A human CD4- T cell leukemia subclone with contact-dependent helper function.

Helper T lymphocytes provide a contact dependent signal to resting B cells that is required for optimal differentiation into Ig-secreting cells. The surface structure(s) on T cells that mediate helper function have not been identified but are known to be induced by T cell activation. A CD4- subclone of the Jurkat leukemic T cell line (D1.1) was isolated and found to be distinct from CD4+ Jurkat clones and a variety of other T and non-T cell leukemic lines in that coculture of D1.1 with resting B cells induced B cells to specifically express surface CD23 molecules, a marker of B cell activation. Furthermore, Jurkat D1.1 induced B cell proliferation and terminal B cell differentiation into IgG-secreting cells in the presence of T cell-dependent B cell mitogens. Similar to the helper effector function of activated T cells, the effects of Jurkat D1.1 were neither Ag nor MHC restricted. Paraformaldehyde fixed Jurkat D1.1 cells remained competent to activate B cells while D1.1 supernatants and diffusible factors were inactive. The effect of Jurkat D1.1 on B cell activation was distinct from that of rIL-4 and was not inhibited by antibodies to IL-4. Together these observations suggested that surface structures on D1.1 and not secreted factors, mediated contact-dependent helper effector function.     

Regulation of gene expression of proteasomes (multi-protease complexes) during growth and differentiation of human hematopoietic cells.

We have reported that proteasomes are expressed at abnormally high levels in various hematopoietic tumor cells (Kumatori, A., Tanaka, K., Inamura, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawa, T., Shin, S., and Ichihara, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7071-7075). In the present study, we examined changes in the expressions of proteasomes during growth of peripheral T-lymphocytes from healthy adults and differentiation of human leukemic cell lines. Up-regulation of mRNAs encoding multiple proteasome subunits was observed during proliferation of resting T-cells induced by mitogens such as phytohemagglutinin and interleukin-2. In contrast, in vitro terminal differentiation into monocytic, granulocytic, and erythroid cells of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells, by various inducing agents caused rapid and marked down-regulation of proteasomes expression, independently of the cell type, direction of differentiation, or type of signal. The syntheses of proteasome subunits of 21-31 kDa and their associated components of 35-110 kDa, measured by [35S]methionine incorporation, were much higher in mitogen-activated T-cells and unstimulated HL-60 cells, which grow rapidly, than in resting and differentiated cells, indicating apparent correlations of the mRNA levels of proteasomes with their translational activities. However, immunochemically, no detectable difference in the cellular contents of proteasomes was found in these cells in induced and uninduced states for proliferation and differentiation, suggesting accelerated turnover of proteasomes in rapidly proliferating cells. Inhibition of proteasome expression by an antisense oligodeoxynucleotide for the largest proteasome subunit, C2, caused partial arrest of cell cycle progression of T-lymphocytes, suggesting that up-regulation of proteasomes is indispensable for proliferation of the cells. We also observed that the nuclear fraction of proteasomes increased in proliferating T-cells and that proteasomes moved rapidly between the nucleus and cytoplasm during differentiation of HL-60 cells.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Mechanisms controlling the kinetics in proliferation and differentiation of populations of mouse myeloid leukemic cells in vitro

The kinetics of differentiation and proliferation of clone cells (B24) of mouse myeloid leukemic Ml cells in vitro were studied by quantitative determination of cellular morphology. B24 cells were induced to differentiate into only macrophagelike cells by an inducer of differentiation in conditioned medium (CM) of embryo cells. During cell differentiation, the ratio of the area of the nucleus to that of the cell (N.C.R.) decreased from about 55% to 10%. Decrease in the N.C.R. was used as an index of cell differentiation in analysis of the kinetics of differentiation of cells treated with various concentrations of CM. The results showed that the process of differentiation was promoted by increasing the concentration of CM, and that the transition of the cells from the undifferentiated state to the differentiated state occurred in a stochastic manner. Comparison of these morphometric results with those of autoradiography showed that the labeling index of the cells decreases gradually in association with decrease in the N.C.R. of the cells from 50% to 30%. A stochastic model for the kinetics of proliferation and differentiation of the cells simulated the experimental observations on the production of differentiated cells.     

A system for monocytic differentiation of leukemic cells HL 60 by a short exposure to 1,25-dihydroxycholecalciferol

The human promyelocytic cell line HL 60 can be induced to differentiate toward more mature myeloid or monocytic forms by a variety of agents. This process is thought to require several days of exposure to the inducer, thus making it difficult to identify the early cellular changes which are fundamental to the differentiation program, and to relate the induction to phases of the cell cycle. In order to study the kinetics of leukemic cell differentiation we have developed a system for the induction of rapid monocytic maturation in a subpopulation of HL 60 cells. The cells are exposed to 10(-7) M 1,25-dihydroxycholecalciferol for 4 hr in serum-free medium. Subsequent incubation in a complete medium results in cellular differentiation recognizable by several criteria (phagocytosis, nonspecific esterase reaction, adherence to substratum, cell morphology) beginning at 10 hr from the exposure to the inducer. Approximately 20 hr later 30-40% of the cells in culture show the differentiated phenotype and are capable of phagocytosis. The proportion of differentiated cells in culture decreases thereafter. This system has been utilized to study the expression of c-myc oncogene in relation to the kinetics of maturation, and it was found that the inhibition of the expression of this gene precedes the onset of phenotypic differentiation by approximately 8 hr, is transient, and is accompanied by a brief retardation of cell proliferation, which resumes the normal rate within 24 hr of the exposure to the inducer.     

Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60)

More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator-the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonaI homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Changes in cell kinetics associated with differentiation of a human promyelocytic cell line (HL60)

Abstract. Terminal cell differentiation results in an irreversible arrest in the G₁ phase of the cell cycle and loss of the capacity for cell renewal. In the murine erythroleukaemia cell line (MELC), commitment to erythroid differentiation was found also to be preceded by an early, transient, phase of inhibition of growth due to prolongation of the G₁ phase. We determined the effect of differentiation-inducing agents on the growth kinetics of a human promyelocytic cell line (HL60) which undergoes differentiation into mature granulocyte. At concentrations of inducers optimal for cell differentiation, an early, transient stimulation of cell multiplication was found. DNA synthesis was enhanced in HL60 cells as early as 5 hr after exposure to inducer. Nevertheless, HL60 cell maturation eventually also resulted in a loss of the multiplication ability. The duration of exposure to inducer required for irreversible loss of the potential for self-renewal was determined by the fall in the cloning efficiency of induced cells; the results indicate that it preceded the switch-off of the replication mechanism; the majority of the cells lost their ability to form large colonies at the time of peak DNA synthesis and were able to complete an additional two to three cell cycles at a rate similar to uninduced cells. These changes occurred before HL60 cells became committed and might play a pivotal role in the process of cell differentiation.     

Dependence of HL-60 myeloid cell differentiation on continuous and split retinoic acid exposures: Precommitment memory associated with altered nuclear structure

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca²⁺ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.     

Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines

Three human leukemia cell lines (TALL-101, AML-193, and MV4-11) that require granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth in a chemically defined medium were examined for their response to recombinant human (rh) cytokines. Either rh interleukin (IL)-3 or rhGM-CSF alone supported the long term growth of all three cell lines, and the two growth factors acted synergistically to stimulate the proliferation of the early T lymphoblastic leukemia (TALL-101) and of the monocytic leukemia (AML-193) cells. However, IL-3 antagonized the proliferation of the biphenotypic B-myelomonocytic leukemia (MV4-11) cells in the presence of GM-CSF when both factors were used at very low concentrations. The rh granulocyte (G)-CSF independently supported the long and short term growth of AML-193 and MV4-11, respectively, and synergized with GM-CSF in inducing proliferation of these cells. By contrast, G-CSF did not stimulate TALL-101 cell growth and antagonized the effect of GM-CSF such that proliferation was arrested. Although neither rh macrophage (M)-CSF nor rhIL-1 alpha independently promoted proliferation of the three leukemia cell lines, these cytokines were able to either up- or down-regulate the GM-CSF-dependent growth of these cells. Taken together, these data demonstrate that leukemic cells often require the synergistic action of several cytokines for optimal growth, whereas other combinations of factors may be growth-inhibitory. This raises the possibility that multiple hemopoietic growth factors sustain or control leukemic cell proliferation also in vivo. In addition, the observation the G-CSF, M-CSF, and IL-1 alpha can, in some cases, arrest cell proliferation without inducing differentiation suggests that the programs of proliferative arrest and differentiation in leukemic cells can be dissociated.     

Isoenzymes of lactate dehydrogenase in human leukemic cells in culture treated with inducers of differentiation

The human leukemic cell lines HL60 and K562, were induced to differentiate terminally by chemical agents. The isoenzyme patterns of lactate dehydrogenase (LD) in the cells before and after differentiation were determined electrophoretically on agarose gels. In general, treatment of the leukemic cells with inducers of differentiation resulted in a quantitative shift of the isoenzyme pattern towards anodic or cathodic forms. This was correlated with the conversion of the chemically treated cells to morphologically more normal cells, as verified by light microscopy and/or synthesis of hemoglobin. The LD isoenzyme patterns of the chemically differentiated cells were: (a) characteristic for the particular cell type obtained rather than for the nature of the inducer used; and (b) not similar to those of normally differentiated cells of the corresponding lineage, indicating that incomplete differentiation had occurred.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

IL-4 regulates differentiation and proliferation of human precursor B cells

The mechanism by which precursor and pre-B cells undergo differentiation is unclear; however, it is known that growth factors play an important role in this maturation process. The lymphokine, IL-4 has been shown to increase expression of class II Ag on B cells and induce B cell proliferation. In the murine system, IL-4 induced differentiation of precursor B cells into pre-B cells. In order to analyze growth factors on B cell development we have established an in vitro culture system for human bone marrow cells. We found that in the presence of IL-4, normal human precursor and pre-B cells can be induced to differentiate in the absence of cell proliferation with four days of culture. Furthermore, IL-4 depressed proliferation induced by supernatant from a T cell line. The differentiation was measured by an increase in both the number of cytoplasmic mu and surface IgM-positive cells. The effect of IL-4 on precursor and pre-B cell differentiation was detected as soon as 14 h of exposure to the lymphokine in the absence of an adherent feeder layer. These data suggest that IL-4 directly affects the differentiation process of normal human precursor and pre-B cells, and may antagonistically affect cell proliferation.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

Efficient differentiation of proadipocyte stem cells on nonadherent surfaces: Evidence for differentiation without DNA synthesis

The differentiation of low density BALB/3T3 T proadipocytes that are cultured in standard tissue culture flasks can be induced by heparinized medium containing human plasma. It has been shown that under these conditions, cells first growth-arrest at a distinct state in the G₁ phase of the cell cycle, designated G_D, and thereafter differentiate within 8 to 12 days. In this paper, we report that the kinetics of proadipocyte differentiation can be significantly accelerated by culture of cells in differentiation-promoting medium on non-adherent surfaces, such as agarose-coated plates or bacteriological Petri dishes. Data also show that in a nonadherent microenvironment extensive differentiation can occur in the absence of DNA synthesis. This was established most convincingly by the demonstration that placement of mitotic cells in heparinized medium containing human plasma and hydroxyurea on agarose-coated Petri dishes induced 70–80% of the cells to G_D arrest and differentiate without traversing the S phase of the cell cycle. It is concluded that under appropriate microenvironmental conditions metabolic events that occur solely in the late M or early G₁ phase of the cell cycle can mediate the integrated control of proadipocyte proliferation and differentiation.     

Enhancement of radiation-induced cell killing and DNA double-strand breaks in a human tumor cell line using nanomolar concentrations of aclacinomycin A.

Several agents that induce differentiation have previously been shown to induce the terminal differentiation of leukemic cells and enhance the radiosensitivity of certain solid tumor cell lines in vitro using millimolar concentrations. We now report that aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation in vitro, can significantly enhance the radiosensitivity of a human colon tumor cell line (Clone A) at a concentration of 10 nM. Based on colony-forming efficiency, the maximum increase in radiosensitivity was found using 15 nM ACM for 3 days with a dose enhancement factor of 1.4 at a surviving fraction of 0.10. This treatment increased cell doubling time, but had no effect on cell-cycle phase distribution. To gain insight into the mechanisms responsible for this radiosensitization, gamma-ray-induced DNA single- and double-strand breaks were examined. Aclacinomycin A had no effect on the induction of DNA single-strand breaks but significantly enhanced the formation of gamma-ray-induced DNA double-strand breaks. The rate or extent of repair of the induced double-strand breaks was not influenced by ACM treatment. These data suggest that ACM, at achievable plasma concentrations, can enhance the radiosensitivity of a human tumor cell line by increasing the initial level of radiation-induced DNA double-strand breaks.     

Retinol bound to physiological carrier molecules regulates growth and differentiation of myeloid leukemic cells

We have tested effects of retinol bound to its physiological carrier molecules, i.e. low density lipoprotein chylomicron remnants, and retinol binding protein (RBP) on differentiation and proliferation of myeloid leukemic cells in concentrations that can be obtained in vivo. Data presented in this study show that physiological concentrations of retinyl ester in chylomicron remnants induce differentiation and inhibit proliferation of the cell line HL-60 and promyelocytic leukemic cells in primary culture. Retinyl ester in low density lipoprotein showed no effect either on cell differentiation or proliferation of any of the myeloid cells tested. Retinol bound to RBP induced differentiation of HL-60 cells only in concentrations above those that can be found in vivo. However, cell proliferation was reduced both in HL-60 cells and in primary culture of leukemic cells using physiological concentrations of holo-RBP. These results suggest that retinyl ester in chylomicron remnants is the most effective vehicle for transport of retinol into leukemic cells in vivo.     

Essential role of IL-21 in B cell activation, expansion, and plasma cell generation during CD4+ T cell-B cell collaboration

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4(+) T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4(+) T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.