Characterization of Blimp-1 function in effector regulatory T cells.

Regulatory T (Treg) cells maintain immunological tolerance in steady-state and after immune challenge. Activated Treg cells can undergo further differentiation into an effector state that highly express genes critical for Treg cell function, including ICOS, TIGIT and IL-10, although how this process is controlled is poorly understood. Effector Treg cells also specifically express the transcriptional regulator Blimp-1 whose expression overlaps with many of the canonical markers associated with effector Treg cells, although not all ICOS+TIGIT+ Treg cells express Blimp-1 or IL-10. In this study, we addressed the role of Blimp-1 in effector Treg cell function. Mice lacking Blimp-1 specifically in Treg cells mature normally, but succumb to a multi-organ inflammatory disease later in life. Blimp-1 is not required for Treg cell differentiation, with mutant mice having increased numbers of effector Treg cells, but regulated a suite of genes involved in cell signaling, communication and survival, as well as being essential for the expression of the immune modulatory cytokine IL-10. Thus, Blimp-1 is a marker of effector Treg cells in all contexts examined and is required for the full functionality of these cells during aging.

Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis: A Study of Humans and a Transgenic Mouse Model.

Importance: Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo. Objective: To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease. Design, Setting, and Participants: This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration. Main Outcomes and Measures: In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice. Results: The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves. Conclusions and Relevance: These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function.

Inclusion of BLIMP-1+ effector regulatory T cells improves the Immunoscore in a cohort of New Zealand colorectal cancer patients: a pilot study.

Analysis of tumour-infiltrating T cells in colorectal cancer can predict disease-free survival. The Immunoscore, obtained by quantifying tumour-infiltrating CD3+ and CD8+ T cells, may improve current staging. Effector regulatory T cells are a potently suppressive subset in mice and, while present in human colorectal cancer, their role in patient outcome is unknown. Immunofluorescence was used to analyse immune cell infiltrates in patients with early (stage II) colorectal cancer with (n = 13) and without (n = 19) recurrent disease. CD3 and CD8 were used for the Immunoscore; FOXP3, BLIMP-1 and CD3 to identify effector regulatory T cells. Patients with high Immunoscores had increased disease-free survival compared to patients with low Immunoscores (Log-rank test p < 0.01). Prediction of outcome was further improved by stratifying patients with a low Immunoscore according to CD3+FOXP3+BLIMP-1+ cell infiltration at the invasive margin. Patients with a low Immunoscore and high infiltrate of CD3+FOXP3+BLIMP-1+ cells tended to have better disease-free survival than patients with low Immunoscore and low infiltrate of CD3+FOXP3+BLIMP-1+ cells. Patients with a high Immunoscore had better disease-free survival than patients with a low Immunoscore and low infiltrate of CD3+ FOXP3+ BLIMP-1+ cells (Log-rank test p < 0.001). These results indicate that tumour infiltration with effector regulatory T cells improves the prognostic value of the Immunoscore and implies that these cells may play a role in colorectal cancer patient outcome.

Th17 cell differentiation proceeds independently of IRF8.

The transcriptional repressor/activator interferon regulatory factor 8 (IRF8) modulates the differentiation of a multitude of hematopoietic lineages. However, the role of IRF8 in CD4(+) T-cell development is less well defined, with a recent study implicating IRF8 as an intrinsic repressor of interleukin-17 (IL-17) expressing T helper type 17 (Th17) cell differentiation. Using an IRF8-EGFP reporter strain we have confirmed that IRF8 is expressed in all T helper lineages, including Th17 cells. The loss of IRF8 did not affect Th17 differentiation in vitro, beyond a small increase in IL-22 expression. Moreover, IRF8 deficiency did not enhance the Th17 immune response in experimental T-cell transfer colitis. Together, these results suggest that IRF8 does not play an essential intrinsic role in Th17 cell differentiation.

Blimp-1-Dependent IL-10 Production by Tr1 Cells Regulates TNF-Mediated Tissue Pathology.

Tumor necrosis factor (TNF) is critical for controlling many intracellular infections, but can also contribute to inflammation. It can promote the destruction of important cell populations and trigger dramatic tissue remodeling following establishment of chronic disease. Therefore, a better understanding of TNF regulation is needed to allow pathogen control without causing or exacerbating disease. IL-10 is an important regulatory cytokine with broad activities, including the suppression of inflammation. IL-10 is produced by different immune cells; however, its regulation and function appears to be cell-specific and context-dependent. Recently, IL-10 produced by Th1 (Tr1) cells was shown to protect host tissues from inflammation induced following infection. Here, we identify a novel pathway of TNF regulation by IL-10 from Tr1 cells during parasitic infection. We report elevated Blimp-1 mRNA levels in CD4+ T cells from visceral leishmaniasis (VL) patients, and demonstrate IL-12 was essential for Blimp-1 expression and Tr1 cell development in experimental VL. Critically, we show Blimp-1-dependent IL-10 production by Tr1 cells prevents tissue damage caused by IFNγ-dependent TNF production. Therefore, we identify Blimp-1-dependent IL-10 produced by Tr1 cells as a key regulator of TNF-mediated pathology and identify Tr1 cells as potential therapeutic tools to control inflammation.

Differentiation and function of Foxp3(+) effector regulatory T cells.

Regulatory T (Treg) cells are essential for immunological tolerance and homeostasis. Although forkhead box (Fox)p3 is continually required to reinforce the Treg cell program, Treg cells can also undergo stimulus-specific differentiation that is regulated by transcription factors typically associated with the differentiation of conventional CD4(+) T cells. This results in effector Treg (eTreg) cells with unique migratory and functional properties matched to the stimulus that elicited the initial response. Despite this functional and transcriptional heterogeneity, expression of the transcription factor B lymphocyte-induced maturation protein (Blimp)-1, a key player in late B cell and conventional T cell differentiation, is common to all eTreg cells. Here, we discuss the factors that control the differentiation of eTreg cells and their importance in disease settings.

The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells.

Regulatory T cells (T(reg) cells) are required for peripheral tolerance. Evidence indicates that T(reg) cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of T(reg) cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector T(reg) cells. Thus, our study defines a differentiation pathway that leads to the acquisition of T(reg) cell effector functions and requires both IRF4 and Blimp-1.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim.

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Age-dependent, polyclonal hyperactivation of T cells is reduced in TNF-negative gld/gld mice.

The generalized lymphoproliferative disorder (gld) mouse strain is characterized by severe splenomegaly/lymphadenopathy, the production of autoimmune antibodies, and the appearance of CD4/CD8-negative T cells. An additional TNF deficiency of gld/gld mice attenuates the course of the disorder through a yet-unknown mechanism. In this study, we could demonstrate that the reduced splenomegaly and lymphadenopathy in B6.gld/gld.TNF-/- mice were correlated with a decreased peripheral T cell proliferation rate and a delayed polyclonal activation. A comparative analysis of naïve T cells and memory/effector T cells showed an age-dependent difference in the T cell activation pattern in the spleen of B6.gld/gld and B6.gld/gld.TNF-/- mice. T cells from B6.gld/gld.TNF-/- spleens and lymph nodes showed significantly higher levels of CCR7 and CD62 ligand on their surface compared with B6.gld/gld mice when mice of the same age were compared. Additionally, we found an increased titer of the Th1 cytokine IFN-gamma in the serum of B6.gld/gld mice, whereas the concentration of IFN-gamma was markedly reduced in the serum of B6.gld/gld.TNF-/- mice. These findings support the hypothesis that increased T cell activation and proliferation in the presence of TNF contribute to the exacerbation of the gld syndrome.

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease.

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

No requirement for TRAIL in intrathymic negative selection.

The contribution of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway to intrathymic negative selection is a controversial subject with two studies suggesting a key role for TRAIL, while others demonstrated normal negative selection, in TRAIL- and TRAIL receptor-deficient mice. The basis of these discrepancies is unclear and may in part reflect differences in the negative selection models under investigation. Considering the importance of the negative selection process in the establishment of a competent immune system, it is essential that these discrepancies be fully resolved. In this study, we failed to identify a role for TRAIL in an acute model of peptide antigen-specific negative selection using a TCR transgenic system as well as antibody-mediated TCR/CD3 ligation in vitro and in vivo. Moreover, thymic dendritic cells, the main cellular mediators of negative selection in the thymus, did not constitutively express TRAIL, and TRAIL receptor (DR5) expression was negative or extremely low on thymocytes. Furthermore, in vitro thymocyte deletion was normal in C57BL/6 TRAIL(-/-) gld mice, suggesting that TRAIL and FasL do not function cooperatively to induce negative selection. These results, combined with the fact that aged C57BL/6 TRAIL(-/-) mice showed no signs of spontaneous autoimmunity, strongly indicate that intrathymic negative selection occurs normally in the absence of TRAIL signaling.

Cancer: novel therapeutic strategies that exploit the TNF-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor pathway.

Cancer is a widespread disease, with half of all men and one-third of all women in the United States developing cancer during their lifetime. The efficacy of many cancer treatments including radiotherapy, chemotherapy and immunotherapy is due to their ability to induce tumor cell apoptosis. Recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is currently being developed as a cancer therapeutic since it selectively induces apoptosis in a variety of transformed cells, but not in most normal cells. Agonistic monoclonal antibodies (mAbs) specific for human death-inducing TRAIL receptors (DR4 or DR5) are also being actively pursued. Importantly, in experimental mice, synergistic anti-tumor effects have been observed with a combination treatment of agonistic mAb against DR5 together with either IL-21 or agonistic mAbs against CD40 and CD137. Together, these findings suggest that antibody-based therapies that cause tumor cell apoptosis and promote T cell memory or function may be effective in fighting cancer.

NK cells use NKG2D to recognize a mouse renal cancer (Renca), yet require intercellular adhesion molecule-1 expression on the tumor cells for optimal perforin-dependent effector function.

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.

IL-21 enhances tumor-specific CTL induction by anti-DR5 antibody therapy.

Tumor cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful anti-tumor immunotherapies. In this study, we show that induction of tumor cell apoptosis by agonistic mAb against DR5, combined with delayed IL-21 treatment, suppressed tumor growth and pre-established tumor metastases. Synergistic effects of the combination were observed in several tumor models where the target tumor was sensitive to DR5-mediated apoptosis. IL-21 promoted tumor-specific CTL activity and enhanced memory responses to tumor rechallenge. These results indicate that a rational combination of Ab-based therapy that causes tumor cell apoptosis and a cytokine that promotes T cell memory is a useful new strategy for cancer immunotherapy.

TNF-related apoptosis-inducing ligand as a therapeutic agent in autoimmunity and cancer.

Recombinant, soluble TNF-related apoptosis-inducing ligand (TRAIL) is currently being developed as a promising natural immune molecule for trial in cancer patients because it selectively induces apoptosis in transformed or stressed cells but not in most normal cells. In cancer patients, phase 1 and 2 clinical trials using agonistic mAbs that engage the human TRAIL receptors DR4 and DR5 have also provided encouraging results. It is now evident that TRAIL suppresses autoimmune disease in various experimental animal models, suggesting that the therapeutic value of recombinant TRAIL and agonistic DR4 and DR5 mAbs might also extend to the suppression of autoimmune disease. This review provides an insight into our current understanding of the role(s) of TRAIL in disease, with a specific focus on cancer and autoimmunity. We also emphasize biological agents and drugs that sensitize tumour cells to TRAIL-mediated apoptosis and discuss the potential molecular basis for their sensitization.

Cutting edge: TRAIL deficiency accelerates hematological malignancies.

TNF apoptosis-inducing ligand is attracting considerable interest as a potential extrinsic tumor suppressor mechanism, although previous reports have conveyed somewhat contrasting views regarding the likely importance of this pathway. In this study, we provide the first evaluation of spontaneous tumor formation over the life span of TRAIL-deficient mice. Interestingly, >25% of these mice do develop lymphoid malignancies after 500 days of life. TRAIL suppressed the initiation and development of both tumors of lymphoid and stromal origin in the context of the loss of at least one p53 allele. Specific examination of the role of TRAIL in Her2/neu oncogene-driven mammary epithelial cancer revealed no critical role for TRAIL despite the inherent TRAIL sensitivity of such mammary carcinomas. Overall, the data indicate an important function of TRAIL in controlling carcinogenesis, but suggest that further examination of this pathway in epithelial malignancies is warranted.

TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L suppresses experimental autoimmune encephalomyelitis in mice.

Studies have suggested that endogenous TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L may suppress the induction of some autoimmune diseases in mice. Here, we show that TRAIL/Apo2L suppresses autoimmune damage in relapsing-remitting, and non-remitting models of experimental autoimmune encephalomyelitis (EAE). TRAIL/Apo2L-deficient mice and wild-type mice treated with neutralizing anti-TRAIL/Apo2L antibody displayed enhanced clinical score, increased T-cell proliferative responses to myelin oligodendrocyte glycoprotein (MOG), and increased numbers of inflammatory lesions in the spinal cord and central nervous system. TRAIL neutralization immediately before disease onset was most effective at exacerbating disease score. More importantly, therapeutic intervention with recombinant soluble TRAIL/Apo2L delayed the onset and reduced the severity of MOG-induced EAE. These data are the first to illustrate the potential therapeutic value of recombinant TRAIL/Apo2L in suppressing T-cell-mediated autoimmune diseases.