Inducing differentiation of transformed cells with hybrid polar compounds: a cell cycle-dependent process.

Transformed cells do not necessarily lose their capacity to differentiate. Various agents can induce many types of neoplastic cells to terminal differentiation. Among such inducers, a particularly potent group consists of hybrid polar compounds; hexamethylene bisacetamide (HMBA) is the prototype of this group. With virus-transformed murine erythroleukemia cells as a model, HMBA was shown to cause these cells to arrest in G1 phase and express globin genes. This review focuses on HMBA-induced modulation of factors regulating G1-to-S phase progression, including a decrease in the G1 cyclin-dependent kinase cdk4, associated with inhibition of phosphorylation of the retinoblastoma protein pRB and possibly other related proteins that, in turn, sequester factors required for initiation of DNA synthesis; this provides a possible mechanism for HMBA-induced terminal cell division. Evidence that hybrid polar compounds have therapeutic potential for cancer treatment will also be reviewed.     

Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo.

Recruitment of antigen-specific tumor-infiltrating lymphocytes (TILs) is a major goal for immunotherapy of malignant tumours. We now describe that T-cell-activating superantigens targeted to a tumor by monoclonal antibodies induced large numbers of pseudospecific TILs and eradication of micrometastases. As a model for tumor micrometastases, syngeneic B16 melanoma cells transfected with the human colon carcinoma antigen C215 were injected intravenously into C57BL/6 mice and therapy with an anti-C215 Fab fragment-staphylococcal enterotoxin A (C215Fab-SEA) fusion protein reacting with the C215 antigen was initiated when visible lung metastases were established. More than 90% reduction of the number of lung metastases was observed when mice carrying 5-day-old established lung metastases were treated with C215Fab-SEA. The antitumor effect of C215Fab-SEA was shown to be T-cell-dependent since no therapeutic effect was seen in T-cell-deficient nude mice. Depletion of T-cell subsets by injection of monoclonal antibody demonstrated that CD8+ cells were the most prominent effector cells although some contribution from CD4+ cells was also noted. C215Fab-SEA treatment induced massive tumor infiltration of CD4+ and CD8+ T cells, while only scattered T cells were observed in untreated tumors. SEA treatment alone induced a slight general inflammatory response in the lung parenchyme, but no specific accumulation of T cells was seen in the tumor. TILs induced by C215Fab-SEA were mainly CD8+ but a substantial number of CD4+ cells were also present. Immunohistochemical analysis showed strong production of the tumoricidal cytokines tumor necrosis factor alpha and interferon gamma in the tumor. Thus, the C215Fab-SEA fusion protein targets effector T lymphocytes to established tumors in vivo and provokes a strong local antitumor immune response.     

A new group of potent inducers of differentiation in murine erythroleukemia cells.

This report identifies a group of compounds, polymethylene bisacetamides (acetylated diamines), which are potent inducers of erythroid differentiation in murine erythroleukemia cells. A known inducing agent, N-methylacetamide, was dimerized through varying numbers of methylenes in an attempt to increase the local effective concentration at adjacent target sites. The simple dimer was no more effective than N-methylacetamide alone; introduction of five to eight methylenes between acetamide groups substantially increased the effectiveness of these compounds. The hexamethylene bisacetamide was active between 0.5 mM and 5 mM; the percentage of cells induced and the rate at which they were recruited to differentiation was dependent upon the concentration of inducer within this range. At 5 mM hexamethylene bisacetamide essentially the entire population (greater than 99%) was induced to a pathway of erythroid differentiation which was greater differentiation of the cultured cells than with any inducer yet tested.     

Erythroid cell differentiation: murine erythroleukemia cell variant with unique pattern of induction by polar compounds.

The murine-virus-infected erythroleukemia cell system provides an opportunity to examine regulatory mechanisms controlling cytodifferentiation. A cloned cell line (DR10c3) resistant to the erythropoiesis-inducing effect of dimethylsulfoxide (Me2SO) was isolated from the Me2SO-sensitive line DS19. DR10c3 is characterized as follows: (1) the uptake of [3H]Me2SO is similar to that in DS19; (2) cell growth with and without Me2SO is similar to that of DS19; (3) resistance is relatively stable; (4) the karyotype of DR10c3 reveals an average loss of five chromosomes per cell, but is otherwise similar to that of DS19; (5) total protein and globin synthesis by cells cultured 4 days with or without Me2SO is similar to these syntheses in DS19 cultured without Me2SO; (6) virtually no globin mRNA is detectable after 3 days in Me2SO, as assayed both by RNA-complementary DNA hybridization and by the heterologous cell-free protein-synthesizing system; (7) other polar compounds, N-methylpyrrolidinone, 1-methyl-2-piperidone, N, N-dimethylacetamide, and N-methylacetamide, induce erythroid differentiation in DR10c3, and the accumulation of alpha- and beta-globin chains is indistinguishable from that in DS19; and (8) the concentration optima for induction of differentiation by all these compounds are identical for DR10c3 and DS19.     

Cryoprotective agents as inducers of erythroleukemic cell differentiation in vitro.

The ability of families of compounds with known and potential cryoprotective properties to induce the differentiation of Friend leukemia cells in vitro was studied. For each agent, both the proportion of differentiated cells in the culture and the total amount of heme/10(7) cells were determined. Within each family of compounds there was a direct correlation between a compound's cryoprotective ability, its ability to donate electron pairs for hydrogen bonding (basicity), and its ability to induce differentiation. While individual agents differed with respect to the proportion of cells which were induced to differentiate, the biology of the process of differentiation appeared to be similar, regardless of the agent used. A cell line which was unresponsive to DMSO was responsive to other inducers, suggesting that this DMSO-resistant cell line differed from its parent DMSO-responsive cell line either in its metabolism of the inducers or in the ability of the inducers to enter the cell. Alternatively, there may be more than one mechanism involved in the chemical induction of differentiation.     

Polar/apolar chemical inducers of differentiation of transformed cells: strategies to improve therapeutic potential.

N,N'-Hexamethylenebisacetamide (HMBA) induces transformed cells to differentiate, accompanied by suppression of oncogenicity. Clinical trials have shown that HMBA can cause positive therapeutic responses in some cancer patients, but clinical efficacy may be limited, in part, by dose-related toxicity. Potential improvements in efficacy may be accomplished by changes in the chemical structure of inducing agents and by increasing the sensitivity of tumor cells to inducers of differentiation. We have previously described an approach to improving tumor cell responsiveness to inducing agents. Transformed cell lines that have acquired low levels of resistance to vincristine display a markedly increased sensitivity to HMBA. We now report on a series of hybrid polar/apolar compounds--some of which are as active as HMBA and several of which are significantly more active than HMBA in vitro--whose chemical structures make it likely that they have different pharmacokinetics. Vincristine-resistant murine erythroleukemia cells also are shown to have marked increased sensitivity to these hybrid polar/apolar compounds. Thus these findings suggest potentially useful strategies for the application of polar/apolar inducers of differentiation to the treatment of cancers. These studies also provide approaches to further understanding of the biological process of terminal differentiation.     

Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro

Basement membranes have been implicated in morphogenesis and cell differentiation. In this study, the effect of basement membrane components on intestinal epithelial cell maturation in a mesenchyme-free environment was investigated. Fetal rat small intestinal epithelial cells (from the 14th-17th day of gestation) were exposed to basement membrane-derived proteins (laminin, collagen type IV, and a complex basement membrane-enriched extract from the Engelbreth-Holm-Swarm sarcoma) and other extracellular matrix proteins (collagen type I and fibronectin) coated onto Petri dishes. The cells attached readily only to fibronectin and basement membrane proteins. For 5 days the developing epithelial colonies were monitored in vitro, assessing morphological and functional parameters of cell maturation. Colonies grown on laminin and the basement membrane extract were larger and of greater cell density. An increase in alkaline phosphatase and lactase activity was observed after 3-4 days in these colonies which could be enhanced to yield 90%-100% positive cells by the addition of dexamethasone to the medium while no sucrase-isomaltase activity was elicited. Electron microscopy confirmed a high degree of cellular polarization illustrated by tight junctions and apical microvilli in epithelial cells grown on a basement membrane-like support. In contrast, none of the other proteins stimulated the cells to mature in vitro. The authors conclude that certain basement membrane components actively promote fetal intestinal epithelial cell differentiation.     

Glycosyltransferase alterations are cell type related when human promyelocytic leukemia (HL-60) cells are treated with various inducers of differentiation

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.     

Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression

OBJECTIVE: To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. MATERIALS AND METHODS: Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of gamma-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. RESULTS: All compounds tested increased gamma firefly luciferase activity, gamma/gamma+beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 microM increased the gamma/gamma+beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 microM increased the gamma/gamma+beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 microM in the luciferase system and 2 to 8 microM in BFU-E cultures; SAHA at 1 to 7.5 microM in the luciferase system and 1 to 2.5 microM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC(50) determinations led to values of 384 microM for propionyl-hydroxamate, 47 microM for butyryl-hydroxamate, 0.93 microM for SBHA, and 0.26 microM for SAHA. CONCLUCION: Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies.     

Somatic cell hybrid analyses of hematopoietic differentiation

A differentiated cell expresses an entire set of specialized features. Somatic cell hybridization provides a method to examine control of gene regulation. We studied the expression of tissue-specific features in hybrids between human promyelocytes (HL-60) and human Burkitt's lymphoma cells (P3HR-1). Two hybrid lines, HP-1 and HP-2, and 18 hybrid clones were established and confirmed by karyotype, isozyme, and surface antigen analyses. The hybrids extinguished the 10 myeloid (HL- 60) features that we examined including myeloid morphology, histochemistry, and functions that included response to colony- stimulating factor and ability to differentiate to granulocytes or macrophages. In contrast, the hybrids synthesized immunoglobulin and expressed Epstein-Barr nuclear, early, and viral capsid antigens similar to the P3HR-1 lymphoid parental line. Results are contrasted to the findings when P3HR-1 lymphocytes are fused to human erythroid- myeloid cells (K562). Taken together, our results suggest that phenotypic differences between human myeloid and lymphoid cells in the hematopoietic lineage involve mutually exclusive programs and may possibly be mediated by the activity of diffusible, transacting molecules.     

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation.

OBJECTIVE. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). METHODS. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. RESULTS. RA SF and ST T cells were found to be markedly enriched in CD45RAdim, CD45RO+, CD45RBdim mature memory cells, whereas in the PB, CD45RAbright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RBdim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3-stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RBdim phenotype and B cell help, CD45RBdim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RBdim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RBdim T cells with mitomycin C. In contrast, healthy control sorted CD45RBbright or sorted CD4+, CD45RO+, CD45RBbright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. CONCLUSION. RA synovium is enriched in differentiated CD45RBdim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.     

HT-29 cells are an in vitro model for the generation of cell polarity in epithelia during embryonic differentiation.

A monoclonal antibody that recognizes a membrane glycoprotein specific for the apical membrane of human colonic epithelial cells has been used to follow the differentiation and polarization of a cell line, HT-29, derived from a human colon adenocarcinoma. When these cells formed a polarized epithelium, the antigen was concentrated at the apical plasma membrane. It was also found intracellularly in vesicles and vacuoles. When HT-29 cells were undifferentiated and unpolarized, the antigen was not expressed significantly at the plasma membrane but was found concentrated in the membranes of intracellular vacuoles. Cells not yet organized into an epithelium may thus synthesize a membrane protein specific for their future apical membranes and store it intracellularly until the polarization process takes place. Intermediary stages of differentiation were occasionally recognized. They are characterized by a small number of cells surrounding an intercellular lumen. These lumina displayed apical membrane features (the presence of the apical antigen, of some microvilli, and of junctional complexes), although the cells were not fully differentiated. The differentiation process in HT-29 cells is apparently similar to that observed during embryonic development of the intestine. Therefore, HT-29 cells represent a useful model system to study epithelial differentiation in vitro.     

T cells induce terminal differentiation of transformed B cells to mature plasma cell tumors.

Major interest in the analysis of mature plasma cell neoplasias of mice and humans has focused on identification of precursor cells that give rise to mature malignant plasma cells. Although several laboratories have recently suggested that such cells are present in the granulomas of pristane-treated mice and the bone marrow of some multiple myeloma patients, the in vivo cellular interactions required for their differentiation into mature plasma cell tumors remains unclear. Given the extensive interactions of peripheral T cells and normal B cells, we assessed the potential role of T cells in plasma-cell tumor development, by using a myc, raf-containing retrovirus, J3V1, to induce plasmacytomas in normal BALB/c mice, T-cell-deficient nude mice, and T-cell-reconstituted nude mice. The B-lineage tumors arising in normal BALB/c mice were uniformly mature plasmacytomas, most of which secreted immunoglobulin. In contrast, nude mice yielded predominantly non-immunoglobulin-secreting B-cell lymphomas with a phenotype characteristic of peripheral B cells. T-cell reconstitution of nude mice prior to tumor induction resulted in a shift from B-cell lymphomas to plasmacytomas. These results imply that transformation can occur prior to terminal differentiation of B cells and that such transformed cells can be driven to terminal differentiation by peripheral T cells. These findings further suggest that, in human multiple myeloma, the ability of T cells to influence the differentiation state of transformed B cells may provide a mechanism by which malignant plasma cells found in the bone marrow could arise from clonotypically related less-mature B cells found in both the bone marrow and periphery.     

Sphingomyelinase converts lipoproteins from apolipoprotein E knockout mice into potent inducers of macrophage foam cell formation

The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may be similarities to chylomicron remnant-induced atherosclerosis in humans. Although the lesions of these mice contain large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell formation. When d<1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages, cellular CE mass and stimulation of the cholesterol esterification pathway were increased approximately 5-fold compared with untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by competition with human S(f) 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis. Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants in humans.     

Conventional type 2 lung dendritic cells are potent inducers of follicular helper T cells in the asthmatic lung

BackgroundFollicular helper T (Tfh) cells represent a unique subset of helper CD4⁺ T cells in lymphoid follicles. Recently, Tfh cells were shown to play an important role in asthma through B cell differentiation. Conventional lung DCs are classified into two major subsets: conventional type 1 (cDC1) and type 2 (cDC2). Although the two subsets are different in driving particular T cell responses, the subset that induces Tfh cells in the asthmatic lung primarily has yet to be fully elucidated.MethodsWe evaluated Tfh cells, defined by the expression of CD4 and CXCR5, in HDM-challenged mice. Next, we characterized cDC1 and cDC2 purified from antigen-primed lung and examined their Tfh cell-inducing capacity. Additionally, the ability of lung DC-induced Tfh cells to cause germinal center B (GCB) cells to produce antigen-specific IgE was assessed.ResultsIn HDM-challenged mice, Bcl-6-expressing Tfh cells were significantly increased in the mediastinal lymph nodes. Lung cDC2, but not lung cDC1, increased after HDM priming, and cDC2 secreted larger amounts of IL-6 with higher ICOS-L expression than cDC1. In the co-cultures with OVA-specific naïve CD4⁺ T cells, cDC2 from OVA-primed lung induced Bcl-6-expressing Tfh cells more efficiently, together with larger amounts of IL-6 and IL-21, than cDC1. Blockage of IL-6 or ICOS-L significantly reduced Tfh cell induction. Finally, cDC2-induced Tfh cells enabled GCB cells to produce OVA-specific IgE.ConclusionsIn asthmatic lung, cDC2 is the primary DC subset responsible for Tfh cell differentiation and plays an important role in humoral immunity in asthma by inducing Tfh cells.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation

Objective. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). Methods. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. Results. RA SF and ST T cells were found to be markedly enriched in CD45RA^dim, CD45RO+, CD45RB^dim mature memory cells, whereas in the PB, CD45RA^bright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RB^dim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3—stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RB^dim phenotype and B cell help, CD45RB^dim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RB^dim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RB^dim T cells with mitomycin C. In contrast, healthy control sorted CD45RB^bright or sorted CD4+, CD45RO+, CD45RB^bright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. Conclusion. RA synovium is enriched in differentiated CD45RB^dim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.