OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.     

B cell activation via CD40 is required for specific antibody production by antigen-stimulated human B cells

Costimulatory signals provided by T cells are required for B cells to produce specific antibody (Ab) to T-dependent antigen (Ag) bacteriophage phi x 174. In this study, we demonstrate that if cultured in the presence of anti-CD40, interleukin 10 (IL-10), and Ag, purified B cells can produce antiphage Ab in quantities comparable to those synthesized by B cells cocultured with Ag and T cells. Isotypes produced by B cells in this culture system correspond to those observed in sera of B cell donors. Culture of immunoglobulin (Ig)D- and IgD+ B cells reveals that Ag-induced production of antiphage Ab is restricted to IgD- subset of B cells. In the absence of Ag, anti-CD40/IL-10- stimulated B cells produce only minute amounts of antiphage Ab, indicating that Ag stimulation is indispensable and provides a signal that is synergistic with anti-CD40 and IL-10. Addition of a soluble form of the CD40 ligand (sgp39) to the culture system has a similar effect on specific Ab synthesis as anti-CD40; addition of the soluble construct, CD40 Ig, known to inhibit gp39/CD40 interaction, suppresses in vitro antiphage Ab production by Ag exposed peripheral blood mononuclear cells. Finally, in vivo requirement of gp39/CD40 interaction for specific Ab production was demonstrated by the finding that activated T cells from patients with x-linked hyper IgM syndrome express functionally defective gp39 and respond with depressed Ab titers and fail to switch from IgM to IgG after multiple phage immunizations. These observations illustrate that in vitro and possibly in vivo Ag-specific Ab synthesis requires the presence of Ag and IL-10, and activation signals via CD40.     

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T- dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.     

CD133+ cell selection is an alternative to CD34+ cell selection for ex vivo expansion of hematopoietic stem cells

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.     

B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells

Dendritic cells comprise a system of highly efficient antigen- presenting cells involved in the initiation of T cell responses. Herein, we investigated the role of the CD28 pathway during alloreactive T cell proliferation induced by dendritic-Langerhans cells (D-Lc) generated by culturing human cord blood CD34+ progenitor cells with granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha. In addition to expressing CD80 (B7/BB1), a subset of D-Lc expressed B70/B7-2. Binding of the CTLA4-Ig fusion protein was completely inhibited by a combination of monoclonal antibodies (mAbs) against CD80 and B70/B7-2, indicating the absence of expression of a third ligand for CD28/CTLA-4. It is interesting to note that mAbs against CD86 completely prevented the binding of CTLA4-Ig in the presence of mAbs against CD80 and bound to a B70/B7-2-transfected fibroblast cell line, demonstrating that the B70/B7-2 antigen is identical to CD86. CD28 triggering was essential during D-Lc-induced alloreaction as it was inhibited by mAbs against CD28 (9 out of 11 tested). However, none of six anti-CD80 mAbs demonstrated any activity on the D-Lc-induced alloreaction, though some were previously described as inhibitory in assays using CD80-transfected cell lines. In contrast, a mAb against CD86 (IT-2) was found to suppress the D-Lc-dependent alloreaction by 70%. This inhibitory effect was enhanced to > or = 90% when a combination of anti-CD80 and anti-CD86 mAbs was used. The present results demonstrate that D-Lc express, in addition to CD80, the other ligand for CTLA-4, CD86 (B70/B7-2), which plays a primordial role during D-Lc-induced alloreaction.     

Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 ligand interaction

To study the role of the CD40-CD40 ligand interaction in the development of memory B cells and its level of action during primary antibody responses in vivo, mice were injected with a soluble CD40 fusion protein (sCD40-gamma 1), so as to block the interaction. The effects of the treatment on the primary antibody response were reminiscent of hyper-immunoglobulin M (IgM) syndrome (HIMG1): antigen- specific IgG responses were grossly inhibited whereas the IgM response was augmented severalfold. The latter observation suggests that there is a T-dependent, CD40 ligand-independent pathway of B cell activation that leads to IgM responses and that a significant component of the IgM in HIMG1 patients is derived from T-dependent responses. The secondary response was not readily blocked by sCD40-gamma 1 treatment, indicating a relative independence of CD40 ligation of antigen-experienced B cells. The most striking finding from these studies is that the development of memory B cell populations (measured by adoptive transfer) is grossly impaired by administration of sCD40-gamma 1 during the early induction phase of the response. It is surprising that although the generation memory is diminished, there is no quantitative difference in the development of germinal centers. Whereas entry of B cells into the memory cell pathway is dependent on CD40 ligation, the clonal expansion of the potential memory precursors in germinal centers seems not to require a CD40 signal.     

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.     

Compromised OX40 function in CD28-deficient mice is linked with failure to develop CXC chemokine receptor 5-positive CD4 cells and germinal centers.

Mice rendered deficient in CD28 signaling by the soluble competitor, cytotoxic T lymphocyte-associated molecule 4-immunoglobulin G1 fusion protein (CTLA4-Ig), fail to upregulate OX40 expression in vivo or form germinal centers after immunization. This is associated with impaired interleukin 4 production and a lack of CXC chemokine receptor (CXCR)5 on CD4 T cells, a chemokine receptor linked with migration into B follicles. Germinal center formation is restored in CTLA4-Ig transgenic mice by coinjection of an agonistic monoclonal antibody to CD28, but this is substantially inhibited if OX40 interactions are interrupted by simultaneous injection of an OX40-Ig fusion protein. These data suggest that CD28-dependent OX40 ligation of CD4 T cells at the time of priming is linked with upregulation of CXCR5 expression, and migration of T cells into B cell areas to support germinal center formation.     

Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.     

CD4(+) T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses.

A major goal of the transplant field is to tolerize donor T cells to prevent graft-versus-host disease (GVHD) (1). We describe an ex vivo approach in which the blockade of CD40 ligand (CD40L:CD154):CD40 interactions, a pathway required for optimal T cell expansion, induces donor CD4(+) T cells to become tolerant to host alloantigens (2). High doses of tolerized cells did not cause GVHD lethality in vivo. T cells had intact responses to antigens not present during tolerization. Tolerance was long lived and not readily reversible in vivo. These data have significant implications for the use of tolerization approaches to prevent human GVHD.     

A multidrug-resistance protein (MRP)-like transmembrane pump is highly expressed by resting murine T helper (Th) 2, but not Th1 cells, and is induced to equal expression levels in Th1 and Th2 cells after antigenic stimulation in vivo.

A transmembrane pump for organic anions was identified in resting murine T helper (Th) 2, but not Th1 lymphocyte cell clones, as revealed by extrusion of a fluorescent dye. Dye extrusion inhibition studies suggested that the pump may be the multidrug-resistance protein (MRP). The different expression of the pump in resting Th1 and Th2 cell clones correlated with their respective levels of MRP mRNA. The pump was inducible in Th1 cells by antigenic stimulation in vitro leading to equal expression in activated Th1 and Th2 cell clones. This suggested that dye extrusion might allow the detection of Th2 (resting or activated) or of activated Th1 cells ex vivo based on a functional parameter. To test this, mice were infected with Leishmania major parasites to activate L. major-specific T cells of either Th1 (C57BL/6 mice) or Th2 (BALB/c mice) phenotype: 2-3% of CD4+ lymph node T cells of both strains of mice extruded the dye, defining a cell subset that did not coincide with subsets defined by other activation markers. Fluorescence-activated cell-sorting revealed that the lymphokine response (Th1 or Th2, respectively) to L. major antigens was restricted to this dye-extruding subset.     

A subset of CD4+ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex

Signaling through surface CD40 is essential for selecting B cells that have mutated their immunoglobulin variable region genes in germinal centers and is an important signal in the early stages of antibody responses to T cell-dependent antigens. It is shown that a subset of CD45RO+, CD4+ T cells isolated from human tonsil contains preformed 30- 35-kD ligand for CD40. This is expressed on their surfaces within 5 min of their antigen-receptor complexes interacting with CD3 epsilon antibodies bound to ox erythrocytes. This surface expression does not require de novo protein synthesis and lasts for only 1-2 h. Preformed CD40 ligand (CD40L) was not detected in any CD4+ CD45RA+ T cells, but > 90% of all CD4+ T cells from the tonsil can be induced to express large amounts of CD40L on culture with phorbol myristate acetate and the calcium ionophore ionomycin. This expression of CD40L starts between 1 and 2 h, peaks at 6 h, and remains at a high level for > 20 h. It is totally prevented by adding a concentration of cycloheximide that inhibits CD25 synthesis by these activated cells. While CD3 epsilon antibody bound to ox red cells is a good inducer of surface expression of CD40L, it is a much less potent inducer of CD40L synthesis than phorbol myristate acetate with ionomycin. Immunohistological analysis of tonsil sections shows that cells containing CD40L are located mainly in the outer zone of germinal centers and the margins of the T zones that are rich in dendritic cells (interdigitating cells). The distribution of these cells is consistent with: (a) their interaction in T zones with B cells that have taken up and processed antigen and (b) their involvement in B cell selection in germinal centers.     

In vivo activity and in vitro specificity of CD4+ Th1 and Th2 cells derived from the spleens of diabetic NOD mice.

CD4+ T cell lines were generated from the spleens of diabetic NOD mice against crude membrane preparations derived from a rat insulinoma. Adoptive transfer of these lines into neonatal mice confirms that overt diabetes is induced by gamma-IFN-secreting Th1 cells, whereas transfer of IL-4-secreting Th2 cells resulted in a nondestructive peri-islet insulitis. Analysis of the antigens recognized by individual T cell clones from the Th1 line included reactivity against an insulinoma membrane fraction enriched in proteins of approximately 38 kD. Immune responses to the same antigen preparation have been associated with T cell clones derived from human insulin-dependent diabetes mellitus. The specificity of Th2 cells includes reactivity to a fraction enriched in proteins of 30 kD. The data suggest that in insulin-dependent diabetes mellitus the balance between beta cell destruction, associated with intra-islet infiltration, and nondestructive (potential protective) peri-islet insulitis may depend on both the antigens recognized, and the prevailing cytokine environment.     

Therapeutic efficacy of experimental rheumatoid arthritis with low-dose methotrexate by increasing partially CD4CD25Treg cells and inducing Th1 to Th2 shift in both cells and cytokines

Low-dose methotrexate (MTX), a traditional folate antagonist and disease-modifying antirheumatic drug administered weekly either alone or as combination therapy, is widely accepted as the gold standard in rheumatoid arthritis (RA) treatment. Although its mechanism of action in RA is still poorly understood, MTX potentially acts via antiproliferative, anti-inflammatory, and/or immunosuppressive means. The therapeutic mechanisms and efficacy of low-dose MTX and the oral tolerance protein natural chicken type II collagen (nCCII) were compared in vitro and in vivo using an established collagen-induced arthritis (CIA) rat model. We used clinical visual scoring, radiographic X-ray analysis, histopathological examination, and sera anti-CII IgG measurements to determine the severity of disease with and without treatment. Low-dose MTX had significant clinical therapeutic efficacy against established CIA. Similar to nCCII, MTX mediated CIA by specific immunotolerant effects and not by nonspecific immunosuppression. The mechanism underlying the therapeutic efficacy could be at least partially attributed to the increased production of CD4⁺CD25⁺ Treg cells. These cells specifically downmodulated the T lymphocyte proliferative response to CCII but not PHA, induced a Th1-to-Th2 shift, downregulated Th1 cytokines, and upregulated both Th2 and Th3 cytokines. To the best of our knowledge, this is the first demonstration that low-dose MTX probably serves as a potent inducer of specific immunotolerance but not of nonspecific immunosuppression in the treatment of RA.     

Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling

The recently described ligand-receptor pair, B7h-inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R-mediated B7h down-regulation. These data suggest that the CD40-CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen-activated T cells through ICOS.