Conventional natural killer cells (NK cells) provide continual surveillance for cancer and rapid responses to infection. They develop in the bone marrow, emerge as either NK precursor cells, immature, or mature cells, and disperse throughout the body. In the periphery NK cells provide critical defense against pathogens and cancer and are noted to develop features of adaptive immune responses. In the tightly regulated and dynamic mucosal tissues, they set up residency via unknown mechanisms and from sources that are yet to be defined. Once resident, they appear to have the ability to functionally mature dependent on the mucosal tissue microenvironment. Mucosal NK cells play a pivotal role in early protection through their cytolytic function and IFNγ production against bacteria, fungi, viruses, and parasitic infections. This review presents what is known about NK cell development and phenotypes of mucosal tissue resident conventional NK cells. The question of how they come to reside in their tissues and published data on their function against pathogens during mucosal infection are discussed. Dissecting major questions highlighted in this review will be important to the further understanding of NK cell homing and functional diversity and improve rational design of NK cell based therapies against mucosal infection.

Apicomplexans are a diverse and complex group of protozoan pathogens including Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., Eimeria spp., and Babesia spp. They infect a wide variety of hosts and are a major health threat to humans and other animals. Innate immunity provides early control and also regulates the development of adaptive immune responses important for controlling these pathogens. Innate immune responses also contribute to immunopathology associated with these infections. Natural killer (NK) cells have been for a long time known to be potent first line effector cells in helping control protozoan infection. They provide control by producing IL-12 dependent IFNγ and killing infected cells and parasites via their cytotoxic response. Results from more recent studies indicate that NK cells could provide additional effector functions such as IL-10 and IL-17 and might have diverse roles in immunity to these pathogens. These early studies based their conclusions on the identification of NK cells to be CD3–, CD49b+, NK1.1+, and/or NKp46+ and the common accepted paradigm at that time that NK cells were one of the only lymphoid derived innate immune cells present. New discoveries have lead to major advances in understanding that NK cells are only one of several populations of innate immune cells of lymphoid origin. Common lymphoid progenitor derived innate immune cells are now known as innate lymphoid cells (ILC) and comprise three different groups, group 1, group 2, and group 3 ILC. They are a functionally heterogeneous and plastic cell population and are important effector cells in disease and tissue homeostasis. Very little is known about each of these different types of ILCs in parasitic infection. Therefore, we will review what is known about NK cells in innate immune responses during different protozoan infections. We will discuss what immune responses attributed to NK cells might be reconsidered as ILC1, 2, or 3 population responses. We will then discuss how different ILCs may impact immunopathology and adaptive immune responses to these parasites.

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination, and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii ( T. gondii ) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and alters cyst burdens in brain. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis in spleen. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell characteristics. NK cells are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. These NK cells are found in both spleen and brain. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced on CD8+ T cells. Blockade of NKp46 rescued the chronically infected mice from death and reduced the number of NKG2A+ cells. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

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Conventional natural killer (cNK) cells, members of group 1 innate lymphoid cells, are a diverse cell subpopulation based on surface receptor expression, maturation, and functional potential. cNK cells are critical for early immunity to Toxoplasma gondii via IFNγ production. Acute cNK cell responses to infection with different strains of T. gondii have not yet been characterized in detail. Here, we comprehensively performed this analysis with Type I virulent RH, Type II avirulent ME49, and fully attenuated Type I cps1-1 strains. In response to these three parasite strains, murine cNK cells produce IFNγ and become cytotoxic and polyfunctional (IFNγ+CD107a+) at the site of infection. In contrast to virulent RH and avirulent ME49 T. gondii strains, attenuated cps1-1 induced only local cNK cell responses. Infections with RH and ME49 parasites significantly decreased cNK cell frequency and numbers in spleen 5 days post infection compared with cps1-1 parasites. cNK cell subsets expressing activating receptors Ly49H, Ly49D, and NKG2D and inhibitory receptors Ly49I and CD94/NKG2A were similar when compared between the strains and at 5 days post infection. cNK cells were not proliferating (Ki67−) 5 days post infection with any of the strains. cNK cell maturation as measured by CD27, CD11b, and KLRG1 was affected after infection with different parasite strains. RH and ME49 infection significantly reduced mature cNK cell frequency and increased immature cNK cell populations compared with cps1-1 infection. Interestingly, KLRG1 was highly expressed on immature cNK cells after RH infection. After RH and ME49 infections, CD69+ cNK cells in spleen were present at higher frequency than after cps1-1 infection, which may correlate with loss of the mature cNK cell population. Cytokine multiplex analysis indicated cNK cell responses correlated with peritoneal exudate cell, spleen, and serum proinflammatory cytokine levels, including IL-12. qPCR analysis of parasite-specific B1 gene revealed that parasite burdens may affect cNK cell responses. This study demonstrates infection with RH and ME49 parasites impacts cNK cell maturation during acute T. gondii infection. Different cNK cell responses could impact early immunity and susceptibility to these strains.

22Using vaccine challenge model of T. gondii infection, we found that treatments with two commonly 23 used for NK cell depletion antibodies resulted in different survival outcomes during secondary 24 infection. Anti-ASGM1 resulted in 100% death and greater parasite burden at the site of infection 25 than anti-NK1.1. Anti-NK1.1 treatment resulted in increased parasite burdens, but animals did not 26 die. Further we found that anti-ASGM1 treatment depleted T cells. CD8+ T cells were more 27 susceptible that CD4+ T cells to the treatment. ASGM1 was expressed on a higher percentage of 28 CD8+ T cells than CD4+ T cells and CD8+ T cells. In T. gondii-immunized animals ASGM1 was 29enriched on effector memory (Tem) and central memory (Tcm) CD8+ T cells. However, Tem were 30 more susceptible to the treatment. After secondary infection, Tem, Tcm, effector (Tef) and naïve (Tn) 31 CD8+ T cells were positive for ASGM1. Anti-ASGM1 treatment during reinfection resulted in 32 greater depletion of activated IFNγ+, Granzyme B+, Tem and Tef than Tcm and Tn CD8+ T cells. 33Anti-ASGM1 also depleted IFNγ+ CD4+ T cells. Recombinant IFNγ supplementation prolonged 34 survival of anti-ASGM1 treated mice, demonstrating that this antibody eliminated IFNγ-producing T 35 and NK cells important for control of the parasite. These results highlight that anti-ASGM1 antibody 36is not an optimal choice for targeting only NK cells and more precise approaches should be used. 37This study uncovers ASGM1 as a marker of activated effector T cells and the potential importance of 38 changes in sialylation in lipid rafts for T cell activation during T. gondii infection. 39 40 establishes a persistent infection in the brain and muscles of the host (Harker et al., 2015, Wohlfert et 46 al., 2017. Recent studies suggest that infection with the parasite correlates with schizophrenia, 47 depression, behavioral changes, and neurodegenerative disorders (Lang et al., 2018, Donley et al., 48 2016. In the patients with weakened immune system, the T. gondii parasites can cause toxoplasmic 49 encephalitis and death (Luft and Remington, 1992, Kodym et al., 2015). Currently, there are no 50 vaccines or treatments that completely clear the infection (Radke et al., 2018). Understanding how 51 the immune system functions in response to the parasite is paramount for the development of novel 52 therapeutic approaches to treat this infection. 53T. gondii infection induces a robust Th1 response that provides long-term protection through 54

Reactivation of chronic Toxoplasma gondii (T. gondii) infection and toxoplasmic encephalitis is a major health concern in immune compromised people. Dysregulated T-cell responses are thought to promote chronic Toxoplasmosis. The non-T-cell factors that contribute to T cell dysregulation and chronic T. gondii infection are unclear. We have discovered that NKp46+ NK cells paradoxically increase the frequency and number of IFNγ+TNFα+CD4+ T-cells in the brain in during chronic T. gondii infection. Depletion of NK cells resulted in a decrease in polyfunctional brain CD4+ T-cells and an increase in IFNγ+Grzb+CD8+ T-cell responses in the brain. This decrease in CD4+ T-cell and increase in CD8+ T-cell responses correlated with better survival of chronically infected mice. This result suggests that NKp46+ NK cells drive an NK cell-dependent CD4+ T cell response that is detrimental to the CD8+ T cell response important for controlling chronic T. gondii infection. In support of this possibility, adoptive transfer of NK cell-dependent CD4+ T cells into chronically infected mice reduced polyfunctional CD8+ T-cell responses in the brain while CD4+ T cells generated in the absence of NK cells during chronic T. gondii infection did not. NKp46+ NK cells were not found in brain tissue, but appear to localize to the vasculature of the brain and periphery during chronic T. gondii infection. Surprisingly, we observe that NKp46+ NK cells increase their expression of MHC Class II. Our results lead us to hypothesize that during chronic T. gondii infection NKp46+ NK cells acquire the ability to prime CD4+ T-cell responses via MHC Class II expression resulting in decreased polyfunctional CD8 T cell responses to promote parasite persistence.

Our lab has developed a subunit vaccination regimen that induces a highly robust T cell response. These vaccine-elicited CD8+ T cells (Tvac) are phenotypically, functionally and metabolically distinct from infection-elicited CD8+ T cells (Tinf). In contrast to Tinf, almost all Tvac rapidly acquire a memory phenotype (CD127hi, KLRG1lo). This suggests that Tvac could be differentially transcriptionally programmed to commit to a memory, rather than effector, cell fate. The transcription factor TCF1 (encoded by tcf7) is essential for CD8+ T cell memory in response to infection. Since high TCF1 expression is also important for the generation of stem-like T cells with long-term survival capacity and anti-tumor properties, it is now recognized as a promising target for immunotherapies. However, little is known about how TCF1 is regulated in response to subunit vaccination. We find that, in striking contrast to Tinf, TCF1 expression is actually increased in Tvac after vaccination. As others have shown, we also find that increasing inflammation during vaccination by the addition of TLR9 agonist CpG reduces TCF1 expression in Tvac, skewing their memory phenotype towards the effector phenotype (CD127lo, KLRG1hi) and compromising their long-term memory. While the loss of TCF1 in this context has conventionally been ascribed to inflammatory receptors, utilizing reporter Nur77GFP mice, we also observe an altered duration and frequency of TCR triggering in the context of inflammation. Our data indicates a complex, previously unappreciated interplay between inflammatory and TCR-mediated signals in the regulation of TCF1.