# STRATEGIES FOR MODULATING T CELL RESPONSES IN AUTOIMMUNITY AND INFECTION

EDITED BY : Maria Florencia Quiroga, María Fernanda Pascutti and Gustavo Javier Martinez PUBLISHED IN : Frontiers in Immunology

#### Frontiers eBook Copyright Statement

The copyright in the text of individual articles in this eBook is the property of their respective authors or their respective institutions or funders. The copyright in graphics and images within each article may be subject to copyright of other parties. In both cases this is subject to a license granted to Frontiers. The compilation of articles constituting this eBook is the property of Frontiers.

Each article within this eBook, and the eBook itself, are published under the most recent version of the Creative Commons CC-BY licence. The version current at the date of publication of this eBook is CC-BY 4.0. If the CC-BY licence is updated, the licence granted by Frontiers is automatically updated to the new version.

When exercising any right under the CC-BY licence, Frontiers must be attributed as the original publisher of the article or eBook, as applicable.

Authors have the responsibility of ensuring that any graphics or other materials which are the property of others may be included in the CC-BY licence, but this should be checked before relying on the CC-BY licence to reproduce those materials. Any copyright notices relating to those materials must be complied with.

Copyright and source acknowledgement notices may not be removed and must be displayed in any copy, derivative work or partial copy which includes the elements in question.

All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. For further information please read Frontiers' Conditions for Website Use and Copyright Statement, and the applicable CC-BY licence.

ISSN 1664-8714 ISBN 978-2-88963-609-9 DOI 10.3389/978-2-88963-609-9

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

#### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. 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<EMAIL_ADDRESS>
# STRATEGIES FOR MODULATING T CELL RESPONSES IN AUTOIMMUNITY AND INFECTION

Topic Editors:

Maria Florencia Quiroga, National Council for Scientific and Technical Research (CONICET), Argentina María Fernanda Pascutti, Sanquin Diagnostic Services, Netherlands Gustavo Javier Martinez, Rosalind Franklin University of Medicine and Science, United States

Citation: Quiroga, M. F., Pascutti, M. F., Martinez, G. J., eds. (2020). Strategies for Modulating T cell Responses in Autoimmunity and Infection. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-609-9

# Table of Contents

*05 Editorial: Strategies for Modulating T Cell Responses in Autoimmunity and Infection*

María Fernanda Pascutti, Gustavo Javier Martinez and Maria Florencia Quiroga

*08 Features of Effective T Cell-Inducing Vaccines Against Chronic Viral Infections*

Eleni Panagioti, Paul Klenerman, Lian N. Lee, Sjoerd H. van der Burg and Ramon Arens

Jimena Tosello Boari, Cintia L. Araujo Furlan, Facundo Fiocca Vernengo, Constanza Rodriguez, María C. Ramello, María C. Amezcua Vesely, Melisa Gorosito Serrán, Nicolás G. Nuñez, Wilfrid Richer, Eliane Piaggio, Carolina L. Montes, Adriana Gruppi and Eva V. Acosta Rodríguez

*91 Alpha2beta1 Integrin (VLA-2) Protects Activated Human Effector T Cells From Methotrexate-Induced Apoptosis* Amna Abderrazak, Mohammed-Amine El Azreq, Dalila Naci, Paul R. Fortin

and Fawzi Aoudjit

Jimena Salido, María Julia Ruiz, César Trifone, María Inés Figueroa, María Paula Caruso, María Magdalena Gherardi, Omar Sued, Horacio Salomón, Natalia Laufer, Yanina Ghiglione and Gabriela Turk

*132 Harnessing the Induction of CD8+ T-Cell Responses Through Metabolic Regulation by Pathogen-Recognition-Receptor Triggering in Antigen Presenting Cells*

Francesco Nicoli, Stéphane Paul and Victor Appay

#### *140 Limited Foxp3+ Regulatory T Cells Response During Acute* Trypanosoma cruzi *Infection is Required to Allow the Emergence of Robust Parasite-Specific CD8+ T Cell Immunity*

Cintia L. Araujo Furlan, Jimena Tosello Boari, Constanza Rodriguez, Fernando P. Canale, Facundo Fiocca Vernengo, Santiago Boccardo, Cristian G. Beccaria, Véronique Adoue, Olivier Joffre, Adriana Gruppi, Carolina L. Montes and Eva V. Acosta Rodriguez

*157 CCL22-Producing Resident Macrophages Enhance T Cell Response in Sjögren's Syndrome*

Aya Ushio, Rieko Arakaki, Kunihiro Otsuka, Akiko Yamada, Takaaki Tsunematsu, Yasusei Kudo, Keiko Aota, Masayuki Azuma and Naozumi Ishimaru

*172 Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione*

Jie Liang, Beate Jahraus, Emre Balta, Jacqueline D. Ziegler, Katrin Hübner, Norbert Blank, Beate Niesler, Guido H. Wabnitz and Yvonne Samstag

Deborah Cluxton, Andreea Petrasca, Barry Moran and Jean M. Fletcher

# Editorial: Strategies for Modulating T Cell Responses in Autoimmunity and Infection

María Fernanda Pascutti 1 , Gustavo Javier Martinez 2 and Maria Florencia Quiroga3 \*

1 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands, 2 Center for Cancer Cell Biology, Immunology and Infection, Discipline of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, United States, 3 Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Investigaciones Biomédicas en Retrovirus y Sida, Universidad de Buenos Aires, Buenos Aires, Argentina

Keywords: T cells, animal studies, human studies, immunemodulation, health, disease

#### **Editorial on the Research Topic**

#### **Strategies for Modulating T Cell Responses in Autoimmunity and Infection**

T-cell differentiation and effector functions are shaped by the integration of different signals from the environment. The process initiated when the T-cell receptor is engaged is then modulated by activating (costimulatory molecules and cytokines) and inhibitory (checkpoint receptors and cytokines) signals, which have a direct impact on the development or function of effector and/or regulatory T (Treg) cells (1, 2). In pre-clinical human or animal studies, the manipulation of T-cell function has been attempted via numerous different approaches, including (i) manipulation of metabolism (3), (ii) modulation of autophagy, (iii) regulation of epigenetic processes (4), (iv) agonistic or antagonistic therapies engaging cellular receptors (5), (v) the use of cytokines/chemokines to skew immune response profiles, (vi) the expansion/depletion of certain cell type (i.e., depletion of Tregs or expansion of effector T cells specific for a certain pathogen) (6), and (vii) the use of diverse immunomodulating natural or synthetic compounds that could regulate any of the above mentioned aspects of the immune response (7), among others. The goal of the current Research Topic is to highlight some of these approaches and to focus on the diverse ways of modulating T-cell function or frequency in health and disease.

Edited and reviewed by:

Wanjun Chen, National Institutes of Health (NIH), United States

> \*Correspondence: Maria Florencia Quiroga<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology

Received: 08 January 2020 Accepted: 27 January 2020 Published: 20 February 2020

#### Citation:

Pascutti MF, Martinez GJ and Quiroga MF (2020) Editorial: Strategies for Modulating T Cell Responses in Autoimmunity and Infection. Front. Immunol. 11:208. doi: 10.3389/fimmu.2020.00208

Within this eBook, some authors addressed the potential of diverse strategies, such as manipulating metabolic pathways, autophagy processes, and/or modification of the redox cellular milieu with an impact on T-cell function. Cluxton et al. proposed that the fundamental difference in metabolic requirements of human Tregs and Th17 cells could be used in order to modulate inflammatory responses in human diseases. Similarly, Liang et al. work focused on the effect of sulforaphane (SFN), a compound derived from certain vegetables, as a modulator of the extracellular redox milieu. They observed that SFN induced a pro-oxidative state, consequently inhibiting Th17-related cytokines via targeting of STAT3. They finally stated that SFN could be a novel option for treatment of inflammatory/autoimmune diseases, such as rheumatoid arthritis. Continuing with the importance of metabolism in mediating T-cell function, Nicoli et al. suggested in their review that antigen-presenting cells could be metabolically manipulated through TLR4, TLR9, or STING to induce metabolic and/or autophagy changes in order to subsequently impact on CD8+ T cell responses, while Merkley et al. revisited the influence of autophagy over T-cell responses by modulating several aspects of antigen-presenting cells' function and the impact of this modulation over T-cell signaling, survival, cytokine production, and metabolism.

The impact of post-transcriptional modifications on T-cell responses was explored in a very nice study by Hao et al.. They unraveled, in a rat model of autoimmune hepatitis, the major role of

**5**

glycosylation on Treg differentiation and the impact of this deficiency on disease development. They demonstrated that deficiency in O-GlcNAc glycosylation affects the Notch function, a signaling pathway previously reported to promote the differentiation and survival of Tregs. As a result, impaired Treg differentiation allowed for increasing CD4+ T cell infiltration in the liver, indicating that modulation of O-GlcNAc glycosylation in this model could be a target for immunotherapy. In a related disease model, Kroger et al. enumerated the various immunotherapies aimed at controlling autoimmunity in type I diabetes, including among other modulation of regulatory T cells and cytokines.

A more traditional approach used by several authors within this eBook consists of underscoring how signaling through various receptors modifies cellular functions in T cells. For instance, Holgado et al. have described for the first time the role of CD32, a low-affinity Fc-gamma receptor, as promoter of human CD4+ T-cell responses that consequently increases cell proliferation and creates a wide pattern of cytokines triggered by polyclonal stimulation, thus enhancing inflammatory responses. Additionally, the work of Jogdand et al. explored the relevance of ICOS signaling in the context of the blood stage of Plasmodium berghei ANKA infection. In this setting, they observed that depletion of ICOS+ cells from infected mice induced longer survival and lower parasitemia. Also, they observed that ICOS+ T cells also expressed IFN-γ and the transcription factor Tbet; both positively correlated with ICOS expression, therefore suggesting a positive regulation loop between ICOS expression and T-cell function. Abderrazak et al. observed in their manuscript that VLA-2 integrin signaling protected T cells from methotrexate-induced apoptosis (a drug used for rheumatoid arthritis treatment), and "rescued" cells producing IL-17 and IFN-γ contributing to the perpetuation of inflammation and the development of methotrexate resistance seen in rheumatoid arthritis. Finally, Tosello Boari et al. demonstrated the importance of IL-17 cytokines and their receptor IL-17RA in a model of chronic infection with Trypanosoma cruzi. In their work, the authors demonstrated that signaling through IL-17RA is required for the maintenance of effector function in CD8+ T cells in a cell-intrinsic manner. In fact, deficiency in IL-17RA rendered CD8+ T cells more dysfunctional, and blockade of PD-L1, a checkpoint inhibitor currently targeted in many cancer models, partially restored the effector function of these cells. In line with this topic, Panagioti et al. discussed in their review article the various T-cell inducing vaccines used in protection against chronic infections, particularly in viral infections.

Chemokines and/or cytokines themselves could serve as therapeutic targets. In their work, Ushio et al. used a murine model for Sjögren's syndrome as a way to understand the role of CCL22 in this autoimmune disease. They observed that CCL22-producing tissue-resident macrophages may control autoimmune lesions by enhancing the migratory capacity of CD4+ T cells to the affected tissue as well as by inducing IFN-γ production of T cells. Another example of how cytokines and their downstream effects could be used as targets for immunotherapy is addressed in the work of Min et al.. The authors clearly showed the relevance of the Type I IFN/IL-10 axis in the induction of antigen-specific T-cell function and the generation of T-cell memory in a mouse model for scrub typhus, an infectious disease caused by the intracellular bacteria Orientia tsutsugamushi. Their work contributes to a better understanding of the mechanisms behind the short longevity of antigen-specific adaptive immunity observed during this human infection (8).

A different approach proposed by some authors consists of expanding effector cells or limiting the function of regulatory cells with the aim to improve immune responses against pathogens. Salido et al. expanded in vitro CD8+ T cells from HIV+ subjects on combination antiretroviral therapy (cART). Their ultimate goal was to explore new strategies aimed at modulating CD8+ T lymphocytes to achieve functional cure of HIV infections. They observed that expanded cells were polyfunctional, skewed toward an effector phenotype, and that PD-1 expression was clustered in HIV-specific effector memory CD8+ T cells, which had the highest cytokine-producing capacity. Antiviral activity was also enhanced in these cells, indicating that, despite being dampened in subjects on cART, the HIV-specific CD8+ T-cell response could be selectively stimulated and expanded in vitro. On the other hand, the work of Araujo Furlan et al. explored the role of Tregs during Trypanosoma cruzi infection and the impact of Treg frequency over pathology. They observed that massive accumulation of effector immune cells resulted in a diminished frequency of Tregs and inversely correlated with the magnitude of the immune response as well as with emergence of acute immunopathology. After transfer of Tregs, CD8+ T-cell function was affected and parasite control in blood and tissues was diminished, highlighting the relevance of Tregs over cytotoxic immunity against the parasite. Another important population of T cells that was reviewed by Shiromizu et al. consists of γδ T cells, whose role during autoimmunity and infection has been analyzed in depth in their work. They have also revised concepts about the mechanisms that modulate γδ T-cell function, giving the framework to consider the modulation of these cells as a tool to control pathological immune responses.

Collectively, the wide spectrum of original papers and reviews presented in this eBook have provided a comprehensive overview of potential strategies aiming to convert a pathological immune response into a protective one through the control or reprogramming of specific T-cell subsets. The insights outlined here will hopefully help shape future therapeutics for the treatment of infectious and autoimmune diseases.

# AUTHOR CONTRIBUTIONS

MQ wrote the first draft of the present Editorial article. MP and GM revised it and provided their valuable and precious comments, remarks, and suggestions. The final Editorial's draft and the current Editorial's text were entirely agreed and approved by all three Guest co-Editors of the present Research Topic, and it details the Strategies for Modulating T cell Responses in Autoimmunity and Infection.

# REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Pascutti, Martinez and Quiroga. 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) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Features of effective T Cell-inducing vaccines against Chronic viral infections

*Eleni Panagioti 1,2, Paul Klenerman3 , Lian N. Lee3 , Sjoerd H. van der Burg1 and Ramon Arens 2 \**

*1Department of Medical Oncology, Leiden University Medical Center, Leiden, Netherlands, 2Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands, 3Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom*

For many years, the focus of prophylactic vaccines was to elicit neutralizing antibodies, but it has become increasingly evident that T cell-mediated immunity plays a central role in controlling persistent viral infections such as with human immunodeficiency virus, cytomegalovirus, and hepatitis C virus. Currently, various promising prophylactic vaccines, capable of inducing substantial vaccine-specific T cell responses, are investigated in preclinical and clinical studies. There is compelling evidence that protection by T cells is related to the magnitude and breadth of the T cell response, the type and homing properties of the memory T cell subsets, and their cytokine polyfunctionality and metabolic fitness. In this review, we evaluated these key factors that determine the qualitative and quantitative properties of CD4+ and CD8+ T cell responses in the context of chronic viral disease and prophylactic vaccine development. Elucidation of the mechanisms underlying T cell-mediated protection against chronic viral pathogens will facilitate the development of more potent, durable and safe prophylactic T cell-based vaccines.

Keywords: T cells, quality, vaccine, prophylaxis, chronic infection, virus

#### INTRODUCTION

Our bodies are persistently exposed to various pathogens present in the environment. The immune system is fortified with physical barriers and with diverse immune cell populations that play an integral role in protection against disease. Long-term immune responses are mediated by antigenspecific lymphocytes and antibodies that are formed upon pathogen entry. Memory B and T cells are numerically and functionally superior to their naïve precursors cells that are present before infection, and upon encounter with the same pathogen memory immune cells are able to induce a more rapid and powerful recall response (i.e., immunological memory) (1, 2).

The majority of prophylactic vaccines against viral infections have focused on the induction of neutralizing antibodies. Indeed, potent antibody inducing vaccines against virally induced diseases are available. Nevertheless, they fail to provide long-term efficacy and protection against a number of chronic viral infections. Studies in mice, non-human primates, and humans provide evidence that effective prophylactic vaccines against chronic (low level and high level) replicating viruses [i.e., herpesviruses, human immunodeficiency virus (HIV), and hepatitis C virus (HCV)] should engage strong cellular T cell immunity (3–5). The development of T cell-eliciting prophylactic vaccines has gained increasing attention, although such vaccines are not always able to provide

#### *Edited by:*

*Maria Florencia Quiroga, Universidad de Buenos Aires, Argentina*

#### *Reviewed by:*

*John J. Miles, James Cook University, Australia Maria Magdalena Gherardi, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina*

#### *\*Correspondence:*

*Ramon Arens<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 21 November 2017 Accepted: 31 January 2018 Published: 16 February 2018*

#### *Citation:*

*Panagioti E, Klenerman P, Lee LN, van der Burg SH and Arens R (2018) Features of Effective T Cell-Inducing Vaccines against Chronic Viral Infections. Front. Immunol. 9:276. doi: 10.3389/fimmu.2018.00276*

**8**

sterilizing immunity. Despite various promising vaccines that are capable of stimulating robust T cell responses, the critical factors of T cell-mediated immune protection against these chronic infections have not been clearly defined. Often, the memory response provoked by vaccines is not sustained and frequently diminishes over time (6, 7). Thus, more knowledge is required to tailor the vaccine's capacity to induce durable CD4+ and/or CD8+ T cell responses of appropriate magnitude and quality to effectively contribute to pathogen clearance. Elucidating the mechanisms through which antigen-specific T cell populations mediate long-term protection against viruses at body surfaces and (lymphoid) tissues remains an important goal, and will facilitate the development of more effective and safe prophylactic T cell-eliciting vaccines. Here, we review determinants and mechanistic factors of effective T cell populations implicated in the vaccine efficacy against chronic viral infections, and discuss how this knowledge can be utilized to maximize the possibility of creating effective vaccine platforms for persistent viral infections.

# THE COMPLEXITY OF THE ANTIGEN-SPECIFIC T CELL RESPONSE DURING INFECTION

The antigen-specific interactions between T cells and DCs resulting in activation may initially be short lived, before stabilizing and may last up to 12 h. During this period, T cells receive their necessary activating signals (8, 9). For proper activation of naïve CD4+ and CD8+ T cells, cognate antigenic signals through the TCR (signal 1), costimulatory signals (signal 2) and signals provided by inflammatory cytokines (signal 3) are required (10, 11). Expression of particular chemokine receptors such as CCL19 and CCL21 enhance immune responses by stimulating the interactions between T cells and DCs during antigen presentation (12–15). In addition, the secretion of chemokines by activated DCs and CD4+ T cells enhances CD8+ T cell accumulation and help attract rare antigen-specific T cells (16, 17). The activation of T cells results in alteration of the expression of various molecules including integrins, selectins, and chemokine receptors, leading to the modulation of key intracellular signaling events that promote proliferation, differentiation, and migration of T cells to inflamed tissues (18–20).

After resolution of the infection, the majority (90–95%) of the effector T cells are eliminated due to programmed cell death and only a small, yet diverse pool of memory cells remains (21, 22). Traditionally, memory T cells were classified into two major categories based on their proliferation capacity, phenotypic features, and migration potential (23). Effector-memory T (TEM) cells are identified based on combined expression and/or lack of certain cell surface markers including KLRG1hi/CD44hi/ CD127lo/CD62Llo. These cells have limited proliferation capacity upon TCR triggering but rapidly produce effector molecules and cytokines such as IFN-γ and TNF (24, 25). Central-memory T (TCM) cells are distinguished by the expression of KLRG1lo/ CD44hi/CD127hi/CD62Lhi surface markers, exhibit a superior proliferation capacity and produce cytokines that are directly associated with better secondary expansion such as interleukin (IL)-2. Secondary lymphoid organs are the main homing sites of TCM cells whereas TEM cells are more dominantly present in (nonlymphoid) tissues (26–29). Both TCM and TEM cells can circulate, whereas a recently discovered new category of T cells present in tissues lacks migratory capacity. These cells, named tissueresident memory T (TRM) cells, permanently reside in peripheral tissues, even after the infection is cleared. TRM cells are present in most organs and tissues and can be defined based on the expression of CD69hi/CD62Llo/CD44hi and other surface markers (e.g., CD11a, CD38, CD49a, CD103, and CXCR3) (30–33). However, the composition of these markers depends on tissue-specific cues, and expression levels vary in different tissues. Besides these three main memory T cell subsets, a small subset of memory T cells exists that exhibit advanced stem cell like qualities and proliferation capacities compared with other T cell subsets (34). These memory T cells, which were designated stem cell memory T cells (TSCM cells), display a phenotype highly similar to naïve T cells (TN cells), being KLRG1lo/CD44lo/CD127hi/CD62Lhi/CD69lo, but also co-express stem cell antigen (Sca-1), the β chain of the IL-2 and IL-15 receptor (CD122 and IL-2Rβ), and the chemokine receptor CXCR3 (34–39). Some studies reported that T cells with an early stage of differentiation can be induced by vaccines (40, 41) but whether this induction is important for vaccine efficacy is unclear. Thus, whether sufficient amounts of TSCM-like T cells able to elicit protection can be generated by vaccines needs further exploration. Notably, humans and mice have broadly analogous T cell biology, and the above described subsets (i.e., TCM, TEM, TRM, and TSCM cells) have been described in both species and share similar characteristics.

Live attenuated as well as synthetic or subunit vaccines are able to elicit TCM, TEM, and TRM cells (30, 32). With respect to live attenuated vaccines, the vaccine-induced T cell subsets can be highly similar to those subsets that develop upon infection (42). However, live vaccines have disadvantages (e.g., transformation to a virulent form and requires refrigeration), which prompts the development of inactivated, synthetic, or subunit vaccines. T cell subsets that develop upon immunization with those vaccines are highly dependent on the addition of adjuvants and on the route of administration (43).

# THE MAGNITUDE OF THE T CELL RESPONSE IS IMPORTANT FOR OPTIMAL PROTECTION

The magnitude of viral-specific T cell responses is highly dictated by the infectious dose and route of infection (44). Higher infectious dosages lead generally to higher peak values of effector T cells, and correspondingly larger amounts of memory T cells in the circulation are found. However, if the immune system is overwhelmed and virus replication remains at a high level, this eventually leads to exhaustion of T cells and poor memory formation (45).

Given the frequently observed correlation between the magnitude of T cell responses and establishment of immunity during infections, simply determining the magnitude of the vaccineelicited T cell response may already serve as a predictor of efficacy in vaccination settings. A number of studies have shown a direct association between the vaccine-elicited T cell response size and the ability for virus control (5, 46–48). Several parameters directly impact the magnitude of the vaccine-induced T cell response. In the case of live (attenuated) viruses, the size of the initial dose of the inoculum correlates to the magnitude of the vaccinespecific T cell response until a threshold is reached (49). To reach similar levels as that elicited by virulent virus, inoculum sizes are generally higher for replication-deficient or single-cycle viral vectors. For synthetic vaccines, however, the saturation threshold may not be reached because of lack of sufficient inflammatory signals. However, recent discoveries in adjuvant development and synthetic (nano)particles provide promising approaches to augment T cell responses (50–52). Besides the initial inoculum dosage, booster vaccine regimens increase the magnitude of the T cell response (43, 53, 54) and are likely essential for the majority of vaccine platforms including live vaccines (55). In this regard, vaccines that prime with DNA or adenoviral vectors and boost with modified vaccinia Ankara are excellent demonstrations that underline the supremacy of prime-boost vaccination regimens (4, 56–64).

#### MEMORY T CELL INFLATION PROVOKED BY RECOMBINANT VACCINES

An alternative mechanism leading to a durable increased magnitude of memory T cells, described as memory "inflation" (65, 66), is observed for certain viral-specific responses following infection by cytomegalovirus (CMV). Here, antigen-specific T cells specific to a subset of viral peptides show an unusual response, whereby they expand gradually over time and are maintained at high frequencies as TEM-like populations—as opposed to the standard expansion and contraction kinetic of conventional memory cells. Critically, and unlike exhausted CD8 T cells that develop during other persistent infections these inflationary responses maintain their effector functions, tissue homing ability and can provide protection against pathogen rechallenge. Memory inflation has also been observed for CMV-specific antibodies, whose levels gradually increase over time (67). Although the rules that determine the onset of memory inflation have not been fully defined, it is clear that for inflation to occur viral antigen must persist long term, a criterion fulfilled by CMV infection through periodic episodes of reactivation from its latent state. Memory T cell inflation appears to require T cell costimulation (68, 69), yet is less dependent on the immunoproteasome (70). Modifying the context of the peptide can convert a classical response to an inflationary one (71).

Recombinant CMVs may provide important vectors for vaccines, although they are highly complex viruses containing multiple immune evasion genes. Nevertheless, in experimental models engineered mouse cytomegalovirus (MCMV)-based vaccine vectors containing foreign viral sequences (e.g., derived from influenza virus, lymphocytic choriomeningitis virus, Ebola virus, herpes simplex virus, and respiratory syncytial virus) provide long-lasting protection (42, 71–73). In rhesus macaques, a recombinant CMV vector expressing simian immunodeficiency virus (SIV) antigens induced in addition to MHC class I-restricted CD8+ T cell responses also MHC class II-restricted and HLA-Erestricted CD8+ T cell responses (74, 75). These unconventional responses are likely to arise because of the restrictions placed on normal antigen presentation by the attenuated CMV vectors used. More work is needed to identify which of these populations is critical for protection, and whether this protection correlates to magnitude, breadth, or effector mechanism.

Memory inflation is not exclusively induced by CMV. Similar phenomena have been observed with other viruses, e.g., Epstein–Barr virus (EBV), herpes simplex virus-1, parvovirus B19, murine polyoma virus, and adenoviral vectors (66, 76). The latter is of interest with respect to vaccine-induced responses. In mouse models, adenovirus-based vectors can lead to induction of inflationary responses, which closely resemble those induced by natural CMV infections (77, 78). Moreover, in this vaccine platform, it is possible to generate inflationary responses against otherwise non-inflationary epitopes by constructing "minigenes," in which only the CD8 T cell epitope of interest is inserted into the vector and expressed, thus bypassing antigen processing requirements (79). Adenoviral vectored vaccines have been developed against many pathogens, including EBV, HCV, HIV, malaria, and Ebola (4, 64, 80–82), and the responses elicited by these vectors in human volunteers are sustained over time. The HCV-specific responses induced in healthy CMV+ volunteers after immunization with a chimpanzee adenovectored-HCV vaccine shared similar phenotype and functionality to their CMV-specific memory populations as well as to inflating memory cells induced after AdHu5 and MCMV infection in mice (78).

#### THE BREADTH OF THE INDUCED T CELL RESPONSE IMPACTS ON PROTECTION

An increased breadth of the vaccine-induced T cell response has been found beneficial against many chronic viral pathogens (5, 54, 83–86). Induction of T cells with multiple antigenspecificities correlates with advanced killing capacity for control of HCV or even complete eradication during primary infection with HCV and superior protection upon reinfection (80, 86, 87). Analysis of CD8+ T cell responses in untreated HIV-infected individuals showed that an increasing breadth of Gag-specific responses is associated with decreased viremia (88).

Successful induction of potent and broad T cell responses has been reported for DNA plasmid vaccines (89, 90) and adenovirus serotype 26 vector-based vaccines (91). The latter approach incorporated a combination of subdominant and dominant epitopes of rhesus macaques SIV in prime-boost vaccination schedules. In parallel with these findings, the efficacy of synthetic long peptide (SLP)-based vaccines to protect against MCMV was significantly improved by combinations of SLPs that increased the breadth of the antigen-specific T cell response (5). These findings indicate that cytotoxic CD8+ T cell populations consisting of a broad repertoire of specificities are better capable to effectively kill virus-infected cells compared with T cell pools with a single specificity. Possible explanations are that T cells of diverse specificity results in enhanced killing of virus-infected cells (compared with T cells with one specificity) or that viral escape mechanisms become restricted. Moreover, an increase in recognition of multiple epitopes may also contribute to protection against infection with heterologous viruses *via* cross-reactive responses (92). Vaccine efficacy is expected to be also dictated by the TCR clonotypes within a polyclonal antigen-specific T cell population, since immune escape during viral infection is linked to conserved TCR motifs while diverse clonotypic repertoires without discernible motifs are not associated with viral escape (93, 94). Hence, the importance of the diversity in the antigenspecific T cell repertoire (with respect to recognition of multiple antigens and diversity in clonotypes specific for the same epitope) should be taken into account while designing prophylactic T cellbased vaccines.

As discussed earlier, both the magnitude and breadth of the T cell response is of importance. However, it should be noted that simply determining the magnitude in the blood is not always valuable, as vaccine efficacy depends also on the type of memory T cell and its location. For example, a direct association between protection and the frequency of the T cells in the circulation does not always exist (95). Actually, depending on the route of infection, T cells present in the mucosal surfaces or in the tissues (TEM and/or TRM) play a dominant role in controlling the infection, and sufficient numbers in these areas rather than in the circulation are likely required to form a robust frontline defense against, e.g., HIV-1 (30, 96). Competition between antigens (e.g., the cellular processing and presentation machinery) is also an important consideration (5), highlighting that antigen selection is not simply a case of "the more the better." Furthermore, not all antigen-specific T cell populations have the same efficacy on a per-cell basis. For example, T cell populations specific for CMV antigens that invoke inflationary responses show superior protective capacity (5). Selection of the correct but also the appropriate quantity of antigens will ultimately steer the immune response and is thus a very critical step of the vaccine development process. Especially, antigens provoking antigen-specific T cell populations with enhanced magnitude, breadth, and diversity in the clonotypic repertoire should be tested and subsequently selected for inclusion when designing vaccine vectors or synthetic vaccines. Furthermore, there is evidence that, besides the quantity and breadth, specific features of antigen-specific T cell populations such as their cytokine polyfunctionality and metabolic properties are also of crucial importance for vaccine efficacy, and this will be further discussed in the next sections.

#### CYTOKINE POLYFUNCTIONALITY OF T CELLS AS PARAMETER OF VACCINE EFFICACY

Cytokine production is an important effector mechanism of T cell-mediated immunity. Upon most viral and bacterial infections protective T cell immunity consists of CD4+ and CD8+ T cells with a "Th1" cytokine profile that is characterized by (co-) production of IFN-γ, TNF, and IL-2 (97).

The frequency of IFN-γ-producing T cells has been widely used as a parameter to assess vaccine-induced responses. In terms of effector function, IFN-γ has been shown to play a role in the clearance of various viral infections (98). However, there are many examples showing that the magnitude of the IFN-γ secreting T cell response is not a sufficient immune correlate of protection. Single positive IFN-γ-producing T cells can comprise a relatively large fraction of the total cytokine-producing CD4+ and CD8+ T cell population after immunization. However, such T cells have a limited capacity to be sustained as memory T cells (99). Hence, prophylactic vaccines that elicit a high proportion of single IFNγ-producing T cells would likely not be protective and provide a clear example for why the quality of the response is far more useful in assessing long-term protection than just measuring the frequency of IFN-γ-producing T cells. Instead, studies characterizing (vaccine-elicited) T cell responses against HIV, HBV, HCV, CMV, influenza, and *Leishmania* revealed a strong correlation between the protection level and the induction of high frequencies of polyfunctional T cells [e.g., coproducing IFN-γ, TNF, and IL-2 (4, 80, 100–107)]. Importantly, some of these studies showed that measuring the magnitude of IFN-γ-producing CD4+ and CD8+ T cells alone was not sufficient to predict protection, and provided evidence that measuring the quality of the CD4+ and CD8+ T cell response, *vis-à-vis* polyfunctional T cells, is required.

The supremacy of the polyfunctional T cells may relate to the superior survival properties of these cells (81, 99, 108) and to a higher level of target killing (109). This may be related to a higher IFN-γ production on a per-cell basis by polyfunctional cells compared with monofunctional cells (110), and to the capacity of TNF that is like IFN-γ also capable of mediating the killing of virus-infected cells (111–113). Moreover, reciprocal production of IFN-γ and TNF leads to synergistic actions (114).

Furthermore, the other cytokine in the panel, IL-2, is decisive as well. Studies analyzing the production of IL-2 and IFN-γ by CD4+ and CD8+ T cells from individuals infected with HIV showed that long-term non-progressors, or individuals on anti-retroviral treatment, had increased frequencies of T cells expressing IL-2 only or both IL-2 and IFN-γ, whereas individuals with high viral loads (progressors) have increased frequencies of T cells producing IFN-γ only (95). Although IL-2 has no direct antiviral function, it promotes proliferation and secondary expansion of antigen-specific T cells (115–120). In addition, IL-2 increases expression of the effector molecules perforin and granzyme, which mediate cytolytic function (121, 122). IL-2 signals may also enhance NK cell activity that could contribute to the early control of infection following challenge (99, 123–126). Taken together, we conclude that cytokine polyfunctionality is of major importance for the efficacy of T cell-based vaccines (**Figure 1**), hence dissecting how cytokine polyfunctionality is regulated during the programming of T cells is of interest and may reveal potential strategies to improve vaccine-mounted T cell responses.

#### IMPROVING VACCINATION BY TARGETING T CELL METABOLISM?

The transition of naïve T cells to active effector cells and memory T cells involves dynamic and coordinated metabolic modifications (129). This reprogramming of the cellular metabolism is

not a consequence of activation but is linked to the differentiation and activation processes and reflects the fuel and substrates necessary to support the differentiation stages of a T cell (130, 131). Both naïve T cells and memory T cells rely primarily on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) for fuel. This reflects the low level yet persistent need for energy as such cells are generally long-lived. Effector T cells on the other hand have particularly high energetic and synthesis demands. These cells have enhanced glycolysis and employ the mitochondrial tricarboxylic acid cycle to support their demand for *de novo* proteins, lipids, and nucleic acids synthesis. It is becoming increasingly clear that metabolic reprogramming plays a critical role in T cell activation, differentiation, and function. The distinct metabolic demands of different T cell subsets make them exquisitely sensitive to pharmacologic inhibitors of metabolism (132). These different metabolic requirements of T cell subsets provide us with a promising therapeutic opportunity to selectively tailor (vaccine-induced) immune responses. Thus, targeting T cell metabolism affords the opportunity to additionally regulate vaccine-induced responses.

Metabolic reprogramming occurs simultaneously with T cell activation and is facilitated by mTOR (mammalian target of rapamycin) (133). mTOR activation promotes glycolysis, fatty acid synthesis, and mitochondrial biogenesis. As such, targets upstream and downstream of the mTOR signaling pathway are potential therapeutic targets. Rapamycin, although known as an "immunosuppressive" drug due to its ability to slow down T cell proliferation, promote robust responses to vaccination by enhancing CD8+ T cell memory formation (134). Correspondingly, deletion of the mTORC1 inhibitory protein TSC2 leads to enhanced mTORC1 activity and increased effector function (135). Targeting of TSC2 or other molecules in the mTOR pathway might accordingly enhance immunity.

Targeting of glycolysis to inhibit immune responses in the setting of autoimmune disease and transplantation rejection is evolving, and this strategy is also used to enhance antitumor immunity by promoting long-lived memory cells *ex vivo* (136). Whether this can be used in vaccination strategies remains to be examined. Although most studies have focused on the critical role of glycolysis in promoting effector T cell generation and function, it has become clear that mitochondrial-directed metabolism also plays an important role. Memory T cells rely for their energy upon OXPHOS and FAO. Because these metabolic pathways are dependent on mitochondria, the abundance and the organization of the mitochondria are instrumental for development of fit memory cells (137). Alterations in the mitochondrial biogenesis can influence the differentiation of T cells, thereby providing opportunity to augment T cell-mediated immunity (138, 139). The transcription factor PGC1α promotes mitochondrial biogenesis and function (140). Hence, pharmacologically or genetically enhancing PGC1α represents a potential strategy for improving vaccine-induced T cell responses. In *ex vivo* systems, it has already been shown that enforced overexpression of PGC1α, leads to improved metabolic fitness and effector cytokine function of CD8+ T cells (141). Finally, the immediate uptake of amino acids such as glutamine and leucine is critical for proper metabolic reprogramming of T cells. This is accompanied with the upregulation of amino acid transporters involved in glutamine (SLC1A5) and leucine (SLC7A5/SLC3A2 heterodimer) (142, 143). Whether *in vivo* targeting of the above described metabolic processes is possible remains to be examined and may depend on the specificity of metabolic inhibitors/enhancers as they could affect many cells of the body. The future will tell if indeed metabolic targeting is possible to enhance vaccines. Nevertheless, the metabolic profiles of (vaccine-induced) T cells are surely of interest and correlate to vaccine-mediated immunity (144).

#### COSTIMULATION EMPOWERS T CELL-ELICITING VACCINES

Targeting costimulatory and inhibitory receptors on the cell surface of T cells has shown efficacy in various preventive and therapeutic preclinical vaccination settings. Costimulatory signals transduced *via* the CD28 family members CD28 and ICOS and *via* the tumor necrosis factor receptor (TNFR) family members CD27, 4-1BB, and OX40 play dominant roles in orchestrating the required "signal 2" for optimal T cell proliferation and survival (127). While CD28 and CD27 are constitutively expressed on naïve T cells, ICOS, 4-1BB, and OX40 are upregulated upon T cell activation (127, 145). Collaboration between costimulatory molecules was expected (127, 146) and confirmed in experimental models (147).

Enforced engagement of costimulatory molecules results in enhanced T cell activation, expansion, survival, and establishment of long-term memory (148–154), and has thus the potential to serve as effective immunomodulatory components of prophylactic vaccines against chronic viruses (127, 151, 155). Indeed, this has already been observed for DNA and adenovirus-based vector vaccines in which enforced expression of costimulatory ligands stimulating CD27, 4-1BB, and OX40 leads to increased T cell expansion, enhanced cytotoxic activity and antibody responses (156, 157). Strikingly, agonistic antibodies to OX40 combined with synthetic peptide vaccines prompt robust effector and memory CD4+ and CD8+ antiviral T cell responses, thereby enhancing the prophylactic vaccine efficacy against lytic MCMV infection (153). Chronic viral infections are characterized by accumulation of functionally impaired antigen-specific CD8+ T cells. Studies have shown that activation *via* 4-1BBL alone or in combination with CD80 can enhance the generation of primary CD8+ T cell responses and induce expansion of the antigenspecific CD8+ T cells from this pool of impaired T cells (145, 158). Similarly, 4-1BB stimulation has been shown to enhance the generation of primary CD8+ T cell responses (148, 159, 160) and synergizes with attenuated vaccinia virus vectors to augment CD8+ T cell responses (148).

Targeting of inhibitory molecules on T cells, such as PD-1 and CTLA-4, has been shown to restore the effector function of (over) activated T cells in settings of chronic viral infections and cancer (161–164). Inhibitor blockade with monoclonal antibodies in combination with therapeutic vaccines synergizes in reinvigorating antitumor and antiviral T cell responses (165, 166). Targeting of inhibitory pathways during primary immunization with prophylactic vaccines may advance the vaccine efficacy as well (167, 168).

Although the use of antibodies targeting costimulatory and inhibitory molecules as immunostimulatory modalities in vaccine approaches can facilitate antigen-specific T cell responses, the use of such Abs, however, is associated with toxicity as demonstrated in rodents and in clinical settings (164, 169–171). Nevertheless, given the potential benefit to significantly increase the effectiveness of vaccines, both the efficacy and safety of targeting costimulation is currently extensively examined in various immunotherapeutic approaches against persistent viral infections. Examining the timing and/or the dosing is in this respect an important aspect, not only to prevent unwanted side effects but also to improve effectiveness. However, mass deployment of antibodies to improve vaccines may be too expensive, hence alternative methods able to target costimulatory and inhibitory molecules are desired.

CD28-mediated costimulation modulates T cell metabolism *via* activation of PI3K pathways, and this is essential to control effector cytokine production (172, 173). Moreover, CD28 signaling leads to PI3K-dependent upregulation of surface GLUT1 to facilitate enhanced glucose influx (172). This upregulation of GLUT1 is critical for T cell function, as genetic deletion of GLUT1 markedly inhibits effector T cells (174). Concomitant with increased expression of glucose transporters is the upregulation of key glycolytic enzymes (175). The inhibitory receptor PD-1 also regulates metabolic activity including glycolytic and mitochondrial processes (139, 176). TNFR family members are also able to metabolically program T cells (177, 178). Another important property of T cell costimulation is its effect on improving the T cell cytokine polyfunctionality. For example CD28 but also the TNFR family members are able to promote IL-2 production (153, 179–181), thereby directly improving the cytokine polyfunctionality (**Figure 1**). The TCR affinity also impacts polyfunctionality (182), and likely the collective signals of the TCR and costimulatory receptors are programming the polyfunctional status of T cells. In conclusion, targeting of T cell costimulation can impact the important quantitative (magnitude, breadth) and qualitative (cytokine polyfunctionality and metabolic fitness) determinants of vaccine-induced T cells, and provides thus major opportunities for further exploration in future vaccine designs.

#### CONCLUSION AND PERSPECTIVES FOR VACCINE DESIGN

The design of vaccines that imprint T cells with the ability to protect against persistent viral pathogens has gained remarkable progress. An understanding of the appropriate initial programming signals is a key step, as is how the route of priming or boosting influences the development of effective memory T cells. A combination of several metrics such as the type of the memory T cell, breadth, polyfunctional quality, and metabolic characteristics demonstrate a valid toolbox to define when a vaccine-elicited T cell response is protective. Information about the anatomic location, activation, and differentiation of memory T cells in lymphoid compared with non-lymphoid organs could be very valuable as well. Costimulatory signaling pathways mediate important T cell memory properties (e.g., programming of cytokine polyfunctionality and metabolism) and may serve as interesting targets for vaccine improvement. Insight into these pathways may identify the requisite pathways and potentially other targets to improve T cell-based immunotherapy. Coupling this to the identification of the best correlates of protection for persistent viral pathogens will foster the development of more effective vaccination regimes.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

All authors contributed to the writing of this review.

#### FUNDING

This work was funded by a grant from the European Commission [FP7 Marie Curie Action, Grant number: 316655, VacTrain (SB and RA)].

proliferation and survival of effector CD8(+) T cells. *Int Immunol* (2002) 14:1155–67. doi:10.1093/intimm/dxf080

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Panagioti, Klenerman, Lee, van der Burg and Arens. 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) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Gajendra M. Jogdand, Soumya Sengupta, Gargee Bhattacharya, Santosh Kumar Singh, Prakash Kumar Barik and Satish Devadas\**

*Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, India*

#### *Edited by:*

*Maria Florencia Quiroga, Universidad de Buenos Aires, Argentina*

#### *Reviewed by:*

*Jason Scott Stumhofer, University of Arkansas for Medical Sciences, United States Shahram Salek-Ardakani, Pfizer, United States*

> *\*Correspondence: Satish Devadas<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 18 January 2018 Accepted: 26 April 2018 Published: 28 May 2018*

#### *Citation:*

*Jogdand GM, Sengupta S, Bhattacharya G, Singh SK, Barik PK and Devadas S (2018) Inducible Costimulator Expressing T Cells Promote Parasitic Growth During Blood Stage Plasmodium berghei ANKA Infection. Front. Immunol. 9:1041. doi: 10.3389/fimmu.2018.01041*

The lethality of blood stage *Plasmodium berghei ANKA* (PbA) infection is associated with the expression of T-bet and production of cytokine IFN-γ. Expression of inducible costimulator (ICOS) and its downstream signaling has been shown to play a critical role in the T-bet expression and IFN-γ production. Although earlier studies have examined the role of ICOS in the control of acute blood-stage infection of *Plasmodium chabaudi chabaudi* AS (a non-lethal model of malaria infection), its significance in the lethal bloodstage of PbA infection remains unclear. Thus, to address the seminal role of ICOS in lethal blood-stage of PbA infection, we treated PbA-infected mice with anti-ICOS antibody and observed that these mice survived longer than their infected counterparts with significantly lower parasitemia. Anti-ICOS treatment notably depleted ICOS expressing CD4+ and CD8+ T cells with a concurrent reduction in plasma IFN-γ, which strongly indicated that ICOS expressing T cells are major IFN-γ producers. Interestingly, we observed that while ICOS expressing CD4+ and CD8+ T cells produced IFN-γ, ICOS−CD8+ T cells were also found to be producers of IFN-γ. However, we report that ICOS+CD8+ T cells were higher producers of IFN-γ than ICOS−CD8+ T cells. Moreover, correlation of ICOS expression with IFN-γ production in ICOS+IFN-γ+ T cell population (CD4+ and CD8+ T cells) suggested that ICOS and IFN-γ could positively regulate each other. Further, master transcription factor T-bet importantly involved in regulating IFN-γ production was also found to be expressed by ICOS expressing CD4+ and CD8+ T cells during PbA infection. As noted above with IFN-γ and ICOS, a positive correlation of expression of ICOS with the transcription factor T-bet suggested that both of them could regulate each other. Taken together, our results depicted the importance of ICOS expressing CD4+ and CD8+ T cells in malaria parasite growth and lethality through IFN-γ production and T-bet expression.

Keywords: malaria, inducible costimulator, T cells, IFN-**γ**, T-bet, CD4, CD8

# INTRODUCTION

Malaria is a major cause of mortality in millions of infected individuals every year, especially children from developing countries. Among all human Plasmodium strains, infection with *Plasmodium falciparum* is the leading cause of death involving severity, cerebral manifestation, and multi-organ dysfunction. To some extent, murine infection of *Plasmodium berghei ANKA* (PbA) can be correlated to human *P. falciparum* infection corresponding to parasite growth, lethality, or severity and immune response (1). As human malaria studies are restricted to only clinical observations, modulation of T cell immune response in murine models can provide a better insight to ameliorate malaria pathology and vaccine design (2, 3). The role of T cells in malaria infection, however, has been controversial as studies have shown both its critical role in protection from the malaria parasite and its direct role exacerbating malaria pathogenesis. As an example, CD4+ T cells play a major role in the clearance of parasite *Plasmodium chabaudi chabaudi* blood stage infection (4). However, during lethal PbA infection, both CD4+ and CD8+ T cells are involved in cerebral manifestation. Moreover, depletion of both the T cells by antibodies before or during infection ameliorated pathology (5). Thus, these studies among others suggested that T cells play both protective as well as pathological roles during malaria infection.

In both human and murine malaria, CD4+ and CD8+ T cells are producers of IFN-γ, which have been shown to play crucial protective and pathological roles (6, 7). During lethal malaria infection, extraneous administration of IFN-γ led to the dosedependent protection of BALB/c mice (8). In contrast, another study suggested that IFN-γ produced by CD4+ T cells enhanced CD8+ T cell accumulation in the brain leading to augmented cerebral malaria (9). Moreover, suppressing IFN-γ production from T-bet positive CD4+ T cells efficiently hampered parasite clearance (10). Also, studies with T-bet knockout mice have demonstrated the role of T-bet in regulating parasite burden as well as its role in pathogenesis during experimental cerebral malaria (11). Taken together, these studies indicated that both IFN-γ and T-bet play a role in malaria parasite growth and lethality.

Along with antigenic stimulation, signaling through CD28 plays a critical role in T cell-mediated immunity in clearance of acute blood-stage *P. chabaudi* infection (12). Inhibiting CD28 signaling by blocking CD86 with anti-CD86 antibody preferentially differentiated T cells toward IFN-γ producing Th1 cells and inhibited the Th2 cytokine IL-4. This Th1 cytokine IFN-γ controlled acute parasite infection but did not play a role in limiting chronic malaria infection. Thus, blockade of CD28 signaling suggested that IL-4 production during malaria infection required CD28 signaling whereas augmentation of IFN-γ illustrated the involvement of other co-stimulatory molecules (13). Moreover, *P. chabaudi* infection in the CD28 knock out mice also demonstrated that redundant CD28 signaling pathway with other costimulatory molecules might play a role in IFN-γ production (14). Thus, these studies suggested that IFN-γ production during malarial infection could involve other co-stimulatory molecules.

Inducible costimulator (ICOS), a CD28 homolog, plays a critical role in T cell proliferation, differentiation, cytokine secretion, cell–cell interaction, and B cell maturation (15–21). Depending on the nature of antigen and chronicity of infection, ICOS signaling mediates differential effector CD4+ T cell response. As an example, during *Schistosome mansoni* infection, Th2 and Tfh response were observed whereas, during *Toxoplasma gondii* and *Mycobacterium tuberculosis* infection (Mtb), it was a Th1 response (22–24). In contrast, during non-lethal blood stage *P. chabaudi chabaudi* infection, in the absence of ICOS, enhanced Th1 response led to reduced peak parasitemia (20). In case of CD8+ T cells, ICOS signaling played a role in its activation, expansion, and enhanced secondary response (21, 25). A substantial defect in antigen-specific CD8+ T cells along with hampered CD4+ T cells response was observed during Salmonella infection in ICOS knockout mice (26). Moreover, during late stage of Mtb, ICOS deficiency was associated with reduced Mtb-specific CD8+ T cell response (27). Taken together, these studies strongly suggest that ICOS plays a critical role in CD4+ and CD8+ T cell response in both intra and extracellular infection. However, during lethal intracellular blood-stage of PbA infection, the role of ICOS in T cell response (CD4+ and CD8+) remains unclear.

In this study, we examined the role of ICOS in parasite growth and lethality during lethal PbA infection in BALB/c mice. We characterized ICOS expression and found that both CD4+ and CD8+ T cells express higher ICOS in the PbA-infected mice. Upon depletion of ICOS-expressing T cells by anti-ICOS antibody treatment, we observed prolonged survival of mice with lower parasitemia, suggesting a positive role for ICOS expressing T cells in PbA parasite growth. Further, we report that these T cells are major producers of IFN-γ as correlated with reduced plasma IFN-γ cytokine level and depleted percentage of ICOS expressing T cells upon anti-ICOS treatment. We found both CD4+ and CD8+ T cells expressed transcription factor, T-bet, which is already known to be involved in malaria pathogenesis. Collectively, our study demonstrates that ICOS expressing CD4+ and CD8+ T cells are involved in malarial parasite growth and lethality through IFN-γ production and T-bet expression.

#### MATERIALS AND METHODS

#### Ethics Statement

The use of animals and animal procedures were approved by the Institutional Animal Ethics Committee, Institute of Life Sciences, Bhubaneswar, India in accordance with the "Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)."

#### Mice, Antibodies, Kits, Reagents, and Malaria Parasite

Male BALB/c mice (6–8 weeks old) used for this study were maintained under pathogen-free condition at institutional animal house facility. Anti-mouse antibodies Alexa Fluor 700-CD8 (Clone 53-6.7), APC-cy7-CD19 (Clone 1D3), APC-cy7-CD45R/B220 (Clone RA3-6B2), were procured from BD Biosciences (San Jose, CA, USA), FITC-CD4 (clone GK 1.5), FITC CD278 (7E.17G9), APC-CD278 (Clone-C398.4A), PerCP-Cy 5.5-CD4 (clone RM4-5), PE-Cy7-IFN-γ (clone XMG 1.2), PE-T-bet (clone eBio 4B10), Alexa Fluor 700-CD8 (53-6.7) were from eBioscience (San Diego, CA, USA), Brilliant violet 605 TCR Vb (clone H57-597), APC-CD278 (clone C398.4A), Anti-CD49b (clone DX5) were from BioLegend (San Diego, CA, USA). Purified antibodies Antimouse CD278 ICOS (clone 7E.17G9), Anti-Rat IgG2b isotype control, and anti-CD16/32 were procured from BioXcell (West Lebanon, USA). Dynabeads untouched mouse CD4+ T cell isolation kit was procured from Life Technologies AS (Oslo, Norway) and CD8+ T cell negative selection kit was from Stem cell technologies (Vancouver, BC, Canada). Phorbol 12-myristate 13-acetate (PMA), Ionomycin, Brefeldin A was procured from Sigma-Aldrich (St. Louis, MO, USA). Malarial parasite *P. berghei ANKA* (MRA-671, MR4, ATCC, Manassas, VA, USA) was obtained from MR4 repository, ATCC, Manassas, VA, USA.

#### Parasite Infection

*Plasmodium berghei* ANKA parasitized red blood cells were stored in liquid nitrogen. The parasitic infection was initiated by thawing *P. berghei* ANKA parasite stabilate and intraperitoneally (i.p.) injecting into a donor mouse. After initial expansion of parasites in the donor mouse, blood was collected from tail vein bleed and serially diluted in PBS. Infection was then induced in experimental mice by intravenous (i.v.) injection of 1 × 104 *P. berghei* ANKA parasitic RBCs. After 3 days of infection, percent parasitemia was enumerated by thin blood films stained with modified Giemsa stain (Sigma-Aldrich, St. Louis, MO, USA).

# *In Vivo* Depletion of ICOS-Positive T Cells

On day 1 of PbA infection, ICOS positive T cells were depleted by administering a single dose of anti-CD278 monoclonal antibody (clone 7E.17G9) and its isotype control (rat IgG2b) at 0.2 mg dose, *via* intraperitoneal (i.p.) injection in 200 µl DPBS. Specific depletion of ICOS positive T cells was analyzed by flow cytometry as shown in figure (**Figure 3H**).

#### Determination of Plasma Cytokines

On day 3, day 5, and day 7, day of PbA infection, murine blood was collected in 15% acid citrate dextrose anticoagulant and centrifuged at 1,000 *g* for 10 min. The collected plasma was stored in −80°C until assayed for cytokines. Cytokine from stored plasma was quantified using the Milliplex mag mouse cytokine/ chemokine kit as per the manufacturer's protocol (Millipore, Billerica, MA, USA). The samples were acquired in Bio-plex200 system, and the concentration of cytokines was calculated using Bio-Plex manager software with a five-parameter curve-fitting algorithm applied for standard curve calculation.

# Intracellular Cytokine Staining

For measuring T cell secreted cytokines, the negatively selected splenic CD4+ T cells (purity was >93%) and splenocyte (for CD8+ T cell analysis) from uninfected and PbA-infected mice were cultured in RPMI 1640 complete medium [RPMI 1640 (# P04- 16500), PAN-Biotech GmbH, Germany] supplemented with 10% FBS US origin (#1302-P100402, PAN-Biotech GmbH, Germany), 50 µM 2-ME, 100 U/ml penicillin and 100 µg/ml streptomycin (# P4333-100 ml, Sigma-Aldrich, USA) and stimulated by 20 ng/ml PMA/1 μg ionomycin for 5 h after with Brefeldin A added in the last 2.5 h of stimulation. Stimulated cells were stained with dead cell discrimination dye for 20 min on the ice and then washed and stained for surface markers. Surface stained cells were fixed with Fixation/permeabilization buffer of BD Biosciences for 20 min at room temperature.

# Intracellular Transcription Factor Staining

FoxP3 transcription factor staining kit protocol (eBiosciences, San Diego, CA, USA) was used for intracellular transcription factor staining. In brief, T cells were stained for desired surface markers after dead cell discrimination and blocking with CD16/32 (2.4 G) antibody. After 20 min of incubation, the cells were washed twice and then fixed and permeabilized at room temperature for 20 min as recommended by the manufacturer. The cells were then washed twice with permeabilization buffer and then stained with transcription factor antibody cocktail for 20–30 min at room temperature.

#### Statistical Analysis

Non-parametric Mann–Whitney test was used for comparisons between two groups. One-way ANOVA with Bonferroni's Multiple Comparison Test was used for multiple comparisons having three or more groups. Two-way ANOVA with Bonferroni posttest was used for comparing weight lost. A log-rank (Mantel–Cox) test was used to determine the significance of survival of PbAinfected mice with or without anti-ratIgG2b and anti-ICOS treatment. Graphs depict mean values ± SEM. *p*-value < 0.05 (\**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001) was considered significant. All graphs were prepared with Prism 5.0 (GraphPad, La Jolla, CA, USA) software.

# RESULTS

#### Anti-ICOS Administration Slows *P. berghei* ANKA Growth and Prolonged Mice Survival

To understand the possible involvement of ICOS in the lethality of blood stage *PbA* infection, we first established parasite infection in BALB/c mice by intravenous injection of 1 × 104 parasitic RBCs. To one group of PbA-infected mice, a single dose of anti-ICOS was given while to another group, rat-anti-IgG2b antibody was injected. On every alternate day starting from day 3, percent parasitemia was ascertained from Giemsa-stained thin blood smear prepared from tail vein blood. On Day 3, the mean parasitemia in untreated (0.0875 ± 0.025%), anti-ICOS (0.1%), and anti-rat IgG2b (0.1%) treated mice were found to be similar, indicating that infection in all the groups started equally. On Day 5 of infection, we observed lowered percent parasitemia albeit non-significant in anti-ICOS treated mice (2.125 ± 0.478%) when compared to untreated (3.62 ± 0.643%) and rat IgG2b-treated (3.5 ± 0.408%) mice. Statistically significant lower parasitemia in anti-ICOS treated (4.16 ± 0.372, *p-*value < 0.001) as compared to untreated PbA group (10.916 ± 1.067) (**Figure 1A**) was established on day 7. Lowered parasitemia in anti-ICOS-treated mice suggested that ICOS indeed played a significant role in parasite growth. Further, analyzing for the survival of the mice, anti-ICOS treated mice survived significantly longer than the rat IgG2b treated and rat IgG2b treated mice survived longer than the non-treated one (**Figure 1B**). Eventually, anti-ICOS treated mice died with hyperparasitemia. Next, we analyzed percent weight loss; a pathological marker in anti-ICOS treated and untreated mice during PbA infection. On day 5 and day 7 of PbA infection, significantly reduced percent weight loss was observed in anti-ICOS treated mice as compared to PbA-infected mice (**Figure 1C**). The above results indicated that ICOS plays a significant role in PbA parasite growth and pathogenesis.

It is well known that regulation of pro-inflammatory (IFN-γ, TNF-α, IL-6, IL-17) and anti-inflammatory (IL-10, IL-4) cytokines play a pivotal role in malaria parasite growth, protection, and/or pathogenesis (28–32). Moreover, previous studies in blood stage infection of PbA have determined the crucial role of IFN-γ in immunopathology and death. However, during acute non-lethal blood-stage malaria infection, IL-27-mediated IL-10 production from IFN-γ-producing CD4+ T cells has been shown to play an essential role in protection from immune pathology (33). Thus, we scored for plasma cytokine IFN-γ, IL-4, IL-10, IL-17, IL-6, IL-27, and TNF-α during PbA infection in BALB/c mice with and without anti-ICOS treatment. In plasma samples of PbAinfected mice, significantly higher IFN-γ levels were observed as compared to other tested cytokines (**Figure 2A**). The ratio of cytokines in infected to uninfected samples (fold change) also suggested that IFN-γ production was higher than other tested cytokines between the groups (**Figure 2B**). Moreover, significantly higher IFN-γ was observed on day 5 and 7 of PbA infection as compared to day 3 (**Figure 2C**). Interestingly, significantly reduced plasma IFN-γ level was found after anti-ICOS treatment (**Figure 2D**). Also, non-significant changes in the levels of cytokine IL-10 (**Figure 2E**), IL-4 (**Figure 2F**), and no difference in the levels of other tested cytokines were observed (data not shown). Altogether, this suggested the critical involvement of ICOS in malaria parasite growth and lethality might be through IFN-γ production.

#### ICOS Expression During Blood Stage *P. berghei* ANKA Infection

To investigate the role of ICOS and cells associated with its expression for *PbA* lethality, we scored ICOS expression on lymphocytes. On day 3 and day 6 of *PbA* infection, mice were sacrificed and their spleens analyzed for surface markers to identify ICOS expression on CD4+, CD8+, NK, NKT, and B cells by flow cytometry. After gating out doublets and dead cells (**Figure 3A**), we observed higher ICOS expression on CD4+ and CD8+ T cells (**Figure 3B**). Minimal ICOS expression was found on B cells, NK cells, and NKT cells in PbA infected and uninfected mice (data not shown) that did not vary with progressive infection. On day 6 of infection, however, we observed an increased percentage of ICOS-positive CD4+ and CD8+ T cells as compared to uninfected mice (**Figures 3C–E**). A significant increase in mean fluorescent intensity for ICOS on CD4+ and CD8+ T cells suggested that expression of ICOS at protein level per cell was also higher on day 6 of PbA infection (**Figures 3F,G**). Anti-ICOS treatment significantly reduced the percentage of ICOS-positive

Figure 2 | Th1 cytokine IFN-γ is highly produced during PbA infection. Plasma was collected on indicated days from anti-inducible costimulator (ICOS), anti-ratIgG2b, untreated PbA infected and uninfected (control) mice (*N* = 3/group). The collected plasma was analyzed for cytokine IFN-γ, IL-4, IL-10, IL-6, TNF-α, IL-27, and IL-17E with bioplex ELISA. (A) Th1 cytokine IFN-γ is significantly increased as compared to Th2 (IL-4), Treg (IL-10), TNF-α, and IL-6 in *Plasmodium berghei* ANKA (PbA)-infected mice. (B) Percent cytokine change as compared to uninfected mice. (C) Kinetics of IFN-γ during PbA infection. (D) Reduction in plasma IFN-γ production by anti-ICOS treatment. (E) Plasma level of IL-10 (F) plasma level of IL-4. Data represent one of the two independent experiments. Bar represent mean ± SEM. Statistics: Mann–Whitney test, one-way ANOVA with Bonferroni's Multiple Comparison Test, *p-*value <0.05 (\**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001) considered significant.

CD4+ and CD8+ T cells (**Figures 3H–J**) while ICOS MFI on nondepleted T cells from anti-ICOS treated mice was significantly lower (**Figures 3K,L**) than untreated and RatIgG-treated mice. Moreover, we observed significant depletion in percent activated (CD44hiCD62Llo) as well as total ICOS expressing CD44hiCD62Llo CD4+ T cells upon anti-ICOS treatment (**Figures 3M,N**). Further, we observed non-significant depletion in percent activated (CD44hiCD62Llo) CD8+ T cells and a significant depletion in ICOS expressing CD44hiCD62Llo CD8+ T cells (**Figures 3O,P**). However, anti-Rat IgG2b administration non-significantly reduced ICOS expressing CD4+ and CD8+ T cells (**Figures 3I,K,N,P**). Collectively, the above data indicated that PbA growth and lethality might be associated with ICOS expressing CD4+ and CD8+ T cells.

#### IFN-**γ**-Producing CD4+ T Cells Are Highly ICOS Positive at Protein Level

Higher ICOS expression on T cells and increased plasma IFN-γ production suggested that ICOS expressing T cells may be the source and major producers of IFN-γ. Thus, to quantify IFN-γ

production by ICOS expressing CD4+ T cells, on day 6, we negatively isolated CD4+ T cells from uninfected and *PbA* infected BALB/c mice and stimulated them with PMA/ionomycin. The stimulated cells were analyzed for ICOS and IFN-γ co-expression by staining cells with anti-ICOS, anti-IFN-γ antibodies after dead cell discrimination and Fc blocking (**Figure 4A**). As expected, we found a significant increase in the percentage of ICOS+IFN-γ+ CD4+

T cells from *PbA-*infected mice as compared to uninfected mice (**Figure 4B**). Increased integrated mean fluorescent intensity for IFN-γ depicted that IFN-γ production was significantly higher in CD4+ T cell from infected mice than in uninfected mice at the protein level (**Figure 4C**). We further analyzed ICOS expression on IFN-γ positive and IFN-γ negative CD4+ T cells from PbA-infected mice. We found that ICOS expression per cell

Figure 3 | Both CD4+ and CD8+ T cells express inducible costimulator (ICOS) during *Plasmodium berghei* ANKA (PbA). A single cell suspension of splenic cells from uninfected and PbA infected (*N* = 3/group) were stained with surface staining with antibodies to anti-TCR-vb, anti-CD4, anti-CD8a, CD19/B220, CD278 (ICOS) after dead cell staining with fixable blue dead cell stain. (A) Gating strategy for selection of live cells after doublet and dead cell discrimination. This gating strategy was used for all flow cytometry analysis. (B) Representative gating strategy for selecting CD4+ and CD8+ T cells and ICOS expression. (C) Representative counter plot showed ICOS expression by CD4+ and CD8+ T cells on day 3 and day 6 of PbA infection. (D) Percentage ICOS expressing CD4+ T cells. (E) Percentage ICOS expressing CD8+ T cells. (F) Expression of ICOS on CD4+ T cells. (G) Expression of ICOS on CD8+ T cells. (H) Representative counter plot showed depletion of ICOS expressing CD4+ and CD8+ T cells upon anti-ICOS treatment. (I) Percentage depletion of ICOS expressing CD4+ T cells. (J) Percentage depletion of ICOS expressing CD8+ T cells. (K) ICOS expression on non-depleted CD4+ T cells. (L) ICOS expression on non-depleted CD8+ T cells. (M) Percentage activated (CD44hiCD62LLo) CD4+ T cells. (N) Total number of ICOS expressing activated (CD44hiCD62LLo) CD4+ T cells. (O) Percentage activated (CD44hiCD62LLo) CD8+ T cells. (P) Total number of ICOS expressing activated (CD44hiCD62LLo) CD8+ T cells. Data represent one of the three independent experiments. Bar represent mean ± SEM. Statistics: Mann–Whitney test, one-way ANOVA with Bonferroni's Multiple Comparison Test, *p*-value <0.05 (\**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001) considered significant.

(as scored with mean fluorescent intensity) was significantly higher on IFN-γ+ cells than the negative ones (**Figure 4D**). Moreover, anti-ICOS treatment significantly reduced percentage as well as total ICOS+IFN-γ+ CD4+ T cells (**Figures 4E,F**). Taken together, our data for CD4+ T cells suggest that ICOS may play a role in IFN-γ production based on correlation of ICOS expression and IFN-γ production. It also clearly indicated that all IFN-γproducing CD4+ T cells were ICOS positive but all ICOS-positive CD4+ T cells were not IFN-γ producers.

#### ICOS Expressing CD8+ T Cells Are Higher IFN-**γ** Producers

To ascertain the role of ICOS in the production of IFN-γ by CD8+ T cells during PbA infection, we stimulated single cell splenocyte with PMA/ionomycin. The stimulated cells were stained with anti-TCR-Vb, anti-CD8a, anti-ICOS, and anti-IFN-γ antibodies. After gating TCR-Vb and CD8+, we observed four populations including ICOS+IFN-γ−, ICOS+IFN-γ+, ICOS−IFN-γ+, and ICOS−IFN-γ− CD8+ T cells (**Figure 4G**). We observed that all ICOSexpressing CD8+ T cells were not IFN-γ producers and all IFN-γ producers were not ICOS expressing (**Figure 4G**). Additionally, we found a significant increase in percent ICOS+IFN-γ+ CD8+ T cells in PbA-infected mice as compared to both the CD8+ T cell populations (ICOS−IFN-γ+, ICOS+IFN-γ+) of uninfected mice (4H). Moreover, in PbA-infected mice, the percentage of ICOS+IFN-γ+ CD8+ T cells was significantly higher than the ICOS−IFN-γ+ population (**Figure 4H**). Increased mean fluorescence intensity of IFN-γ in ICOS+ CD8+ T cells than ICOS− depicted that ICOS expressing CD8+ T cells were higher IFN-γ producers (**Figure 4I**). Moreover, ICOS expression in PbA-infected mice was higher on ICOS+IFN-γ+ CD8+ T cells than ICOS+IFN-γ− CD8+ T cells as determined by the increased mean fluorescent intensity (**Figure 4J**). In addition, we found anti-ICOS treatment significantly reduced percentage as well as total ICOS+IFN-γ+ CD8+ T cells (**Figures 4K,L**). Taken together, the above results suggested that during PbA infection, IFN-γ was produced by

Figure 4 | Higher inducible costimulator (ICOS) expression on IFN-γ producing T cells. Negatively isolated CD4+ T cells and single cell splenocyte from *Plasmodium berghei* ANKA infected and uninfected mice (*N* = 5/group) were stimulated by Phorbol 12-myristate 13-acetate/Ionomycin for 5 h with Brefeldin A at last 2.5 h. Stimulated CD4+ T cells were stained with anti-ICOS and anti-IFN-γ after dead cell discriminator staining and Fc blocking. The stimulated single cell splenocytes were stained with anti-TCR-vb, anti-CD8a, anti-ICOS, and IFN-γ. (A) Representative counter plot show co-expression of ICOS and IFN-γ by CD4+ T cells. (B) Percentage increase in ICOS+IFN-γ+ CD4+ T cells. (C) Increased expression of IFN-γ in CD4+ T cells. (D) ICOS expression on IFN-γ+ and IFN-γ− CD4+ T cells. (E) Anti-ICOS treatment reduced percentage ICOS+IFN-γ+ CD4+ T cells. (F) Anti-ICOS treatment depleted total number of ICOS+IFN-γ+ CD4+ T cells after anti-ICOS treatment. (G) Representative counter plot show expression of ICOS and IFN-γ in CD8+ T cells. (H) Percentage IFN-γ positive CD8+ T cells in ICOS+ and ICOS− population. (I) Expression of IFN-γ by ICOS+ and ICOS− CD8+ T cells. (J) ICOS expression on IFN-γ+ and IFN-γ− CD8+ T cells. (K) Anti-ICOS treatment reduced percentage ICOS+IFN-γ+ CD8+ T cells. (L) Anti-ICOS treatment depleted total number of ICOS+IFN-γ+ CD8+ T cells. Data represent one of the two independent experiments. Bar represent mean ± SEM. Statistics: Mann–Whitney test, one-way ANOVA with Bonferroni's multiple comparison test, *p*-value <0.05 (\**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001) considered significant.

ICOS+ and ICOS− CD8+ T cells; however, ICOS+ CD8+ T cells were higher producers of IFN-γ than the ICOS− ones.

#### T-Bet Expressing CD4+ and CD8+ T Cells Have Higher ICOS

The master transcription factor T-bet importantly involved in regulating IFN-γ has been shown to play a paradoxical role during blood-stage malaria infection. As an example, in non-lethal blood stage infection, transcription factor T-bet plays a role in parasite growth wherein T-bet knock out mice infected with *Plasmodium yoelii yoelii* exhibited lower parasitemia (34). Conversely, during PbA infection, while T-bet regulated parasite burden, it also promoted ECM pathology (11). Moreover, T-bet expression critically depends on ICOS expression and its downstream signaling (35). Therefore, we hypothesized that ICOS expressing CD4+ and CD8+ T cells promote PbA parasite growth through T-bet expression. Thus, we analyzed co-expression of ICOS and T-bet by CD4+ and CD8+ T cells during blood stage PbA infection. To ascertain ICOS and T-bet co-expression, we negatively isolated CD4+ T cells and stimulated with PMA/ionomycin for 5 h. The stimulated CD4+ T cells were stained with anti-ICOS and anti-T-bet antibody. We observed low (Lo), intermediate (Int), and high (Hi) ICOS+T-bet+ and ICOS+T-bet− CD4+ T cell population (**Figure 5A**) in *PbA*infected mice. Percent ICOS+T-bet+ (Lo), ICOS+T-bet+ (Int) CD4+ T cells were significantly higher than ICOS+T-bet+(Hi) in uninfected mice. Whereas in PbA infected mice, percent ICOS+T-bet+(Hi) and ICOS+T-bet+(Int) were significantly higher than ICOS+T-bet+(Lo) of *PbA*. Percent ICOS+T-bet+ (Hi,Int,Lo) CD4+ T cells of infected mice was higher than ICOS+T-bet+ (Hi,Int,Lo) of the uninfected (**Figure 5B**). We observed significantly higher T-bet expression in CD4+ T cells from PbA-infected mice as compared to the uninfected (**Figure 5C**). Also, we analyzed ICOS expression on T-bet-positive CD4+ T cells from infected and uninfected mice. We observed significantly higher ICOS expression on T-bet (Int, Hi) positive CD4+ T cells of PbA-infected mice as compared to T-bet+ (Hi,Int,Lo) of uninfected and T-bet (low) of infected mice (**Figure 5D**). ICOS expression was significantly higher in T-bet+ (Hi) than T-bet− CD4+ T cells of PbA-infected mice (**Figure 5E**). Thus, our data suggested that all T-bet expressing CD4+ T cells were ICOS positive, but all ICOS-positive CD4+ T cells were not expressing T-bet.

Similar to CD4+ T cells, we determined the role of ICOS for T-bet expression in CD8+ T cells during PbA infection. The splenocytes were stimulated as described earlier and stained with TCR-Vb, CD8a, ICOS, and T-bet antibodies. After gating TCR-vb and CD8+, we observed four populations; ICOS+T-bet+ (Hi), ICOS+T-bet+ (Lo), ICOS+T-bet− (Hi), and ICOS+T-bet− (Lo) of CD8+ T cells (**Figure 5F**). A significant increase in percent ICOS+T-bet+(Hi) CD8+ T cells was observed as compared to ICOS+T-bet+ (Lo) of infected mice. Also, it was higher than the uninfected (ICOS+T-bet+ (Hi, Lo)) CD8+ T cell population (**Figure 5G**). Expression of T-bet at protein level per cell was significantly higher in ICOS+T-bet+ (Hi) than ICOS+T-bet+ (Lo) in PbA infected mice and ICOS+T-bet+ (Hi), ICOS+T-bet+ (Lo) CD8+ T cells in uninfected mice (**Figure 5H**). Also, we analyzed ICOS expression on T-bet positive cells from uninfected and PbA infected mice and found that ICOS expression was higher on ICOS+T-bet+ (Hi) (**Figure 5I**). Moreover, ICOS expression on T-bet positive CD8+ T cells was higher than T-bet negative cells in infected mice (**Figure 5J**). Thus, our data suggested that all T-bet expressing CD8+ T cells were ICOS positive, but all ICOS positive CD8+ T cells were not expressing T-bet. Taken together, our data indicated that the transcription factor T-bet was expressed by ICOS positive CD4+ as well as CD8+ T cells during PbA infection.

#### DISCUSSION

Most symptoms such as fever, anemia, and cerebral manifestations occur during the asexual erythrocytic or blood-stage malaria parasite infection and have been shown to be strongly associated with IFN-γ production by various cells. The produced IFN-γ is involved in modulation of parasite growth leading to protection as well as pathology (7, 8, 29, 36–38). In experimental allergic encephalomyelitis, IFN-γ-mediated immunopathology is associated with ICOS expression and its signaling (39). Further, impaired IFN-γ production and reduced CD4+ and CD8+ T cell response was observed in ICOS-deficient patients leading to immunodeficiency and autoimmunity (40, 41). Moreover, during non-lethal blood-stage malaria infection, ICOS plays an essential role as a regulator of IFN-γ production and also in distributing parasite-specific CD4+ T cells to both lymphoid and non-lymphoid organs (20, 42). Thus, these studies suggested that ICOS could play a role in IFN-γ production during infectious and autoimmune diseases (43). ICOS signaling regulate transcription factor T-bet, which is also involved in regulation of malaria parasite growth and pathogenesis (11, 34, 35). Taking these studies into consideration, we tried to demonstrate the role of ICOS in parasite growth and lethality through T-bet expression and IFN-γ production, during lethal blood stage of PbA infection. Thus, in our initial experiment, the depletion of ICOS-expressing cells resulted in significantly lowered parasitemia and prolonged mice survival, suggesting that ICOS positive cells are required for PbA growth. Lowered parasitemia due to substantial slowing of parasite maturation, was observed during intraerythrocytic *P. berghei* ANKA infection in Rag(−/−) (lacks T and B cells and mount weak inflammatory response) mice (44). Thus, in our study, we speculate that impaired T cell response leading to diminished parasitemia might be associated with parasite maturation.

stained with anti-ICOS and anti-T-bet antibodies after dead cell staining and Fc blocking. The stimulated single cell splenocytes were stained with anti-TCR-vb, anti-CD8a, anti-ICOS, and T-bet. (A) Representative counter plot show co-expression of ICOS and T-bet by CD4+ T cells. (B) Percentage increase in ICOS+T-bet+ CD4+ T cells. (C) Increased expression of T-bet in ICOS+CD4+ T cells. (D) Increased ICOS expression on T-bet positive CD4+ T cells. (E) ICOS expression on T-bet+ and T-bet− CD4+ T cells. (F) Representative counter plot show expression of ICOS and T-bet in CD8+ T cells. (G) Percentage T-bet positive CD8+ T cells in ICOS+ and ICOS population. (H) Expression of T-bet by ICOS+CD8+ T cells. (I) ICOS Expression on T-bet expressing CD8+ T cells. (J) ICOS expression on T-bet+ and T-bet− CD8+ T cells. Data represent one of the two independent experiments. Bar represent mean ± SEM. Statistics: Mann–Whitney test, one-way ANOVA with Bonferroni's multiple comparison test, *p-*value < 0.05 (\**p* < 0.05; \*\**p* < 0.01; \*\*\**p* < 0.001) considered significant.

Further, reduced percent weight loss (a marker of pathology) in anti-ICOS treated mice as compared to untreated suggested that ICOS expressing cells are also involved in PbA pathology. The role of ICOS in parasite growth and pathology, thus, led us to further characterize its expression during PbA infection.

During blood-stage malarial infection, helper CD4+ and cytotoxic CD8+ T cells contribute to both protection and pathology (45–48). For activation of both T cells, ICOS expression, and its downstream signaling is critical (21, 40, 41, 49, 50). Consistent with this, in our experiment during blood stage PbA infection, ICOS may be involved in the activation of splenic CD4+ and CD8+ T cells as both these cells express significantly higher ICOS. A study involving depletion of these T cells with antibodies prevented pathology of experimental cerebral malaria (5). Similarly, in this study, upon anti-ICOS administration, depletion of ICOS expressing CD4+ and CD8+ T cells led to lower parasitemia, longer survival, and ameliorated pathology. Thus our study suggested that depletion of ICOS expressing CD4+ and CD8+ T cell was sufficient to control parasite growth and lethality as compared to total T cell depletion.

IFN-γ has been known to play a critical role in lethality during *P. berghei* ANKA infection (9) and consistent with these earlier findings, increased plasma IFN-γ production in our study may be correlated with its role in PbA pathogenesis. Lowered parasitemia, reduced IFN-γ, and increased survival upon anti-ICOS treatment suggest that ICOS plays a role in PbA growth and lethality through IFN-γ production. Further, characterization of CD4+ and CD8+ T cells for IFN-γ production showed that both these cells were indeed IFN-γ producers. Moreover, depletion of ICOS-expressing CD4+ and CD8+ T cells and reduction of plasma IFN-γ upon anti-ICOS treatment suggested that in blood stage of PbA infection, ICOS expressing CD4+ and CD8+ T cells were the major producers of IFN-γ. Further, earlier studies also demonstrated the role of ICOS in modulating cytokines of naïve and activated T cells. Moreover, during *Mycobacterium tuberculosis*, ICOS signaling controlled antigen-specific protective IFN-γ production and the produced IFN-γ, in turn, regulated ICOS expression (24). Similarly, in our study, higher ICOS expression on IFN-γ producing T cells and higher IFN-γ production in ICOS expressing T cells suggested that both ICOS and IFN-γ could regulate each other.

On similar line master transcription factor T-bet has shown to be involved in malaria parasite growth and pathogenesis. For example, lower parasite burden was observed in T-bet knock out (Tbx21−/−) mice than T-bet wild-type mice suggesting that T-bet promotes malaria parasite growth (34). In another study, T-bet regulated PbA growth but has also been shown to promote the pathogenesis of ECM (11). Interestingly, ICOS expression and its downstream signaling have been shown to be critical for T-bet expression (35). In our study, ICOS expressing CD4+ and CD8+

T cells, which were involved in PbA growth has also been shown to co-express T-bet. Our results also demonstrated that ICOS and T-bet might regulate each other's expression as correlated with higher ICOS expression on T-bet positive cells and *vice versa*.

The malaria parasite has evolved with multiple mechanisms such as changing shapes, motility, metabolic requirement, and immune evasion strategies to survive inside the host (51). In correlation with this, we demonstrated that malaria parasite utilizes ICOS-expressing T cells for their growth through T-bet expression and IFN-γ production. Other studies have depicted that altering ICOS signaling leads to modulated T cell response, which in turn is involved in infection clearance, tumor regression, and ameliorated pathology (52–54). Thus, modulation of ICOS expression and its signaling might be helpful in altering parasite growth and lethality, which might be valuable in preventing severe malaria in humans.

#### ETHICS STATEMENT

The use of animals and animal procedures were approved by the Institutional Animal Ethics Committee, Institute of Life Sciences, Bhubaneswar, India in accordance with the "Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)."

#### AUTHOR CONTRIBUTIONS

GJ conceived the study and discussed with SD. GJ planned and performed experiments, analyzed data, and drafted manuscript.

#### REFERENCES

SS, GB, SS, and PB helped to perform components of some of the experiments. SD arranged the grants for the study and edited manuscript. All authors contributed intellectual content and approved it for publication.

#### ACKNOWLEDGMENTS

We thank MR4 for providing us with malaria parasite *P. berghei ANKA* contributed by Mark Weiser M. Hollingdale. We thank Dr. Brian deSouza (London School of Hygiene and Tropical Medicine London) and Dr. Suchitra Mohanty for their critical suggestions. We also thank Dr. Ajay Parida (Director), Dr. B. Ravindran (Former Director), Dr. Durg V. Singh, Dr. Vivek Rai, and Dr. Madhumita Panda from Institute of Life Sciences, Bhubaneswar, for their kind support. We are thankful to the ILS animal facility Staff for supplying mice as required.

#### FUNDING

This work was supported in part by core funds from the Institute of Life Sciences and from the Basic Research in Modern Biology Task Force, Department of Biotechnology, Govt. of India (Grant number-BT/PR11259/BRB/10/646/2008 and BT/ PR14425/BRB/10/839/2010). GJ was supported with Senior Research Fellowship of Council of Scientific and Industrial Research (CSIR).

control during blood-stage *Plasmodium* infection. *Eur J Immunol* (2011) 41(9): 2688–98. doi:10.1002/eji.201141539

polarization after virus and parasite infection. *J Exp Med* (2000) 192(1):53–61. doi:10.1084/jem.192.1.53

**Conflict of Interest Statement:** The authors declare that the research conducted have no commercial or financial involvement that could be considered as potential conflict of interest.

*Copyright © 2018 Jogdand, Sengupta, Bhattacharya, Singh, Barik and Devadas. 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) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Therapies to Suppress **β** Cell Autoimmunity in Type 1 Diabetes

*Charles J. Kroger1,2, Matthew Clark1,2, Qi Ke1,2 and Roland M. Tisch1,2\**

Keywords: autoimmunity, diabetes, immunoregulation, immunotherapy

*1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, 2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States*

Type 1 diabetes (T1D) is an autoimmune disease that is generally considered to be T celldriven. Accordingly, most strategies of immunotherapy for T1D prevention and treatment in the clinic have targeted the T cell compartment. To date, however, immunotherapy has had only limited clinical success. Although certain immunotherapies have promoted a protective effect, efficacy is often short-term and acquired immunity may be impacted. This has led to the consideration of combining different approaches with the goal of achieving a synergistic therapeutic response. In this review, we will discuss the status of various T1D therapeutic strategies tested in the clinic, as well as possible combinatorial approaches to restore β cell tolerance.

#### *Edited by:*

*Gustavo Javier Martinez, Rosalind Franklin University of Medicine and Science, United States*

#### *Reviewed by:*

*Abdel Rahim A. Hamad, Johns Hopkins University, United States Maja Wallberg, University of Cambridge, United Kingdom Rachel Friedman, National Jewish Health, United States*

#### *\*Correspondence:*

*Roland M. Tisch<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology*

> *Received: 14 June 2018 Accepted: 31 July 2018 Published: 16 August 2018*

#### *Citation:*

*Kroger CJ, Clark M, Ke Q and Tisch RM (2018) Therapies to Suppress β Cell Autoimmunity in Type 1 Diabetes. Front. Immunol. 9:1891. doi: 10.3389/fimmu.2018.01891*

# INTRODUCTION

Type 1 diabetes (T1D) is an autoimmune disease marked by the dysfunction and/or destruction of the insulin-producing β cells found in the pancreatic islets of Langerhans (1–4). T1D most often arises in children but also develops in adults. To compensate for impaired β cell function, T1D individuals require daily insulin therapy. Despite insulin administration, however, establishing optimal glycemic control long-term is often problematic resulting in a number of complications, including retinopathy, nephropathy, vasculopathy, and neuropathy. This morbidity coupled with an increasing incidence of T1D world-wide, underscores an urgent need for effective immunotherapies for T1D prevention and treatment.

The goal of an immunotherapy is threefold: (1) selectively suppress ongoing autoimmunity, (2) reestablish self-tolerance long-term, and (3) preserve acquired immunity. This review will focus on the different strategies of immunotherapy being tested experimentally and in the clinic to suppress β cell autoimmunity, and prevent clinical onset and/or treat T1D.

# IMMUNOPATHOLOGY OF T1D

The loss of β cell tolerance involves both genetic and ill-defined environmental factors (5–16). A strong genetic association with specific human leukocyte antigen haplotypes, coupled with several variants of genes expressed by β cells, T cells, and other immune effectors underscore the complexity of the autoimmune response of T1D (17–23). β cell autoimmunity is typically viewed as a chronic inflammatory response characterized by the progressive infiltration of the pancreatic islets with various immune effectors (24). In the nonobese diabetic (NOD) mouse, a spontaneous model of T1D, islet infiltration is initiated at an early age by macrophages and dendritic cells (DC), CD4+ and CD8+ T cells, and B cells. This insulitis initially exhibits benign properties with little effect on β cell viability or function. However, at a late preclinical stage, an ill-defined qualitative change occurs within the islet infiltrate, which promotes efficient β cell destruction leading to the onset of overt diabetes.

**33**

In NOD mice and other rodent models of T1D, pathogenic β cell-specific CD4+ and CD8+ effector T cells (Teff) are essential drivers of autoimmunity. Diabetogenic CD4+ and CD8+ T cells target several β cell autoantigens and related peptide epitopes, including proinsulin/insulin, glutamic acid decarboxylase 65 (GAD65), chromogranin A (ChgA), zinc transporter 8 (ZnT8), glucose-6-phosphatase catalytic subunit-related protein (IGRP), and hybrid insulin peptides (HIPs) among others (25–34). It is thought that early in the disease process only few β cell autoantigens and epitopes are targeted by T cells. However, over time additional epitopes within a given autoantigen as well as new autoantigens are recognized. Furthermore, chronic inflammation negatively affects β cells in part by generating neo-autoantigens, such as HIPs, *via* post-translational modification events (32–39). These neo-autoantigens further diversify the diabetogenic response. Together this epitope spread effectively amplifies the β cell-specific T cell response (28, 40–42).

Analyses of human cadaveric T1D pancreases have also demonstrated islet infiltrates consisting of CD8+ T cells and macrophages, and to a lesser extent CD4+ T cells, and B cells (29, 31, 43–52). However, T1D pancreases have been reported that lack T cell infiltrates suggesting that the immunopathology of human T1D is heterogeneous (53, 54). The prevalence of T cell-independent subsets of T1D is unclear, and thought to be primarily associated with adult T1D onset. On the other hand, evidence indicates that the rapid and severe T1D that develops in children and adolescents is T cell-mediated (44). For instance, recent reports show that childhood onset is marked by a broader and more aggressive β cell-specific T cell response compared to adult T1D (29, 31, 43–52, 55–57). Multiple β cell autoantigens are recognized by human CD4+ and CD8+ T cells found in peripheral blood, as well as the islets of T1D subjects; many of which are also targeted in the NOD mouse diabetogenic response (e.g., insulin, GAD65, IGRP, and ZnT8) (4, 25, 28, 57).

Pathogenic β cell-specific CD4+ and CD8+ Teff in NOD and human T1D typically exhibit a type 1 or T helper 1 (Th1) phenotype marked by IFNγ production (47, 58, 59). IL-17 producing CD4+ Th17 cells have also been implicated in mediating β cell destruction (60–62). Differentiation and expansion of pathogenic Teff are in part attributed to aberrant peripheral immunoregulation (63–68). An impaired pool of thymic-derived FoxP3-expressing immunoregulatory T cells (Foxp3+Treg) has been linked to T1D (68–70). In general, Foxp3+Treg play an essential role in maintaining peripheral self-tolerance through cytokine and contact-dependent mechanisms of suppression (71). Decreased survival of islet-resident Foxp3+Treg is thought to be a key factor in promoting the progression from benign to pathogenic insulitis in NOD mice (69). Failure to maintain islet Foxp3+Treg numbers in NOD mice is due to insufficient local levels of IL-2, a critical cytokine needed for Foxp3+Treg survival, fitness, and function (69, 72–74). FOXP3+Treg from T1D subjects have defective IL-2 receptor (R) signaling which limits fitness and function of FOXP3+Treg (66, 75). Additionally, production of the proinflammatory cytokine IL-21, which is critical for T1D development, can inhibit IL-2 expression by T cells which negatively impacts Foxp3+Treg viability and function (76). Human T1D is also marked by deficiencies in non-FoxP3-expressing adaptive (a) Treg. For example, the frequency of β cell-specific IL-10-secreting Tr1 cells is reduced in T1D versus healthy subjects (77–79). In both NOD and human T1D, Teff exhibit a reduced sensitivity to Treg-mediated suppression, which further permits expansion of the diabetogenic Teff pool (63, 64).

Dysregulation among antigen-presenting cells (APC), such as DC, macrophages, and B cells, has also been reported to contribute to T1D (80–85). Although detection of autoantibodies is a key indicator of β cell autoimmunity, B cells are thought to play a critical role in the development of T1D by functioning primarily as an APC (86–88). APC exhibiting proinflammatory properties also skew differentiation of naïve β cell-specific T cells toward pathogenic Teff, as well as amplify islet inflammation and β cell destruction. For instance cytokines, such as IFNγ, TNFα, and IL-1β secreted by islet APC are cytotoxic to β cells *in vitro* (89). The culmination of the adaptive and innate effector immune response within the islets results in β cell destruction/dysfunction and elevated blood glucose levels (**Figure 1**).

Aberrant peripheral immunoregulation has been the focus of most T1D immunotherapies. Different strategies have attempted to selectively suppress the pathogenic proinflammatory events that affect β cells, while reestablishing protective immunoregulation that persists long-term without altering acquired immunity (**Figure 1**). Achieving these goals in the clinic, however, has proven to be highly challenging.

#### IMMUNOTHERAPY OF T1D

The progression of T1D affords three windows of therapeutic intervention to alter disease outcome (90, 91). The first is during the prodromal stage of T1D, which may persist for a number of years. At-risk individuals with ongoing β cell autoimmunity, identified by serum autoantibodies specific for various β cell autoantigens, are candidates for prevention of clinical onset of T1D (90–92). The second window of intervention is at the time of clinical onset. The goal here is to suppress β cell autoimmunity, rescue residual β cell mass, and ideally reverse clinical diabetes. Early studies provided proof of principle that sufficient β cell mass exists at the time of diagnosis to reverse diabetes (93, 94). Administration of the immunosuppressive drug cyclosporine A (CsA) induces remission in new onset children. However, CsA results in severe kidney toxicity, and once therapy is halted, patients develop recurrent diabetes (95, 96). The third window of intervention is in long-standing T1D patients to protect residual β cell function. Evidence indicates that after T1D onset low but sustained C-peptide levels are observed (93, 94). Therefore, maintenance of a small amount of functional β mass may aid in the control of glycemia as well as limit morbidity.

Two general approaches of immunotherapy have been tested experimentally and in the clinic for the prevention and treatment of T1D. The first approach entails manipulating the autoimmune response independent of β cell specificity. Typically, antibodies (Ab) targeting specific immune effectors or effector molecules have been employed for this approach. The second approach makes use of β cell antigen-specific strategies. Here, β cell autoantigens or corresponding peptides are administered under various conditions. In the following sections, we will discuss

Figure 1 | The progression and treatment of β cell autoimmunity. *Top panel*: In general, overt diabetes results from the gradual loss of functional insulin-producing β cells due to the inflammatory environment driven by infiltrating self-reactive T cells and antigen-presenting cells (APC). Although β cell-specific T cell clones are detected in both healthy and type 1 diabetes (T1D) susceptible individuals, a number of factors promote T1D development in the latter population. Decreased efficiency of negative selection in the thymus allows for the increased escape of β cell-specific T cell clones into the periphery. In the periphery, β cell-specific T cells are stimulated in the pancreatic lymph nodes (PLN) by APC derived from the islets, leading to effector T cells (Teff) differentiation due to suboptimal Foxp3+Treg suppression. These pathogenic Teff then infiltrate the islets and drive inflammation leading to reduced β cell function and/or survival. *Bottom panel:* Several distinct therapeutic interventions have been tested for the prevention or treatment of T1D. The major approaches have included: administration of β cell autoantigens, expansion of Foxp3+Treg, Ab therapies that alter T cell responses, therapeutic reagents that tolerize APC function, neutralization of proinflammatory molecules, or treatments that expand or enhance β cell function and/or survival. Some of these approaches have had clinical, albeit limited, success. Future therapeutic interventions should look toward refinement in the specificity of these treatments, and the development of combinatorial therapies targeting multiple arms of the immune system, as T1D is a multi-pronged autoimmune disease.

different strategies of immunotherapy that are included in these two general approaches, highlighting related strengths and weaknesses (**Figure 1**).

#### ANTIGEN-INDEPENDENT IMMUNOTHERAPIES

Ab and cytokines have been administered to modulate or block the function of immune effector cells and/or molecules. The general approach is advantageous, since pathogenic effectors can be targeted *en masse*, often resulting in rapid and robust outcomes in experimental settings. However, the limited specificity of these therapeutics can lead to unwanted systemic effects impacting acquired immunity for instance. Nevertheless, a sizable body of work has demonstrated clinical efficacy for certain Ab therapies, whereas promising early results are being seen with cytokine- and cell-based strategies.

#### Antibody-Based Therapies

Arguably, the most effective strategy to clinically alter T1D progression has been to target diabetogenic Teff with anti-CD3 monoclonal Ab (mAb) (97, 98). In new onset NOD mice anti-CD3 mAb therapy reverses diabetes in part by driving apoptosis of isletinfiltrating Teff. Engulfment of the apoptotic T cells induces TGFβ secretion by APC that promotes differentiation of Foxp3+Treg. Once established this pool of induced (i) Foxp3+Treg mediate long-lasting β cell tolerance (99–101). In addition, peripheral Teff exhibit reduced IFNγ production, and increased markers of exhaustion and anergy (102). Interestingly, anti-CD3 mAb therapy given at a preclinical stage fails to prevent diabetes onset in NOD mice (98, 103). This indicates that a given immunotherapy may be effective only at particular stages of T1D progression.

For clinical trials, humanized anti-CD3 mAb hOKT3γ1 (Ala-Ala) (teplizumab) or CCHAglyCD3 (otelixizumab) have been engineered to prevent binding to Fc receptor, and minimize proinflammatory cytokine release by APC (97, 100, 104–112). Anti-CD3 mAb therapy administered to new onset T1D patients reduces the rate of loss of β cell function in the majority of individuals (113). The mechanism of protection is ill-defined, although expansion of CD8+ Treg and CD8+ Teff exhibiting an exhausted phenotype has been reported (109). Despite this efficacy, diabetes reversal is not achieved, and protection of residual β cell mass is short-term, lasting up to 4 years in some individuals (113). Furthermore, anti-CD3 mAb binding can activate T cells resulting in cytokine release and unwanted inflammation, albeit transient. Moreover, the transient depletion of T cells systemically by anti-CD3 mAb is linked to recurrent viral infections (100, 104). Nevertheless, these findings provide direct evidence that progression of β cell autoimmunity can be modulated by targeting T cells. However, further refinement of the approach is required to enhance β cell protection while minimizing effects on acquired immunity. This may in part be achieved by using anti-CD3 mAb in combination with other therapeutics (114–116). For instance, treatment of NOD mice with anti-CD3 mAb and the β cell trophic growth factor prolactin, increases both diabetes reversal and β cell proliferation relative to anti-CD3 mAb alone (116).

The aim of Ab-based combinatorial strategies is to establish a synergistic or additive effect to enhance both efficacy and safety. An increased tolerogenic response may permit reduced dosing and, therefore, minimize unwanted complications with a given therapeutic. One combinatorial strategy being tested in the clinic is the application of antithymocyte globulin (ATG) and granulocyte colony stimulating factor (G-CSF). Low dose ATG induces apoptosis and transiently depletes T cells, whereas G-CSF promotes mobilization of aTreg and induction of tolerogenic DC. In preclinical studies, ATG alone induces ~30% remission in newly diabetic NOD mice, whereas the combination of ATG and G-CSF increases diabetes reversal >twofold (117). Initial phase I clinical results for low dose ATG plus G-CSF therapy given to recent onset T1D patients indicate that β cell function can be maintained up to 2 years in a group of responders (118). This protective effect correlates with quantitative and qualitative changes in the conventional T cell pool, coupled with an increase in the frequency of FOXP3+Treg (118).

Although most T1D immunotherapies have targeted T cells, success has also been demonstrated by altering the B cell response in various autoimmune diseases including T1D, both in mice and humans (88, 119, 120). As previously mentioned, serum autoantibodies indicate B cells also target β cell antigens, including GAD65 and insulin, which serve as a biomarker for individuals with an increased risk for developing T1D (90–92). NOD mice lacking B cells are protected from insulitis and diabetes onset (86, 87). Several molecules, such as CD20, CD22, BAFF, and BCMA have been targeted to modulate the B cell pool (121–125). In different murine models of T1D, anti-CD20 mAb suppresses the progression of β cell autoimmunity as well as reverses diabetes onset, by promoting Foxp3+Treg and regulatory B cell populations (121, 122). In recent onset T1D patients, anti-CD20 mAb (Rituximab), which has also been used in the treatment of rheumatoid arthritis (RA), transiently depleted B cells and prolonged β cell function (126, 127). However, this protective effect was only transient, diminishing after 1 year, suggesting brief deletion of B cells alone is not sufficient to restore β cell tolerance.

The depleting effects of a given Ab make dosing problematic, as well as limit the frequency of treatments. On the other hand, nondepleting (ND) Ab provide a strategy to modulate the function of Teff and other immune cells without systemic cell depletion. Human IgG4 is intrinsically ND due to a decreased capacity to fix complement and bind to Fc receptor, and, therefore, is well suited for this purpose. Other IgG isotypes can be engineered accordingly to establish a ND property. A striking example of altering the activity of β cell-specific Teff is observed with the application of ND anti-CD4 and -CD8α Ab. A shortcourse of ND anti-CD4 and -CD8α Ab induces remission in the majority of new onset diabetic NOD mice without affecting systemic T cell numbers (128–131). Induction of remission is due to Ab-mediated crosslinking of the CD4 and CD8 molecules, which reduces TCR signaling as well as upregulates additional signaling pathways (128, 131–133). The result is suppressed expression of proinflammatory cytokines by Teff combined with the induction of a promigratory phenotype that drives pancreatic egress of Teff (128, 133).

ND Ab and immunoglobulin (Ig) fusion molecules can also serve to block the function of key surface effector molecules. For instance, CTLA-4-Ig (Abatacept)-mediated blockade of the CD80 and CD86 co-stimulatory molecules inhibits APC function and alters the Teff pool in recent onset T1D patients. Abatacept therapy administered monthly for 24 months extends β cell function up to 30 months (134, 135). Examination of peripheral blood shows a decrease in the central memory CD4+ T cell population with a concomitant increase in naïve T cells in Abatacept-treated individuals, supporting the notion that Teff expansion is limited (135).

Inhibiting cytotoxic pathways with ND blocking Ab offer another option to protect β cell viability. NOD mice deficient in Fas lack islet-infiltrating effector cells and fail to develop T1D (136, 137). Ab blockade of FasL between 2 and 4 weeks of age prevents T1D in NOD mice. Interestingly, FasL blockade results in the presence of protective IL-10-producing B cells in the pancreas of NOD mice after treatment (138). These results suggest the targeting the Fas–FasL pathway may be a viable therapeutic avenue for the treatment of clinical T1D.

In addition to directly targeting immune effector cells, Ab or Ig-fusion molecules have been used to neutralize secreted effector molecules, such as proinflammatory cytokines. Neutralization of TNFα with several antagonists including mAb (infliximab, adalimumab) and a recombinant TNFα receptor fusion protein, etanercept, have been widely used to treat the autoimmunity driving RA (139). Although TNFα is directly cytotoxic to β cells, its role in the pathogenesis of T1D is controversial. In NOD mice, for instance, TNFα accelerates the progression of β cell autoimmunity when given at a young but not older age (140, 141). In a 24-week clinical study, anti-TNFα therapy in human T1D patients was seen to reduce levels of glycated hemoglobin A1C (HbA1c), a marker indicative of blood glucose values over a 3-month period, as well as increase C-peptide levels suggesting preservation of β cell function (142). In a case report, treatment with infliximab was also found to reduce HbA1c levels, increase insulin secretion, and alleviate insulin resistance in a T1D patient also diagnosed with Crohn's disease (143). Anti-TNFα therapy, however, fails to prevent the development of T1D in at-risk patients, and may in fact accelerate disease progression (144). These results further underscore how efficacy (or lack of) is dependent on the stage of disease progression at which a given therapeutic is administered.

IL-21 is an appealing cytokine to target and suppress β cell autoimmunity. IL-21 is chiefly produced by T follicular helper cells (Tfh) and Th17 cells, and has diverse roles that include regulating B cell and CD8+ Teff function (145–147). The emerging role for both Tfh and Th17 cells in the progression of T1D, and the protective phenotype seen in NOD mice deficient of IL-21 suggests that neutralizing human IL-21 is a promising approach (60, 148–152). Indeed, neutralization of IL-21 with an IL-21R-Ig prevents diabetes in NOD mice (153).

#### Cytokine-Based Immunotherapies

Administration of cytokines offers an approach to reestablish peripheral immunoregulation and β cell tolerance. One such strategy currently being investigated is administration of low dose IL-2 (65, 154–156). Conventional CD4+ and CD8+ T cells but not Foxp3+Treg are producers of IL-2, which plays a central role in driving both pro- and anti-inflammatory responses (157, 158). IL-2 for instance is needed for conventional T cell activation and expansion, and IL-2 drives proinflammatory responses by other immune effectors (158). IL-2 also plays an essential role in Foxp3+ Treg survival, expansion, and suppressor function (159–161). Constitutive expression of the high affinity IL-2R (CD25) allows Foxp3+Treg to out-compete conventional T cells for limiting amounts of IL-2. The latter provides rationale for using low dose IL-2 to preferentially affect Foxp3+Treg versus conventional T cells (154). Indeed, low dose IL-2 has been effective in the clinic for the treatment of graft-versus-host disease, and systemic vasculitis (162, 163). In NOD mice, low-dose IL-2 prevents clinical onset and reverses diabetes *via* an expanded pool of Foxp3+ Treg in the islets and draining pancreatic lymph nodes (69, 164, 165). Similarly, low-dose IL-2 in combination with rapamycin in recent onset T1D patients increases the frequency of Foxp3+Treg in blood (166). However, these patients also exhibit an accelerated rate of β cell loss (166), suggesting an enhanced pathogenic response, and highlighting the key problem of administering a cytokine with pleiotropic effects (167, 168).

Different strategies are being developed to enhance the efficacy of IL-2 (and other cytokines), while avoiding unwanted systemic effects (169, 170). One approach is to promote selective binding of IL-2 to Foxp3+Treg *via* IL-2-Ab complexes (IL-2C) (170–172). Targeting particular epitopes on IL-2 with anti-IL-2 Ab can favor binding to the high affinity IL-2R constitutively expressed by Foxp3+Treg (173). Administration of IL-2C readily expands Foxp3+Treg in mice and prevents autoimmunity (170–172). While promising, polyclonal expansion of Foxp3+Treg by IL-2C may compromise protective immunity against pathogens.

An additional strategy to minimize the systemic effects of IL-2 while expanding β cell-specific Foxp3+Treg is to target cytokine expression to β cells *in vivo*. Adeno-associated virus (AAV) vectors have been used to deliver and target expression of an insulin promoter-driven IL-2 transgene (AAViP-IL2) to β cells *in vivo* (72–74, 174). In general, AAV vectors are appealing for *in vivo* gene delivery due to limited immunogenicity, lack of integration into the genome, and efficient transduction of non-proliferating cells (175). Notably, treatment of NOD mice at a late preclinical T1D stage with AAViP-IL2 results in the expansion of islet-resident Foxp3+Treg, suppression of β cell-specific Teff, and prevention of diabetes onset (72–74). AAV vectors can be further exploited by co-delivering genes encoding other anti-inflammatory cytokines (e.g., IL-10, TGFβ1, and IL-35) and/or pro-survival proteins to enhance both the tolerogenic effect and maintenance of β cell mass *in vivo* (72, 174, 176–179).

#### Foxp3**+**Treg-Mediated Therapy

An alternative strategy to manipulate the Foxp3+Treg population *in vivo* is to transfer Foxp3+Treg that have been expanded *in vitro* (180–182). The approach is effective in preventing diabetes onset in NOD mice (183). However, *in vitro* expansion of a homogeneous and stable pool of Foxp3+Treg, particularly when starting from relatively few cells, has been technically difficult. Expansion protocols are being devised using drugs such as rapamycin to prevent outgrowth of Teff, as well as DNA methyltransferase and histone deacetylase inhibitors to enhance Foxp3+Treg stability (184, 185). Despite these hurdles, clinical studies have shown Foxp3+Treg therapy is well tolerated and therapeutic efficacy is observed for various diseases (180, 181). Furthermore, a phase I trial has demonstrated that *in vitro*-expanded Foxp3+Treg persist long-term and exhibit a stable phenotype when transferred back into T1D subjects (180). Notably, loss of C peptide is also limited in some patients. These results provide justification for additional clinical studies to directly assess the efficacy of Foxp3+Treg transfer for T1D treatment.

#### Microbiome Interventions in T1D

In recent years, the importance of the gut microbiota in maintaining the normal function of the immune system has emerged (186). Reports have documented that changes in gut microbiota can markedly influence T1D development, and that differences in the colonizing microflora contributes to variations in T1D onset among NOD mouse colonies (187, 188). Toll-like receptors (TLRs) play a crucial role in the innate immune system's recognition of common bacterial and viral components. TLR signaling triggers APC maturation and production of proinflammatory mediators (189). One of the major TLR signaling adaptor molecules driving APC maturation is MyD88, which activates several downstream signaling pathways (190). NOD mice lacking MyD88 expression and housed under normal specific-pathogenfree conditions exhibit a reduced T1D incidence. In contrast, MyD88-deficient NOD mice housed in germ-free conditions develop robust diabetes (16). Interestingly sex differences in the gut microbiota also influence T1D susceptibility. For example, young female NOD mice receiving gut microbiota from male NOD mice exhibit a reduced T1D incidence (191). These results demonstrate that the gut microbiota can have a protective role against autoimmunity, and specifically T1D. Furthermore, differences in the biodiversity of the gut microbiome are seen between T1D patients and healthy individuals (192–196). Similarly, the microbiome within the gut of T1D infants fails to diversify during development in comparison to healthy individuals, potentially due to hyperglycemia (197). Here, changes in the production of microbial metabolites such as short-chain fatty acids found in the colonic lumen and peripheral blood are thought to impact immunoregulatory networks. Indeed, a recent study showed that diets that enhance production of acetate and butyrate by the gut microbiome, effectively prevent diabetes in NOD mice (198). Increased acetate is seen to reduce β cell-specific Teff, whereas elevated butyrate expands Foxp3+Treg. Together these results indicate an association between modified gut microbiota and compromised self-tolerance. Manipulating the gut microbiota may prove to be an effective adjuvant therapy to limit the progression of β cell autoimmunity in at-risk individuals.

#### ANTIGEN-DEPENDENT IMMUNOTHERAPIES

The goal of antigen-based therapies is to selectively tolerize the autoreactive Teff pool and induce or expand autoantigen-specific Treg (199–202). The approach is appealing, since β cell antigen vaccination is expected to have no effect on acquired immunity. Teff are tolerized *via* whole antigen or peptide by various mechanisms, including: (i) clonal anergy, (ii) clonal deletion, or (iii) exhaustion. This has classically been achieved by administration of high doses of soluble antigen or peptide. On the other hand, antigen vaccination has been used to induce differentiation of naïve β cell-specific T cells into aTreg, including iFoxp3+Treg. Depending on the conditions of antigen vaccination, distinct subsets of aTreg can be induced. Expanding the aTreg pool is advantageous, since local cytokine-mediated suppression is independent of Teff antigen-specificity; this bystander-mediated suppression also can downregulate the activity of proinflammatory APC. Success of either tolerizing Teff or inducing/expanding Treg is dictated by multiple factors including dose, the frequency, and route of antigen vaccination. Also important is the context in which the antigen is delivered. Antigen administration in the context of an adjuvant, microparticles, or DC can significantly alter the nature of the T cell response. The most critical factor determining efficacy, however, is the identity of the autoantigen (or peptide) used for vaccination. To effectively block the progression of β cell autoimmunity, prevalent, or immunodominant Teff clonotypes need to be targeted. Ongoing efforts defining the specificity of islet-resident Teff are expected to identify immunodominant Teff clonotypes (31). Furthermore, promising antigen-based immunotherapies are exploiting advancements in targeted drug delivery systems to effectively tolerize Teff and promote Treg-mediated suppression.

# **β** Cell-Autoantigen Vaccination