Chakraborty S, Sa G. J Immunol Sci. (2018); 2(2): 8-12 Journal of Immunological Sciences

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Development, maintenance and functions of CD8+ T-regulatory cells: Molecular orchestration of FOXP3 Sreeparna Chakraborty1 & Gaurisankar Sa1* 1Division of Molecular Medicine, Bose Institute, P-1/12, Calcutta Improvement Trust Scheme VII M, Kolkata 700054, India

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Article Notes Modulation of immune cells to rejuvenate the immune responses Received: January 10, 2018 against becomes a promising strategy for cancer therapy. T-regulatory Accepted: March 23, 2018 cells are one of the major hurdles in successful cancer immunotherapy. Recent + + *Correspondence: studies discovered that apart from CD4 Treg cells, CD8 Tregs also play roles in Prof. Gaurisankar Sa, Division of Molecular Medicine, Bose tumor immune evasion. Moreover, CD8+ Tregs shows synergistic immunosup- Institute, P-1/12, Calcutta Improvement Trust Scheme VII M, pression with CD4+ Treg cells in . Several phenotypic Kolkata 700 054, India; markers have been described for peripherally induced CD8+ Treg cells, but Tel: +91-33-2569-3258; till now no universal phenotypic signature has yet established. FOXP3 is the Fax: +91-33-2355-3886, E-mail: [email protected] of Treg cells and its transcription is critically regulated by pro- © 2018 Sa G. This article is distributed under the terms of the moter region as well as three intronic conserved non-coding regions, viz; CNS Creative Commons Attribution 4.0 International License. 1, 2 and 3. In this review, we have described the transcriptional networking associated with the regulation of FOXP3 in tumor-CD8+ Treg cells along with Keywords: CD4+ nTreg and iTreg cells. Intervention of the intensive transcriptional machin- Cancer ery of FOXP3 regulation may aid to target Treg cells and thus could potentiate Immunotherapy Treg immunotherapy of cancer. FOXP3 Transcription Introduction A new tide in cancer therapy encourages the immunotherapy to become - vances several immunotherapeutic strategies are evolving which include cancera clinically vaccines, potential adoptive way totransfer fight against of ex vivo this activateddeadly disease. T and naturalIn recent killer ad cells, CART cell and administration of antibodies or recombinant - ecules1 The tumor microenvironment is enriched with cancer-associated that could block the immune checkpoint pathways i.e. co-stimulatory mol . andfibroblasts, thereby myeloid-derived act as a self-check suppressor mechanism cells, to maintainand T- regulatory the peripheral (Treg) toler cells.- In normal physiology Treg cells are potent suppressors of effector T cells tumor-associated Treg cells have been related to poor prognosis in various cancersance and1 homeostaticThe predominance in immune of Treg response. cells in However,the tumor expanded microenvironment densities ofis found to be one of the main impediments to successful cancer immunother- . insusceptibility is to selective depletion of differentiated Treg along with apy. Therefore, one methodology for initiating compelling hostile to tumor or immune checkpoint blockade and these might unleash potent antitu- the activation of tumor-specific effector T cells by using cancer vaccine Till now the involvement of FOXP3-positive CD4+ Treg cells with the ad- morvancement responses of cancer to make is well the establishedcurrent cancer2, 3 immunotherapy more effective. of CD8+ Treg cells in tumor immunomodulation was resurrected, in part, by the occurrence of this suppressor cells . inHowever, the tumor the microenvironmentinterest in the role along with CD4+

Treg cells.

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CD8+ Treg cell: a new recruitment to T-regulatory cell is reported11 family CD8+CD25+FOXP3+ T cells is profound12 Moreover, the role of CD8+Foxp3+ . In the predominance13 and of CD8+ + . +FoxP3+CCL4 CD8+ Treg cells4 Treg in GVHD+14 (Graft-versus-host disease) In the 1970s Grehson et al. first reported the existence of LAG-3 (-activation--3)CD25 and helps to maintain. Tregs the are homeostasis, generally classified while iTregs as either arise ‘natural’ in the prevalence(Chemokine-ligand-4) of CD8+ Treg in inflammatory infectious diseases+ + or nTreg or ’inducible or iTreg’. The nTregs develop in the cellsis already in ovarian documented. cancer whichRecent suppress advances the in researchanti-tumor suggest immune the nCD8+ Tregs share functional and phenotypic similarities with CCR7(C-C chemokine--type-715 and CD8+CD28 ) IL10 cells CD4periphery+ following stimulation from microenvironment.5 The in non-small cell lung carcinoma16 On the other hand, in prostate− for peripherally induced CD8+ Treg cells, till now no universal responsecancer, viaCD8 TGFβ1+CD25 +andCD122 IFNg+FOXP3 production+ Tregs and express CTLA4, GITR and FOXP3 . However, + show their. suppressive functions via Toll- CD8+ Treg cells have been discussed in many autoimmune GITR(glucocorticoid-inducedwhereas CD8+CD25+FOXP3+ phenotypic emblem has yet established. The involvement of TNFR-relatedcells in colon cancer ) that inhibit CD417 + proliferation and Th1 biology6 The predominance of suppressor CD8+CD28 CD56+ cytokinelike receptor-8 production (TLR8)18 Our signaling research reported the occurrence of cells,diseases, CD8 + inflammatoryCD11c+ cells diseasesand CD8 as+CD25 well+ Foxp3 as in + transplantation Tregs −in RA CD8+CD25+FOXP3+ Treg cells in breast tumor microenvironment . , CD8+CD122+ and CD8+LAP+ . +/PD-119+/ 7-9 - in nature These CD8+ . (rheumatoid arthritis), CD8+CD28 T cells in Myasthenia( latency- gravis inFurther the tumor study microenvironment identified19 that thisto suppress subset the is CTLA4effector T cells associated-peptide)9,10 Treg in EAE (Experimental-− autoimmune- CD127 . Treg cells secrete IL10 and TGFβ encephalomyelitis)

Figure: The transcription of FOXP3 in nTreg and tumor-Treg: an integrative model. Expression of the FOXP3 gene is controlled by promoter and several regulatory regions. Several transcription factors viz; RUNX3, NFAT, AP1, STAT5 associated with FOXP3-promoter activity. Three conserved non-coding sequences (CNS1, CNS2 and CNS3) have been identified. NFAT, AP1 and SMAD3 bind to CNS1, which are required for chromatin modification and FOXP3 induction during tumor-Treg cell differentiation. CNS2 region associated with FOXP3 maintainence by GATA3, RUNX1–CBFβ complex and FOXP3. CNS3 region activity asgined to nTreg cells by binding of NFκB and c-Rel.

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+ cells Treg showed synergistic along with CD4+ Tregproliferation cells which and functions.further emphasize Moreover, their it was importance observed thatin tumor CD8 , whereas CNS3 activity is profound in thymus-derived nTreg immune evasion cells. Upon TCR stimulation NFκB and c-Rel bind to the CNS3- 19 maintenanceresponsive elements of FOXP3 and expression induce in FOXP3 both nTreg transcription. and iTreg Oncells the28 FOXP3: the ace. controller of Treg cell other hand, the CNS2 region is exclusively associated with the . The FOXP3 gene is found on the X and mutation with tumor-induced CD8+ as well as CD4+ or loss of function of FOXP3 leads to severe autoimmune or Research showed that CNS1 region is profoundly associated iTreg generation. master regulator of Treg cells and the stable FOXP3 expression The CNS1 region is extended from −372 bp upstream of the isinflammatory critical for diseases.proper differentiation Research established and immunosuppressive that FOXP3 is the transcription start site to +1746 bp of the human FOXP3 gene function of Treg cells expressionlocus. It contains in iTreg cells TGFβ responsive element-SMAD3 (at +1544 20 bpis a region) polypeptide which growth is essential19, factor 25 forwhich peripheral regulates induction divergent of cellular FOXP3 FOXP3 acts as a transcription. regulator and switches on a complex transcriptional network that ensures the Treg processes like proliferation,. Transforming differentiation, growth and factor-β apoptosis (TGF-β) 29 and regulates the insensitivity to antigrowth signals or evasion. and functions. In one hand, FOXP321 On acts the as other a transcriptional hand, it acts TGF-β applies a multifaceted impact on malignancy pathogenesis activator to induce the expression of CD25 (IL2-Rα) and CTLA4 (negative Co-stimulatory molecule) . of apoptosis. It induces the tumor angiogenesis, invasion, as a repressor to inhibit IL17 transcription by binding to21, RORg 22 activationand metastasis. of SMAD3 TGF-β by isphosphorylation abundantly expressed at the C-terminal in most serine of the or FOXP3 competes with AP-1 for cooperative binding with NFAT residueshuman tumors and binds to TGFβ-R1 which further triggers the andFOXP3 binds promoteron the IL2 promoter is tightly to repressregulated IL2 transcriptionby a number .of studied3130, transcription factors . The functions of SMAD3 in promoting EMT is well and causeshowever, phosphorylation it has a profound of SMAD3 role which in immune further system translocate also. The human FOXP3 promoter is stretched from -6221 bp toIt isnucleus observed that tumor-secreted TGFβ acts on responder T cells region of FOXP33, 19. gene and causes permissive histone acetylation 23 The eukaryotic promoter contains several consensus SMAD3 binds to the responsive element in CNS1+ upstreamsequences ofviz; the TATAAA TSS but sequence is interrupted known by as a TATA 6000-bp box, located at 5’ UTR . Treg cells in H4 that19 leads to the induction of FOXP3 transcription in CD8 box located at 138bp upstream of the TSS and CAAT box contains . Thus, in TGFβ-induced Treg cells SMAD3 plays an at -30 to -25 bp upstream of the TSS, GGGCGG sequence i.e. GC Bi-functionalindispensable role role in FOXP3 of induction.GATA3 in transcription of Likewise in the human FOXP3 genome, TATA box is situated GGCCAATCT consensus sequence (218bp upstream of the TSS). FOXP3 23 atnTreg -211/ cells, 176 TCR-mediated bp whereas signaling GC box is plays at -307/ critical 176 roles bp andin FOXP3 have specific transcription factors binding motif . In thymus-derived of biologicalGATA3 is processes a member viz; of development, a GATA-binding differentiation, protein family and transcription factors that are induced following TCR engagement tumorigenesisthat interacts 32, with 33 GAATA motif. GATA3 regulates a variety andtranscription ensure the initiation. T cell activationNFκB, NFAT,23-25 andFOXP3 AP1 arepromoter well-recognized contains . Moreover, GATA-334 is critically involved the trans-activation of FOXP323 . infunction immune is also regulation. constitutively GATA-3 expressed is known in CD8 as + the T cells ‘‘master also threein the NFAT putative and cis-elemenAP1 binding in sites the which FOXP3 play promoterdefinitive roles region in regulator’’and very crucial for Th2 to cellmaintain differentiation their functions. However,35 the GATA3 and induce FOXP3 transcription. NFκB in also nTreg has potentialcells24 Moreover, binding that . It is reported36 positive trans-activator of FOXP326 . Th2 cell polarizing condition antagonizes TGFβ-induced IL2 activated STAT5 binds to the FOXP3 promoter and acts as a Foxp3 expression and promotes the Th9 cell differentiation . . Human FOXP3 promoter also In silico analysis showed that the CNS1 region in FOXP3 gene19 playscontains a decisive three conserved role in the putative differentiation RUNX3 of responsive CD8+ T cells elements from responsivecontains a conserved element and GATA3-binding represses the site FOXP3 at +1448 transcription bp region in. CD4/CD8-double(at −720 bp, −670 positive bp and −441progenitor bp upstream cells of the TSS). RUNX3 tumor-CD8On contrary+ to SMAD3, GATA3 binds on the specific CNS1- induced CD8+ Treg cells this runt domain27 , roles in tumor-induced FOXP3 induction in CD8+ Treg cells via . However, in tumor- Treg cells. Thus, SMAD3 and GATA3 play opposing at the putative site in the promoter region and causes permissive RUNX3, acts as positive trans-activator of FOXP3 by binding regulatingThe theme the CNS1-genomicof Treg cell stability . and plasticity has been 19 + + FOXP3chromatin inductionmodification and. maintenance is regulated by FOXP3 CD4 T cells may lose FOXP3 expression and acquire debatable for a long time. Scientists have found that some conserved non-coding sequences effector Th cell function depending on their microenvironment Our research shows that tumor-induced CD8+ Treg cells are with37 Apart from the promoter region, alignment of the FOXP3 stable FOXP3 expression over time which certainly indicates its. gene from different species has revealed the presence of a few gene locus is known to be very crucial for sustaining a stable FOXP3distinctive expression transcriptional in Treg cells or 28 epigenetic regulation. The CNS2 monitored conserved non-coding regions viz; CNS1, CNS2, and CNS3. These conserved non-coding DNA sequence (CNS) elements FOXP3 via auto-regulation38 . In association with Cbfβ–Runx1, encode information that defines the differentiation and stability FOXP3 binds to CNS2 and helps to maintain stable expression of of the Treg cells. The CNS1 region is exclusively associated with . However, the recruitment of FOXP3– TGFβ-induced FOXP3 induction in peripherally induced Treg Runx1–Cbfβ complexes to CNS2 occurs after and is dependent

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38 67 12. Correale J, Villa A. Role of CD8+ CD25+ Foxp3+ regulatory T cells in criticalon demethylation role in Th2 ofcell the differentiation CpG island located at this region . multiple sclerosis. Ann Neurol. 2010; : 625-638. GATA3 induces DNA demethylation in 39Th2 gene locus and play a + . Apart from CNS1, CNS219 13. 189Beres AJ. CD8+ Foxp3+ regulatory T cells are induced during graft- regionresponsive also sitecontains and helps a GATA3-responsive to maintain stable element FOXP3 at expression +3488 bp . versus-host disease and mitigate disease severity. J Immunol. 2012; In tumor-induced CD8 Treg cells, GATA3 binds to the CNS2-19 : 464-474. that mediates suppression through the chemokine CC chemokine . 14. Joosten SA. Identification of a human CD8+104 subset Thus, it seems probable that GATA3 helps in maintenance of an active state of the FOXP3 via inducing DNA-demethylation ligand 4. Proc Natl Acad Sci U S A. 2007; : 8029-8034. in CNS2 and further helps in the FOXP3–Runx1–Cbfβ mediated 15. Zhang 12S. Analysis of CD8+ Treg cells in patients with ovarian cancer: auto-regulationConclusion of FOXP3. a possible mechanism for immune impairment. Cell Mol Immunol. As illustrated in this review, Treg cells are a heterogeneous 2015; : 580-591. population and their stability and plasticity depend on their 16. Chen C. Changes of CD4+CD25+FOXP3+18 and CD8+CD28- regulatory T cells in non-small cell lung cancer patients undergoing surgery. Int CD4+ and CD8+ Treg cells have overlapping but ndependen Immunopharmacol. 2014; : 255-261. microenvironment. It has become clearer with the time that 13 17. Kiniwa Y. CD8+ Foxp3+ regulatory T cells mediate immunosuppression + + between CD4 Tregs and CD8 Tregs might play critical roles in in prostate cancer. Clin Cancer Res. 2007; : 6947-6958. tumordifferentiation immune mechanismsevasion which in certainly different indicates . the A necessity crosstalk 58 18. Chaput N. Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue. Gut. 2009; : 520-529. accumulation of acquaintance on the molecular mechanism of Tregto acquire cell generation knowledge and on their both immunosuppressive the Treg cells subsets. functions, With thewe 19. Chakraborty S. Transcriptional regulation7 of FOXP3 requires expect that in the near future, the hurdles of successful cancer integrated activation of both promoter and CNS regions in tumor- induced CD8(+) Treg cells. Sci Rep. 2017; : 1628. 4 20. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development immunotherapyAcknowledgement could be achieved. and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003; : 330-336. 241 grant commission and Department of Science and Technology, 21. Rudensky AY. 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