Stabilized beta-catenin potentiates Fas-mediated T cell apoptosis

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) T cells. Using transgenic mice expressing a stabilized beta-catenin (beta-cat(Tg)), we show here that beta-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, beta-cat(Tg) mice exhibited accelerated deletion of CD4(+)Vbeta8(+) T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from beta-cat(Tg) mice than that from wild type mice. Additionally, T cells from beta-cat(Tg) mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, beta-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the beta-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.     

Antigen-driven interactions with dendritic cells and expansion of Foxp3+ regulatory T cells occur in the absence of inflammatory signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.     

Lenalidomide enhances antigen-specific activity and decreases CD45RA expression of T cells from patients with multiple myeloma

The aim of this study was to investigate whether the specific T cell response against the multiple myeloma Ag HM1.24 is enhanced by the immunomodulatory drug lenalidomide (Revlimid). Ag-specific CD3(+)CD8(+) T cells against the HM1.24 Ag were expanded in vitro by dendritic cells in 29 healthy donors and 26 patients with plasma cell dyscrasias. Ag-specific activation was analyzed by IFN-γ, granzyme B, and perforin secretion using ELISA, ELISPOT assay, and intracellular staining, and generation of Ag-specific T cells was analyzed by tetramer staining. Expression of T cell maturation markers (CD45RA, CD45R0, CCR7, and CD28) was investigated by flow cytometry. We found that activation of HM1.24-specific T cells from healthy donors and patients with plasma cell dyscrasias was enhanced significantly by lenalidomide and furthermore that the impact of lenalidomide on T cells depends on the duration of the exposure. Notably, lenalidomide supports the downregulation of CD45RA on T cells upon activation, observed in healthy donors and in patients in vitro and also in patients during lenalidomide therapy in vivo. We showed for the first time, to our knowledge, that lenalidomide enhances the Ag-specific activation of T cells and the subsequent downregulation of CD45RA expression of T cells in vitro and in vivo.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

A bone marrow-derived APC in the gut-associated lymphoid tissue captures oral antigens and presents them to both CD4+ and CD8+ T cells

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer's patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.     

Mannose receptor expression and function define a new population of murine dendritic cells

In vitro the mannose receptor (MR) mediates Ag internalization by dendritic cells (DC) and favors the presentation of mannosylated ligands to T cells. However, in vivo MR seems to play a role not in Ag presentation but in the homeostatic clearance of endogenous ligands, which could have the secondary benefit of reducing the levels of endogenous Ag available for presentation to the adaptive immune system. We have now observed that while MR(+) cells are consistently absent from T cell areas of spleen and mesenteric lymph nodes (LN), peripheral LN of untreated adult mice contain a minor population of MR(+)MHCII(+) in the paracortex. This novel MR(+) cell population can be readily identified by flow cytometry and express markers characteristic of DC. Furthermore, these MR(+) DC-like cells located in T cell areas can be targeted with MR ligands (anti-MR mAb). Numbers of MR(+)MHCII(+) cells in the paracortex are increased upon stimulation of the innate immune system and, accordingly, the amount of anti-MR mAb reaching MR(+)MHCII(+) cells in T cell areas is dramatically enhanced under these conditions. Our results indicate that the MR can act as an Ag-acquisition system in a DC subpopulation restricted to lymphoid organs draining the periphery. Moreover, the effect of TLR agonists on the numbers of these MR(+) DC suggests that the immunogenicity of MR ligands could be under the control of innate stimulation. In accordance with these observations, ligands highly specific for the MR elicit enhanced humoral responses in vivo only when administered in combination with endotoxin.     

Nonproliferating bystander CD4+ T cells lacking activation markers support HIV replication during immune activation

HIV replicates primarily in lymphoid tissue and immune activation is a major stimulus in vivo. To determine the cells responsible for HIV replication during Ag-driven T cell activation, we used a novel in vitro model employing dendritic cell presentation of superantigen to CD4(+) T cells. Dendritic cells and CD4(+) T cells are the major constituents of the paracortical region of lymphoid organs, the main site of Ag-specific activation and HIV replication. Unexpectedly, replication occurred in nonproliferating bystander CD4(+) T cells that lacked activation markers. In contrast, activated Ag-specific cells were relatively protected from infection, which was associated with CCR5 and CXC chemokine receptor 4 down-regulation. The finding that HIV replication is not restricted to highly activated Ag-specific CD4(+) T cells has implications for therapy, efforts to eradicate viral reservoirs, immune control of HIV, and Ag-specific immune defects.     

Langerhans cells require MyD88-dependent signals for Candida albicans response but not for contact hypersensitivity or migration

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88(fl) mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88(fl) mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.     

Low-affinity, Smith antigen-specific B cells are tolerized by dendritic cells and macrophages

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.     

TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses

Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-alphabeta receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.     

Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo

We previously showed that immunization with a combination of TLR and CD40 agonists (combined TLR/CD40 agonist immunization) resulted in an expansion of Ag-specific CD8 T cells exponentially greater than the expansion observed to immunization with either agonist alone. We now show that the mechanism behind this expansion of T cells is the regulated expression of CD70 on dendritic cells. In contrast to previous results in vitro, the expression of CD70 on dendritic cells in vivo requires combined TLR/CD40 stimulation and is not significantly induced by stimulation of either pathway alone. Moreover, the exponential expansion of CD8(+) T cells following combined TLR/CD40 agonist immunization is CD70 dependent. Thus, the transition from innate stimuli (TLRs) to adaptive immunity is controlled by the regulated expression of CD70.     

Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor

TLR are evolutionarily conserved molecules that play a key role in the initiation of innate antimicrobial immune responses. Through their influence on dendritic cell maturation, these receptors are also thought to indirectly shape the adaptive immune response. However, no data are currently available regarding both TLR expression and function in human CD8+ T cell subsets. We report that a subpopulation of CD8+ T cells, i.e., effector, but neither naive nor central memory cells, constitutively expresses TLR3. Moreover, the ligation of the receptor by a specific agonist in TLR3-expressing CD8+ T cells increased IFN-gamma secretion induced by TCR-dependent and -independent stimulation, without affecting proliferation or specific cytolytic activity. These results thereby suggest that TLR3 ligands can not only indirectly influence the adaptive immune response through modulation of dendritic cell activation, but also directly increase IFN-gamma production by Ag-specific CD8+ T cells. Altogether, the present work might open new perspectives for the use of TLR ligands as adjuvants for immunotherapy.     

Functional human antigen-specific T cells produced in vitro using retroviral T cell receptor transfer into hematopoietic progenitors

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRalphabeta gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRalphabeta-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.     

Plasmacytoid dendritic cells synergize with myeloid dendritic cells in the induction of antigen-specific antitumor immune responses

Plasmacytoid dendritic cells (pDC) are capable of producing high levels of type I IFNs upon viral stimulation, and play a central role in modulating innate and adaptive immunity against viral infections. Whereas many studies have assessed myeloid dendritic cells (mDC) in the induction of antitumor immune responses, the role of pDC in antitumor immunity has not been addressed. Moreover, the interaction of pDC with other dendritic cell subsets has not been evaluated. In this study, we analyzed the capacity of pDC in stimulating an Ag-specific T cell response. Immunization of mice with Ag-pulsed, activated pDC significantly augmented Ag-specific CD8(+) CTL responses, and protected mice from a subsequent tumor challenge. Immunization with a mixture of activated pDC plus mDC resulted in increased levels of Ag-specific CD8(+) T cells and an enhanced antitumor response compared with immunization with either dendritic cell subset alone. Synergy between pDC and mDC in their ability to activate T cells was dependent on MHC I expression by mDC, but not pDC, suggesting that pDC enhanced the ability of mDC to present Ag to T cells. Our results demonstrate that pDC and mDC can interact synergistically to induce an Ag-specific antitumor immune response in vivo.     

Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3(flox/flox) mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNA were also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.     

Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.     

Follicular dendritic cell secreted protein (FDC-SP) regulates germinal center and antibody responses

We previously identified follicular dendritic cell secreted protein (FDC-SP), a small secreted protein of unknown function expressed in human tonsillar germinal centers (GC). To assess potential in vivo activities of FDC-SP, transgenic mice were generated to constitutively express FDC-SP in lymphoid tissues. FDC-SP transgenic mice show relatively normal development of immune cell populations, with the exception of a small increase in mature follicular B cells, and normal lymphoid tissue architecture. Upon immunization with a T-dependent Ag, FDC-SP transgenic mice were capable of producing an Ag-specific Ab; however, the titers of Ag-specific IgG2a and IgE were significantly reduced. GC responses after immunization were markedly diminished, with transgenic mice showing decreased numbers and sizes of GCs but normal development of follicular dendritic cell networks and normal positioning of GCs. FDC-SP transgenic mice also showed reduced production of Ag-specific IgG3 Ab after immunization with a type II T-independent Ag, suggesting that the FDC-SP can also regulate the induction of B cell responses outside the GC. Purified FDC-SP transgenic B cells function normally in vitro, with the exception of blunted chemotaxis responses to CXCL12 and CXCL13. FDC-SP can induce the chemotaxis of CD40-stimulated nontransgenic B cells and can significantly enhance B cell migration in combination with chemokines, indicating that FDC-SP may function in part by regulating B cell chemotaxis. These results provide the first evidence for immunomodulatory activities of FDC-SP and implicate this molecule as a regulator of B cell responses.     

Antigen targeting to plasmacytoid dendritic cells via Siglec-H inhibits Th cell-dependent autoimmunity

Plasmacytoid dendritic cells (PDCs) have been shown to present Ags and to contribute to peripheral immune tolerance and to Ag-specific adaptive immunity. However, modulation of adaptive immune responses by selective Ag targeting to PDCs with the aim of preventing autoimmunity has not been investigated. In the current study, we demonstrate that in vivo Ag delivery to murine PDCs via the specifically expressed surface molecule sialic acid binding Ig-like lectin H (Siglec-H) inhibits Th cell and Ab responses in the presence of strong immune stimulation in an Ag-specific manner. Correlating with sustained low-level MHC class II-restricted Ag presentation on PDCs, Siglec-H-mediated Ag delivery induced a hyporesponsive state in CD4(+) T cells leading to reduced expansion and Th1/Th17 cell polarization without conversion to Foxp3(+) regulatory T cells or deviation to Th2 or Tr1 cells. Siglec-H-mediated delivery of a T cell epitope derived from the autoantigen myelin oligodendrocyte glycoprotein to PDCs effectively delayed onset and reduced disease severity in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis by interfering with the priming phase without promoting the generation or expansion of myelin oligodendrocyte glycoprotein-specific Foxp3(+) regulatory T cells. We conclude that Ag delivery to PDCs can be harnessed to inhibit Ag-specific immune responses and prevent Th cell-dependent autoimmunity.