In vivo 4-1BB deficiency in myeloid cells enhances peripheral T cell proliferation by increasing IL-15.

4-1BB signals are considered positive regulators of T cell responses against viruses and tumors, but recent studies suggest that they have more complex roles in modulating T cell responses. Although dual roles of 4-1BB signaling in T cell responses have been suggested, the underlying mechanisms are still not fully understood. In this study, we tested whether 4-1BB expression affected T cell responses differently when expressed in myeloid versus lymphoid cells in vivo. By assessing the proliferation of 4-1BB(+/+) and 4-1BB(-/-) T cells in lymphocyte-deficient RAG2(-/-) and RAG2(-/-)4-1BB(-/-) mice, we were able to compare the effects on T cell responses of 4-1BB expression on myeloid versus T cells. Surprisingly, adoptively transferred T cells were more responsive in tumor-bearing RAG2(-/-)4-1BB(-/-) mice than in RAG2(-/-) mice, and this enhanced T cell proliferation was further enhanced if the T cells were 4-1BB deficient. Dendritic cells (DCs) rather than NK or tissue cells were the myeloid lineage cells primarily responsible for the enhanced T cell proliferation. However, individual 4-1BB(-/-) DCs were less effective in T cell priming in vivo than 4-1BB(+/+) DCs; instead, more DCs in the secondary lymphoid organs of RAG2(-/-)4-1BB(-/-) mice appeared to induce the enhanced T cell proliferation by producing and transpresenting more IL-15. Therefore, we conclude that in vivo 4-1BB signaling of myeloid cells negatively regulates peripheral T cell responses by limiting the differentiation of DCs and their accumulation in secondary lymphoid organs.

Immune evasion in cancer: Mechanistic basis and therapeutic strategies.

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immunity, measures to counteract tumor escape have not kept pace. There are a number of factors that contribute to tumor persistence despite having a normal host immune system. Immune editing is one of the key aspects why tumors evade surveillance causing the tumors to lie dormant in patients for years through "equilibrium" and "senescence" before re-emerging. In addition, tumors exploit several immunological processes such as targeting the regulatory T cell function or their secretions, antigen presentation, modifying the production of immune suppressive mediators, tolerance and immune deviation. Besides these, tumor heterogeneity and metastasis also play a critical role in tumor growth. A number of potential targets like promoting Th1, NK cell, γδ T cell responses, inhibiting Treg functionality, induction of IL-12, use of drugs including phytochemicals have been designed to counter tumor progression with much success. Some natural agents and phytochemicals merit further study. For example, use of certain key polysaccharide components from mushrooms and plants have shown to possess therapeutic impact on tumor-imposed genetic instability, anti-growth signaling, replicative immortality, dysregulated metabolism etc. In this review, we will discuss the advances made toward understanding the basis of cancer immune evasion and summarize the efficacy of various therapeutic measures and targets that have been developed or are being investigated to enhance tumor rejection.

Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity: implications for possible immune adjuvant therapy.

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC - high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

4-1BB signal stimulates the activation, expansion, and effector functions of γδ T cells in mice and humans.

We show here that the expression of 4-1BB is rapidly induced in γδ T cells following antigenic stimulation in both mice and humans, and ligation of the newly acquired 4-1BB with an agonistic anti-4-1BB augments cell division and cytokine production. We further demonstrate that γδ rather than αß T cells protect mice from Listeria monocytogenes (LM) infection and 4-1BB stimulation enhances the γδ T-cell activities in the acute phase of LM infection. IFN-γ produced from γδ T cells was the major soluble factor regulating LM infection. Vγ1(+) T cells were expanded in LM-infected mice and 4-1BB signal triggered an exclusive expansion of Vγ1(+) T cells and induced IFN-γ in these Vγ1(+) T cells. Similarly, 4-1BB was induced on human γδ T cells and shown to be fully functional. Combination treatment with human γδ T cells and anti-hu4-1BB effectively protected against LM infection in human γδ T cell-transferred NOD-SCID mice. Taken together, these data provide evidence that the 4-1BB signal is an important regulator of γδ T cells and induces robust host defense against LM infection.

4-1BB triggering ameliorates experimental autoimmune encephalomyelitis by modulating the balance between Th17 and regulatory T cells.

Agonistic anti-4-1BB Ab is known to ameliorate experimental autoimmune encephalomyelitis. 4-1BB triggering typically leads to the expansion of CD8(+) T cells, which produce abundant IFN-γ, and this in turn results in IDO-dependent suppression of autoimmune responses. However, because neutralization of IFN-γ or depletion of CD8(+) T cell only partially abrogates the effect of 4-1BB triggering, we sought to identify an additional mechanism of 4-1BB-triggered suppression of autoimmune responses using IFN-γ- or IFN-γR-deficient mice. 4-1BB triggering inhibited the generation of Th17 cells that is responsible for experimental autoimmune encephalomyelitis induction and progression, and increased Foxp3(+)CD4(+) regulatory T (Treg) cells, particularly among CD4(+) T cells. This was not due to a direct effect of 4-1BB signaling on CD4(+) T cell differentiation: 4-1BB signaling not only reduced Th17 cells and increased Treg cells in wild-type mice, which could be due to IFN-γ production by the CD8(+) T cells, but also did so in IFN-γ-deficient mice, in that case by downregulating IL-6 production. These results show that although secondary suppressive mechanisms evoked by 4-1BB triggering are usually masked by the strong effects of IFN-γ, 4-1BB signaling seems to modulate autoimmune responses by a number of mechanisms, and modulation of the Th17 versus Treg cell balance is one of those mechanisms.

Targeting TNF superfamily members for therapeutic intervention in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory disease is one of the most serious medical problems, affecting ∼1% of all people worldwide, irrespective of race. The disease is autoimmune in nature and characterized by chronic inflammation of the synovial tissues in multiple joints that leads to joint destruction. Although T cells are central players in RA development, B cells are required for full penetrance of disease largely via their production of autoantibodies against Fc domain of IgG rheumatoid factor (RF). Treatment options for RA are limited and if any, are inadequate due to associated side effects. Members of the tumor necrosis factor (TNF) superfamily play important roles in a number of autoimmune diseases, including RA. In this review, we briefly summarize key features of the superfamily, we will consider how the well-characterized members concerned with immune regulation are coordinated and their roles in rheumatoid arthritis.

PDCA expression by B lymphocytes reveals important functional attributes.

We have demonstrated in this study the existence of a PDCA-expressing functional B cell population (PDCA+ B lymphocytes), which differentiates from activated conventional B (PDCA-IgM+) lymphocytes. Stimulation with anti-micro, LPS, CpG oligodeoxynucleotide, HSV-1, or CTLA-4 Ig activates the PDCA+ B lymphocytes, leading to cell division and induction of type I IFNs and IDO. Notably, the PDCA+ B lymphocytes are capable of Ag-specific Ab production and Ig class switching, which is corroborated by transfer experiments in B- and PDCA+ B lymphocyte-deficient microMT mice. Importantly, in lupus-prone MRL-Fas(lpr) mice, PDCA+ B lymphocytes remain the principal source of autoantibodies. The PDCA+ B lymphocytes have phenotypes with plasmacytoid dendritic cells, but are a distinct cell population in that they develop from C-kit+B220+ pro-B precursors. Thus, our data suggest that not all PDCA+ cells are dendritic cell-derived plasmacytoid dendritic cells and that a significant majority is the PDCA+ B lymphocyte population having distinct phenotype and function.

Peripheral 4-1BB signaling negatively regulates NK cell development through IFN-gamma.

Stimulation of 4-1BB (CD137) was shown to produce strong anticancer effects in vivo. In contrast, 4-1BB-deficient (4-1BB(-/-)) B6 mice are remarkably resistant to tumor growth. We set out to determine the mechanisms involved in these seemingly contradictory observations. We found that the therapeutic effects of 4-1BB triggering were mainly dependent on CD8(+) T cells and partially on NK cells, whereas CD8(+) T and NK cells were equally needed to suppress tumor growth in 4-1BB(-/-) mice. Cellular analysis showed that the frequency and number of NK cells in the spleen and bone marrow were decreased by 4-1BB triggering but were increased in the absence of 4-1BB signaling in tumor-challenged mice. The 4-1BB-mediated downregulation of NK cell development was primarily dependent on IFN-gamma, which was produced by peripheral CD8(+) T and NK cells. The suppression of NK cell development by 4-1BB-mediated IFN-gamma production occurred in the bone marrow. As 4-1BB signaling increased in the periphery, more CD8(+) T cells but fewer NK cells contributed to the antitumor immunity. As 4-1BB signaling decreased, more NK cells participated in the antitumor immunity. We conclude that 4-1BB signaling results in a shift of the dominant type of immune cell in antitumor immunity from the innate NK cell to the adaptive CD8(+) T cell and that the level of IFN-gamma is critical for this 4-1BB-mediated shift.

4-1BB-mediated immunotherapy of rheumatoid arthritis.

Collagen type II-induced arthritis is a CD4(+) T-cell-dependent chronic inflammation in susceptible DBA/1 mice and represents an animal model of human rheumatoid arthritis. We found that development of this condition, and even established disease, are inhibited by an agonistic anti-4-1BB monoclonal antibody. Anti-4-1BB suppressed serum antibodies to collagen type II and CD4(+) T-cell recall responses to collagen type II. Crosslinking of 4-1BB evoked an antigen-specific, active suppression mechanism that differed from the results of blocking the interaction between 4-1BB and its ligand, 4-1BBL. Anti-4-1BB monoclonal antibodies induced massive, antigen-dependent clonal expansion of CD11c(+)CD8(+) T cells and accumulation of indoleamine 2,3-dioxygenase in CD11b(+) monocytes and CD11c(+) dendritic cells. Both anti-interferon-gamma and 1-methyltryptophan, a pharmacological inhibitor of indoleamine 2,3-dioxygenase, reversed the anti-4-1BB effect. We conclude that the suppression of collagen-induced arthritis was caused by an expansion of new CD11c(+)CD8(+) T cells, and that interferon-gamma produced by these cells suppresses antigen-specific CD4(+) T cells through an indoleamine 2,3-dioxygenase-dependent mechanism.

Origins and functional basis of regulatory CD11c+CD8+ T cells.

Previously, we showed that CD11c defines a novel subset of CD8(+) T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11c(surface-)CD8(+) T cells and undergoes robust expansion in an antigen- and 4-1BB (CD137)-dependent manner. CD11c(+)CD8(+) T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4(+) T cells in vivo and in vitro. Suppression of CD4(+) by CD11c(+)CD8(+) T cells is related to an increase in IDO activity induced in competent cells via the general control non-derepressible-2 pathway. CD11c(+)CD8(+) T cells are refractory to the effect of IDO but constrict in a novel 1-methyl D,L-tryptophan-dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c(+)CD8(+) T-cell subpopulation that has many signature features of Treg.

CD11c+CD8+ T cells: two-faced adaptive immune regulators.

Regulatory cells, important controllers of immune homeostasis, carry out a multi-pronged attack by deleting overactive pathogenic immune cells, by supporting anergy, and by blocking effector functions, thereby contributing to the amelioration of disease. CD8+ T cells co-expressing CD11c are a new addition to the growing list of regulatory cells. Naïve mice harbor CD11c-expressing CD8+ T cells (<3%) that expand further in an antigen-dependent manner. Although activated CD11c+CD8+ T cells express suppressive cytokines such as IL-10 and TGF-beta, their production of IFN-gamma is central to their immune suppressive potential. The CD11c+CD8+ T cells target pathogenic CD4+ T cells in a cell-cell contact dependent manner via IDO- and GCN2-dependent mechanisms. Adoptive transfer of activated CD11c+CD8+ T cells halts the progression of autoimmune rheumatoid arthritis and colitis. However, in certain virus and cancer models the CD11c+CD8+ T cells assume the role of immune effectors, boosting immune potential. This seemingly dual nature of these cells--exerting regulatory vs. effector activities--makes them an attractive therapeutic target. In this review, we discuss the discovery, origins and developmental requirements of CD11c+CD8+cells, and the basis of their immuno-suppressive and effector potentials.

TNF superfamily: costimulation and clinical applications.

The molecules concerned with costimulation belong either to the immunoglobulin (Ig) or tumor necrosis factor (TNF) superfamily. The tumor necrosis superfamily comprises molecules capable of providing both costimulation and cell death. In this review we briefly summarize certain TNF superfamily receptor-ligand pairs that are endowed with costimulatory properties and their importance in health and disease.

Harnessing immune checkpoints for cancer therapy.

Immunomodulatory antibodies that directly trigger and reawaken suppressed T-cell effector function are termed 'checkpoint inhibitors'. CTLA-4 and PD-1/PD-L1 molecules are the most studied inhibitory immune check points against cancer and because of this therapeutic property have entered the clinic for treating a variety of tumor types. The results so far demonstrate a positive impact on cancer remission. Preclinical studies have demonstrated that targeting a number of other T-cell surface molecules including both positive and negative immune regulators, also possesses strong antitumor activity. Some of these molecules have already entered clinical trials. In this report, we briefly highlight the status of these immune checkpoint inhibitors and discuss their side effects and future directions for their use.

Absence of 4 1BB gene function exacerbates lacrimal gland inflammation in autoimmune-prone MRL-Faslpr mice.

PURPOSE: To define the role of endogenous 4-1BB (an important T-cell costimulatory molecule) in the regulation of ocular disease, MRL-Fas(lpr) mice deficient in 4-1BB were generated, and their lacrimal gland function was studied. METHODS: 4-1BB(-/-)MRL/MpJ-Tnfrs(lpr)/Tnfrs(lpr) (lpr/4-1BB(-/-)) mice were generated and used at the ninth backcross. Mice were killed at various times, and lacrimal gland cellularity was analyzed by flow cytometry. Tear and tissue samples were analyzed by Western blotting for the presence of aquaporin 5 (AQP5) and 120-kDa fragments of alpha-fodrin. Cytokine expression of lacrimal glands was assessed by flow cytometry and RT-PCR analysis. RESULTS: Absence of the 4-1BB gene function in lpr mice resulted in early and increased infiltration of mononuclear cells into lacrimal glands compared with 4-1BB intact lpr mice. The severity of lesions in lpr/4-1BB(-/-) mice was closely associated with enhanced accumulation of primarily CD4(+) T cells within the lacrimal glands and with increased expression of IL-4. Elevated levels of AQP5 and cleaved 120-kDa fragments of alpha-fodrin were found in tears and lacrimal gland lysates, respectively, of lpr/4-1BB(-/-) but not lpr/4-1BB(+/+) mice. CONCLUSIONS: Deletion of 4-1BB in lpr mice accelerates lacrimal gland lesions through increased CD4(+) T-cell infiltration and their production of immune modulators.

Dual immunoregulatory pathways of 4-1BB signaling.

It is perhaps rare to encounter among the various immunologically competent receptor-ligand pairs that a single cell surface determinant unleashes both a hidden suppressive function and costimulation. 4-1BB, an activation-induced tumor necrosis factor receptor family member chiefly viewed as a powerful T-cell costimulatory molecule, is one such example. Accumulated evidence in recent years uncovered an unknown facet of in vivo 4-1BB signaling (i.e., "active suppression"). Although in vitro signaling via 4-1BB is shown to support both CD4(+) and CD8(+) T-cell responses, the same induces a predominant CD8(+) T-cell response suppressing CD4(+) T-cell function when applied in vivo. How, when, and why such dual immunoregulatory effect of anti-4-1BB monoclonal antibody (MAB) comes into play is currently the focus of intense research. Existing data, although not complete, uncover several important aspects of in vivo 4-1BB signaling in the amelioration or exacerbation of various immune disorders. Despite minor disagreements, a majority agree that upregulation of interferon (IFN)-gamma is critical to anti-4-1BB MAB therapy in addition to immune modulators such as interleukin 2, transforming growth factor beta, and indolamine 2,3-dioxygenase(5), all of which contribute greatly to the success of anti-4-1BB MAB-based immunotherapy. Anti-4-1BB MAB-mediated expansion of novel CD11c(+)CD8(+) T cells is additional weaponry that appears critical for its in vivo suppressive function. These CD11c(+)CD8(+) T cells express high levels of IFN-gamma, become effective killers, and mediate selective suppression of CD4(+) T cells. In this review, we discuss the dual nature (costimulatory and suppressive) of 4-1BB-mediated immune regulation, its current status, future direction, and its impact on the immune system, with special reference to its immunotherapy.

Immunotherapy targeting 4-1BB and its ligand.

T-cell activation in the absence of costimulation is futile because T-cells deprived of costimulatory signals enter a state of unresponsiveness or anergy. The interaction of 4-1BB and 4-1BB ligand (4-1BBL) activates an important costimulatory pathway with diverse and important roles in immune regulation. Signals relayed through 4-1BB generate strong CD8(+) T-cell responses rather than CD4(+) T-cell responses; this action results in cytokine induction and promotes T-cell survival. In recent years, 4-1BB-mediated immune regulation has gained great significance because of the seemingly contradictory dual roles of agonistic anti-4-1BB in vivo disease models. To date, agonistic 4-1BB monoclonal antibody has shown therapeutic potential against a variety of tumors, CD4(+) T-cell-mediated autoimmune diseases, and chronic graft-versus-host disease. In addition, blockade of 4-1BB/4-1BBL interaction has produced therapeutic effects against coxsackievirus-induced myocardial inflammation, herpetic stromal keratitis, and graft rejection. We propose that the dual roles of agonistic anti-4-1BB--an enhanced effector function and a suppressor function--are mediated by a novel CD11c(+)CD8(+) T-cell population.

Therapeutic potential of anti-CD137 (4-1BB) monoclonal antibodies.

INTRODUCTION: 4-1BB (CD137) is an important T-cell stimulating molecule. The 4-1BB mAb or its variants have shown remarkable therapeutic activity against autoimmunity, viral infections, and cancer. Antibodies to 4-1BB have recently entered clinical trials for the treatment of cancer with favorable toxicity profile. In this article, we present a review documenting the efficacy and pitfalls of 4-1BB therapy. AREAS COVERED: An extensive literature search has been made on 4-1BB, spanning two decades, and a comprehensive report is presented here highlighting the origins, biological effects, therapeutic potential, and mechanistic basis of targeting 4-1BB as well as the side effects associated with such therapy. EXPERT OPINION: Research so far indicates that 4-1BB is highly protective against various pathological conditions including cancer. However, a few important side effects of 4-1BB therapy such as liver toxicity, thrombocytopenia, anemia, and suppressive effects on certain immune competent cells should be taken into consideration before it is used for human therapy.

4-1BB-dependent inhibition of immunosuppression by activated CD4+CD25+ T cells.

4-1BB (CD137) is a costimulatory molecule involved in the activation and survival of CD4, CD8, and natural killer cells. Although a great deal has been learned as to how 4-1BB-mediated signaling governs the immunity of conventional T cells, the functional role of 4-1BB in the context of CD4(+)CD25(+) regulatory T cell (Tr) activation is largely unknown. Using 4-1BB-intact and -deficient mice, we investigated the effect of the 4-1BB/4-1BB ligand pathway on the suppressive function of Tr cells. Our data indicate that although 4-1BB is expressed on Tr cells, its contribution to their proliferation is minimal. We also showed that signaling through the 4-1BB receptor inhibited the suppressive function of Tr cells in vitro and in vivo. It is interesting that anti-4-1BB-mediated but not anti-GITR-directed inhibition was more potent when Tr cells were preactivated. Collectively, these data indicate that 4-1BB signaling is critical in Tr cell immunity.

Herpesvirus infection of ICAM-1-deficient mice.

PURPOSE: To determine the effect of ICAM-1 deficiency on viral infection of the cornea. MATERIALS AND METHODS: Wild-type and intercellular adhesion molecule 1 (ICAM-1)-deficient mice were infected with the RE strain of herpes simplex virus type 1 (HSV-1). Corneal swabs and trigeminal ganglia were obtained and analyzed for infectious virus. Corneas and trigeminal ganglia were evaluated for signs of inflammation by immunohistochemical staining and for interferon-gamma (IFN-gamma)-producing cells by enzyme-linked immunospot assay (ELISPOT). Serum anti-HSV-1 antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Viral titers in corneal swabs from the wild-type and ICAM-1-deficient mice were not significantly different during the 21-day study. Infectious virus was present in the trigeminal ganglia of wild-type and ICAM-1-deficient mice through day 6 after infection. Serum anti-HSV-1 antibody titers were significantly higher in wild-type mice 6 days after infection, compared with ICAM-1-deficient mice; by day 8 and thereafter, however, antibody titers were not significantly different. Production of interferon gamma was greater in trigeminal ganglion cells from wild-type mice stimulated with interleukin 12 and interleukin 18 on days 4, 6, and 8 after infection compared with cells from ICAM-1-deficient mice. Histopathologic analysis of corneal and ganglion sections from wild-type and ICAM-1-deficient mice showed no significant differences in the time-course of appearance or the intensity of the inflammatory infiltrate. Immunohistochemical staining for CD3(+) T-lymphocytes and CD11b(+) neutrophils and macrophages demonstrated equivalent numbers of these cells in the corneas and trigeminal ganglia of wild-type and ICAM-1-deficient mice. CONCLUSIONS: The results of these experiments indicate that ICAM-1 deficiency has only a modest effect on viral infection of the cornea and the development of an acquired immune response.

4-1BB (CD137), an inducible costimulatory receptor, as a specific target for cancer therapy.

Although considerable progress has been made in understanding how tumors evade immune surveillance, measures to counter the same have not kept pace with the advances made in designing effective strategies. 4-1BB (CD137; TNFRS9), an activation-induced costimulatory molecule, is an important regulator of immune responses. Targeting 4-1BB or its natural ligand 4-1BB ligand (4-1BBL) has important implications in many clinical conditions, including cancer. In depth analysis revealed that 4-1BB-mediated anti-cancer effects are based on its ability to induce activation of cytotoxic T lymphocytes (CTL), and among others, high amounts of IFN-γ. In this review, we will discuss the various aspects of 4-1BB-mediated anti-tumor responses, the basis of such responses, and future directions.