supernatants and Northern blot analysis of lymphokine mRNA, demonstrates that TGF-beta 2 markedly inhibits interleukin (IL)-4 and IL-5 synthesis by polyclonally activated human T cells in the absence of any significant effect on (IFN)-gamma, lymphotoxin or IL-2, suggesting a modulatory effect of TGF-beta 2 on the interferon Th1/Th2) balance of immune responses. The inhibitory effect of TGF-beta on IFN-gamma production by unfractionated peripheral blood mononuclear cells is likely to reflect the blunting of natural killer cell activation by TGF-beta.

Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed alpha, beta T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 micrograms/ml of PLA, and the addition of interleukin (IL)-2 and/or IL-4 increased their antigen-dependent proliferation. Following antigen stimulation, the clones produced IL-4, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor. Most clones also produced tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta), and some produced IL-5 and/or IL-2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL-4 was produced but little or not IFN-gamma, whereas at higher PLA concentrations significant amounts of both IL-4 and IFN-gamma were obtained. Thus, these PLA-specific T cell clones could be classified according to the changes in the ratio of IL-4/IFN-gamma production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN-gamma induction, and expressed increasing IL-4/IFN-gamma ratios with increasing concentrations of PLA. Modulation of cytokine patterns by the dose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.

The effects of prostaglandin E2 (PGE2) and other eicosanoids were determined on tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT; TNF-beta) production by murine Th1, antigen-specific major histocompatibility complex class II-restricted T cell clones. T cells activated with immobilized anti-CD3 or with soluble concanavalin A (ConA) produced approximately 100-1,000 units/ml TNF-alpha/LT as determined by cytotoxicity against the WEHI-164 murine fibrosarcoma cell line. TNF-alpha/LT biological activity was induced rapidly with significant production evident approximately 4 h after stimulation with either anti-CD3 or ConA. PGE2 inhibited the anti-CD3- and ConA-induced production of TNF-alpha/LT bioactivity in a dose-dependent manner. Incubation with 1 x 10(-9) M PGE2 inhibited production of TNF-alpha/LT biological activity from anti-CD-activated F1.28, or 450A.1 T cell clones by approximately 50%. Incubation with 1 x 10(-7) M PGE2 resulted in 90% inhibition of biological activity. PGE2 also inhibited both TNF-alpha and LT mRNA accumulation by more than 90%. These results suggest that the reduced production of cytotoxic activity in the presence of PGE2 was caused by the inhibition of both TNF-alpha and LT. No inhibition of T cell TNF-alpha or LT production was observed when anti-CD3- or ConA-activated cells were incubated with PGD2, PGF2 alpha, 5-hydroxyeicosatetraenoic acid or leukotriene C4. Nuclear run-on experiments indicated that the PGE2-mediated decrease of TNF-alpha and LT mRNA accumulation was caused, in part, by an inhibitory effect on the transcription of these genes. This is the first report of PGE2-mediated inhibition of TNF-alpha in T cells and PGE2-mediated inhibition of LT production in any cell type.

A human T cell hybridoma, II-23.D7, was induced with phorbol ester to express a surface form of lymphotoxin (LT, TNF-beta) and an associated 33-kDa glycoprotein. The LT epitopes were detected by surface immunofluorescence staining and by immunoprecipitation from radioiodinated or biosynthetically labeled cells with the use of anti-rLT polyclonal and monoclonal antibodies. The epitopes detected by the antibody were related to LT because adsorption of the anti-rLT with PMA-activated II-23.D7 cells resulted in the removal of the neutralizing titer of the anti-rLT antiserum. Immunoprecipitation of surface radioiodinated II-23.D7 cells revealed two bands of 25 kDa and 33 kDa that were specifically precipitated with anti-rLT, but not anti-rTNF antibodies. Enzymatic digestion with glycanases showed both proteins to have N-linked carbohydrate, with O-linked sugar limited to the 25-kDa protein. To determine the biochemical relationship between these proteins, the two LT-like forms were purified from detergent-solubilized II-23.D7 cells by immunoaffinity chromatography. Peptide mapping using CNBr cleavage showed the 25-kDa surface form to be identical to rLT, whereas the 33-kDa protein was different. Biosynthetic labeling studies showed that p33 contained both methionine and cysteine, whereas the p25 contained only methionine. Thus, the surface LT form lacks a leader peptide indicating an anchoring mechanism distinct from that described for membrane TNF. The nature of the attachment of this LT form to the membrane surface is not clear, however, neither TNF receptor binding nor lipid linkages appear to be involved. The accessory protein, p33, may anchor LT to the surface. These findings identify a new characteristic of LT and point toward an additional pathway by which T lymphocytes may mediate cytolytic activity and regulate inflammatory processes.

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.

Production and release of lymphotoxin (LT) was studied by metabolic labeling of human B- and T-cell lines with 14C-leucine and 35S-methionine. LT was immunoprecipitated with antiserum to LT and separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) followed by fluorography. Two molecular weight forms of LT with different rates of release were found both in cell supernatants and cell extracts. Monensin, a sodium ionophore, inhibited the release of LT. LT still appeared in two molecular weight forms after deglycosylation with N-glycanase. Treatment of cells with swainsonine followed by digestion of released LT with endoglycosidase H (endo H) demonstrated that the oligosaccharides were of the complex type. Subcellular fractionation of cells on Percoll density gradients demonstrated that intracellular LT is located to intermediate density fractions. No LT was found in the high density fractions corresponding to lysosomes. Phorbol 12-myristate 13-acetate induced production of tumor necrosis factor (TNF) in the B-lymphoblastoid cell line RPMI-1788. In conclusion, we have demonstrated the presence of two distinct molecular weight forms of LT, which contain N-linked oligosaccharides of the complex type.

Among the myelin basic protein (MBP)-specific T-cell clones mediating experimental allergic encephalomyelitis (EAE), which were established from SJL/J mice, one clone was found to have lost its encephalitogenicity during long-term passages in vitro, although the clone keeps its specific reactivity to the encephalitogenic determinant lying in the sequence of guinea pig MBP 89-101. To clarify the difference between the encephalitogenic T-cell clone (4b.14a) and non-encephalitogenic T-cell clone (4b.14a/n), we examined various lymphokines secreted into the culture media of 4b.14a and 4b.14a/n. The results show that the activities of lymphotoxin, interferon-gamma or interleukin-2 were not different between encephalitogenic clones and 4b.14a/n, whereas the activity of tumor necrosis factor-alpha, possibly secreted from antigen-presenting cells, was higher in culture media of 4b.14a/n. Moreover, the culture fluid of both 4b.14a/n and 4b.14a revealed suppressive effect on the proliferation of 4b.14a stimulated by MBP 89-101, but the effect was not different between the two clones. Thus, it is suggested that neither production of lymphokines examined so far nor soluble suppressive substance is related to the loss of encephalitogenicity of the T-cell clone.

Staphylococcal enterotoxin at concentrations of less than 1 pg/ml induces significant TNF activity in human peripheral blood T cells and monocytes. Maximal TNF activity is routinely detected after 48 to 72 h of culture. IL-2 and IL-4 were both growth promoting for human T cells but only IL-2 could efficiently induce TNF production. The production of TNF-alpha and TNF-beta differed greatly in kinetics. An early intracytoplasmatic production of TNF-alpha after 6 h was detected in both monocytes and T cells whereas a late production of TNF-beta (lymphotoxin) after 48 h, occurred in the T cell population. Induction of TNF-alpha and TNF-beta production by Staphylococcal enterotoxin requires the presence of both monocytes and T cells. The CD4+45R- but not CD4+45R+ and CD8+ cells supported TNF-alpha production in monocytes. The main lytic component from Staphylococcal enterotoxin-activated mononuclear cells is TNF-beta. CD4+ and CD8+ T cells produced about equal amounts of biologically active TNF into the culture supernatants but a fourfold higher frequency of TNF-beta producing cells was demonstrated among CD4+ vs CD8+ cells. The CD4+45R- T cell subset was an efficient producer of TNF-beta and IFN-gamma whereas the CD4+45R+ T cell subset produced significant amounts of TNF-beta but only marginal amounts of IFN-gamma.

Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE.

Stable human T cell hybridomas were produced by cell fusion between PHA-activated human peripheral blood lymphocytes and human acute lymphatic leukemia cells of a CEM cell line whose proliferation had been inhibited by treatment with an irreversible protein synthesis inhibitor (emetine) and an irreversible RNA synthesis inhibitor (actinomycin D). Two hybrid cell lines (E10, F8) thus produced expressed OKT3-reactive antigen, HLA-A1 and -B8 antigens derived from CEM cells, and HLA-A2 antigen derived from PBL, and they have 10 more chromosomes than CEM cells. Furthermore, these cell lines continuously secreted lymphotoxin over 3 mo. E10 was found to produce MIF in addition to lymphotoxin. Cloning of E10 and the relationship between the functions and surface phenotypes of E10 sublines were examined. Lymphotoxin-producing hybrids (E10-15 and E10-37) express more OKT8-reactive antigen than OKT4-reactive antigen, while lymphotoxin and MIF-producing hybrids (E10-25, E10-42, and E10-43) express more OKT4-reactive antigen than OKT8-reactive antigen.

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.

The appearance of lymphotoxin in the culture fluid of phytohemagglutinin (PHA)-stimulated mouse spleen cells required two cell fractions that were separated by adherence to plastic. Upon stimulation with PHA, neither cell fraction alone produced significant amounts of lymphotoxin; however, when the cell fractions were combined and then stimulated with PHA, full activity was produced. Cytotoxic activity was not fully restored by combining PHA-stimulated cultured fluids from adherent and nonadherent cell fractions. This indicated that the cytotoxic activity was not the result of two factors, one produced by each cell fraction, that acted on the target cells, but rather, two cells interacted to produce lymphotoxin. Treatment of the unfractionated spleen cells with monoclonal anti-Thy1.2 and complement before PHA stimulation greatly reduced the production of lymphotoxin and indicated that at least one of the cells was a T cell. Lymphotoxin production was partially restored by the addition of nonadherent cells to the anti-Thy1.2-treated cells, suggesting that the T cell was nonadherent. Treatment of unfractionated cells with either silica or carrageenan had no effect on the subsequent production of lymphotoxin by PHA, suggesting that the adherent cell was not actively phagocytic.

Seventy-eight patients with acute myocardial infarction (MI) were examined in order to assess the place of immune response caused by myocardial necrosis in the clinical pattern of the disease. The extent of autosensitization was estimated on the basis of E-RFC inhibition and lymphotoxin production in lymphocyte cultures in the presence of myocardial antigen. Sensitized lymphocytes emerge in the blood during the second week in primary acute MI, and during the first day in repeated acute MI. Patients with marked autosensitization more frequently developed recurrent anginal attacks and cardiovascular insufficiency; they were the only ones to show Dressler's syndrome and fatal outcomes. A positive correlation between cell sensitization and elevated IgG was seen during the third week of the disease. Lymphotoxin production tended to be increased in patients with repeated acute MI and Dressler's syndrome.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.

It is known that two major cytotoxins, lymphotoxin (LT) and tumour necrosis factor (TNF), are produced by T cells and macrophages, respectively. We report in this paper that a human T-cell hybridoma, AC5-8, produces TNF in addition to LT depending on the kind of stimuli used. When phorbol myristate acetate (PMA) and concanavalin A (Con A) or PMA alone were used to induce AC5-8 cells to secrete a cytotoxin, we found that the cytotoxin secreted was antigenically indistinguishable from LT. On the other hand, when AC5-8 cells were activated by PMA and A23187, they secreted another cytotoxin which was antigenically indistinguishable from TNF, in addition to LT. These two cytotoxins could be separated by affinity chromatography on a column of Con A-Sepharose. A cytotoxin, which did not bind to the Con A-Sepharose column and showed about 70-95% of the total cytotoxic activity secreted from AC5-8 cells stimulated with PMA and A23187, was found to be antigenically identical to TNF. The other cytotoxin, which bound to the Con A-Sepharose column and showed only 5-30% of the total activity induced by PMA and A23187, was found to be antigenically identical to LT.

The PCl-6 T cell line, derived from mice sensitized by skin painting with picryl chloride (PCl), shows antigen dependence for DNA synthesis and for lymphotoxin (LT) production. These cells produce LT, but not interferon (IFN), when exposed to 2,4,6-trinitrobenzene sulfonic acid- (TNBS) coupled syngeneic spleen cells. Concanavalin A (Con A) induces IFN production by PCl-6 cells, and IFN levels, but not LT titers, are increased by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). These results support the noncoordinate regulation of these two lymphokines. Line 32, a T cell growth factor- (TCGF) dependent T cell line and its Ly-1.2+, 2.2- derivative clone, 32H1, produce both antiviral and antiproliferative activity after exposure to several different mitogens. Tests for acid lability, sensitivity to anti-mouse IFN-alpha, beta antisera, and antiproliferative activity on non-mouse target cells indicates that an Ly-1+ clone, in the absence of both TCGF and accessory cells, can produce at least three separate lymphokine activities after Con A exposure: IFN-gamma (Type II), IFN-alpha, beta (Type I), and LT.

Normal human T cells purified from the peripheral blood and cultured for 6 days in the presence of OKT3 and IL-2 were shown to produce simultaneously TNF and lymphotoxin (LT) mRNA. Both biologically active TNF and LT were detected in supernatants of the cultured T cells using WEHI 164 as target cells. The cytotoxic activities of both cytokines could be inhibited with specific antibodies against TNF or LT. The T cell cultures were free of macrophages, NK cells, or B lymphocytes as assessed by flow cytometric analysis.

Human peripheral blood lymphocytes (PBL) were evaluated by their responses to phytohemmagglutinin (PHA-P), concanavallin A (con-A), and pokeweed mitogen (PWM), both before and after treatment with an antiserum against human thymic lymphocyte antigens (HTLA) that had been made T-cell-specific by multiple absorptions with immunoglobulin EAC-positive lymphoblast cell lines (B cells). Cells treated with HTLA were examined for their ability to react in a mixed lymphocyte culture (MLC) and to form killer cells in a cell-mediated lymphocytotoxicity (CML) system. Sensitized cells were also examined for their ability to respond to purified protein derivative (PPD) by blastogenesis, migration inhibitory factor release (MIP), and lymphotoxin (LT) production, both before and after treatment with HTLA and complement. The HTLA was in itself highly stimulatory to PBL. However, with the addition of complement and subsequent cell destruction, a marked decrease in its stimulatory response was noted. PBL treated with HTLA and complement exhibited marked inhibition of responsiveness to con-A with little decrease in PHA-P -OR PWM stimulation except at very high concentration of HTLA. MLC reaction was inhibited only when responder cells were treated with HTLA + C'. Treatment of stimulator cells with HTLA + C' did not significantly alter the MLC response. The HTLA + C'-treated cells failed to form killer cells in the CML reaction and inhibited PPD-induced blasto-genesis from PPD-sensitized individuals; however, treatment of sensitized cells with HTLA + C' had little effects on the release of MIF and LT. It is suggested that subpopulations of T-cells carry surface antigens that bind with this specific antisera, and that the con-A-responsive cells, the responder cells in the MLC, and killer T-cells comprise a separate subset from cells responding to PHA-P or PWM, OR THE MIF-and LT-producing cells.

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.

Mononuclear cells from human blood were stimulated to tumor necrosis factor alpha (TNF alpha) or beta (TNF beta) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNF alpha- or TNF beta-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNF alpha or -beta in the Golgi system. TNF alpha-producing cells appeared 2-3 h after activation of the cultures and increased up to 6 h. The majority of these early TNF alpha-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNF beta production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNF beta-producing cells was much larger than that of TNF alpha-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNF beta production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNF beta at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.

Human T cells were isolated from peripheral blood lymphocytes (PBL) and sensitized to allogeneic PBL in a one-way mixed-lymphocyte culture. These sensitized T cells were fractionated on the basis of their possession of Fc receptors for IgG (TG+) or IgM (TM+), or the absence of both IgG and IgM receptors (TG-M-). When restimulated with alloantigen of the same derivation, TG+, TM+, and TG-M- cells yielded almost equal amounts of cytotoxin. Anti-alpha-lymphotoxin serum neutralized most of this cytotoxic activity indicating that alpha-lymphotoxin (alpha-LT) constituted most of this activity. Although TG-M- cells function as effectors in allogeneic cytotoxicity, TG+ cells lyse IgG-coated targets in an antibody-dependent cell-mediated cytotoxic (ADCC) reaction, which has been shown to be mediated in part by alpha-LT. Whether TM+ cells can be cytotoxic is not clear. In addition, freshly isolated human T-cell subsets were stimulated with phytohemagglutinin-P (PHA-P). After PHA stimulation, TG+, TM+, and TG-M- cells produced similar amounts of soluble cytotoxin, which was largely neutralized by anti-alpha-LT. The TG+ cells incorporated less thymidine than the TM+ or TG-M- cells. Likewise, OKT4+ and OKT8+ subsets, isolated with the aid of monoclonal OKT8 or OKT4 antibody and complement, yielded lymphotoxin after stimulation with PHA. It is shown that all T-cell subsets, as defined here, can produce lymphotoxin. Furthermore, depending on the assay system, cytotoxicity can be clearly demonstrated in all of these subsets, except in TM+ cells, where positive and negative results have been reported.

The immunosuppressive action of cyclosporine in transplantation (Tx) is thought to be due to its potent inhibition of lymphokine production by T cells. Several studies have shown a decrease in interleukin 2 (IL-2) and interferon-Gamma (IFN-G) production of renal Tx recipients on CsA treatment and have suggested that increases in lymphokine production can be correlated with rejection episodes. In this study we measured IL-2, IFN-G, and lymphotoxin (LT) production by mitogen-stimulated peripheral blood lymphocytes in eight renal Tx recipients before and at various times after Tx. IL-2 production was significantly (P less than 0.05) decreased by one week post-Tx compared with pre-Tx and normal levels. IFN-G production was significantly (P less than 0.05) decreased by one week post-Tx, after which time it returned to normal. LT production was not decreased post-Tx compared with pre-Tx or normal levels. Lymphokine production was measured every 48-72 hr in the first month post-Tx, when we failed to detect any correlation between increases in production in any of the three lymphokines and rejection episodes. A further group of patients were studied in whom the production of all three lymphokines was measured at the time of diagnosis of rejection and after treatment for rejection. In only 3/5 patients did IL-2 production decrease with a return to stable graft function, while IFN-G production did not alter in these patients. Interestingly LT production increased significantly (P less than 0.05) after treatment. We conclude from these studies that the usefulness of lymphokine determinations for the diagnosis of allograft rejection remains unproved.

T cell clones derived from patients with autoimmune diseases were found to be capable of producing tumor necrosis factor (TNF). This was demonstrated by stimulating the clones, in the absence of accessory cells, with antibodies against the Ti/T3 complex and with recombinant interleukin 2 (IL2). Analysis of RNA extracted from these clones showed that TNF mRNA was more abundant than lymphotoxin (LT) mRNA. We also found that TNF protein in the supernatants of these clones was generally more abundant than LT as assessed by using the murine L929 cell assay. TNF production was not limited to T cells from autoimmune individuals, since the T cell tumor HUT78 and T cells purified from the peripheral blood of healthy individuals also made TNF. Unlike the T cell clones, HUT78 produced greater amounts of LT mRNA than TNF mRNA. Induction of TNF mRNA in T cells from healthy individuals displayed a two-signal requirement (phorbol myristate 13-acetate and phytohemagglutinin or OKT3 and phorbol myristate 13-acetate), similar to that described for the induction of the T cell lymphokines IL 2 and interferon-gamma (IFN-gamma). Additionally we found that IL2 alone was sufficient to induce TNF in these cells when they had been precultured with phytohemagglutinin for 7 days to express IL 2 receptors. The cloned T cells we have characterized also produce IFN-gamma which was detected in the supernatants of the clones using a radioimmunoassay. The evidence suggests that T cells can produce TNF and have the potential to deliver by themselves the dual and synergistic signals of TNF/LT and IFN-gamma to target cells, a process which may be of importance in the pathogenesis of human autoimmunity.

The capacity of human peripheral blood-derived T cell clones to carry out a variety of functions was examined. T cell clones were generated by stimulating individual peripheral blood T cells with PHA by a procedure that yielded a growing clone from a mean of greater than 92% of the cultured cells. A total of 65 T cell clones (44 CD4+ and 21 CD8+) generated from two individual donors were examined for their functional capabilities. All T cell clones examined secreted IL-2, IFN-gamma, and lymphotoxin/tumor necrosis factor like activity when stimulated with immobilized mAb to the CD3 complex (64.1). When 54 additional T cell clones from a third donor were analyzed, all were found to produce IL-2. Upon activation with immobilized 64.1, all CD4+ clones and 91% of the CD8+ clones induced the generation of Ig-secreting cells from purified B cells. The CD8+ clones that did not serve as Th cells alone were able to augment the capacity of fresh CD4+ cells to generate Ig-secreting cells. Each of these clones was also found to effect MHC-unrestricted cytotoxicity upon activation with immobilized 64.1. The CD8+ clones were somewhat more effective killers than CD4+ clones, although there was considerable overlap. A total of 18 clones was analyzed for TCR beta-chain gene rearrangement. Of the clones exhibiting rearrangements of the beta-chain gene, 94% were found to have a single rearrangement pattern. Finally, the detailed phenotype of 15 (11 CD4+ and 4 CD8+) of these clones was examined. Variable numbers of cells of each of the clones expressed Ag identified by mAb 4B4 (CD29), Leu 8, Leu 15 (CD11b), and NKH1. Moreover, cells of 6 of 11 CD4+ clones and 4 of 4 CD8+ clones also expressed CD45R in addition to CD29; expression of CD45R and CD29 varied with the activation status of the clone. The current data demonstrate that nearly all of the T cell clones were able to accomplish each of the functions examined regardless of the surface phenotype. Inasmuch as the clones were generated using a technique that expanded more than 92% of the circulating T cells, the data imply that the progeny of the vast majority of T cells may have the inherent capacity to exert a wide array of functional activities.

Stereological data of phytohaemagglutinin (PHA)-activated human T-lymphocytes were recorded at intervals (12 to 72 h) together with biochemical (isotope-uptake, lymphotoxin-release) and morphological measurements. About 98% of the cells were activated 12 h after PHA-stimulation. The activation phase lasted less than 48 h, i.e., cells entering the activation phase within 12 h were at their activation maximum by 48 h. The activated cell increased in size. The nuclear/cytoplasmic-ratio decreased. Most of the cytoplasmic organelles developed in phase with the increase of cytoplasmic volume. After 48 h, mitotic figures were frequently seen. Due to the increasing number of secondary, activated daughter cells, parameters of most cytoplasmic components declined between 48 and 72 h. Structural changes in the nucleus preceded the 3H-leucine uptake, which had not reached its maximum after 72 h of incubation. The 3H-leucine uptake started as early as 12 h after culture initiation, and its increase was proportional to the increasing polyribosome density. No maximum uptake was reached up to 72 h, but the development of structural components related to this uptake was at its maximum at the end of the activation phase (48 h). The formation of bound ribosomes occurred subsequent to the enlargement of the surface of the rough endoplasmic reticulum. Initial polysome formation occurred at the expense of existing free ribosomes.

Lymphotoxin is produced by T cells sensitized to antigen upon re-exposure in vitro. It is also elicited by mitogen treatment. Its production has been correlated with delayed-type hypersensitivity and it may be a mediator of that phenomenon. We have examined the Ly phenotype of the subset(s) of T cells that produce lymphotoxin in order to investigate the relationship of lymphotokin killing to allo-killing mediated by Ly2+ T cells. We have found that Ly1 T cells sensitized to ovalbumin secrete more lymphotoxin than Ly2 cells. The ovalbumin-sensitized T cells do not lyse their target in a short term 51Cr-release assay even when "glued" to the target with Con A. Thus, lymphotoxin-producing cells differ phenotypically from the previously defined cytotoxic T cells that bear the Ly2 differentiation marker.

Identification of cells producing ATL was carried out on suspensions of cells enriched with lymphocytes T, B and macrophages. The cells were isolated from the spleen of syphilitic rabbits and from their testes when the rabbits were infected intratesticularly. The isolation of lymphocytes and macrophages was performed on different days after infection. It has been found that all examined cells are able to produce ATL but most active are T lymphocytes. These cells isolated from the testes were able to produce ATL already 6 days after infection, whereas lymphocytes B and macrophages acquired this capability not before day 10-13. The ability of cells to produce ATL was followed by marked reduction of treponemes from testes. The isolated treponemes were also immobilized and destroyed. Dependence between capability of cells to produce ATL and disappearance of treponemes from infected organ indicated that ATL kills treponemes also in vivo.