Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

Metabolic carbohydrate-labelling of glycolipids from mouse splenocytes. Mitogen-stimulated B and T cells show different labelling patterns.

Splenic lymphocytes from CBA/J, AKR/A/J, BALB/c/A, C57/BL/6J, C3H/HeJ and C3H/Tif nu/nu mice and B lymphocyte or T lymphocyte preparations derived from CBA/J mouse spleen were cultivated in the presence of either concanavalin A, phytohemagglutinin, Salmonella minnesota R595 lipopolysaccharide or Proteus mirabilis soluble lipoprotein. The mitogens stimulated the incorporation of [14C]galactose into acid-insoluble cell material with the same specificity for B or T cells as that known for thymidine incorporation. The glycolipids extracted from mitogen-activated, carbohydrate-labelled B or T cells were compared by thin-layer chromatography and characteristic differences between B and T cells were noted in the ganglioside as well as in the neutral glycolipid fractions. In addition, subsets of B or T cells, namely lipopolysaccharide-responsive or lipoprotein-responsive B-cell populations or nylon-purified T cells may be recognized by characteristic neutral glycolipid bands.     

Syndecan-2 expression enhances adhesion and proliferation of stably transfected Swiss 3T3 cells

Syndecans (heparan sulfate proteoglycans) participate in cell-cell and cell-matrix adhesion and are co- and low-affinity receptors for growth factors and enzymes, respectively. We examined the influence of stable syndecan-2 expression in Swiss 3T3 cells on cell-adhesion and proliferation. Higher syndecan-2 expression changed cell morphology and increased spreading and adhesion in these cells and proliferation induced by FCS and FGF-2. This emphasizes the role of syndecan-2 in the integration of signals from soluble and insoluble factors.     

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.     

Suppressor T cells activated by lymphoblastoid cell lines inhibit pokeweed mitogen-lnduced immunoglobulin synthesis

EBV-transformed B-cell lines of normal or malignant origin suppressed pokeweed mitogen-induced immunoglobulin synthesis of normal B cells, measured by the protein A plaque assay method. Autologous and allogeneic LCL were equally effective. Allogeneic irradiated B cells were ineffective or slightly enhancing. EBV-negative hematopoietic cell lines derived from myeloid leukemia (K562 and HL-60) were ineffective or slightly enhancing. The suppressive effect of the Burkitt lymphoma line Raji was partly due to released soluble suppressor factors, but to a larger extent to the generation of radiosensitive suppressor T cells in the responder population. Autologous and allogeneic LCL were equally effective in generating suppressor T cells. It is postulated that the suppressor circuit reflects the existence of regulatory mechanisms that govern the proliferation of B lymphocytes.     

IgD-receptor-positive human T lymphocytes. II. Identification and partial characterization of human IgD-binding factor.

Membrane receptors specific for IgD (IgD-R) have been identified on murine CD4+ and human CD4+ and CD8+ T lymphocytes. Up-regulation of these IgD-specific receptors can be achieved by exposure of such T cells to various stimuli, including oligomeric or Ag cross-linked IgD, IL-2, IL-4, and T cell mitogens, such as PHA. Previous studies with murine IgD-R+ splenic T cells and IgD-R+ T hybridoma cells have demonstrated the existence of soluble IgD-binding factors (IgD-BF) that are shed or released into the medium in which these cells are grown. In our study, human peripheral blood T cells and IgD-R+ T hybridoma cells were examined for their ability to produce human IgD-BF. PHA stimulation of peripheral blood T cells results in their release of an IgD-specific factor with an apparent Mr of 70 kDa. IgD- Sepharose-purified IgD-BF was able to competitively inhibit rosetting of IgD-R+ T cells with IgD-coated RBC. Immunoblot assays in which alkaline phosphatase-conjugated human IgD myeloma protein was used as a probe, confirmed the IgD specificity of IgD-BF. An IgD-BF-specific mAb (LTB9) that also reacts with membrane IgD-R was produced after immunization of BALB/c mice with this factor. LTB9 was able to detect IgD-BF in the supernatants derived from human IgD-R+, tetanus toxoid-specific T hybridoma cells, H9-CEM1, and to stain membrane IgD-R by indirect immunofluorescence. Stimulation of H9-CEM1 cells with immobilized IgD resulted in up-regulation of membrane IgD-R expression, as measured cytofluorometrically with LTB9-stained cells, and potentiated release of IgD-BF from these cells. Finally, LTB9 as well as IgD-Sepharose, immunoprecipitated a 70-kDa protein from the lysates of biosynthetically labeled H9-CEM1 cells. Similar immunoprecipitation results were obtained with H9-CEM1-derived supernatants containing IgD-BF. Taken together, these results support the hypothesis that human T cell membrane IgD-R are released as soluble IgD-BF.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Filarial parasite-specific T cell lines: induction of IgE synthesis

The development of T lymphocyte lines and clones of defined specificity has provided an important method for investigating T cell recognition of foreign antigens as well as T cell influence on B cell activity. We described previously a parasite-specific T cell line (TCL) derived from a patient with a naturally acquired filarial infection and elevated levels of serum IgE. The TCL is composed of Leu-3+ helper cells and is maintained independent of exogenous growth factors. In the present study, we used these T cells to investigate their immunoregulatory function on the in vitro IgE response. These parasite-specific T cells can provide isotype-specific help for antigen-induced IgE production by B cells in vitro. Autologous T cells profoundly suppress IgE production in a concentration-dependent manner. Furthermore, soluble factors generated from these filarial-specific TCL after antigen stimulation are able to induce the production of IgE in normal human cells not already synthesizing measurable amounts of IgE in vitro. Partial physicochemical characterization of this factor has shown that it is heat labile, has an m.w. between 10,000 and 30,000 M(r), and is a mannose-rich glycoprotein.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.     

Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus.

Actin and tubulin are major protein constituents of 3T3 and SV40 virus-transformed 3T3 cells. We have fractionated growing, confluent and SV403T3 cells into particulate and soluble fractions using conditions designed to sediment microtubules, actin filaments or membrane associated actin or tubulin. The ratio of particulate to soluble actin synthesized in growing or confluent 3T3 cells is 2 to 1, while the ratio is reversed in transformed cells. There is also a 60% decrease in particulate tubulin synthesis in SV403T3 cells when compared with that in normal cells. Similar results are obtained when total actin and tubulin amounts are determined. The half-lives of actin, tubulin and total protein are over 3 days in growing 3T3 and SV40 cells and decrease over two-fold in confluent 3T3 cells. The significance of these results with respect to loss of contact inhibition and development of malignancy by these cells after transformation is discussed.     

Distinct T cell/renal tubular epithelial cell interactions define differential chemokine production: implications for tubulointerstitial injury in chronic glomerulonephritides

Chemokines can promote interstitial fibrosis that is, in turn, a strong predictor of renal failure in chronic glomerulonephritides (GN). Resident renal cells, including renal tubular epithelial cells (RTEC), represent a prominent source of chemokine expression. Evaluating those factors responsible for sustained chemokine production by RTEC during GN is therefore crucial. The contribution of interstitial T cells to such expression, and in particular the precise nature of their interactions with RTEC, are poorly understood. Activated T cell/RTEC coculture induced production of high levels of monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-inducible protein-10 from RTEC. Using double-chamber cultures and activated T cell plasma membrane preparations we demonstrated that both cell contact and soluble factors contributed to RTEC chemokine production. Importantly, different chemokines exhibited distinct activation requirements. Thus, for RANTES cell contact was essential, but not sufficient. In contrast, either soluble factors or cell contact induced MCP-1 and IFN-inducible protein-10 production, although both pathways were required for a maximal response. Neutralization experiments identified critical roles in this process for proinflammatory cytokines such as TNF-alpha, IL-1beta, and IFN-gamma as well as membrane molecules such as LFA-1, CD40 ligand, and membrane bound TNF-alpha. Finally, chemotactic bioassays of T cell/RTEC coculture supernatants demonstrated 80% reduction of monocyte migration following MCP-1 neutralization, indicating a dominant role for this chemokine. In summary, activation of renal tubular cells by infiltrating T cells can amplify and perpetuate local inflammatory responses through chemokine production differentially mediated by soluble and cell contact-dependent factors. Recognition of this regulatory diversity has important implications in the choice of potential therapeutic targets in GN.     

Cloned, Ia-restricted T cells that do not produce interleukin 4(IL 4)/B cell stimulatory factor 1(BSF-1) fail to help antigen-specific B cells

T cells can be subdivided based on cell surface markers, MHC restriction, function, and production of soluble factors. Analysis of the ability of cloned, Ia-restricted, L3T4+ T cells to induce an in vitro anti-hapten antibody response to hapten-carrier conjugates allowed the definition of three functional subtypes. To examine whether these functional subtypes also differed in the production of soluble mediators, supernatants of the cloned lines were examined for the production of T cell growth factors and factors inducing increased expression of Ia glycoproteins on small resting B cells. All of the cloned lines produced T cell growth factors that could be further differentiated by inhibition with monoclonal antibodies. None of the Ia-restricted, L3T4+ cloned T cell lines that failed to produce IL 4/BSF-1 could provide helper function. Thus, the activation of antigen-specific B cells by helper T cells appears to require IL 4/BSF-1 as a necessary but not sufficient signal for differentiation into antibody-forming cells.     

Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes

The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.     

Sphingomyelinase and cell-permeable ceramide analogs stimulate cellular proliferation in quiescent Swiss 3T3 fibroblasts.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.     

Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixed lymphocyte reaction systems

BACKGROUND AIMS. Studies performed using human and animal models have indicated the immunoregulatory capability of mesenchymal stromal cells in several lineages. We investigated whether human dental pulp-derived stem cells (hDP-SC) have regulatory effects on phytohemagglutinin (PHA)-activated CD3(+) T cells. We aimed to define the regulatory mechanisms associated with hDP-SC that occur in mixed lymphocyte reaction (MLR) and transwell systems with PHA-CD3(+) T cells and hDP-SC at a ratio of 1:1. METHODS. Proliferation, apoptosis and pro- and anti-inflammatory cytokines of PHA-CD3(+)T cells, the expression of Regulatory T cells (Treg) markers and some regulatory factors related to hDP-SC, were studied in Both transwell and MLR are co-cultures systems. RESULTS. Anti-proliferative and apoptotic effects of hDP-SC were determined in co-culture systems. Elevated expression levels of human leukocyte antigen (HLA)-G, hepatocyte growth factor (HGF)-β1, intracellular adhesion molecule (ICAM-1)-1, interleukin (IL)-6, IL-10, transforming growth factor (TGF)-β1, vascular adhesion molecule (VCAM)-1 and vascular endothelial growth factor (VEGF) by hDP-SC were detected in the co-culture systems. We observed decreased expression levels of pro-inflammatory cytokines [interferon (IFN)-γ, IL-2, IL-6 receptor (R), IL-12, Interleukin-17A (IL-17A), tumor necrosis factor (TNF)-α] and increased expression levels of anti-inflammatory cytokine [inducible protein (IP)-10] from PHA-CD3(+) T cells in the transwell system. Expression of Treg (CD4(+) CD25(+) Foxp3(+)) markers was significantly induced by hDP-SC in both co-culture systems. We observed apoptosis of PHA-CD3(+) T cells with 24 h using time-lapse camera photographs and active caspase labeling; it is likely that paracrine soluble factors and molecular signals secreted by hDP-SC led this apoptosis. CONCLUSIONS. We suggest that hDP-SC have potent immunoregulatory functions because of their soluble factors and cytokines via paracrine mechanisms associated with PHA-CD3(+) T cells, which could contribute to clinical therapies.     

Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

Interactions between Trypanosoma brucei and CD8+ T cells

The mechanisms for several important features o f African trypanosomiasis are still largely unexplained. These include (1) the occurrence of parasite growth-promoting molecules provided by the host, (2) polyclonal T- and B-cell activation and the development of immunosuppression and (3) neuropsychiatric signs. Here Tomas Olsson, Moiz Bakhiet and Krister Kristensson focus on interactive events between Trypanosoma brucei and CD8(+) T cells, and the release of a diffusible molecule from T. brucei that triggers CD8(+) T cells to produce interferon-gamma (IFN-gamma). This cytokine provides a growth stimulus for the parasite and modulates events in the host's immune and nervous systems.