In vivo accumulation of T cells in response to IL-2/anti-IL-2 mAb complexes is dependent in part on the TNF family ligand 4-1BBL

Immune complexes combining IL-2 with particular anti-IL-2 antibodies can be used to selectively expand regulatory T cells or memory T cells. Combining IL-2 with anti-IL-2 (Clone S4B6) greatly enhances the biological potency of IL-2 in vivo leading to selective expansion of CD8 memory T cells and NK cells compared with regulatory T cells. Here we show that in vivo administration of IL-2/anti-IL-2 mAb (IL-2/mAb) complexes induces 4-1BB expression on both adoptively transferred antigen-specific memory CD8 T cells as well as on endogenous memory phenotype cells. Remarkably, the accumulation of adoptively transferred memory CD8 T cells following in vivo IL-2/mAb-complex treatment was found to be dependent in part on the presence of 4-1BBL in the host. These effects were independent of IL-2-induced cell division, suggesting that 4-1BBL-induced survival signals contribute to IL-2/mAb-complex-induced T-cell accumulation in vivo.     

The role of cytokines in lymphocyte homeostasis

The size of lymphocyte populations is regulated by replication and death. Cytokines produced by non-lymphoid cells provide key survival and replication signals for several lymphocyte subpopulations. The availability of these cytokines thus serves as a homeostatic regulatory mechanism by determining the upper limit of the population size. IL-7 is required for survival of naive CD4+ and CD8+ cells and memory CD8+ cells. IL-15 is required for survival of memory CD8+ cells. IL-12 and IL-4 also promote memory CD8+ survivaL BAFF is required for survival of mature B cells. Antigen receptor signals, together with these cytokine signals, are required for survival of mature B cells and naive T cells. The list of extracellular survival signals for lymphocytes remains incomplete, and the intracellular pathways leading to survival are poorly understood.     

CD40 ligation in vivo induces bystander proliferation of memory phenotype CD8 T cells

Injection of agonistic anti-CD40 Abs into mice has been shown to amplify weak CD8 T cell responses to poorly immunogenic compounds and to convert T cell tolerance to T cell priming. In this study we demonstrate that anti-CD40 treatment of C57BL/6 mice, without Ag delivery, led to a marked increase in the number of memory phenotype CD4 and CD8 T cells. Adoptive transfer experiments using CD40-deficient hosts further revealed that the proliferative response of memory T cells, induced by systemic CD40 signaling, was dependent on CD40 expression of host APCs. CD40 ligation in vivo induced vigorous cell division of both memory phenotype and bona fide virus-specific memory CD8 T cells in a partially IL-15-dependent manner. However, only memory phenotype, but not Ag-experienced memory CD8 T cells increased in cell number after anti-CD40 treatment in vivo. Taken together our data show that activation of APC via CD40 induces a marked bystander proliferation of memory phenotype T cells. In addition, we demonstrate that bona fide Ag-experienced memory CD8 T cells respond differently to anti-CD40-induced signals than memory phenotype CD8 T cells.     

Impaired SLAM-SLAM homotypic interaction between invariant NKT cells and dendritic cells affects differentiation of IL-4/IL-10-secreting NKT2 cells in nonobese diabetic mice

The regulatory function of invariant NKT (iNKT) cells for tolerance induction and prevention of autoimmunity is linked to a specific cytokine profile that comprises the secretion of type 2 cytokines like IL-4 and IL-10 (NKT2 cytokine profile). The mechanism responsible for iNKT cell differentiation toward a type 2 phenotype is unknown. Herein we show that costimulatory signals provided by the surface receptor signaling lymphocytic activation molecule (SLAM) on myeloid dendritic cells (mDC) to iNKT cells is crucial for NKT2 orientation. Additionally, we demonstrate that the impaired acquisition of an NKT2 cytokine phenotype in nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes is due to defective SLAM-induced signals generated by NOD mDC. Mature mDC of C57BL/6 mice express SLAM and induce C57BL/6 or NOD iNKT cells to acquire a predominant NKT2 cytokine phenotype in response to antigenic stimulation with the iNKT cell-specific Ag, the alpha-galactosylceramide. In contrast, mature NOD mDC express significantly lower levels of SLAM and are unable to promote GATA-3 (the SLAM-induced intracellular signal) up-regulation and IL-4/IL-10 production in iNKT cells from NOD or C57BL/6 mice. NOD mice carry a genetic defect of the Slamf1 gene that is associated with reduced SLAM expression on double-positive thymocytes and altered iNKT cell development in the thymus. Our data suggest that the genetic Slamf1 defect in NOD mice also affects SLAM expression on other immune cells such as the mDC, thus critically impairing the peripheral differentiation of iNKT cells toward a regulatory NKT2 type.     

Identification of cellular intermediates and molecular pathways induced by IL-21 in human B cells

The complex process of B cell development is controlled by multiple factors from the surrounding microenvironment including cytokines. IL-21 is a recently identified type I cytokine, mainly produced by activated CD4(+) T cells. It has been shown to promote differentiation of human primary B cells into Ig-secreting plasma cells. The objective of our study was to describe cellular intermediates that exist during IL-21-induced transition from an activated B cell to an Ig-secreting cell and to identify molecular mechanisms involved in this process. Novel Epstein-Barr Virus-positive human B cell lines with phenotypes characteristic of Ag-activated IgG(+) B cell blasts were used as a model system to study IL-21 effects in vitro. We show that IL-21 increased both proliferation and survival of B cell lines during the first 3 days of in vitro culture. This process was associated with CD38(low/int)CD23(int)HLA-DR(high)CD19(high)CD20(int) cell surface phenotype. Continued culture with IL-21 resulted in accumulation of cells in G(0)/G(1) stage of the cell cycle and increased apoptosis. This coincided with differentiation into small, CD38(high)CD23(low/-)HLA-DR(int)CD19(int)CD20(low) late plasmablasts/early plasma cells that expressed lower levels of c-Myc protein, and secreted greater amounts of Ig than the control cells. Partial inhibition of IL-21-induced JAK/STAT signaling by the low-dose pharmacological agent, JAK inhibitor I, did not prevent the initial increase in proliferation. However, decrease in c-Myc protein expression and subsequent differentiation to late plasmablasts/early plasma cells were strongly inhibited. Our study is the first to show the link between IL-21-induced JAK/STAT signaling, c-Myc regulation, and differentiation of human B cells.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Differential roles of galectin-1 and galectin-3 in regulating leukocyte viability and cytokine secretion

Galectin-1 (Gal-1) and galectin-3 (Gal-3) exhibit profound but unique immunomodulatory activities in animals but their molecular mechanisms are incompletely understood. Early studies suggested that Gal-1 inhibits leukocyte function by inducing apoptotic cell death and removal, but recent studies show that some galectins induce exposure of the common death signal phosphatidylserine (PS) independently of apoptosis. In this study, we report that Gal-3, but not Gal-1, induces both PS exposure and apoptosis in primary activated human T cells, whereas both Gal-1 and Gal-3 induce PS exposure in neutrophils in the absence of cell death. Gal-1 and Gal-3 bind differently to the surfaces of T cells and only Gal-3 mobilizes intracellular Ca2+ in these cells, although Gal-1 and Gal-3 bind their respective T cell ligands with similar affinities. Although Gal-1 does not alter T cell viability, it induces IL-10 production and attenuates IFN-gamma production in activated T cells, suggesting a mechanism for Gal-1-mediated immunosuppression in vivo. These studies demonstrate that Gal-1 and Gal-3 induce differential responses in T cells and neutrophils, and identify the first factor, Gal-3, capable of inducing PS exposure with or without accompanying apoptosis in different leukocytes, thus providing a possible mechanism for galectin-mediated immunomodulation in vivo.     

Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells

Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.     

Galectin-1 triggers an immunoregulatory signature in Th cells functionally defined by IL-10 expression

Galectin-1 (Gal-1), a β-galactoside-binding protein, can alter fate and effector function of Th cells; however, little is known about how Gal-1 induces Th cell differentiation. In this article, we show that both uncommitted and polarized Th cells bound by Gal-1 expressed an immunoregulatory signature defined by IL-10. IL-10 synthesis was stimulated by direct Gal-1 engagement to cell surface glycoproteins, principally CD45, on activated Th cells and enhanced by IL-21 expression through the c-Maf/aryl hydrocarbon receptor pathway, independent of APCs. Gal-1-induced IL-10(+) T cells efficiently suppressed T cell proliferation and T cell-mediated inflammation and promoted the establishment of cancer immune-privileged sites. Collectively, these findings show how Gal-1 functions as a major glycome determinant regulating Th cell development, inflammation, and tumor immunity.     

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.     

Defects in the acquisition of CD8 T cell effector function after priming with tumor or soluble antigen can be overcome by the addition of an OX40 agonist

Several members of the TNFR superfamily, including OX40 (CD134), 4-1BB (CD137), and CD27 provide critical costimulatory signals that promote T cell survival and differentiation in vivo. Although several studies have demonstrated that OX40 engagement can enhance CD4 T cell responses, the mechanisms by which OX40-mediated signals augment CD8 T cell responses are still unclear. Previously, we and others have shown that OX40 engagement on Ag-specific CD8 T cells led to increased CD8 T cell expansion, survival, and the generation of greater numbers of long-lived memory cells. Currently, we demonstrate that provision of an OX40 agonist during the activation of naive CD8 T cells primed in vivo with either soluble or tumor-associated Ag significantly augments granzyme B expression and CD8 T cell cytolytic function through an IL-2-dependent mechanism. Furthermore, augmented CTL function required direct engagement of OX40 on the responding CD8 T cells and was associated with increased antitumor activity against established prostate tumors and enhanced the survival of tumor-bearing hosts. Thus, in the absence of danger signals, as is often the case in a tumor-bearing host, provision of an OX40 agonist can overcome defective CD8 T cell priming and lead to a functional antitumor response in vivo.     

Distinct role of follicular dendritic cells and T cells in the proliferation, differentiation, and apoptosis of a centroblast cell line, L3055

Germinal center (GC) B cells undergo complex interactions with follicular dendritic cells (FDC) and T cells in the course of differentiation into memory B and plasma cells. To delineate the individual roles of FDC and T cells at each stage of GC B cell differentiation at the clonal level and to analyze the signals involved, we adopted a unique experimental model using an FDC line, HK, and a lymphoma cell line, L3055, that resembles centroblasts. A detailed phenotypic analysis revealed L3055 cells to be a clonal population originating from the GC. Like freshly isolated centroblasts, L3055 cells underwent spontaneous apoptosis when cultured in the absence of fresh FDC or HK cells. L3055 cells proliferated continuously in the presence of HK cells, while they differentiated into a population with the phenotype of centrocytes after stimulation with CD40 ligand (CD40L) and IL-4. The CD40L-stimulated L3055 cells underwent CD95-mediated apoptosis, which was reminiscent of the feature of CD40L-stimulated tonsillar GC B cells. In contrast to HK cells that did not protect L3055 cells from anti-Ig killing, CD40L plus IL-2, IL-4, and IL-10 prevented anti-Ig-induced apoptosis. These experimental results demonstrate a distinct function of FDC and activated T cells, in that FDC provide signals for rapid proliferation of centroblasts, whereas T cells confer signals for differentiation of centroblasts into centrocytes and resistance to B cell receptor-mediated apoptosis. T cells collaborate with FDC in the protection and expansion of the Ag-specific GC B cells.     

SDF-1α Degrades whereas Glycoprotein 120 Upregulates Bcl-2 Interacting Mediator of Death Extralong Isoform: Implications for the Development of T Cell Memory

After a primary immune response, T cell memory occurs when a subset of Ag-specific T cells resists peripheral selection by acquiring resistance to TCR-induced death. Recent data have implicated Bcl-2 interacting mediator of death (Bim) as an essential mediator of the contraction phase of T cell immunity. In this article, we describe that stromal-derived factor-1α (SDF-1α) ligation of CXCR4 on activated T cells promotes two parallel processes that favor survival, phospho-inactivation of Foxo3A, as well as Bim extralong isoform (Bim(EL)) degradation, both in an Akt- and Erk-dependent manner. Activated primary CD4 T cells treated with SDF-1α therefore become resistant to the proapoptotic effects of TCR ligation or IL-2 deprivation and accumulate cells of a memory phenotype. Unlike SDF-1α, gp120 ligation of CXCR4 has the opposite effect because it causes p38-dependent Bim(EL) upregulation. However, when activated CD4 T cells are treated with both gp120 and SDF-1α, the SDF-1α-driven effects of Bim(EL) degradation and acquired resistance to TCR-induced death predominate. These results provide a novel causal link between SDF-1α-induced chemotaxis, degradation of Bim(EL), and the development of CD4 T cell memory.     

Galectin 3 acts as an enhancer of survival responses in <Emphasis Type="Italic">H. pylori</Emphasis>-infected gastric cancer cells

Galectin 3 (Gal-3) is upregulated in gastric epithelial cells as a host response to Helicobacter pylori infection. However, the significance of Gal-3 expression in H. pylori-infected cells is not well established. We analyzed Gal-3 intracellular expression, localization, and its effects in H. pylori-infected gastric epithelial cells. The predominantly nuclear confined Gal-3 was shown to be upregulated and exported out to the cytoplasm in H. pylori-infected AGS cells. The nuclear export was channeled through CRM-1 (exportin-1) protein. Interestingly, knock down of Gal-3 expression led to reduced NF-κB promoter activity and interleukin-8 (IL-8) secretion, suggesting its pro-inflammatory roles. Furthermore, Gal-3 was found to be pro-proliferative and anti-apoptotic in nature, as its knock down caused a reduction in cell proliferation and an increase in apoptosis, respectively. Taken together, our data suggest the expression and upregulation of Gal-3 as a critical endogenous event in H. pylori infection that interferes with various intracellular events, causing prolonged cell survival, which is characteristic in carcinogenesis.     

Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets--neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells--into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7- T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.     

Divergent generation of heterogeneous memory CD4 T cells

Mechanisms for the generation of memory CD4 T cells and their delineation into diverse subsets remain largely unknown. In this study, we demonstrate in two Ag systems, divergent generation of heterogeneous memory CD4 T cells from activated precursors in distinct differentiation stages. Specifically, we show that influenza hemagglutinin- and OVA-specific CD4 T cells activated for 1, 2, and 3 days, respectively, exhibit gradations of differentiation by cell surface phenotype, IFN-gamma production, and proliferation, yet all serve as direct precursors for functional memory CD4 T cells when transferred in vivo into Ag-free mouse hosts. Using a conversion assay to track the immediate fate of activated precursors in vivo, we show that day 1- to 3-activated cells all rapidly convert from an activated phenotype (CD25(high)IL-7R(low)CD44(high)) to a resting memory phenotype (IL-7R(high)CD25(low)CD44(high)) 1 day after antigenic withdrawal. Paradoxically, stable memory subset delineation from undifferentiated (day 1- to 2-activated) precursors was predominantly an effector memory (CD62L(low)) profile, with an increased proportion of central memory (CD62L(high)) T cells arising from more differentiated (day 3-activated) precursors. Our findings support a divergent model for generation of memory CD4 T cells directly from activated precursors in multiple differentiation states, with subset heterogeneity maximized by increased activation and differentiation during priming.