Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora

We showed previously that cecal bacterial Ag (CBA)-specific CD4(+) T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4(+) T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10-14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-gamma, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4(+) Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-gamma production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFbeta1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4(+) T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4(+) T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.     

Functional dichotomy in CD40 reciprocally regulates effector T cell functions

Activation of T cells requires signals through Ag-specific TCR and costimulatory molecules such as CD40L. Although the use of defined tumor Ags for the induction of protective T cells met with limited success, the CD40-CD40L interaction that was proposed to induce antitumor T cells did not prevent tumor growth completely. Using a model for prostate tumor, a leading cause of tumor-induced mortality in men, we show that the failure is due to a novel functional dichotomy of CD40 whereby it self-limits its antitumor functions by inducing IL-10. IL-10 prevents the CD40-induced CTL and TNF-alpha and IL-12 production, Th1 skewing, and tumor regression. Priming mice with tumor lysate-pulsed IL-10-deficient dendritic cells (DCs) or wild-type DC plus anti-IL-10 Ab establishes antitumor memory T cells that can transfer the protection into syngenic nude mice. Infusion of Ag-pulsed IL-10-deficient but not wild-type DCs back into syngenic mice results in successful therapeutic autovaccination. Thus, we demonstrate the IL-10-sensitive antitumor T cell memory formulating a novel prophylactic and therapeutic principle.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

Paradoxical effect of reduced costimulation in T cell-mediated colitis

B7-1 and B7-2 play different roles in the pathogenesis of autoimmunity, but this is controversial. We analyzed colitis induced by transfer of CD45RB(high)CD4(+) T cells to RAG(-/-) recipients lacking B7-1 and/or B7-2. Surprisingly, disease was greatly accelerated in RAG(-/-) recipients deficient for either B7-1 or B7-2, especially in the B7-2(-/-) recipients. This accelerated colitis induction correlated with increased T cell division in vivo and production of Th1 cytokines. Although colitis pathogenesis following T cell transfer was inhibited in the absence of CD40L expression, CD40-CD40L interactions were not required in the B7-2(-/-) RAG(-/-) recipients. In vitro priming by APCs lacking either B7-1 or B7-2 caused decreased IL-2 production, which led to decreased CTLA-4 expression, although T cells primed in this way could respond vigorously upon restimulation by producing increased IL-2 and proinflammatory cytokines. Consistent with this mechanism, we demonstrate that blocking IL-2 early after T cell transfer accelerated colitis. Our data therefore outline a mechanism whereby synergistic costimulation by B7-1 and B7-2 molecules during priming is required for optimal IL-2 production. The consequent inhibitory effect of full CTLA-4 expression, induced by IL-2, may slow colitis, even in the absence of regulatory T cells.     

Differential CD40/CD40L expression results in counteracting antitumor immune responses

Establishment of host-protective memory T cells against tumors is the objective of an antitumor immunoprophylactic strategy such as reinforcing T cell costimulation via CD40-CD40L interaction. Previous CD40-targeted strategies assumed that T cell costimulation is an all-or-none phenomenon. It was unknown whether different levels of CD40L expression induce quantitatively and qualitatively different effector T cell responses. Using mice expressing different levels of CD40L, we demonstrated that the greater the T cell CD40L expression the less tumor growth occurred; the antitumor T cell response was host-protective. Lower levels of CD40L expression on T cells induced IL-10-mediated suppression of tumor-regressing effector CD8(+) T cells and higher productions of IL-4 and IL-10. Using mice expressing different levels of CD40 or by administering different doses of anti-CD40 Ab, similar observations were recorded implying that the induction of protumor or antitumor T cell responses was a function of the extent of CD40 cross-linking. IL-10 neutralization during priming with tumor Ags resulted in a stronger tumor-regressing effector T cell response. Using IL-10(-/-) DC for priming of mice expressing different levels of CD40L and subsequent transfer of the T cells from the primed mice to nu/nu mice, we demonstrated the protumor role of IL-10 in the induction of tumor-promoting T cells. Our results demonstrate that a dose-dependent cross-linking of a costimulatory molecule dictates the functional phenotype of the elicited effector T cell response. The T cell costimulation is a continuum of a function that induces not only graded T cell responses but also two counteracting responses at two extremes.     

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.     

Activation of APCs through CD40 or Toll-like receptor 9 overcomes tolerance and precipitates autoimmune disease

Some autoreactive T cells normally escape thymic selection and persist in the periphery. This is true of myelin-reactive CD4(+) T cells, the effectors of experimental autoimmune encephalomyelitis (EAE) in laboratory animals and the presumed mediators of multiple sclerosis in humans. Nonetheless, most individuals do not succumb to autoimmune disease. There is growing evidence that while peripheral APCs stimulate immune responses against foreign Ags in the setting of tissue destruction and "danger," they actually maintain tolerance against self Ags under steady state conditions. We hypothesized that tolerance against candidate autoantigens could be reversed by activation of APCs via CD40 or Toll-like receptor 9 signaling. Adult SJL mice injected i.p. with a peptide fragment of proteolipid protein (a candidate autoantigen in multiple sclerosis) emulsified in IFA fail to mount lymphoproliferative or cytokine responses and are protected from EAE upon subsequent challenge with the Ag combined with adjuvants. Here we report that tolerized proteolipid protein-specific lymph node cells regain the ability to divide, differentiate along a Th1 lineage, and transfer EAE when reactivated in the presence of agonistic Abs against CD40 or CpG oligonucleotides. The effects of both anti-CD40 and CpG oligonucleotides are dependent upon induction of IL-12. Our findings suggest two mechanisms to explain the well-documented association between infectious illnesses and flare-ups of multiple sclerosis. Microbial pathogens could 1) release molecules that bind Toll-like receptors, and/or 2) stimulate microbe-specific T cells to express CD40 ligand, thereby licensing APCs that bear both microbial and autoantigens to break tolerance.     

Quantities of interleukin-12p40 in mature CD8alpha negative dendritic cells correlate with strength of TCR signal and determine Th cell development

Strength of T cell antigen receptor (TCR) signaling drives the development of Th1 and Th2 subsets from naive T helper precursors. The quantity of interleukin-12 (IL-12) from antigen presenting cells (APC) is also profoundly involved in Th development. TCR signal strength and IL-12 production from dendritic cells (DCs) are linked by CD40 ligand (CD40L) expression on activated T cells. CD40L on the activated T cells interacts with CD40 on DC, resulting in induction of IL-12 from DCs. However, the subsets of DC in spleen that produce the IL-12 have not been clearly identified. Purification of DC subsets itself may provide maturation signals to immature DCs. Thus, we used non-purified mouse spleen cells to analyze IL-12 producing cells, near to steady states, during the interaction of naive T cells either with or without agonist. Mature CD86highCD8alpha- DCs in spleen mainly produced IL-12p40 after stimulation of high dose agonist. The ratio of CD40L positive T cells and IL-12p40 secreting CD86high DCs correlated with the concentration of agonist and Th1 development. However, anti-IL-12 did not completely inhibit the Th1 development. Altogether, strength of TCR signaling directs Th cell development by regulating CD40L expression on T cells which determines production of IL-12p40 from CD86high CD8alpha- DC via CD40.     

Disruption of CD154:CD40 blocks generation of allograft immunity without affecting APC activation.

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-gamma, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154-/- mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.     

Th2-dependent B cell responses in the absence of CD40-CD40 ligand interactions

CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of beta-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.     

B cells capturing antigen conjugated with CpG oligodeoxynucleotides induce Th1 cells by elaborating IL-12

APCs initiate T cell-mediated immune responses against foreign Ags. Dendritic cells are professional APCs that play unique roles, including Ag-nonspecific capture, priming of naive T cells, and Th1 induction, whereas B cells generally lack these functions. In this study we uncovered novel aspects of murine B cells as APCs using CpG oligodeoxynucleotides (CpG) conjugated with an Ag. B cells served as efficient APCs independently of surface Igs. This characteristic was underlaid by the CpG-mediated Ag uptake and presentation, which were functional only when CpG were covalently conjugated to Ag. The B cells cultured with CpG-conjugated Ag not only enhanced IFN-gamma formation by Th1 cells, but also induced Th1 differentiation from unprimed T cells. These effects paralleled with the increase in the expression of CD40, CD86, and class II molecules on B cells and the coordinated production of IL-12 by the cells. To our knowledge this is the first report revealing that B cells share with dendritic cells common intrinsic characteristics, such as the Ag-nonspecific capture and presentation, and the induction of Th1 differentiation from unprimed T cells.     

CD40 ligand (CD154) enhances the Th1 and antibody responses to respiratory syncytial virus in the BALB/c mouse

CD40 ligand (CD40L) is a cell surface costimulatory molecule expressed mainly by activated T cells. CD40L is critically important for T-B cell and T cell-dendritic cell interactions. CD40L expression promotes Th1 cytokine responses to protein Ags and is responsible for Ig isotype switching in B cells. Respiratory syncytial virus (RSV) is an important pathogen of young children and the elderly, which causes bronchiolitis and pneumonia. Studies of mice infected with RSV suggest that a Th2 cytokine response may be responsible for enhanced pulmonary disease. To investigate the effect CD40L has on RSV immunity, mice were infected simultaneously with RSV and either an empty control adenovirus vector or one expressing CD40L or were coimmunized with plasmid DNA vectors expressing CD40L and RSV F and/or G proteins and subsequently challenged with RSV. The kinetics of the intracellular and secreted cytokine responses, the cytotoxic T lymphocyte precursor frequency, NO levels in lung lavage, rates of virus clearance, and anti-RSV Ab titers were determined. These studies show that coincident expression of CD40L enhances the Th1 (IL-2 and IFN-gamma) cytokine responses, increases the expression of TNF-alpha and NO, accelerates virus clearance, and increases the anti-F and anti-G Ab responses. These data suggest that CD40L may have the adjuvant properties needed to optimize the safety and efficacy of RSV vaccines.     

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.     

A role for CD40-CD40 ligand interactions in the generation of type 1 cytokine responses in human leprosy

The interaction of CD40 ligand (CD40L) expressed by activated T cells with CD40 on macrophages has been shown to be a potent stimulus for the production of IL-12, an obligate signal for generation of Th1 cytokine responses. The expression and interaction of CD40 and CD40L were investigated in human infectious disease using leprosy as a model. CD40 and CD40L mRNA and surface protein expression were predominant in skin lesions of resistant tuberculoid patients compared with the highly susceptible lepromatous group. IL-12 release from PBMC of tuberculoid patients stimulated with Mycobacterium leprae was partially inhibited by mAbs to CD40 or CD40L, correlating with Ag-induced up-regulation of CD40L on T cells. Cognate recognition of M. leprae Ag by a T cell clone derived from a tuberculoid lesion in the context of monocyte APC resulted in CD40L-CD40-dependent production of IL-12. In contrast, M. leprae-induced IL-12 production by PBMC from lepromatous patients was not dependent on CD40L-CD40 ligation, nor was CD40L up-regulated by M. leprae. Furthermore, IL-10, a cytokine predominant in lepromatous lesions, blocked the IFN-gamma up-regulation of CD40 on monocytes. These data suggest that T cell activation in situ by M. leprae in tuberculoid leprosy leads to local up-regulation of CD40L, which stimulates CD40-dependent induction of IL-12 in monocytes. The CD40-CD40L interaction, which is not evident in lepromatous leprosy, probably participates in the cell-mediated immune response to microbial pathogens.     

B cells are crucial for determinant spreading of T cell autoimmunity among beta cell antigens in diabetes-prone nonobese diabetic mice

The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in the protective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularly B cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Igmu(-/-) NOD mice reconstituted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Igmu(-/-) NOD), but not mice that lacked mature B cells (T NOD/scid and BM Igmu(-/-) NOD), spontaneously developed Th1 autoimmunity, which spread sequentially among different beta cell Ags. Immunization of T NOD/scid and BM Igmu(-/-) NOD mice with a beta cell Ag could prime Ag-specific Th1 or Th2 responses, but those T cell responses did not spread to other beta cell Ags. In contrast, immunization of TB NOD/scid and BMB Igmu(-/-) NOD mice with a beta cell Ag in IFA induced Th2 responses, which spread to other beta cell Ags. Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, B cells significantly enhanced the detection of spontaneously primed and induced Th1 responses to beta cell Ags. Our data suggest that B cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-based immunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancing Ag-based immunotherapy and controlling autoimmune diseases.     

Ox40 costimulation enhances the development of T cell responses induced by dendritic cells in vivo.

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4(+) and CD8(+) T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-gamma(-/-) mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-gamma during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4(+) T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-gamma secretion, and cytotoxic activity of Ag-specific CD8(+) T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.     

CD40-CD40 ligand interaction between dendritic cells and CD8+ T cells is needed to stimulate maximal T cell responses in the absence of CD4+ T cell help

Stimulation of CD40 on APCs through CD40L expressed on helper CD4+ T cells activates and "licenses" the APCs to prime CD8+ T cell responses. Although other stimuli, such as TLR agonists, can also activate APCs, it is unclear to what extent they can replace the signals provided by CD40-CD40L interactions. In this study, we used an adoptive transfer system to re-examine the role of CD40 in the priming of naive CD8+ T cells. We find an approximately 50% reduction in expansion and cytokine production in TCR-transgenic T cells in the absence of CD40 on all APCs, and on dendritic cells in particular. Moreover, CD40-deficient and CD40L-deficient mice fail to develop endogenous CTL responses after immunization. Surprisingly, the role for CD40 and CD40L are observed even in the absence of CD4+ T cells; in this situation, the CD8+ T cell itself provides CD40L. Furthermore, we show that although TLR stimulation improves T cell responses, it cannot fully substitute for CD40. Altogether, these results reveal a direct and unique role for CD40L on CD8+ T cells interacting with CD40 on APCs that affects the magnitude and quality of CD8+ T cell responses.     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.