Pathogen-induced memory Tfh cells are important to maintain high-affinity antibodies against pathogens. We have now discovered Tfh cells with a similar memory phenotype (MP) that develop in pathogen-free conditions. These MP Tfh cells are similar to pathogen-induced memory Tfh in both phenotype and function. They express FR4 and Egr2, which are both found in pathogen-induced memory Tfh cells. FR4+Egr2+ CD4 MP cells express genes involved in the development of Tfh cells and homeostatic proliferation, as well as key metabolic pathways discovered in pathogen-induced memory Tfh cells. MP Tfh cells can support B cell-mediated IgG production in vitro and induce germinal center formation and anti-viral antibodies in response to virus infection. These mouse MP Tfh cells share a similar phenotype to human circulating Tfh cells that are increased in Sjögren's syndrome patients. Although Foxp3-positive circulating T follicular regulatory (Tfr) cells are normal, a proportion of circulating Tfh cells from patients express increased levels of T-bet, which is associated with high levels of inflammatory pathology. Thus, although they do not require overt infection for their development, MP Tfh cells are important for protective immune responses, and dysregulated MP Tfh responses may play a role in autoimmunity.

Memory phenotype (MP) CD4 T cells that develop in the absence of overt antigen stimulation are important for both innate and adaptive immune responses. Although pathogen specific MP CD4 T cells have been found to function effectively against infection, it is not known if MP Tfh cells can also be developed in the absence of overt antigen stimulation. Here, we demonstrate that MP Tfh cells develop in the absence of overt antigen stimulation. In addition to FR4+, a mark found in pathogen induced memory Tfh cells, MP Tfh expressed high levels of Egr2. The development of MP Tfh are defective in CD2-Egr2/3−/− mice. MP Tfh cells share similar features in phenotypes and epigenetics with antigen induced memory Tfh cells and can effectively induce germinal center formation and B cells responses. Thus, MP Tfh cells regulated by Egr2/3 pathway are important component of adaptive immunity. Medical Research Council UK (MR/N00096X/1), Barts Charity (MGU0463)

Although T cells can develop into an exhausted state in the tumour microenvironment, tumour infiltrating T cells (TILs) are important to control tumour growth. By analysing single cell RNA-sequencing data from human tumours, we found that the transcription factors Early Growth Response 2 (EGR2) and 3 were highly induced in TILs, but not peripheral CD8 + T cells, in multiple patient cohorts. We found that deficiency of Egr2 and 3 in T cells resulted in enhanced tumour growth and fewer TILs in mouse models. Egr2 is highly expressed together with checkpoint molecules in a proportion of CD8 + TILs and Egr2high cells exhibit better survival and proliferation than Egr2-/-Egr3-/- and Egr2low TILs. Anti-PD-1 treatment increases Egr2 expression in CD8 + TILs and reduces tumour growth, while anti-PD-1 efficacy is abrogated in the absence of Egr2 and 3. Thus, Egr2 and 3 are important for maintaining anti-tumour responses of exhausted CD8 + TILs.

The transcription factors Egr2 and 3 are essential for controlling inflammatory autoimmune responses of memory phenotype (MP) CD4 T cells. However, the mechanism is still unclear. We have now found that the Egr2 + subset (PD-1 high MP) of MP CD4 T cells expresses high levels of checkpoint molecules (PD-1 and Lag3) and also markers of effector T cells (CXCR3 and ICAM-1). Egr2/3 are not required for PD-1 high MP CD4 cell development but mediate a unique transcriptional programme that effectively controls their inflammatory responses, while promoting homeostatic proliferation and adaptive responses. Egr2 negative PD-1 high MP CD4 T cells are impaired in homeostatic proliferation and adaptive responses against viral infection but display inflammatory responses to innate stimulation such as IL-12. PD-1 high MP CD4 T cells have recently been implicated in rheumatoid arthritis pathogenesis, and we have now found that Egr2 expression is reduced in PD-1 high MP CD4 T cells from patients with active rheumatoid arthritis compared with healthy controls. These findings demonstrate that Egr2/3 control the inflammatory responses of PD-1 high MP CD4 T cells and maintain their adaptive immune fitness.

The immune system is a host defence system to protect the body against foreign invaders. T cells are one of the major components of the immune cells and they are essential for immune responses. Early growth response gene (Egr2) in T cells is important for maintaining immune functions of T cells by promoting adaptive immune responses while controlling inflammation and preventing the development of autoimmune diseases. A study by our group demonstrated the function of Egr2 as a checkpoint regulator controlling the proliferation and differentiation of the T cells. In association, Egr2 and 3 play indispensable role in T cell immune response, but the mechanism regulating Egr2 expression in T cells is still unclear. In this study, we analysed the Egr2 expression mechanism in CD4 T cells under antigen stimulation. We found that Egr2 expression is regulated by different cytokines including IL-2 and IL-4, which increased Egr2 induction in activated T cells. However, inflammatory cytokines, including INFγ and IL-6, suppressed Egr2 expression through STAT1 and STAT3 signalling pathway respectively, highlighting a mechanism for tolergenic immune response on T cells.

INTRODUCTION:Impaired proliferation and production of IL2 are the hallmarks of experimental T cell tolerance. However, in most autoimmune diseases, auto-reactive T cells do not display hyper proliferation, but inflammatory phenotypes. METHODS:We have now demonstrated that the transcription factors Egr2 and 3 are important for the control of inflammatory cytokine production by tolerant T cells, but not for tolerance induction. RESULTS:In the absence of Egr2 and 3, T cell tolerance, as measured by impaired proliferation and production of IL2, can still be induced, but tolerant T cells produced high levels of inflammatory cytokines. Egr2 and 3 regulate expression of differentiation repressors and directly inhibit T-bet function in T cells. Indeed, decreased expression of differentiation repressors, such as Id3 and Tcf1, and increased expression of inflammatory transcription factors, such as RORγt and Bhlhe40 were found in Egr2/3 deficient T cells under tolerogenic conditions. In addition, T-bet was co-expressed with Egr2 in tolerant T cells and Egr2/3 defects leads to production of high levels of IFNγ in tolerant T cells. CONCLUSIONS:Our findings demonstrated that despite impaired proliferation and IL2 production, tolerant T cells can display inflammatory responses in response to antigen stimulation and this is controlled at least partly by Egr2 and 3.

T-bet is important for differentiation of cytotoxic CD8 and Th1 CD4 T cells. We have discovered that Egr2 and 3 are potent inhibitors of T-bet function in CD4 and CD8 effector T cells. Egr2 and 3 were essential to suppress Th1 differentiation in Th2 and Th17 conditions in vitro and also to control IFN-γ–producing CD4 and CD8 T cells in response to virus infection. Together with Egr2 and 3, T-bet is induced in naive T cells by Ag stimulation, but Egr2 and 3 expression was inhibited by Th1–inducing cytokines. We found that Egr2 and 3 physically interact with the T-box domain of T-bet, blocking T-bet DNA binding and inhibiting T-bet–mediated production of IFN-γ. Thus, Egr2 and 3 are antagonists of T-bet function in effector T cells and are important for the control of inflammatory responses of T cells.

Egr2 and 3 are important for maintaining immune homeostasis. Here we define a fundamental function of Egr2 and 3 operating as a checkpoint that controls the transition between clonal expansion and differentiation of effector T cells. Egr2 and 3 deficiency resulted in defective clonal expansion but hyperactivation and excessive differentiation of T cells in response to viral infection. Conversely, sustained Egr2 expression enhanced expansion but severely impaired effector differentiation. Egr2 bound to and controlled the expression of genes regulating proliferation (Myc and Myb) and differentiation repressors (Bcl6, Id3), while repressing transcription factors required for effector function (Zeb2, RORa, RORc, and Bhlhe40). Egr2 and 3 expression in T cells was regulated reciprocally by antigen and IFNγ, providing a mechanism for adjusting proliferation and differentiation of individual T cells. Thus, Egr2 and 3 are upstream regulators of effector CD4 and CD8 T cells that are essential for optimal responses with limited immunopathology.

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GCs), and Tfh differentiation requires the function of B cell lymphoma 6 (BCL6). Wehave now discovered that early growth response gene 2 (EGR2) and EGR3 directly regulate the expression of Bcl6 in Tfh cells, which is required for their function in regulation of GC formation. In the absence of EGR2 and -3, the expression of BCL6 in Tfh cells is defective, leading to impaired differentiation of Tfh cells, resulting in a failure to form GCs following virus infection and defects in production of antiviral antibodies. Enforced expression of BCL6 in EGR2/3-deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of EGR2/3 that is important for Tfh cell development and Tfh cell-mediated B cell immune responses.

T-cell responses are induced by antigen presenting cells (APC) and signals from the microenvironment. Antigen persistence and inflammatory microenvironments in chronic infections and cancer can induce a tolerant state in T-cells resulting in hyporesponsiveness, loss of effector function, and weak biochemical signaling patterns in response to antigen stimulation. Although the mechanisms of T-cell tolerance induced in chronic infection and cancer may differ from those involved in tolerance to self-antigen, the impaired proliferation and production of IL-2 in response to antigen stimulation are hallmarks of all tolerant T cells. In this review, we will summarize the evidence that the immune responses change from non-self to "self"-like in chronic infection and cancer, and will provide an overview of strategies for re-balancing the immune response of antigen-specific T cells in chronic infection and cancer without affecting the homeostasis of the immune system.

T cells robustly respond to virus infection. Egr-2 and -3 are induced in both naïve and tolerant T cells (PMC2652694, PMID15585857, PMID15834410). The deficiency of Egr-2 and -3 specifically in B and T cells results in autoimmunity (PMID23021953). It is however unknown whether they also play roles in protective immmunoresponses. We have now shown that Egr-2 and -3 are required for naïve T cell activation in response to virus infection. Defect in Egr-2 and -3 restrains the IL-2 production and the expansion of viral specific CD4 and CD8 T cells, leading to the development of chronic infection despite active phenotypes and excessive production of IFNg. The induced Egr-2 and -3 in naïve T cells are essential for the maintenance of optimal AP-1 activation and IL-2 production leading to the rapid expansion of viral specific T cells. In addition to the proliferation of viral specific T cells, Egr-2 and -3 control the production of inflammatory cytokines and the activation phenotypes of viral specific T cells at early stages of infection to limit inflammatory pathology and build a pool of responding T cells for a robust anti-viral effect with minimum immunopathology. The functions of Egr-2 and -3 are limited in naïve T cells in primary immunoresponses and are not involved in the development of the memory pool and secondary responses. In conclusion, in addition to the maintenance of immunohomeostasis, Egr-2 and -3 are important to primary immune responses against virus infection.

Event Abstract Back to Event Early growth response gene 2 and 3 are essential for the regulation of tumour suppressor genes, Ikaros, Aiolos and FOXO3. Punamdip K. Bhullar1*, Tizong Miao2, Alistair L. Symonds2, Emma Ghaffari1, Su-Ling Li1 and Ping Wang2 1 Brunel University, Bioscience, United Kingdom 2 Barts and London School of Medicine and Dentistry, Blizard Institute, United Kingdom Non-Hodgkin lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases and is associated with inflammatory autoimmunity. The understanding of lymphomagenesis still remains a challenge. We have discovered that early growth response gene (Egr) -2 and -3 are essential for the control of inflammatory responses of lymphocytes and also involve in the regulation of antigen-mediated proliferation. In addition to the control of inflammatory responses, here we report a novel role of Egr- 2 and/or -3, in controlling the development of lymphoma. The deletion of Egr-2 and Egr-3 in lymphocytes results in spontaneous B or T lymphoma. Despite poor responses to antigen stimulation, Egr-2 and -3 deficient B and T cells are resistant to apoptosis and survival longer in vitro in the absence of overt stimulation. We have discovered that Egr-2 and/or -3 directly bind to the promoter regions on Ikaros, Aiolos and FOXO3. The deletion of Egr-2 and -3 down regulates Ikaros, Aiolos and FOXO3 in T and B cells. The impaired expression associates with proliferative disorder and the development of T and B cell lymphoma. Thus, Egr-2 and -3 are suppressors for lymphomagenesis through the regulation of tumour suppressor genes Ikaros, Aiolos and FOXO3. Keywords: Egr-2, Egr-3, lymphocyte, tumour suppressor genes, Lymphoma Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Bhullar PK, Miao T, Symonds AL, Ghaffari E, Li S and Wang P (2013). Early growth response gene 2 and 3 are essential for the regulation of tumour suppressor genes, Ikaros, Aiolos and FOXO3.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00554 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Mrs. Punamdip K Bhullar, Brunel University, Bioscience, Uxbridge, Middelsex, UB8 3PH, United Kingdom, punamdip.bhullar@brunel.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Punamdip K Bhullar Tizong Miao Alistair L Symonds Emma Ghaffari Su-Ling Li Ping Wang Google Punamdip K Bhullar Tizong Miao Alistair L Symonds Emma Ghaffari Su-Ling Li Ping Wang Google Scholar Punamdip K Bhullar Tizong Miao Alistair L Symonds Emma Ghaffari Su-Ling Li Ping Wang PubMed Punamdip K Bhullar Tizong Miao Alistair L Symonds Emma Ghaffari Su-Ling Li Ping Wang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Early growth response gene (Egr)-2 is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. We have now discovered that Egr-2, which is induced by TGF-β and IL-6, negatively regulates the expression of IL-17, but not IL-2 or IFN-γ, in effector T cells. In the absence of Egr-2, CD4 T cells produce high levels of Th17 cytokines, which renders mice susceptible to experimental autoimmune encephalomyelitis induction. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation. Control of IL-17 expression and Th17 differentiation by Egr-2 is due to inhibition of Batf, a transcription factor that regulates IL-17 expression and Th17 differentiation. Egr-2 interacts with Batf in CD4 T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, whereas the activation of STAT3 and expression of retinoic acid-related orphan receptor γt are unchanged in Th17 cells in the absence of Egr-2. Thus, Egr-2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4 T cells from multiple sclerosis patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation, which may be important for the control of multiple sclerosis development.

Lymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.

Impaired function of virus-specific T cells resulting from virus persistence is one of the major mechanisms underlying the development of chronic hepatitis B viral infection. Previously, we found that IL-2 can restore the effector function of T cells rendered tolerant by Ag persistence. However, systemic administration of IL-2 induces organ pathology and expansion of T regulatory cells. In this study, we show that nano-APC with engineered HLA alleles and IL-2 deliver peptide–MHC complexes, costimulatory molecules, and IL-2 to Ag-responding T cells, resulting in enhanced expression of CD25 and activation of TCR signaling pathways, while suppressing PD-1 expression on viral-responding CD8 T cells from chronic hepatitis B virus patients. The enhanced activation of CD4 and CD8 T cells induced by IL-2–nano-APC was Ag dependent and IL-2–nano-APC did not affect T regulatory cells. At a size of 500 nm, the nano-APC effectively induce immune synapse formation on Ag-specific T cells and accumulate as free particles in the lymphoid organs. These attributes of IL-2–nano-APC or other bioadjuvant-engineered nano-APC have profound implications for their use as a therapeutic strategy in the treatment of chronic hepatitis B virus infection or other chronic viral diseases.

Abstract Antigen presenting cells (APC) initiate immune responses against pathogens by presenting antigen and costimulatory signals to T lymphocytes. APCs loaded with antigens in vitro have been used as therapeutic vaccines to induce antiviral or tumour immune responses in chronic infectious diseases or cancer. However, the live APCs are unstable in vivo and cannot overcome the immune tolerance induced by persistent viral infection or tumour microenvironment. We have developed nano-sized APCs that are at the size of ~ 500nM and can present antigen, costimulatory molecules and bioengineered IL-2 to antigen specific T cells directly. The nano-APCs are prepared from isolated endoplasmic reticulum membranes of APCs engineered with an ER-retained IL-2. Assembled with specific antigenic peptides, the nano-APC effectively induce responses of CMV or HBV specific CD4 and/or CD8 T cells responses in peripheral blood lymphocytes (PBL) of CMV serum converted or chronic HBV infected patients. With selected antigens, the bio-engineered nano-APCs will be used as therapeutic vaccines for the treatment of chronic HBV, HCV or HIV infection and also cancer.

Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been showed that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2+ lymphocytes resulted in a severe reduction of CD4+CD8+ (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells

Background Understanding of how transcription factors are involved in lymphocyte development still remains a challenge. It has been shown that Egr-2 deficiency results in impaired NKT cell development and defective positive selection of T cells. Here we investigated the development of T, B and NKT cells in Egr-2 transgenic mice and the roles in the regulation of distinct stages of B and T cell development. Methods and Findings The expression of Egr1, 2 and 3 were analysed at different stages of T and B cell development by RT-PCT and results showed that the expression was strictly regulated at different stages. Forced expression of Egr-2 in CD2+ lymphocytes resulted in a severe reduction of CD4+CD8+ (DP) cells in thymus and pro-B cells in bone marrow, which was associated with reduced expression of Notch1 in ISP thymocytes and Pax5 in pro-B cells, suggesting that retraction of Egr-2 at the ISP and pro-B cell stages is important for the activation of lineage differentiation programs. In contrast to reduction of DP and pro-B cells, Egr-2 enhanced the maturation of DP cells into single positive (SP) T and NKT cells in thymus, and immature B cells into mature B cells in bone marrow. Conclusions Our results demonstrate that Egr-2 expressed in restricted stages of lymphocyte development plays a dynamic, but similar role for the development of T, NKT and B cells.