SEARCHING FOR IMMUNE TOLERANCE MANIPULATING NEW MOLECULES AND EXPLOITING NEW CONCEPTS ON LYMPHOCYTE BIOLOGY EDITED BY : Karina Pino-Lagos, Sergio Quezada, Diego Francisco Catalán and Juan C. Aguillon PUBLISHED IN : Frontiers in Immunology 1 Frontiers in Immunology July 2016 | Searching for Immune Tolerance Manipulating New Molecules Frontiers Copyright Statement © Copyright 2007-2016 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. 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With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: researchtopics@frontiersin.org SEARCHING FOR IMMUNE TOLERANCE MANIPULATING NEW MOLECULES AND EXPLOITING NEW CONCEPTS ON LYMPHOCYTE BIOLOGY Topic Editors: Karina Pino-Lagos, Universidad de los Andes, Chile Sergio Quezada, University College London, UK Diego Francisco Catalán, Universidad de Chile, Chile Juan C. Aguillon, Universidad de Chile, Chile The break on immune tolerance is a common point between autoimmune diseases and the uncontrolled effector immune responses against allo-antigens in transplantation. Among the past years, several approaches to restore a suppressive immune state have included the targeting of co-stimulatory/inhibitory molecules on immune cells, the promotion or blockade of pivotal cytokines, and the extensive study on how to isolate and expand suppressive cells with the purpose to re-infuse them in patients. To date, the availability of new technologies has permitted to learn, in a more detailed way, the immune mechanisms carried out by suppressive lymphocytes, together with the identification of new potential candidates to target in our quest for immune tolerance. For example, the attractive concepts of lymphocyte plasticity and function stability, supported by the finding of new transcription factors, have opened a new window in the understanding of T cell differentiation, effector cell commitment and immune regulatory function. On the other hand, the discovery of new members of the Ig superfamily ligand, VISTA; the intriguing role of modulatory molecules like Retinoic Acid, Neuropilin-1, Fc gamma receptors, or cytokines such as IL-33, among others, are revealing new possibilities in the development of new strategies to conquer our obsession: immune tolerance. Here, we gather the latest information regarding new targets and cellular processes, including an update on current cellular therapies and the exciting coming approaches to cure autoimmunity and permit transplant acceptance. Citation: Pino-Lagos, K., Quezada, S., Catalán, D. F., Aguillon, J. C., eds. (2016). Searching for Immune Tolerance Manipulating New Molecules and Exploiting New Concepts on Lymphocyte Biology. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-951-8 2 Frontiers in Immunology July 2016 | Searching for Immune Tolerance Manipulating New Molecules 05 Editorial: Searching for Immune Tolerance Manipulating New Molecules and Exploiting New Concepts on Lymphocyte Biology Diego Catalán and Karina Pino-Lagos 07 Novel immune check-point regulators in tolerance maintenance Yanxia Guo and Adele Y. Wang 16 Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators Isabelle Le Mercier, J. Louise Lines and Randolph J. Noelle 31 Alarmin’ immunologists: IL-33 as a putative target for modulating T cell-dependent responses Tania Gajardo Carrasco, Rodrigo A. Morales, Francisco Pérez, Claudia Terraza, Luz Yáñez, Mauricio Campos-Mora and Karina Pino-Lagos 39 Role of dendritic cells in the induction of lymphocyte tolerance Fabiola Osorio, Camila Fuentes, Mercedes N. López, Flavio Salazar-Onfray and Fermín E. González 50 Therapeutic potential of hyporesponsive CD4 + T cells in autoimmunity Jaxaira Maggi, Carolina Schafer, Gabriela Ubilla-Olguín, Diego Catalán, Katina Schinnerling and Juan C. Aguillón 57 Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance Paulina Ruiz, Paula Maldonado, Yessia Hidalgo, Daniela Sauma, Mario Rosemblatt and Maria Rosa Bono 66 Regulatory T cells: serious contenders in the promise for immunological tolerance in transplantation Niloufar Safinia, Cristiano Scotta, Trishan Vaikunthanathan, Robert I. Lechler and Giovanna Lombardi 82 Clinical Outlook for Type-1 and FOXP3 + T Regulatory Cell-Based Therapy Silvia Gregori, Laura Passerini and Maria-Grazia Roncarolo 90 Opposing roles of interferon-gamma on cells of the central nervous system in autoimmune neuroinflammation Payton A. Ottum, Gabriel Arellano, Lilian I. Reyes, Mirentxu Iruretagoyena and Rodrigo Naves 98 Stage-specific role of interferon-gamma in experimental autoimmune encephalomyelitis and multiple sclerosis Gabriel Arellano, Payton A. Ottum, Lilian I. Reyes, Paula I. Burgos and Rodrigo Naves 107 Immune response modulation by vitamin D: role in systemic lupus erythematosus Mirentxu Iruretagoyena, Daniela Hirigoyen, Rodrigo Naves and Paula Isabel Burgos Table of Contents 3 Frontiers in Immunology July 2016 | Searching for Immune Tolerance Manipulating New Molecules 114 Systemic sclerosis patients present alterations in the expression of molecules involved in B-cell regulation Lilian Soto, Ashley Ferrier, Octavio Aravena, Elianet Fonseca, Jorge Berendsen, Andrea Biere, Daniel Bueno, Verónica Ramos, Juan Carlos Aguillón and Diego Catalán 123 Breast milk and solid food shaping intestinal immunity Sara M. Parigi, Maria Eldh, Pia Larssen, Susanne Gabrielsson and Eduardo J. Villablanca 4 Frontiers in Immunology July 2016 | Searching for Immune Tolerance Manipulating New Molecules February 2016 | Volume 7 | Article 27 5 Editorial published: 01 February 2016 doi: 10.3389/fimmu.2016.00027 Frontiers in Immunology | www.frontiersin.org Edited and Reviewed by: Luis Graca, University of Lisbon, Portugal *Correspondence: Karina Pino-Lagos karina.p.lagos@gmail.com Specialty section: This article was submitted to Immunological Tolerance, a section of the journal Frontiers in Immunology Received: 06 January 2016 Accepted: 18 January 2016 Published: 01 February 2016 Citation: Catalán D and Pino-Lagos K (2016) Editorial: Searching for Immune Tolerance Manipulating New Molecules and Exploiting New Concepts on Lymphocyte Biology. Front. Immunol. 7:27. doi: 10.3389/fimmu.2016.00027 Editorial: Searching for immune tolerance Manipulating New Molecules and Exploiting New Concepts on lymphocyte Biology Diego Catalán 1 and Karina Pino-Lagos 2 * 1 Programa Disciplinario de Inmunología, Facultad de Medicina, Instituto de Ciencias Biomédicas, Universidad de Chile e Instituto Milenio en Inmunología e Inmunoterapia, Santiago, Chile, 2 Facultad de Medicina, Centro de Investigación Biomédica, Universidad de los Andes, Santiago, Chile Keywords: immune tolerance, regulatory t cells, B cell tolerance, dendritic cells, transplantation, autoimmunity, costimulatory molecules, cytokines The Editorial on the Research Topic Searching for Immune Tolerance Manipulating New Molecules and Exploiting New Concepts on Lymphocyte Biology This research topic was inspired in those still non-curable inflammatory conditions, such as autoim- mune diseases and transplant rejection, based on the fact that immunologists worldwide are still searching for strategies to restore long-term immune tolerance. Thus, we gathered several research- ers whom actively base their investigation lines in novel molecules and immune cell populations that can be exploited to design new strategies for the establishment or recovery of tolerance. In the context of autoimmunity, intriguing is the role of interferon (IFN)- γ in the pathogenesis of multiple sclerosis (MS) and its animal model, the experimental autoimmune encephalomyelitis, which is reviewed in two articles contained in this edition (Ottum et al.; Arellano et al.). They focus on new evidence that help to explain the seemingly opposing effects of this cytokine over different central nervous system cells and on different stages of the disease, giving some important clues that can help to guide the potential therapeutic use of IFN- γ in MS patients. Other cytokine that recently has been focus of interest is IL-33, a molecule first described as an alarmin, but Gajardo-Carrasco et al. detail the plethora of now recognized functions in which IL-33 is involved, with special atten- tion in T cell biology, adaptive immunity, tolerance, and immunological disorders. Continuing with an update on molecules with pivotal immune function, Le Mercier et al. and Guo and Wang deliver us a solid snap shot on receptors and ligands with stimulatory and inhibitory immune activity, revising both classic and newest members, their contribution to disease and how they have been currently targeted to utilize them for therapeutic purposes. Special attention received the new Ig family member VISTA, which is presented as an interesting modulator of the immune response and with high potential for its exploitation in the clinic. Similarly, the article by Iruretagoyena et al. addresses the immune regulatory aspects of vitamin D and its importance in controlling the development of autoimmune diseases. This review has a particular emphasis on the participation of this vitamin in the physiopathology of systemic lupus erythematosus (SLE) and gives an update on the latest data about vitamin D supplementation in SLE patients. Regarding the use of immune cells with therapeutic purposes, this research topic contains five reviews that put the spotlight over the use of dendritic cells (DCs) and regulatory T cells (Tregs) as tools to treat immune-related conditions (including autoimmunity and transplant rejection). The February 2016 | Volume 7 | Article 27 6 Catalán and Pino-Lagos New Insights in Immune T olerance Frontiers in Immunology | www.frontiersin.org article by Schinnerling et al. summarizes the recent advances in the description of intracellular pathways and transcriptional regulators that command the monocyte-derived tolerogenic human DCs differentiation program and propose candidate molecules that could be regarded as key in their tolerogenic func- tions. On the other hand, Maggi et al. examine one of the putative mechanisms of action of tolerogenic DCs; this is the induction of hyporesponsive or anergic CD4 + T cells. The authors review recent findings in the impact of CD4 + T cells anergy induction in animal models of autoimmune diseases development and progression, and discuss on the potential benefits of exploiting this mechanism for therapeutic purposes in humans. Similarly, Osorio et al. present a complete revision on DCs nature, from their origin, lineages, differentiation process, subtypes, and physi- ological role, linking these observations with diseases and men- tioning current technological approaches to use them as a source for cellular therapy. On the other hand, Safinia et al. and Gregori et al. targeted human Tregs, describing extensively all their phe- notypic characteristics, the different subpopulations identified to date based on certain surface markers and their mechanisms to drive immune suppression, and compiling simultaneously all the results from finished and ongoing clinical trials. In addition, both works discuss different aspects of human Tregs clinical grade manufacture and the variables that need to be improved to perfect the protocol, such as viability, antigen-specificity, cell expansion efficiency, and phenotypic/functional stability. In parallel, the original article by Ruiz et al. proposes a modi- fied protocol to favor mixed chimerism and further transplant acceptance in a preclinical model. Their novelty bases in the use of antigen-specific Tregs generated in vitro in the presence of IL-2, TGF- β , and retinoic acid (RA), in conjunction with previ- ously established procedures as non-myeloablative irradiation and administration of immunosuppressant drugs. This group observed that the transfer of RA-Tregs facilitates donor-cells engraftment and allows for the acceptance of skin allografts, proposing the inclusion of Tregs as co-therapeutic tool. Another article reports how Soto et al. pinpoint at another cell population, frequently overlooked when it comes to tolerance mechanisms: B cells. Using systemic sclerosis (SSc) as a paradigmatic autoim- mune disease with cellular and humoral components, the authors describe alterations in the expression levels of activator and inhibitor receptors on B cells from SSc patients that could con- tribute with the hyperactivated phenotype of these cells. They also demonstrate that IL-10-producing B cells and IL-10 secretion by stimulated B cells are reduced in SSc patients, which can imply that these patients have an impaired anti-inflammatory function on regulatory B cells, a subset specifically dedicated to promote tolerance to innocuous antigens. The restoration of the capacity of these cells to express adequate levels of protolerogenic molecules and regain their regulatory capability through novel or current B cell-targeted therapies could be a promising therapy for SSc or related autoimmune diseases. Finally, the review by Parigi et al. brings us to a different face of immune tolerance, the one that keep us from mounting exacerbated immune responses against food antigens and com- mensal microbiota. Disruptions of the tolerogenic mechanisms displayed by a normal intestinal immune system can lead to severe conditions, such as food allergy or inflammatory bowel diseases. This review deals with the way how diet, breast milk, and solid food shape the immune system of newborns and defines the homeostasis in the intestinal microenvironment, thus conferring risk or protection for the future development of immune medi- ated diseases. Overall, we achieved putting together a nice compilation on the current molecules and cell populations that are being aggres- sively targeted to restore immune tolerance in diseased patients. While many efforts are put in translational immunology, basic science immunologists continue working to satisfy these goals. aUtHor CoNtriBUtioNS DC wrote the editorial for this research topic. KP-L coordinated this research topic and wrote the editorial. FUNdiNG DC was funded by FONDECYT Grants 1121100 and 11121497, and Millennium Institute on Immunology and Immunotherapy P09-016-F. KP-L was funded by FONDECYT Grant 11121309 and PMI/UAN1301. Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2016 Catalán and Pino-Lagos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic prac- tice. No use, distribution or reproduction is permitted which does not comply with these terms. REVIEW published: 18 August 2015 doi: 10.3389/fimmu.2015.00421 Edited by: Sergio Quezada, University College London Cancer Institute, UK Reviewed by: Vigo Heissmeyer, German Research Center for Environmental Health, Germany Anne Cooke, University of Cambridge, UK *Correspondence: Yanxia Guo, Merck Research Laboratories, 901 South California Avenue, Palo Alto, CA 94304, USA yanxia.guo@merck.com Specialty section: This article was submitted to Immunological Tolerance, a section of the journal Frontiers in Immunology Received: 01 May 2015 Accepted: 02 August 2015 Published: 18 August 2015 Citation: Guo Y and Wang AY (2015) Novel immune check-point regulators in tolerance maintenance. Front. Immunol. 6:421. doi: 10.3389/fimmu.2015.00421 Novel immune check-point regulators in tolerance maintenance Yanxia Guo* and Adele Y. Wang Merck Research Laboratories, Palo Alto, CA, USA The great success of anti-cytotoxic lymphocyte antigen 4 (CTLA4) and anti-programed cell death protein 1 (PD1) in cancer treatment has encouraged more effort in harnessing the immune response through immunomodulatory molecules in various diseases. The immunoglobulin (Ig) super family comprises the majority of immunomodulatory molecules. Discovery of novel Ig super family members has brought novel insights into the function of different immune cells in tolerance maintenance. In this review, we discuss the function of newly identified B7 family molecules, B7-H4 and V-domain Ig Suppressor of T cell Activation (VISTA), and the butyrophilin/butyrophilin-like family members. We discuss the current stages of immunomodulatory molecules in clinical trials of organ transplantation. The potential of engaging the novel Ig superfamily members in tolerance maintenance is also discussed. We conclude with the challenges remaining to manipulate these molecules in the immune response. Keywords: B7, butyrophilin, immune tolerance Introduction The immune system consists of various cell types to mount both innate and adaptive immunity against pathogens. As an important component of adaptive immunity, T cell activation requires three signals. Signal 1 is acquired through the interaction between T cell receptor (TCR) and peptide-loaded major histocompatibility complex (MHC) molecules. Signal 2 is induced through the interaction between co-stimulatory/co-inhibitory molecules on antigen-presenting cells (APC) and their receptors on T cells, thus enhancing/inhibiting T cell activation. Signal 3 is mediated by cytokines, such as IL2 to promote T cell proliferation. Co-stimulatory/co-inhibitory molecules are the most well-studied molecules and fall into immunoglobulin (Ig) super family members, including B7 family and tumor necrosis factor (TNF) family proteins. These molecules play essential roles in maintaining the balance between sufficient immune response against pathogenic antigens (Ag) as well as limited response against self-antigens or allogenic transplant graft. We identify this family of molecules as “immune modulatory molecules.” The potential receptors and ligands for many novel molecules remain unknown and may serve as both receptors and ligands in different cells. In this review, we define the potential receptors/ligands of these novel molecules as “binding partners” for simplicity. Over the past decade, a large number of B7 and TNF family molecules have been discovered and actively evaluated in clinic. The clinical success of anti-CTLA4 (1, 2) and anti-PD1 blocking antibody (Ab) (3, 4) in various cancers encourages more active research and drug development effort. This effort aims to enhance co-stimulatory or dampening co-inhibitory molecules effect to achieve robust anti-tumor immune response. In parallel, studies are also carried out to dampen the immune response by enhancing the effect of co-inhibitory molecules in treatment of autoimmune or inflammatory diseases (5–7). In addition, organ transplantation is still a great challenge in Frontiers in Immunology | www.frontiersin.org August 2015 | Volume 6 | Article 421 7 Guo and Wang Immune check-point regulators the clinic as the allogenic transplant rejection is a common prob- lem in patients. There is great need for inhibiting the host rejection of donor organs (8). Undoubtedly, the discovery of new immune modulatory molecules has offered new opportunities to manip- ulate the host immune system for tolerance maintenance and graft acceptance. In this article, we review recent discoveries of novel Ig B7 family members VISTA and B7-H4. We also review the butyrophilin (BTN) family members, namely “old molecules with novel immunomodulatory functions.” Learning from the on- going clinical trials on well-described molecules, we discuss the potential application of these new molecules. B7 Family B7 family proteins are among the best-described immunomodu- latory molecules so far. This family consists of both co-inhibitory and co-stimulatory molecules. This family includes B7.1 (CD80), B7.2 (CD86), CD28, CTLA4, PD1, PD1 ligand (PDL1), PDL2, B7RP1 (ICOSL), B7-H3, B7-H4, and VISTA. The growing list of B7 family members offers great opportunities to modulate immune response therapeutically in treating different diseases. This family of molecules was reviewed elsewhere thoroughly (9, 10). In the following section, we will discuss the progress in understanding two new molecules in the family, B7-H4 and VISTA. B7-H4 B7-H4 (B7x, B7S1, VTCN1), identified in 2003 (11), is a highly evolutionarily conserved molecule that shares 87% amino acid identity between human and mouse. Mature B7-H4 is a 50–80 kDa transmembrane protein with one IgV domain and one IgC domain. The mRNA expression of B7-H4 is found on both lymphoid and non-lymphoid tissues. However, B7-H4 protein expression is more restricted and not expressed on ex vivo human immune cells (T, B, DC, and monocytes), but can be induced after in vitro stimulation. Endocrine cells in pancreas have also been shown to express a moderate level of B7-H4. Signaling through B7-H4 pathway leads to the inhibition of TCR- mediated CD4 + and CD8 + T cell proliferation, cell-cycle progres- sion, and IL-2 production (11–13). In experimental autoimmune encephalomyelitis (EAE) model, blocking B7-H4 on host cells by a monoclonal antibody accelerates T cell responses and enhances disease severity (11). However, B7-H4-deficient mice only mount mildly increased Th1 response and intact cytotoxic T-lymphocyte reactions against viral infections (14). The study indicated that B7-H4 is a negative regulator of T cell activation but only plays a minor role in fine-tuning T cell immunity. However, elevated soluble form of B7-H4 in the serum of rheumatoid arthritis (RA) patients is associated with increased disease severity. In a collagen-induced arthritis mouse model, both overexpression of soluble B7-H4 and genetic deletion of B7-H4 enhanced disease severity, due to enhanced B cell, T cell, and neutrophil response (15). This severity suggests that soluble B7-H4 acts as a decoy molecule to block the inhibitory function of B7-H4. Further con- firmation of the correlation between soluble B7-H4 and disease severity in RA patients may indicate a new target pathway in autoimmune diseases. Although soluble B7-H4-immunoglobulin (B7-H4-Ig) fusion protein binds to activated T cells, the binding partner for B7-H4 has not been identified and does not bind to known CD28 family members, such as CD28, CTLA4, ICOS, and PD1. On the other hand, overexpression of B7-H4 on the pancre- atic islets can protect mice from T cell-mediated autoimmunity and these islet allograft increased survival after transplantation (16). The immunosuppressive function of B7-H4 has also been investigated in experimental type I diabetes models using B7- H4-Ig. Pre-diabetic non-obese diabetes mice treated with B7-H4- Ig displayed lower incidence of diabetes by suppressing immune infiltrates into the islets (17). Lee et al. found that B7-H4-Ig treatment decreased Th17 cell whereas promoting CD4 + IFN γ + T cells development (18). These findings suggest a possible role of B7-H4 in maintaining the balance between Th1 and Th17 cells. However, more studies are warranted to verify this observation and elucidate the molecular mechanism. The impact of B7-H4 on T cells is summarized in Figure 1 FIGURE 1 | B7-H4 is a co-inhibitory molecule on APC (blue) and interacts with its unknown receptor (receptor X) on T cells (green) to deliver negative signaling during T cell activation . Left: in the presence of B7-H4-receptor X interaction, T cells proliferate at a lower rate and produce lower amount of IL2 (purple), lower IFN γ (black), and higher IL17 (red). Middle: antagonistic anti-B7-H4 (magenta triangle) blocks the interaction between B7-H4 and receptor X. T cells proliferate at a higher rate and produce more IL2. Right: B7-H4 overexpression leads to lower T cell proliferation, lower IL2 and IL17 production, and higher IFN γ production. Frontiers in Immunology | www.frontiersin.org August 2015 | Volume 6 | Article 421 8 Guo and Wang Immune check-point regulators In addition to the direct impact of B7-H4 inhibition on T and B cell immune response, B7-H4 also acts as a negative regulator for neutrophil response. B7-H4-deficient mice are more resistant to infection by Listeria monocytogenes due to augmented neutrophil accumulation (19). Within 3 days after infection, bacterial colonies in liver and spleen are significantly lower in B7-H4-deficient mice than controls, concomitant with increased number of neutrophils in the spleen of infected mice. B7-H4 inhibits the growth of bone marrow-derived neutrophil progenitors in vitro , demon- strating an inhibitory function of B7-H4 in neutrophil expansion. Enhanced neutrophil growth in B7-H4-deficient mice also occurs in the background of recombination activation gene (RAG) knock out mice, independent of adaptive immunity. The inhibition of neutrophil expansion shows that B7-H4 directly regulates innate immunity. Furthermore, Pawar et al. discovered B7-H4 expres- sion on kidney resident cells and could be induced further with inflammatory stimuli. In an antibody-mediated nephritis model, B7-H4-deficient mice showed more T, B, macrophage, and neu- trophil infiltration into kidney. In addition, macrophages in B7- H4-deficient mice demonstrate a more inflammatory phenotype. Whether B7-H4 modulates other immune cells remains unknown. V Domain-Containing Ig Suppressor of T-Cell Activation V domain-containing Ig suppressor of T-cell activation (VISTA), also named as PD1 homolog (PD1H), is expressed in both hematopoietic and non-hematopoietic tissues. Among immune cells, VISTA is highly expressed on mature CD11b high myeloid- derived APCs and to a less extent on CD4 + , CD8 + , and Tregs (20). VISTA is a 55- to 65-kDa type I Ig membrane protein with the extracellular domain homologous to PD-L1. In vitro , VISTA-Ig inhibits T cell activation, proliferation, and production of cytokines, such as IL2 and IFN γ , during anti-CD3 activation. VISTA overexpression on APC also suppressed T cell prolifera- tion, which was rescued by neutralizing anti-VISTA monoclonal Ab (13F3). More importantly, VISTA overexpression on tumor cells overcame the protective anti-tumor immunity. These studies collectively suggest that VISTA on APC delivers negative signaling to T cells during activation via a yet-to-identify binding partner on T cells. The same Ab administration in vivo exacerbated disease progression in a passive EAE model. Further studies demonstrated that mice deficient in VISTA display normal hematopoietic devel- opment. These mice do not succumb to obvious organ-specific autoimmune diseases, despite chronic inflammation in multiple organs over age. Consistently, spontaneously activated T cells accumulate and produce inflammatory cytokines and chemokines in aged VISTA-deficient mice (21). 2D2 T cell receptor mice overexpress T cell receptor that recognizes myelin oligodendro- cyte glycoprotein (MOG 35–55 ). VISTA deletion in 2D2 transgenic mice significantly enhanced EAE severity. Disease progression is due to an increase in activated encephalitogenic T cells in the periphery and greater infiltration into the central nervous system. The analysis of both VISTA-Ig and genetic ablation demonstrated that VISTA is a negative check-point regulator of T cell activation. Blocking VISTA’s function promotes the pro- inflammatory cytokines production and enhances autoimmune diseases development under susceptible conditions. However, another anti-VISTA Ab seemed to inhibit the progression of acute graft-versus-host-diseases (GVHD). This inhibition indicates the agonistic effect of this Ab in the regard of suppressing T cell activation (22). Even though the binding partner of VISTA remains unknown, Flies et al. demonstrated that VISTA is a co-inhibitory receptor on CD4 + T cells. VISTA − / − CD4 + T cells showed stronger Ag-specific proliferation and cytokine production than wild-type CD4 + T cells. In an APC-T co-culture study, VISTA deficiency on both APC and T cells resulted in the strongest proliferation response, whereas VISTA-sufficient APC and T cells generated the poorest response. VISTA deficiency on either APC or T cells yielded an intermediate response. Taken this data together with previous studies (20), VISTA negatively regulates T cell immunity via direct impact on T cells by engaging different receptor/ligand (illustrated in Figure 2 ). In a murine model of acute hepatitis, a different agonistic VISTA Ab administration suppressed CD4 + T cell and NK cell-mediated acute inflammation. The data suggested that VISTA − / − mice showed survival benefits during GL261 glioma growth, mediated by enhanced CD4 + T cell anti-tumor immunity (22). The bidirectional inhibition of VISTA on APC and T cells suggests that VISTA is a valuable target for immunological diseases. The studies in mice are reinforced by characterization of human VISTA expression and function (23). More studies are needed to characterize the correlation between VISTA expression and autoimmune diseases, such as RA, to establish the rationale for targeting VISTA in clinic. Moreover, it is worthwhile evaluat- ing the potential of agonistic anti-VISTA Ab in tolerance induc- tion during organ transplantation given the preclinical evidences in GVHD. Furthermore, the identification of VISTA’s binding partner will certainly offer great insight into the molecular mech- anism of VISTA signaling and ensure better understanding of agonistic Ab safety in development. Butyrophilin Family in Immune Tolerance As B7 family member proteins, BTN family members have extra- cellular IgV and IgC domain, but most BTN family members have an intracellular B30.2 domain. Therefore, BTN family members belong to the Ig superfamily. Since the discovery of BTN family FIGURE 2 | VISTA is a co-inhibitory molecule on both APC (blue) and T cells (green) . VISTA is expressed on APC and interacts with the partner (receptor X) on T cells. VISTA can also be a co-inhibitory receptor on T cells and binds to its unknown ligand (ligand Y) on APC. Both interactions deliver negative signaling to T cell activation. Frontiers in Immunology | www.frontiersin.org August 2015 | Volume 6 | Article 421 9 Guo and Wang Immune check-point regulators members in the regulation of immune response, butyrophilin- like (BTNL), family members have also been identified in both human and mouse genome. The extracellular structural similarity between BTN/BTNL and B7 family suggested that BTN/BTNL family molecules may have immune regulatory functions as B7 family members. In this section, we discuss the genomic distribu- tion, function, and clinical evidence between BTN/BTNL family proteins and immune tolerance. Genomic Distribution of BTNs The first BTN member was discovered in milk in which BTN sub- family 1 member A1 (BTN1A1) is essential for the secretion, lacta- tion, and stabilization of milk-fat globules (24–26). Further studies discovered more BTN proteins with immune-regulatory func- tions. Thus far, 13 human BTNs and BTNLs have been described. Human BTNs include BTN1A1, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, and BTN3A3 (27). Human BTNLs include BTNL2, BTNL3, BTNL8, BTNL9, BTNL10, and SKINT-like (SKINTL). In mice, 11 genes have been identified to encode BTN and BTNL molecules. These include BTN1A1, BTN2A2, BTNL1, BTNL2, BTNL4, BTNL5, BTNL6, BTNL7, BTNL9, BTNL10, and SKINT. The majority of these proteins are diverse between human and mice, making it difficult to study their function. As mentioned above, the BTN family members have extracellular IgV and IgC domains as B7 family proteins (28–31). Extracellular IgV and IgC domains are essential for the interaction among B7 family members. However, it remains unknown whether BTN/BTNL family proteins bind to their counter-receptors via the IgV and IgC domain. In addition, it remains unknown how intracellular B30.2 domain binds to other intracellular signaling proteins to deliver the function of BTNs and BTNLs. Regarding the expression pat- tern of BTN/BTNL family molecules, there is some inconsis- tency among existing data when comparing mRNA with protein expression. Abeler-Dorner et al. summarized the mRNA and protein expression of each BTN/BTNL family member in another review (27). Function of Butyrophilin in T cell Immunity The wide distributions of certain BTN/BTNLs determine the functions of these molecules in different immune cells. As BTN/BTNLs are expressed on APCs or T cells, studies have been carried out to determine the function of these molecules in the T cell immunity in vitro and in vivo . In this section, we will focus on the function of these “old molecules” in regulation of T cell immunity, in the regard of both conventional αβ T cells and γδ T cells. Butyrophilin in Regulation of Conventional T Cell Response As B7 family members, some BTN/BTNLs are co-inhibitory whereas others are co-stimulatory in the regard of conven- tional T cell function regulation. The function characterization of BTN/BTNL family members depends on the administration of recombinant protein and antagonistic/agonistic Ab in T cell activation, differentiation, and cytokine production in vitro and diseases progression in vivo . In the following section, we will focus on the delineation of co-inhibitory and co-stimulatory function of various BTN/BTNL family members. BTNs as co-inhibitory molecules Nguyen et al. first demonstrated the co-inhibitory role of mouse BTNL2 in T cell immunity (32). Mouse BTNL2-Ig fusion pro- tein inhibited anti-CD3- and anti-CD28-stimulated CD4 + T cell proliferation and IL2 production via reducing activating protein- 1 (AP-1), nuclear factor of activating T cells (NFAT), and NF- κ B activity. Arnett et al. further illustrated the upregulation of BTNL2 in inflamed colons in the mouse Mdr1a-deficient model of inflammatory bowel diseases. BTNL2 conveys its inhibitory signal also by counteracting the co-stimulatory effect of B7RP1 (ICOS ligand) on T cell activation (33). No binding partner has been successfully identified as BTNL2 failed to bind known B7 family proteins, such as CD28, ICOS, PD1, CTLA4, and HVEM. Further studies elucidated the similar co-inhibitory function of murine BTNL1 (34). In vitro studies showed that BTNL1-Ig fusion protein suppressed low-concentration anti-CD3-induced T cell activation and IL2 production by cell-cycle arrest. Like BTNL2, BTNL1 does not bind CD28, CTLA4, triggering recep- tor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2, TREML2), B7-H3, B7-1, or other B7 family members. Antago- nistic anti-BTNL1 Ab-induced strong Th2 response after keyhole limpet hemocyanin (KLH) and complete Freund’s adjuvant (CFA) immunization in vivo , demonstrated by enhanced T cell prolif- eration and higher production of Th2 cytokines (IL-4, IL-5, and IL-13), but lower IFN γ . Antagonistic anti-BTNL1 Ab treatment also exacerbated EAE via promoting Th17 response. Similarly, α -BTNL1 Ab exacerbated Th2-mediated allergy response. These studies indicate that BTNL1 may fine tune Th1 versus Th2 and Th17 development. No potential BTNL1 binding partner has been identified so far. Human BTN3A1, also known as CD277, is highly upregu- lated in myeloid DCs and macrophages by vascular endothe- lial growth factor (VEGF) and CCL3 in human ovarian cancer. BTN3A1 inhibited T cell proliferation and Th1 cytokine pro- duction through preventing the upregulation of cellular caspase- 8 (FLICE)-like inhibitory protein (cFLIP) and the consequently enhanced caspase-8 activity. Caspase-8 activity is necessary to initiate the activation of NF- κ B and promote T cell proliferation (35). A recent study, however, extended the function of BTN3A1 beyond the co-inhibition to presenting phosphorylated antigen (discussed in more details below). Even though the preclinical studies highlighted the importance of these co-inhibitory BTN/BTNLs in in