CD4+ Th1-cells predominate in low-grade B-cell lymphoma of gastric mucosa-associated lymphoid tissue (MALT type).

BACKGROUND: Little is known about the function of T cells in the inflammatory infiltrate in Helicobacter pylori-associated gastritis and B-cell lymphoma of mucosa-associated lymphoid tissue (MALT type). Previous studies have proposed a dominant Th1-type response in low-grade MALT lymphoma consistent with the Th1 response observed in H. pylori-associated gastritis. METHODS: We performed a novel flow cytometric approach in which CD3 panning for enrichment and activation of small numbers of T cells and intracellular cytokine analysis were combined to selectively characterize the cytokine profile of T cells (IFN-gamma for Th1) derived from the gastric mucosa of 23 patients with low-grade MALT lymphoma stage IEI1 (lymphoma infiltration of mucosa/submucosa sparing the muscularis). Endosonography was performed in each case to control the depth of lymphoma infiltration. For comparison, 19 patients with H. pylori-positive gastritis were also analysed. RESULTS: There was a CD4/CD8 ratio of 4 in patients with MALT lymphoma and of 2 in chronic gastritis. The proportion of IFN-gamma producing cells within the CD4-positive T-cell population in MALT lymphoma was 22%; in chronic gastritis it was 13% while no such difference could be encountered in CD8-positive T cells. CONCLUSIONS: The data point towards a dominant intratumoral IFN-gamma dominated T-cell response associated with early low-grade MALT lymphoma. A polarized IFN-gamma dominated Th1-type response may either contribute to the inability of the immune system to eradicate H. pylori infection, thereby promoting the activation status of the lymphocytic infiltrate in low-grade MALT lymphoma, or may mirror a concomitant tumor-specific T-cell response accompanying early stages of tumor progression.

Infliximab induces apoptosis in monocytes from patients with chronic active Crohn's disease by using a caspase-dependent pathway.

BACKGROUND & AIMS: Treatment with a chimeric anti-tumor necrosis factor (TNF) antibody (infliximab) has been shown to be highly efficient for patients with steroid-refractory Crohn's disease (CD). However, the mechanism of action remains largely unknown. As monocytopenia is commonly observed after treatment with infliximab, we investigated the role of infliximab-induced monocyte apoptosis. METHODS: Peripheral blood monocytes from healthy volunteers and patients with chronic active CD (CDAI > 250) were isolated by density gradient centrifugation methods. Apoptosis was determined by annexin V staining DNA-laddering, and transmission electron microscopy. Activation of caspases and mitochondrial release of cytochrome C was determined by immunoblotting. Transcriptional activation of members of the Bcl-2 family have been analyzed by ribonuclease protection assay. RESULTS: Treatment with infliximab at therapeutic concentrations resulted in monocyte apoptosis in patients with chronic active CD in a dose-dependent manner. Infliximab-induced monocyte-apoptosis required the activation of members of the caspase-family since activation of caspase-8, -9, and -3 could be determined. Caspase activation was induced by a CD95/CD95L independent signaling pathway with mitochondrial release of cytochrome C. Cytochrome C release seemed to be triggered by transcriptional activation of Bax and Bak. Monocyte apoptosis in vivo as determined by annexin-V binding and caspase-3 activation could be shown in patients with chronic active CD as soon as 4 hours after treatment with infliximab. CONCLUSIONS: Monocyte apoptosis induced by infliximab may be an important mechanism that could explain the powerful anti-inflammatory properties of infliximab in patients with chronic active CD.

Aspirin effects on gastric epithelial cell proliferation and cytokine expression.

Aspirin is known to cause gastric injury and to delay ulcer healing. The effects of aspirin on gastric epithelial cell function are heterogeneous; in contrast to injuring the mucosa, aspirin may also act beneficially by inducing adaptation; a mechanism that is poorly understood. We aimed to document the effects of different doses of aspirin on gastric epithelial cell function defined as proliferation, and secretion as well as mRNA expression of cytokines. Furthermore, we studied the effects of aspirin pretreatment on cytokine secretion as a potential element of gastric adaptation. The proliferative activity of three different gastric epithelial cell lines (AGS, KATO III, RGM-1) was assessed by (3)H-thymidine incorporation; secretion of growth factors PDGF-AB and VEGF into culture supernatant was documented by ELISA. mRNA transcripts of both cytokines were quantified by real time RT-PCR. Low doses of aspirin did not alter the proliferative dynamics in two of the three studied cell lines; high doses abolished proliferation. Secretion of PDGF-AB and VEGF increased during the first days of low dose aspirin exposition; higher concentrations led to a depletion of cytokines after an initial liberation in the case of VEGF, mRNA of which was also dose-dependently increased by aspirin. Seven-day pretreatment with low amounts of aspirin did not alter the secretory response of the epithelia caused by higher doses of this drug. The secretion of cytokines and proliferation of gastric epithelial cells are adversely effected by aspirin in a similarly dose-dependent fashion as the intended effects of this drug on platelet function and pain relief.

Prostaglandins, but not tumor-derived IL-10, shut down concomitant tumor-specific CTL responses during murine plasmacytoma progression.

IL-10 is assumed to be a major immunosuppressive factor produced by most B-cell tumors. The immunosuppressive role of tumor-derived IL-10 was analyzed using the MHC class II-negative BALB/c plasmacytoma ADJ-PC-5 as a model tumor. Immune monitoring of tumor-bearing mice was based on the measurement of tumor burden, tumor-specific CTL cytotoxicity and intracellular cytokine staining using FACS. ADJ-PC-5 tumor progression in syngeneic recipients is associated with strong, concomitant, tumor-specific CTL responses during early stages of tumor progression which are sufficient to cause rejection of small s.c. autologous test tumors. These initial CTL responses gradually decline during later tumor stages. Blocking of IL-10 in vivo did not abolish CTL suppression or retard tumor growth. More strikingly, application of anti-IL-10 antibodies during early tumor stages abrogated CTL induction and markedly accelerated tumor growth. In contrast to anti-IL-10 treatment, application of cyclo-oxygenase inhibitors to ADJ-PC-5 tumor-bearing mice led to enhanced tumor-specific CTL responses throughout all stages of tumor progression, paralleled by retarded tumor growth and a significantly delayed onset of suppression. Both findings contradict a dominant immunosuppressive role of IL-10 during B-cell tumor progression. Tumor-derived IL-10 must therefore be considered an immunostimulating factor, which accounts for the high immunogenicity of B-cell tumors, whereas prostaglandins, which are not produced by the tumor cells themselves, are the dominant immunosuppressors in this system.

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-gamma) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells.

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll-Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-gamma and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-gamma-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-gamma+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial-lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD-even in an inactive state of disease-exert an increased capacity for IFN-gamma induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-gamma-producing CD8+ lymphocytes.

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis.

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N:-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.

Differential expression of heat shock protein 70 (hsp70) in human monocytes rendered apoptotic by IL-4 or serum deprivation.

Apoptosis of monocytes is regulated by the balance between pro- and antiapoptotic triggers and pathways and may strongly influence inflammatory disorders. The major heat shock protein, hsp70, is an effective inhibitor of apoptosis in lymphocytic and monocytic tumor cell lines, but the implications in the regulation of apoptosis of freshly isolated human monocytes have not been elucidated. In this study, we examined whether two different triggers of monocyte apoptosis, serum deprivation and IL-4, respectively, altered hsp70 expression and whether expression levels correlated with monocyte survival. Monocyte apoptosis was determined quantitatively by flow cytometry detecting annexin V binding or nuclear stainability with propidium iodide (PI). Hsp70 expression was analyzed by semiquantitative RT-PCR and immunoblotting. Exposing monocytes to heat shock (47 degrees C, 20 min) induced a rapid and marked upregulation of hsp70 without evoking injury or apoptosis, suggesting that hsp70 conferred protection and survival. In accordance, when monocytes were rendered apoptotic by serum deprivation, a drastic downregulation of hsp70 occurred, which was accompanied by a reduced synthesis of the constitutive family member hsc70. However, induction of monocyte apoptosis by IL-4 increased hsp70 expression in a concentration and time-dependent fashion. A neutralizing antibody against IL-4 abolished hsp70 expression and apoptosis induction after IL-4 treatment and so excluded indirect effects. LPS rescued monocytes from apoptosis but did not alter hsp70 formation significantly. These findings suggest that, in monocytes, distinct apoptotic triggers induce different responses of hsp70 so that this molecule does not exert protection against cell death directly or in general.

Regulation of mesangial cell apoptosis and proliferation by intracellular Ca(2+) signals.

BACKGROUND: In inflammatory glomerular diseases, proliferation, as well as apoptosis of mesangial cells (MCs), has been shown histomorphologically. Both processes may regulate the cellular content of the mesangium by closely influencing each other. In the present study, we examined whether the cytoplasmic free Ca(2+) concentration [Ca(2+)](i) is involved as a key second messenger in the regulation of proliferative and apoptotic events. METHODS: Thapsigargin, an inhibitor of the endoplasmic Ca(2+)-Mg(2+)-ATPase, was used as a test substance to investigate the role of [Ca(2+)](i) in signaling MC apoptosis and growth in vitro. Apoptosis was determined by nuclear chromatin staining with Hoechst 33258, by a [3H]-thymidine-based DNA fragmentation assay or by flow cytometry detecting binding of FITC-conjugated annexin V. Proliferation was measured by [3H]-thymidine incorporation into acid-precipitable material and corroborated by cell counting. RESULTS: Thapsigargin significantly induced apoptosis and inhibited proliferation dose dependently in nanomolar concentrations without evoking necrotic damage when administered not longer than 12 hours. Significant apoptosis was measurable after a six-hour treatment of MCs with thapsigargin. Determination of [Ca(2+)](i) by fura-2-dependent spectrofluorometry showed that thapsigargin was able to induce prolonged [Ca(2+)](i) rises that could be prevented by preincubation with the intracellular Ca(2+) chelator 1, 2-bis(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid (BAPTA) acetomethyl ester (AM). BAPTA had no influence on MC viability but reversed thapsigargin-induced apoptosis to control levels. After thapsigargin treatment (100 nmol/L, 12 hours), apoptotic MCs had a significantly higher [Ca(2+)](i) of 251 +/- 25 nmol/L (N = 41) as compared with MCs that were not or not yet apoptotic ([Ca(2+)](i) of 116 +/- 20 nmol/L, N = 26, P < 0,05). Platelet-derived growth factor (PDGF), a well-characterized growth factor for MCs, reversed the effects of thapsigargin on proliferation and apoptosis in a similar fashion as BAPTA. PDGF acutely stimulated increases of [Ca2+]i but abolished thapsigargin-dependent, but not angiotensin II- or ATP-induced Ca(2+) rises when administered during a 12-hour preincubation. CONCLUSIONS: Our data suggest that a sustained increase of [Ca(2+)](i) may serve as a signal to trigger MC apoptosis. Growth factors such as PDGF can abolish apoptosis induced by elevations of [Ca(2+)](i) by altering intracellular Ca(2+) signaling.

IL-10 induces apoptosis in human monocytes involving the CD95 receptor/ligand pathway.

The cytokine IL-10 exerts potent immunosuppressive and anti-inflammatory effects, although the mechanisms of this action remain largely unknown. In the present study, we investigated the effects of IL-10 in human peripheral blood monocytes. We were able to demonstrate that IL-10 dose- and time-dependently triggers apoptosis in these cells as detected by annexin-V staining, the nick end labeling (TUNEL) procedure, electron microscopy and analysis of DNA laddering. IL-10-induced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death and, in addition, the proteolytic activation of caspase-8 was observed. Since caspase-8 has been implicated as a regulator of apoptosis mediated by death receptors, we investigated a potential involvement of the CD95 receptor/ligand system. Indeed, treatment of monocytes with IL-10 induced a dose-dependent up-regulation of CD95 receptor and ligand expression on the monocyte surface. Furthermore, a CD95 ligand-neutralizing antibody significantly inhibited IL-10-induced apoptosis. In summary, our data show that IL-10 triggers monocyte apoptosis involving the CD95 system via an autocrine or paracrine process. Therefore, at least part of the anti-inflammatory properties of IL-10 may involve induction of apoptosis in monocytes.

Glucocorticoids induce apoptosis in human monocytes: potential role of IL-1 beta.

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including monocytes and macrophages. However, the exact cellular mechanisms underlying this anti-inflammatory capacity are still unknown. In our study, we determined the induction of apoptosis by GC in human monocytes. Peripheral blood monocytes were isolated by density centrifugation methods with a purity of >90% and were cultured in RPMI 1640 medium. Monocyte apoptosis was determined by four independent methods, including annexin-V staining, TUNEL, DNA-laddering, and typical morphology by means of transmission electron microscopy. TNF-alpha and IL-1beta were measured by ELISA. GC receptor was blocked with mifepristone. Caspase 3 was inhibited with caspase-3 inhibitor (DEVD-CHO). Stimulation with different GC at therapeutic concentrations resulted in monocyte apoptosis in a time- and dose-dependent manner. Necrosis was excluded by propidium iodide staining. Proinflammatory cytokines such as IL-1beta and TNF-alpha were down-regulated by GC treatment. Continuous treatment of monocytes with IL-1beta, but not with TNF-alpha, could almost completely prevent GC-induced cell death. The addition of mifepristone or caspase-3 inhibitor could partially abrogate GC-induced apoptosis as well as GC-induced inhibition of IL-1beta. This is the first study to demonstrate induction of apoptosis by GC in human monocytes. GC-induced monocyte apoptosis may be partially mediated through effects on IL-1beta production. It is conceivable that GC exert their anti-inflammatory capacity in various diseases, at least in part, by the induction of apoptosis in monocytes.

Differential activation of CD8+ tumor-specific Tc1 and Tc2 cells by an IL-10-producing murine plasmacytoma.

The involvement of counteractive CD8+ T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model. CD8+ Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cells in vivo and in vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumor-induced CD8+ T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-gamma as a suppressive factor. Whereas most long-term cultivated CD8+ ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10. CD8+ Tc cells that are induced in a primary MLTC do not produce IFN-gamma, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-gamma or IL-12. In contrast, ADJ-PC-5-specific CD8+ Tc cells from immunized mice are IFN-gamma producing Tc1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-gamma, these Tc1 cells behave similar to the suppressive CD8+ T cells that are induced during early stages of ADJ-PC-5 tumorigenesis.

IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells.

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP-1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4, IL-10 and IL-13 have been described to exert anti-inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP-1 by activated intestinal epithelial cells. We examined Caco-2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP-1 was determined under stimulated and non-stimulated conditions. IL-4, IL-10 and IL-13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL-1beta or tumour necrosis factor-alpha (TNF-alpha), we observed markedly increased concentrations of MCP-1 in supernatants of Caco-2 cells and intestinal epithelial cells. IL-4, IL-10 and IL-13 all had the capacity to down-regulate the production of MCP-1 in Caco-2 cells as well as in freshly isolated epithelial cells. Caco-2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL-1beta or TNF-alpha for MCP-1 production. As MCP-1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP-1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.

Regulation of human monocyte apoptosis by the CD14 molecule.

Bacterial products such as LPS have been shown to activate monocytes and to increase CD14 expression, while anti-inflammatory cytokines, i.e., IL-4, down-regulate CD14. Furthermore, activation of monocytes increases survival, whereas deactivation evokes apoptosis (programmed cell death, PCD). This correlation among activation, CD14 expression, and the lifespan of the cells prompted us to investigate the role of CD14 in monocyte apoptosis. The effects of LPS and IL-4 on the expression of CD14, indicated by binding of Leu M3 Ab, and PCD of monocytes were studied simultaneously and in a kinetic fashion by multiparameter flow cytometry. Monocyte PCD was determined by binding of FITC-conjugated annexin V, which indicates apoptotic cell death in early stages, and was confirmed using well-established detection methods, i.e., DNA electrophoresis, electron microscopy, or colorimetric DNA staining. The present study shows that the LPS-induced increase in CD14 expression rescued monocytes from apoptosis, whereas IL-4 treatment first down-regulated CD14 expression and consecutively evoked apoptosis. CD14-/annexin V- monocytes were not apoptotic as confirmed by DNA electrophoresis, whereas CD14-/ annexin V+ monocytes showed clear apoptotic features. Kinetic studies ruled out that monocytes first bound annexin V and later lost the CD14 Ag. Other molecules, such as HLA-A, -B, and -C Ags, were not down-regulated during apoptosis. Enzymatic removal of membrane-bound CD14 by phosphatidylinositol-specific phospholipase C evoked PCD similarly to IL-4. These results suggest that regulation of CD14 receptor expression is an early effector mechanism mediating life or death of monocytes. Down-regulation or removal of the receptor triggers apoptosis, whereas up-regulation promotes survival.

CD8+ tumor-specific Tc cells primed in vivo or in vitro against the BALB/c plasmacytoma ADJ-PC-5 use the same TcR V beta families but display distinct TC1 or TC2 characteristics.

The involvement of counteractive CD8+ T cell subsets in tumor-specific unresponsiveness was analyzed in a syngeneic murine tumor model. CD8+ cytotoxic T cells against the IL-10 producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced in vitro, in a primary syngeneic mixed lymphocyte tumor cell culture (MLTC), or in vivo, by repeated immunization of syngeneic BALB/c mice with high doses of X-irradiated ADJ-PC-5 tumor cells. Long term cultivated CD8+ ADJ-PC-5-specific Tc lines use either TcR of the V beta 6 or V beta 8.1/8.2 type, irrespective if the lines were derived from a primary MLTC or from immunized mice. While most of the Tc lines produce type-1 cytokines (IFN-gamma, no IL-4) upon stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum (IL-4, no IFN-gamma). The primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a TC2 response: CD8+ Tc cells which are induced in a primary MLTC do not produce IFN-gamma, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-gamma or IL-12. In contrast, ADJ-PC-5-specific CD8+ Tc cells from immunized mice are IFN-gamma producing TC1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by lowest numbers of irradiated ADJ-PC-5-specific TC1 cells via IFN-gamma, these TC1 cells behave similar to a previously described regulatory subset of IFN-gamma producing CD8+ T cells, which are induced during early stages of ADJ-PC-5 tumorigenesis and inhibit the induction of a tumor-specific Tc response from naive BALB/c spleen cells in vitro.

Suppression of tumour-specific cytotoxic T-cell responses against the syngeneic BALB/c plasmacytoma ADJ-PC-5 by tumour-induced CD8+ regulatory T cells via IFN-gamma.

The mechanisms of tolerance induction by tumour cells during early stages of tumourigenesis were analysed in a murine model system using the highly immunogenic BALB/c plasmacytoma ADJ-PC-5. Early stages of tumourigenesis were simulated in syngeneic BALB/c mice by repeated intraperitoneal injections with subimmunogenic doses of X-irradiated ADJ-PC-5 tumour cells. This treatment causes a state of tumour-specific tolerance in a high percentage of mice, involving a population of CD8+ peritoneal T cells which are able to suppress a protective tumour-specific Tc response against this tumour. Using a primary mixed lymphocyte tumour cell culture (MLTC) as an in vitro system to study suppressive mechanisms of such regulatory T cells, the role of production or consumption of a number of cytokines was analysed. The data presented here demonstrate that inhibition of a protective Tc response against ADJ-PC-5 tumour cells is due to IFN-gamma production by suppressive T cells from tolerized mice, but not to IL-2 consumption. In contrast to typical CD8+ Tc cells, ADJ-PC-5-specific CD8+ Tc cells do not produce IFN-gamma and are furthermore suppressed by IFN-gamma. Thus, tumour-induced suppressive T cells and tumour-specific Tc cells seem to represent functionally and phenotypically different subsets of CD8+ T cells, possibly pointing towards a differential activation of type-1 and type-2 CD8+ T cells depending on the dose of tumour cells.

T helper target cell DNA fragmentation through a CD4-positive T suppressor cell clone inducing specific unresponsiveness.

The CD4-positive bovine serum albumin (BSA)-specific Ts cell clone BVI/5 from a CBA/J mouse tolerized to low doses of BSA induces specific unresponsiveness in the T helper (Th) cell population. Tolerance induction can be measured in vitro in proliferation assays using specific Th cell clones or antigen-primed lymph node cells (LNC) and determined in vivo by the failure to produce hapten-specific antibodies. Using the BSA-specific Th cell clone 83/1 as a target one observes in addition 51Cr-release in a 16-hr long-term assay but finds no effect in a typical 6-hr T cell cytotoxicity test. BVI/5 Ts cells do not produce interleukin-2 but otherwise express a Th1 profile. The suppression of proliferation of 83/1 Th cells is partly due to interferon-gamma (IFN-gamma). But lysis of 83/1 Th cells as well as suppression of BSA-specific LNC proliferation needs direct cell contact between BVI/5 Ts cells and their targets. Cell lysis and suppression of LNC cannot be simulated by IFN-gamma, by the combination of IFN-gamma and TNF, or by BVI/5 supernatants. Thus mediators cannot account for specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from LNC and are probably not responsible for the induction of in vivo unresponsiveness. Instead the data show that BVI/5 Ts cells induce apoptosis-like DNA fragmentation in cloned BSA-specific 83/1 Th cells and in LNC from BSA-primed mice. Apoptosis can also be visualized as chromatin condensation in the LNC population. Macrophages have been excluded as targets. It can further be demonstrated that BVI/5 Ts cells express perforin and granzyme A on activation. Thus they are equipped with the effector molecules for target cell destruction. We consider BVI/5 Ts cells to be representative of a regulatory T cell inducing specific unresponsiveness in peripheral lymphoid organs.

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.

T cell receptor alpha and beta gene expression in a murine antigen-specific T suppressor lymphocyte clone with cytolytic potential.

The composition of alpha and beta TCR genes was analyzed in a murine BSA-specific Ts cell clone with cytolytic potential. The isolated poly(A)+ mRNA from Ts cell clone BVI/5 was used to construct a cDNA library in the bacteriophage lambda gt11. Full-length cDNA clones specific for TCR alpha and TCR beta genes have been detected and isolated by hybridization with specific oligonucleotide probes. The functional rearranged TCR alpha gene is composed of a member of the V alpha 1 family, the junctional gene J alpha TT11 and C alpha. The gene segments V beta 13, D beta 2.1, J beta 2.4, and C beta 2 form the functional rearranged TCR beta gene. Furthermore, a nonfunctional TCR alpha gene transcript has been detected, where a V alpha 8 gene is rearranged to a so far not described J alpha gene segment (J alpha BVI). A stop codon in its junctional region is responsible for this non-functional transcript. By using Southern blot analysis, the described rearranged TCR genes can be detected in the J alpha junctional region and in the J beta 2 cluster on the genomic DNA level. Immunoprecipitation studies with the KT3 anti-CD3 mAb and flow microfluorimetry analysis with the H57-597 anti-TCR-alpha/beta mAb show that TCR/CD3 complexes are synthesized and expressed on BVI/5 Ts cells. Taken together, the cDNA sequencing data, the protein studies, and the specificity of Ag recognition demonstrate that the BVI/5 Ts cell clone not only transcribes the TCR alpha and beta genes but also expresses a functional BSA-specific receptor.