During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.
CD40; CD40L; tumor necrosis family; TRAF proteins; humoral immunity; cellular immunity; graft tolerance; tumor immunity
Our knowledge on the function of mast cells (MC) as part of the immune system has expanded from ‘key cells in mediating allergy’ to ‘tunable regulators of the immune response’. Over the past years however, a large body of evidence has been presented indicating a more regulatory role for MC in the immune system by both contact dependent and independent mechanisms. Considering the vast amount of soluble mediators released by MC, it is not surprising that some are involved in the maintenance of peripheral tolerance and the control or even help to resolve ongoing inflammation. In this review we will focus on the immunosuppressive function of some of these mediators produced by MC in a wide variety of disease models.
Mast cells have emerged as critical intermediaries in the regulation of peripheral tolerance. Their presence in many precancerous lesions and tumors is associated with a poor prognosis, suggesting mast cells may promote an immunosuppressive tumor microenvironment and impede the development of protective anti-tumor immunity. The studies presented herein investigate how mast cells influence tumor-specific T cell responses. Male MB49 tumor cells, expressing HY antigens, induce anti-tumor IFN-γ+ T cell responses in female mice. However, normal female mice cannot control progressive MB49 tumor growth. In contrast, mast cell-deficient c-KitWsh (Wsh) female mice controlled tumor growth and exhibited enhanced survival. The role of mast cells in curtailing the development of protective immunity was shown by increased mortality in mast cell-reconstituted Wsh mice with tumors. Confirmation of enhanced immunity in female Wsh mice was provided by (1) higher frequency of tumor specific IFN-γ+ CD8+ T cells in tumor-draining lymph nodes compared with WT females and (2) significantly increased ratios of intratumoral CD4+ and CD8+ T effector cells relative to tumor cells in Wsh mice compared to WT. These studies are the first to reveal that mast cells impair both regional adaptive immune responses and responses within the tumor microenvironment to diminish protective anti-tumor immunity.
While vitamin A has been implicated in host resistance to infectious disease, little is known about the role of vitamin A and its active metabolite, retinoic acid (RA) in host defenses against cancer. Here, we show that local RA production within the tumor microenvironment (TME) is increased up to 5-fold as compared with naïve surrounding tissue, with a commensurate increase in RA signaling to regionally infiltrating tumor-reactive T cells. Conditional disruption of RA signaling in CD8+ T cells using a dominant negative retinoic acid receptor α (dnRARα) established that RA signaling is required for tumor-specific CD8+ T-cell expansion/accumulation and protective antitumor immunity. In vivo analysis of antigen-specific CD8+ T-cell responses revealed that early T-cell expansion was RA-independent; however, late T-cell expansion and clonal accumulation was suppressed strongly in the absence of RA signaling. Our findings indicate that RA function is essential for the survival of tumor-reactive CD8+ T cells within the TME.
Long-term humoral immunity elicited by pathogens and vaccines alike relies upon the generation of both memory B cells (Bmem) and long-lived plasma cells (PCs). Virtually all vaccine formulations induce the concomitant emergence of both Bmem and PCs, suggesting that the emergence of these two differentiated B cells subsets is commonly controlled. Evidence presented shows specific Toll-like Receptor (TLR) agonists coupled with soluble protein antigen (sAg) can selectively induce the expansion of antigen specific Bmem in the absence of PC generation. The co-administration of either TLR 3 or 9 agonists with sAg induced germinal centre (GC) formation, antigen-specific Bmem, but failed to substantively induce the generation of long-lived bone marrow (BM) PCs. Upon re-challenge, high levels of PCs were induced with concomitant high titres of antigen-specific serum IgG. Hence, vaccines can be developed that can prime and protect the host to subsequent infectious agents without initial, high levels of antibody production. Furthermore, these studies suggest that the signals that govern the expansion and differentiation of Bmem can be uncoupled from those that induce long-lived BM PCs.
Memory B cell; Vaccine; Adjuvant; TLRs; B Cell Differentiation
A plethora of work implicates important effects of the Vitamin A derivative, retinoic acid (RA), in myeloid differentiation, while fewer studies explore the role of RA on lymphoid cells. Most work on lymphoid cells has focused on the influence of RA on CD4 T cells. There is little information about the role of RA in CD8 T cell differentiation, and even less on cell-intrinsic effects in the CD8 T cell. This study explores the role of RA on effector and memory differentiation in a cell intrinsic manner in the context of vaccinia virus infection. We observed the loss of the short-lived effector cell phenotype (reduced KLRG1+, T-bethi, granzyme Bhi), accompanied by an enhanced memory precursor phenotype at the effector (increased CD127hi, IL-2+) and contraction phase (increased CD127hi, IL-2+, eomesoderminhi) of the CD8 response in the absence of RA signaling. The lack of RA also increased the proportion of central memory CD8s. Collectively; these results introduce RA in a new role in CD8 T cell activation and differentiation. This may have significant implication in optimal vaccine design where Vitamin A supplementation is used to augment effector responses, but this may be to the detriment of the long-term central memory response.
Tryptophan hydroxylase deficiency in mast cells breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation.
Nutrient deprivation based on the loss of essential amino acids by catabolic enzymes in the microenvironment is a critical means to control inflammatory responses and immune tolerance. Here we report the novel finding that Tph-1 (tryptophan hydroxylase-1), a synthase which catalyses the conversion of tryptophan to serotonin and exhausts tryptophan, is a potent regulator of immunity. In models of skin allograft tolerance, tumor growth, and experimental autoimmune encephalomyelitis, Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation, respectively. All of these effects of Tph-1 deficiency are independent of its downstream product serotonin. Because mast cells (MCs) appear to be the major source of Tph-1 and restoration of Tph-1 in the MC compartment in vivo compensates for the defect, these experiments introduce a fundamentally new mechanism of MC-mediated immune suppression that broadly impacts multiple arms of immunity.
On occasion, emerging scientific fields intersect and great discoveries result. In the last decade, the discovery of regulatory T cells (Treg) in immunity has revolutionized our understanding of how the immune system is controlled. Intersecting the rapidly emerging field of Treg function, has been the discovery that retinoic acid (RA) controls both the homing and differentiation of Treg. Instantly, the wealth and breadth of knowledge of the molecular basis for RA action, its receptors, and how it controls cellular differentiation can and will be exploited to understand its profound effects on Treg. Historically, vitamin A deprivation and repletion and RA agonists have been shown to profoundly affect immunity. Now these findings can be interpreted in light of the revelations that RA controls leukocyte homing and Treg function.
vitamin A; retinoic acid; immune system; tolerance
Peripheral tolerance orchestrated by regulatory T cells, dendritic cells (DCs), and mast cells (MCs) has been studied in several models including skin allograft tolerance. We now define a role for MCs in controlling DC behavior (“conditioning”) to facilitate tolerance. Under tolerant conditions, we show that MCs mediated a marked increase in tumor necrosis factor (TNFα)-dependent accumulation of graft-derived DCs in the dLN compared to nontolerant conditions. This increase of DCs in the dLN is due to the local production of granulocyte macrophage colony-stimulating factor (GM-CSF) by MCs that induces a survival advantage of graft-derived DCs. DCs that migrated to the dLN from the tolerant allograft were tolerogenic; i.e., they dominantly suppress T cell responses and control regional immunity. This study underscores the importance of MCs in conditioning DCs to mediate peripheral tolerance and shows a functional impact of peripherally produced TNFα and GM-CSF on the migration and function of tolerogenic DCs.
CFA is a strong adjuvant capable of stimulating cellular immune responses. Paradoxically, adjuvant immunotherapy by prior exposure to CFA or live mycobacteria suppresses the severity of EAE and spontaneous diabetes in rodents. Here we investigated immune responses during adjuvant immunotherapy of experimental autoimmune encephalomyelitis (EAE). Induction of EAE in CFA-pretreated mice resulted in a rapid influx into the draining lymph nodes (dLNs) of large numbers of CD11b+Gr-1+ myeloid cells, consisting of immature cells with ring-shaped nuclei, macrophages, and neutrophils. Concurrently, a population of mycobacteria-specific IFN-γ-producing T cells appeared in the dLNs. Immature myeloid cells in dLNs expressed the chemokines CXCL10 and CXCL16 in an IFN-γ-dependent manner. Subsequently, CD4+ T cells co-expressing the cognate chemokine receptors, CXCR3 and CXCR6, and myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ T cells accumulated within the chemokine-expressing dLNs, rather than within the CNS. Migration of CD4+ T cells toward dLN cells was abolished by depleting the CD11b+ cells and was also mediated by the CD11b+ cells alone. In addition to altering the distribution of MOG-specific T cells, adjuvant-treatment suppressed development of MOG-specific IL-17. Thus, CFA-adjuvant immunotherapy of EAE requires IFN-γ, which suppresses development of the Th17-response, and diverts autoreactive T cells away from the CNS towards immature myeloid cells expressing CXCL10 and CXCL16 in the lymph nodes.
We demonstrate that all-trans retinoic acid (RA) induces FoxP3+ adaptive T regulatory cells (A-Tregs) to acquire a gut-homing phenotype (α4β7+ CC chemokine receptor 9+) and the capacity to home to the lamina propria of the small intestine. Under conditions that favor the differentiation of A-Tregs (transforming growth factor–β1 and interleukin 2) in vitro, the inclusion of RA induces nearly all activated CD4+ T cells to express FoxP3 and greatly increases the accumulation of these cells. In the absence of RA, A-Treg differentiation is abruptly impaired by proficient antigen presenting cells or through direct co-stimulation. In the presence of RA, A-Treg generation occurs even in the presence of high levels of co-stimulation, with RA attenuating co-stimulation from interfering from FoxP3 induction. The recognition that RA induces gut imprinting, together with our finding that it enhances A-Treg conversion, differentiation, and expansion, indicates that RA production in vivo may drive both the imprinting and A-Treg development in the face of overt inflammation.
Immune cell activation induces concurrent temporal and spatial retinoic acid signaling, and CD4+ T cell–specific loss of RA signals reduces effector function, migration, and polarity.
It is known that vitamin A and its metabolite, retinoic acid (RA), are essential for host defense. However, the mechanisms for how RA controls inflammation are incompletely understood. The findings presented in this study show that RA signaling occurs concurrent with the development of inflammation. In models of vaccination and allogeneic graft rejection, whole body imaging reveals that RA signaling is temporally and spatially restricted to the site of inflammation. Conditional ablation of RA signaling in T cells significantly interferes with CD4+ T cell effector function, migration, and polarity. These findings provide a new perspective of the role of RA as a mediator directly controlling CD4+ T cell differentiation and immunity.
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries causing a major leak of plasma proteins. This disruption results in early death of animals due to severe non-inflammatory protein loosing enteropathy. Deletion of PV1 in endothelium, but not the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Interleukin-9 (IL-9) has attracted renewed interest owing to the identification of its expression by multiple T helper (TH) cell subsets, including TH2 cells, TH9 cells, TH17 cells and regulatory T (TReg) cells. Here, we provide a broad overview of the conditions that are required for cells to produce IL-9 and describe the cellular targets and nature of the immune responses that are induced by IL-9.
For the past 100 years, vitamin A has been implicated as an essential dietary component in host resistance to infectious disease. However, only recently have studies begun to elucidate the cellular and molecular mechanisms of how vitamin A regulates cell-mediated and humoral-mediated immunity. In this review, we present an overview of the recent discoveries of the role that vitamin A and its metabolite, retinoic acid (RA), play in the regulation of immune cells. How RA impacts on leukocyte growth, differentiation, and homing is discussed with special attention to inflammatory responses and solid tumor microenvironment.
retinoic acid; vitamin A; inflammation
Purpose of review
The role of regulatory T cells (Treg) in peripheral tolerance has been studied extensively in transplantation research. Recently, mast cells have been shown to play an indispensable role in allograft tolerance. The purpose of this review is to inform the reader on the current standings of the role of mast cells in dominant tolerance with an emphasis on the interaction of mast cells with Treg.
Mast cells are required to sustain peripheral tolerance via Treg. Treg can stabilize mast cells degranulation by contact-dependent mechanisms through the interaction of OX40 and its ligand OX40L, and by production of soluble factors, such as interleukin-10 and transforming growth factor-β. Conversely, the activation and subsequent degranulation of mast cells break peripheral tolerance.
Both mast cells and Treg are needed to create a local immunosuppressive environment in the transplant. Treg are not only necessary to suppress effector T-cell responses but also to stabilize mast cells. Mast cells in return could contribute to the immunosuppressive state by release of transforming growth factor-β, interleukin-10 and specific proteases. However, the molecular basis for mast cells control of Treg suppression in organ transplantation is still unresolved.
dominant tolerance; mast cells; regulatory T cells
VISTA suppresses T cell proliferation and cytokine production and can influence autoimmunity and antitumor responses in mice.
The immunoglobulin (Ig) superfamily consists of many critical immune regulators, including the B7 family ligands and receptors. In this study, we identify a novel and structurally distinct Ig superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated V-domain Ig suppressor of T cell activation (VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTA expression on APCs inhibits T cell proliferation and cytokine production in vitro. A VISTA-specific monoclonal antibody interferes with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. Furthermore, anti-VISTA treatment exacerbates the development of the T cell–mediated autoimmune disease experimental autoimmune encephalomyelitis in mice. Finally, VISTA overexpression on tumor cells interferes with protective antitumor immunity in vivo in mice. These findings show that VISTA, a novel immunoregulatory molecule, has functional activities that are nonredundant with other Ig superfamily members and may play a role in the development of autoimmunity and immune surveillance in cancer.
The tumor microenvironment (TME), which is composed of stromal cells such as endothelial cells, fibroblasts, and immune cells, provides a supportive niche promoting the growth and invasion of tumors. The TME also raises an immunosuppressive barrier to effective antitumor immune responses and is therefore emerging as a target for cancer immunotherapies. Mast cells (MCs) accumulate in the TME at early stages, and their presence in the TME is associated with poor prognosis in many aggressive human cancers. Some well-established roles of MCs in cancer are promoting angiogenesis and tumor invasion into surrounding tissues. Several mouse models of inducible and spontaneous cancer show that MCs are among the first immune cells to accumulate within and shape the TME. Although MCs and other suppressive myeloid cells are associated with poor prognosis in human cancers, high densities of intratumoral T effector (Teff) cells are associated with a favorable prognosis. The latter finding has stimulated interest in developing therapies to increase intratumoral T cell density. However, cellular and molecular mechanisms promoting high densities of intra-tumoral Teff cells within the TME are poorly understood. New evidence suggests that MCs are essential for shaping the immune-suppressive TME and impairing both antitumor Teff cell responses and intratumoral T cell accumulation. These roles for MCs warrant further elucidation in order to improve antitumor immunity. Here, we will summarize clinical studies of the prognostic significance of MCs within the TME in human cancers, as well as studies in mouse models of cancer that reveal how MCs are recruited to the TME and how MCs facilitate tumor growth. Also, we will summarize our recent studies indicating that MCs impair generation of protective antitumor T cell responses and accumulation of intratumoral Teff cells. We will also highlight some approaches to target MCs in the TME in order to unleash antitumor cytotoxicity.
Mast cells; Cancer; Tumor microenvironment; T cells; AllergoOncology Symposium-in-Writing
Mast cells (MC) have been shown to mediate regulatory T-cell (Treg) dependent, peripheral allograft tolerance in both skin and cardiac transplants. Furthermore, Treg have been implicated in mitigating IgE mediated MC degranulation, establishing a dynamic, reciprocal relationship between MC and Treg in controlling inflammation. In an allograft tolerance model, it is now shown that intragraft or systemic MC degranulation results in the transient loss of Treg suppressor activities with the acute, T-cell dependent rejection of established, tolerant allografts. Upon degranulation, MC mediators can be found in the skin, Treg rapidly leave the graft, MC accumulate in the regional lymph node and the Treg are impaired in the expression of suppressor molecules. Such a dramatic reversal of Treg function and tissue distribution by MC degranulation underscores how allergy may causes the transient breakdown of peripheral tolerance and episodes of acute T-cell inflammation.
To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-α 2a in metastatic renal cell carcinoma patients.
Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 × 107 cells/1 mL Lactated Ringer’s solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-α 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti–renal cell carcinoma antibody and cytokine levels.
All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4+ T regulatory cells had a strong association with outcome. Pre–IP-10 serum levels approached significance for predicting outcome.
The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.
The hypothesis that bystander inflammatory signals promote memory B cell (BMEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220+IgG+ BMEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent BMEM clonally expand. Surprisingly, proliferating BMEM do not acquire germinal center (GC) B cell markers before generating daughter BMEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of BMEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of BMEM occurred. The absence of a BMEM response to nonspecific inflammatory signals clearly shows that BMEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.
Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway.