Invariant Natural Killer T cells. Student Presenter Jessy Slota Lecturer Dr. Catherine Card MMIC 7050 written report Presentation on October 23, 2018

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1 Invariant Natural Killer T cells Student Presenter Jessy Slota Lecturer Dr. Catherine Card MMIC 7050 written report Presentation on October 23, 2018

2 Overview of invariant Natural Killer T (inkt) cells Natural killer T (NKT) cells are a specialized subset of T cells that recognize lipid antigens presented on the non-classical MHC I-like molecule, CD1d. This contrasts with classical MHC restricted T cells, which recognize peptide antigens loaded on MHC I or MHC II. NKT cells possess the same cytotoxic machinery as CD8 + T cells, and therefore can kill target cells (1). However, this does not seem to be their main effector mechanism (2), as NKT cells rapidly release copious amounts of immuno-regulatory cytokines upon activation, allowing them to shape the immune response in the same way as CD4 + T helper cells (3). The term Natural Killer T cells was coined in 1995, to describe a subset of T cells that expressed NK1.1, a receptor associated with natural killer cells (1, 4). The suitability of this nomenclature is disputed because not all CD1d restricted T cells express NK1.1, and those that do only express this receptor following maturation, yet downregulate it upon activation (reviewed in (1)). Three types of NKT cells exist: Type I or Invariant NKT cells, type II or Diverse NKT cells and NKT-like cells (1, 3). Invariant NKT (inkt) cells are the best understood, owing in part to the discovery of α-galactosyl Ceramide (α- GalCer). α-galcer is a glycolipid antigen that strongly activates inkt cells; it was originally discovered in a marine sponge (5) and has been used as a tool to identify and stimulate NKT cells (3). The following discussion is focused entirely on inkt cells. inkt cells are referred to as invariant because they possess a limited repertoire of T cell receptors (3). T cell receptors (TCRs) consist of two chains (α and β chain), each of which contains a variable region that forms the antigen binding site of the receptor. TCRs are randomly generated during the development of thymocytes through a process called V(D)J recombination (6). This involves somatic recombination events which work to form the variable regions of the T cell receptor (or antibodies) from 2 or 3 randomly selected gene segments (named V, D and J segments; the D segment is only present in one of the 2 chains). Through these recombination events, billions of possible antigen receptors can be generated, with each one recognizing a unique antigen. In the case of inkt cells, the TCR is formed from a specific TCR α chain (Vα14Jα18 in mice and Vα24Jα18 in humans) paired with a couple possible β chains (3). The mode of antigen recognition by inkt cells is different than for MHC restricted T cells, and this may explain the requirement of the invariant TCR expressed by inkt cells. Only the α chain of the inkt cell TCR (i.e. Vα14Jα18) makes contacts with the lipid antigen loaded on CD1d (7). While both the TCR α and β chains contact CD1d, and these interactions are responsible for most of the binding affinity (3, 8), the β chain makes no contact with the lipid antigen. The TCR of inkt cells can recognize both microbial and mammalian host lipid antigens. Many if these lipid antigens are glycolipids, with the glyosidic linkage between lipid and sugar existing in either an α or β conformation. Although many of the microbial antigens that strongly activate inkt cells are α-linked glycolipids, mammalian β-linked glycolipids (mammals do not have α-linked glycolipids) have also been known to activate inkt cells (reviewed in (3)). The mode of recognition of lipid antigens by the inkt cell TCR is the same for all antigenic lipids. This is because lipids can either fit, or be induced to fit the TCR binding site, and the structure of the TCR is the same regardless. However, the change in structure of the antigen that results from an induced fit incurs an energetic penalty that weakens the binding interaction, and limits the TCR signal (3, 9). Therefore, the inkt cell TCR can bind and recognize various endogenous and microbial lipid

3 antigens, and the strength of the binding interaction will in part determine whether or not the inkt cell gets activated. In addition to a TCR signal, cytokine signals are also required for the productive activation of inkt cells (3, 10). In the case of a strong TCR signal, only a weak cytokine signal is required for the activation of inkt cells. However, a strong cytokine signal can overcome a weak TCR signal leading to the activation of inkt cells in the absence of a highly antigenic microbial lipid (3). In this case, the engagement of Pattern Recognition Receptors (PRRs) on Antigen Presenting Cells (APCs) results in the enhanced secretion of cytokines by the APC which can work to activate inkt cells (3). Therefore, inkt cells can take advantage of the innate immune receptor system when there is an absence of strongly activating microbial antigens. inkt cells can be thought of as having an innate-like mode of activation (3): they have an invariant antigen receptor which can respond to an assortment of different lipids, and they can take advantage of the innate receptor system in the case of cytokine driven activation. This allows inkt cells to respond to various danger signals instead of specific antigens. inkt cells mature in the thymus from the same pool of CD4/CD8 double positive thymocytes as CD4 and CD8 T cells, however they arise from a distinct developmental pathway (1). Double positive thymocytes express CD1d loaded with self-lipid antigens and unlike for MHC restricted T cells, these thymocytes are the cells that select for inkt cells based on their ability to bind CD1d (3). The development of inkt cells imparts them with an antigen experienced effector phenotype before they leave the thymus (3). This means that inkt cells can respond rapidly the first time they encounter antigen. This rapid response is another feature inkt cells share with innate immunity. Following activation, inkt cells help shape the immune response via the release of cytokines. inkt cells can be categorized into distinct effector subtypes based on the profiles of cytokines they release (Table 1). These cytokine profiles mimic the cytokines released by CD4 + helper T cells (3, 11). For example, NKT1 cells release the Th1 cytokine interferon-gamma (IFN-γ) while NKT2 cells release the Th2 cytokine interleukin-4 (IL-14) and NKT17 cells release the Th17 cytokine IL-17A. The cytokines released by inkt cells allow them to help shape the immune response in a multitude of different ways (Figure 1). For example, the interactions between inkt cells and APCs results in bi-directional activation of each cell type. inkt cells can also boost T-cell immunity indirectly via the activation of APCs, and directly via the cytokines released from inkt cells. B-cell activation can be enhanced through cognate and non-cognate interactions with inkt cells. Innate immunity can also be enhanced through inkt cells, for example some of the cytokines released by inkt cells can lead to the recruitment of neutrophils. These roles for inkt cells are all reviewed in (3). Summary 1: The characteristics of inkt cells allow them to bridge the adaptive and innate arms of the immune system. The innate like mode of recognition, and immediate effector abilities of inkt cells allow them to respond very quickly to the first signs of danger, at the cost of lacking antigen specificity. Following activation, inkt cells shape the ensuing immune response via the release of cytokines, in a way that is very similar to the effector functions of CD4 + T cells. Examples of inkt cells contributing to protective immunity and pathogenesis of microbial infections One role of inkt cells is in the initiation of B cell antibody responses to viral infection. B cells can undergo a process called affinity maturation within structures called germinal centers in the lymph nodes. This involves rounds of somatic hyper-mutation, followed by selection for B cells that express

4 higher affinity antibodies, resulting in a highly effective antibody response (reviewed in (12)). This process depends on the initial seeding of germinal centers by antigen experienced B cells. The formation of germinal centers requires IL-4, and although this cytokine can be provided by T follicular helper (Tfh) cells, seeding of germinal centers precedes a Tfh response (13). inkt cells within B cell follicles provide this early IL-4 signal in the context of viral infection (13). Furthermore, inkt cells are spatially positioned at the follicular borders, and this allows them to receive activating signals from macrophages within the lymph nodes, leading the pre-tfh IL-4 response by the inkt cells. The activation of inkt cells by these lymph-node resident macrophages is mediated through CD1d interactions and IL-18 (13). Viruses do not possess their own lipid antigens, so this IL-4 response by inkt cells must depend on host lipid interactions and strong cytokine signals which originate from TLR signaling. inkt cells can also be important for driving an innate response, contributing to anti-bacterial immunity. One such example is during infection with Streptococcus pneumonia (pneumococcus). During pneumococcal infection of the lungs, inkt cells can become activated following TCR stimulation with pneumococcal lipid antigens and stimulation by IL-12. IL-12 is released from APCs in response to stimulation of TLRs by pneumococcus (14). The activation of inkt cells leads to the release of IFN-γ and IL-17A, which work to recruit neutrophils (14, 15). Neutrophils are the main effector cells against pneumococcal infection which help clear the bacterial infection via phagocytosis (15). Influenza A virus infection can lead to increased susceptibility towards superinfection with pneumococcus and this is due to a hampered ability of inkt cells to release IFN-γ (15). Infection with Influenza A virus leads to a release of IL-10 in the lungs (15). IL-10 is an anti-inflammatory cytokine that prevents the APCs from releasing IL-12 which is vital for activation of inkt cells during pneumococcal superinfection. Without the IL-12 signal, the inkt cells are unable to release IFN-γ and therefore do not contribute to protective immunity against pneumococcus (15). In other instances, inkt cells can contribute to the pathogenesis of certain infections. One such example is during Candida albicans infection. C. albicans is an opportunistic fungal pathogen that normally resides in the gut, but can sometimes enter the blood causing an often-lethal systemic infection (14, 16). The main effector cells which work to clear C. albicans infection are macrophages and neutrophils. Macrophages contribute to defense against C. albicans via direct phagocytosis of the pathogen, and via the release of pro-inflammatory cytokines such as IL-1β and IL-18 (16). These cytokines work to recruit more effector cells, such as neutrophils, to sites of infection and increases the ability of the innate immune system to clear the pathogen. inkt cells can exacerbate C. albicans infection by releasing IL-10. IL-10 works to prevent the activation of macrophages, limiting the release of pro-inflammatory cytokines and phagocytosis of the fungal pathogen (14, 16). In this case inkt cells are the source and not the target of IL-10 and therefore are contributors to the pathogenesis of C. albicans. The beneficial roles inkt cells play towards protective immunity against certain pathogens has led to the design of vaccine adjuvants that target an inkt cell response to boost immunity. Examples of vaccine adjuvants targeting inkt cells are summarized in (14) and select examples are highlighted in (Table 2). The inclusion of an inkt-based adjuvant can have a different effect based on the vaccine it is used with. For example, T cell immunity or antibody responses have been boosted when inkt adjuvants have been used in different vaccines. The identity of the glycolipid used as an adjuvant can also affect the quality of the immune response elicited by targeting inkt cells. As inkt cells can exacerbate infections under certain conditions, the use of vaccine adjuvants targeting inkt cell responses must be carefully assessed.

5 Summary 2: inkt cells can help shape the immune response to an infection and affect the outcome of the response. inkt cells can trigger different arms of the immune system under different conditions, for example inkt cells can boost B cell immunity in response to viral infection or help elicit an innate immune response to pneumococcal infection of the lungs. inkt cells can even contribute to the pathogenesis of a microbe, as seen in the case of C. albicans infection. The beneficial roles of inkt cells has led to their use as targets for vaccine adjuvants, although this must be carefully considered. The tissue specific distribution of inkt cell subsets is related to their function inkt cells are present in the blood and reside within tissues. As previously mentioned, inkt cells can be classified into subsets based on the profile of cytokines they release upon activation, the transcription factors they express and their cell surface markers (Table 1). The subsets of inkt cells mirror those of CD4 + T helper cells, and thus they can be seen as having an analogous function (11). Therefore, the distribution of each inkt cell subset within tissues will determine the role they play within each tissue, and how they may respond to infection. The distribution of the three main inkt cell subsets (NKT1, NKT2 and NKT17) have been characterized in several tissues and compared between three strains of mice (Figure 2). It was revealed that each tissue had a specific distribution of inkt cell subsets, and that this distribution varied quite substantially between each mouse strain (17). Of note the liver had the highest content of NKT cells; almost 10% of all leukocytes within the liver were attributed to NKT1 cells (17). inkt content of all other tissues was drastically lower compared to the liver. Also, BALB/c mice had a much higher content of NKT2 cells compared to the other mouse strains, especially within the mesenteric lymph nodes (mln) (17). One consequence of the tissue specific distribution of inkt cells is that NKT cells in different tissues will respond to different routes of infection. For example, intravenous injection of α-galcer led to an NKT1 response in the liver and spleen, whereas oral administration of α-galcer resulted in an NKT2 response in the mln (17). This is unsurprising as the liver and spleen are highly vascularized, whereas the mln is associated with the gut. The spatial distribution of inkt subsets within each tissue also affects the way inkt cells may respond. For example, a large amount of NKT2 cells was found to be localized to the T-cell zone in the mln of BALC/c mice. It was determined that antigen stimulation of these NKT2 cells resulted in release of IL-4 which acted on the surrounding T cells, and contributed to the homeostasis of this cell population (17). This is in contrast to the previously discussed example where NKT cells were found to be spatially localized to the follicular borders of lymph nodes, and contributed to anti-viral B cell immunity via release of IL-4 (13). It was mentioned that the liver contained an over-abundance of NKT1 cells compared to other tissues. NKT1 cells within the liver have been associated with several roles (Figure 3). For example, NKT1 cells patrol throughout the liver sinusoids under normal conditions where they can be activated either by TCR or cytokine signals. Upon activation NKT1 cells release the pro-inflammatory cytokine IFN-γ. NKT1 cells can contribute to anti-bacterial immunity, as in the case of B. burgdorferi infection. NKT1 cells can become activated via a bacterial lipid antigen, which results in their activation and clustering on Kupffer cells (a type of liver resident macrophage). Following activation, the IFN-γ released by NKT1 cells can help promote anti-bacterial immunity. inkt cells can also contribute to pathogenic inflammation within the liver. In the case of chronic inflammation, the population of NKT1 cells in the liver expands. These NKT1 cells can then cause hepatocyte death directly via their own cytotoxicity, and indirectly via release of IFN-γ. The roles of inkt cells within the liver are reviewed in (11).

6 inkt cells can also be recruited to sites of infection. We have already discussed the protective roles of inkt cells during pneumococcal infection within the lungs. inkt cells mediate the recruitment of neutrophils via the release of pro-inflammatory cytokines such as IFN-γ and IL-17 (14). Furthermore, additional NKT1 and NKT17 cells are recruited to the lungs during infection, resulting in the expansion of the inkt cell population within the lungs (11). This highlights the important role inkt cells play in protective immunity against pneumococcus and the need for tissue resident populations of inkt cells. Summary 3: The tissue specific distribution of inkt cell subsets reflects their function. For example, inkt cell populations within different tissues will respond to different routes of infection and the spatial distribution of inkt cells will determine the role they play within each tissue. NKT1 cells were especially abundant in the liver where they can contribute to protective immunity or pathogenic inflammation. inkt cells can also be recruited to sites of infection, allowing them to have an enhanced role in immunity. Table 1. Characteristics of inkt cell subsets (11). inktsubset Transcription Factors Cell surface markers Cytokines secreted NKT1 PLZF (low) T-bet CD49a CD122 CXDR3 CD4 (+/-) NKT2 PLZF (high) ICOS CD4 NKT17 PLZF (medium) ROR-gamma-t ICOS Syndecan1 CCR6 CD103 NRP1 NKTfh BCL-6 CXCR5 PD1 CD4 NKT10 E4BP4 CD49d PD1 NRP1 IFN-gamma IL-4 TNF IL-4 IL-5 IL-13 IL-17 IL-22 GM-CSF TNF IL-21 IL-10 IL-2

7 Figure 1. Roles of inkt cells. Image accessed from (3). Table 2. Select examples of vaccine adjuvants targeting inkt cells and their responses (14). Vaccine Adjuvant Response Malaria α-galcer Increased CD8 T cells Tubercolosis α-galcer Increased CD4 and CD8 T cells Influenza α-galcer Incresed IgA and IgG Bacillus Calmette-Guerin α-galcer Increased CD8 T cells Bacillus Calmette-Guerin α-c-galcer Better increase in CD8 T cells

8 Figure 2. Tissue specific distribution of inkt cells and their subsets. CD45 is a cell surface marker common to all leukocytes. Image accessed from (17).

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