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Neha N Parayath,1 Hayley Nehoff,1 Samuel E Norton,2 Andrew J Highton,2 Sebastien Taurin,1,3 Roslyn A Kemp,2 Khaled Greish1,4 1Department of Pharmacology and Toxicology, 2Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand; 3Department of Obstetrics and Gynecology, University of Utah, Salt Lake City, UT, USA; 4Princess Al-Jawhara Centre for Molecular Medicine, Arabian Gulf University, Manama, Kingdom of Bahrain Abstract: Oral administration of paclitaxel (PTX), a broad spectrum anticancer agent, is challenged by its low uptake due to its poor bioavailability, efflux through P-glycoprotein, and gastrointestinal toxicity. We synthesized PTX nanomicelles using poly(styrene-co-maleic acid) (SMA). Oral administration of SMA-PTX micelles doubled the maximum tolerated dose (60 mg/kg vs 30 mg/kg) compared to the commercially available PTX formulation (PTX [Ebewe]). In a murine orthotopic colon cancer model, oral administration of SMA-PTX micelles at doses 30 mg/kg and 60 mg/kg reduced tumor weight by 54% and 69%, respectively, as compared to the control group, while no significant reduction in tumor weight was observed with 30 mg/kg of PTX (Ebewe). In addition, toxicity of PTX was largely reduced by its encapsulation into SMA. Furthermore, examination of the tumors demonstrated a decrease in the number of blood vessels. Thus, oral delivery of SMA-PTX micelles may provide a safe and effective strategy for the treatment of colon cancer. Keywords: oral delivery, anticancer nanomedicine, CT-26, enhanced permeability and retention (EPR) effect, HUVEC, antiangiogenic

Background: Immune dysfunction often persists in people living with human immunodeficiency virus (HIV) who are on antiretroviral therapy (ART), clinically manifesting as HIV-1-associated comorbid conditions. Early ART initiation may reduce incidence of HIV-1-associated immune dysfunction and comorbid conditions. Immunometabolism is a critical determinant of functional immunity. We investigated the effect of HIV-1 infection and timing of ART initiation on CD4+ T cell metabolism and function., Methods: Longitudinal blood samples from people living with HIV who initiated ART during hyperacute HIV-1 infection (HHI; before peak viremia) or chronic HIV-1 infection (CHI) were assessed for the metabolic and immune functions of CD4+ T cells. Metabolite uptake and mitochondrial mass were measured using fluorescent analogues and MitoTracker Green accumulation, respectively, and were correlated with CD4+ T cell effector functions., Results: Initiation of ART during HHI prevented dysregulation of glucose uptake by CD4+ T cells, but glucose uptake was reduced before and after ART initiation in CHI. Glucose uptake positively correlated with interleukin-2 and tumor necrosis factor-α production by CD4+ T cells. CHI was associated with elevated mitochondrial mass in effector memory CD4+ T cells that persisted after ART and correlated with PD-1 expression., Conclusions: ART initiation in HHI largely prevented metabolic impairment of CD4+ T cells. ART initiation in CHI was associated with persistently dysregulated immunometabolism of CD4+ T cells, which was associated with impaired cellular functions and exhaustion., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objectives: Lenalidomide (LEN) is used to treat multiple myeloma (MM) and shows in vitro synergy with KappaMab (KM), a chimeric antibody specific for Kappa Myeloma antigen, an antigen exclusively expressed on the surface of kappa-restricted MM cells. Lenalidomide, dexamethasone (DEX) and KM control MM via multiple immunomodulatory mechanisms; however, there are several additional effects of the drug combination on immune cells. Lenalidomide can increase T cell and NKT cell cytotoxicity and dendritic cell (DC) activation in vitro . We investigated the immune cell populations in bone marrow of patients treated with KM, LEN and low-dose DEX in kappa-restricted relapsed/refractory MM ex vivo and assessed association of those changes with patient outcome., Methods: A cohort ( n  = 40) of patients with kappa-restricted relapsed/refractory MM, treated with KM, LEN and low-dose DEX, was analysed using a mass cytometry panel that allowed identification of immune cell subsets. Clustering analyses were used to determine significant changes in immune cell populations at time periods after treatment., Results: We found changes in five DC and 17 T-cell populations throughout treatment. We showed an increase in activated conventional DC populations, a decrease in immature/precursor DC populations, a decrease in activated CD4 T cells and an increase in effector-memory CD4 T cells and effector CD8 T cells, indicating an activated immune response., Conclusion: These data characterise the effects of LEN, DEX, and KM treatment on non-target immune cells in MM. Treatment may support destruction of MM cells by both direct action and indirect mechanisms via immune cells., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Objectives: Crohn's disease (CD) initiation and pathogenesis are believed to involve an environmental trigger in a genetically susceptible person that results in an immune response against commensal gut bacteria, leading to a compromised intestinal epithelial barrier and a cycle of inflammation. However, it has been difficult to study the contribution of all factors together in a physiologically relevant model and in a heterogenous patient population., Methods: We developed an autologous colonic monolayer model that incorporated the immune response from the same donor and a commensal bacteria, Faecalibacterium prausnitzii . Two-dimensional monolayers were grown from three-dimensional organoids generated from intestinal biopsies, and the epithelial integrity of the epithelium was measured using transepithelial electrical resistance. We determined the effect of immune cells alone, bacteria alone and the co-culture of immune cells and bacteria on integrity., Results: Monolayers derived from CD donors had impaired epithelial integrity compared to those from non-inflammatory bowel disease (IBD) donors. This integrity was further impaired by culture with bacteria, but not immune cells, despite a higher frequency of inflammatory phenotype peripheral T cells in CD donors. Variability in epithelial integrity was higher in CD donors than in non-IBD donors., Conclusion: We have developed a new autologous model to study the complexity of CD, which allows for the comparison of the barrier properties of the colonic epithelium and the ability to study how autologous immune cells directly affect the colonic barrier and whether this is modified by luminal bacteria. This new model allows for the study of individual patients and could inform treatment decisions., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

The liver is an important immunological site that can promote immune tolerance or activation. Natural killer (NK) cells are a major immune subset within the liver, and therefore understanding their role in liver homeostasis and inflammation is crucial. Due to their cytotoxic function, NK cells are important in the immune response against hepatotropic viral infections but are also involved in the inflammatory processes of autoimmune liver diseases and fatty liver disease. Whether NK cells primarily promote pro-inflammatory or tolerogenic responses is not known for many liver diseases. Understanding the involvement of NK cells in liver inflammation will be crucial in effective treatment and future immunotherapeutic targeting of NK cells in these disease settings. Here, we explore the role that NK cells play in inflammation of the liver in the context of viral infection, autoimmunity and fatty liver disease., (© 2021. The Author(s).)

Gastrointestinal viral infections are a major global cause of disease and mortality in infants. Cytotoxic CD8 + T cells are critical to achieve viral control. However, studies investigating the development of CD8 + T cell immunity in human tissues early in life are lacking. Here, we investigated the maturation of the CD8 + T cell compartment in human fetal, infant and adult intestinal tissues. CD8 + T cells exhibiting a memory phenotype were already detected in fetal intestines and increased after birth. Infant intestines preferentially harbored effector CCR7 - CD45RA - CD127 - KLRG1 +/- CD8 + T cells compared to tissue-resident memory CD69 + CD103 + CD8 + T cells detected in adults. Functional cytotoxic capacity, including cytokine and granzyme B production of infant intestinal effector CD8 + T cells was, however, markedly reduced compared to adult intestinal CD8 + T cells. This was in line with the high expression of the inhibitory molecule PD-1 by infant intestinal effector CD8 + T cells. Taken together, we demonstrate that intestinal CD8 + T cell responses are induced early in human development, however exhibit a reduced functionality. The impaired CD8 + T cell functionality early in life contributes to tolerance during foreign antigen exposure after birth, however functions as an immune correlate for the increased susceptibility to gastrointestinal viral infections in infancy.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Natural killer (NK) cells are an important component of the innate immune system for the control of intracellular pathogens and cancer cells. NK cells demonstrate heterogeneous expression of inhibitory surface receptors. Signaling through these various receptors during NK cell development promotes functionality, referred to as NK cell education. Here we investigated the impact of education on NK cell metabolism through functional assessment of critical metabolic pathways and calcium signaling. Educated NK cells had an increased uptake of the metabolic substrates 2-NBDG, a fluorescent glucose analog, and BODIPY FL C 16 , a fluorescent palmitate, compared to uneducated NK cells. Comparison of NK cells educated via KIRs or NKG2A showed that NKG2A-educated NK cells were the main contributor to these differences in uptake of metabolites, and that NKG2A-educated NK cells were functionally more resilient in response to metabolic blockade of oxidative phosphorylation. Furthermore, NKG2A-educated NK cells exhibited higher peak calcium concentration following stimulation, indicating stronger signaling events taking place in these educated NK cells. These results demonstrate that cellular metabolism plays an important role in the functional differences observed between educated and uneducated NK cells, and show that NKG2A-educated NK cells remain more functionally competent than KIR-educated NK cells when oxidative phosphorylation is restricted. Understanding metabolic programming during NK cell education may unveil future targets to manipulate NK cell function for use in clinical settings, such as cancer therapies., (Copyright © 2020 Highton, Diercks, Möckl, Martrus, Sauter, Schmidt, Bunders, Körner, Guse and Altfeld.)

Background : Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8 + T-cell responses, described as memory "inflation". These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods : To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results : We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (T RM ) in the gut. Conclusions : These data indicate that CD8 + T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and "inflationary" memory which may be relevant to protection against mucosal infections., Competing Interests: No competing interests were disclosed.

NK cells expressing self-inhibitory receptors display increased functionality compared to NK cells lacking those receptors. The acquisition of functional competence in these particular NK-cell subsets is termed education. Little is known about the underlying mechanisms that lead to the functional differences between educated and uneducated NK cells. An increasing number of studies suggest that cellular metabolism is a determinant of immune cell functions. Thus, alterations in cellular metabolic pathways may play a role in the process of NK-cell education. Here, we compared the glycolytic profile of educated and uneducated primary human NK cells. KIR-educated NK cells showed significantly increased expression levels of the glucose transporter Glut1 in comparison to NKG2A-educated or uneducated NK cells with and without exposure to target cells. Subsequently, the metabolic profile of NK-cell subsets was determined using a Seahorse XF Analyzer. Educated NK cells displayed significantly higher rates of cellular glycolysis than uneducated NK cells even in a resting state. Our results indicate that educated and uneducated NK cells reside in different metabolic states prior to activation. These differences in the ability to utilize glucose may represent an underlying mechanism for the superior functionality of educated NK cells expressing self-inhibitory receptors.

Cytomegalovirus (CMV) and non-replicating adenoviral vectors can induce expanded, sustained effector-memory CD8 + T-cell responses, termed "memory inflation". During murine CMV (MCMV) infection, CD4 + Tcells maintain inflationary virus-specific CD8 + T-cell responses via IL-2 but not IL-21. Adenovirus vector vaccination can induce phenotypically, functionally and transcriptionally similar inflationary responses, but it is not known how IL-21 influences the inflating memory response to adenoviral vaccination. Here, we show that IL-21 is an absolute requirement for induction and maintenance of vaccine-derived inflationary CD8 + T-cell responses. These data indicate that the induction pathway of inflationary Ad-LacZ T-cells is distinct from inflationary MCMV-specific T-cells and is highly dependent on IL-21. Our observations highlight a fundamental difference in the mechanism by which adenovirus vectors and MCMV drive inflationary T-cell responses., (Copyright © 2018. Published by Elsevier Ltd.)

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( K d = 5 nM), potent anti-HCV 2a activity (EC 50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Mucosal-associated invariant T (MAIT) cells are a well-characterized innate-like T cell population abundant in the human liver, peripheral tissues and blood. MAIT cells serve in the first line of defense against infections, through engagement of their T cell receptor, which recognizes microbial metabolites presented on MR1, and through cytokine-mediated triggering. Typically, they show a quiescent memory phenotype but can undergo rapid upregulation of effector functions including cytolysis upon stimulation. T cells profoundly change their cellular metabolism during their maturation and activation. We sought to determine how MAIT cell metabolism may facilitate both the long-term memory phase in tissue and the transition to rapid effector function. Here, we show, by flow cytometric metabolism assays and extracellular flux analysis that, despite an effector-memory profile, human MAIT cells are metabolically quiescent in a resting state comparable to naïve and central memory T cells. Upon stimulation, they rapidly increase uptake of glucose and show a concomitant upregulation of the effector molecules notably granzyme B, which is impaired by inhibition of glycolysis with 2-deoxyglucose. These findings suggest that MAIT cells share some metabolic characteristics of both resting and effector T cell subsets, with a rapid transition upon triggering. Metabolic programming of this cell type may be of interest in understanding and modulating their function in infectious diseases and cancer., (© 2018 The AuthorsImmunology and Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australasian Society for Immunology Inc.)

The induction and maintenance of T cell memory is critical to the success of vaccines. A recently described subset of memory CD8 + T cells defined by intermediate expression of the chemokine receptor CX3CR1 was shown to have self-renewal, proliferative, and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells when memory is sustained at high levels: memory inflation induced by cytomegalovirus (CMV) and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T cells showed sustained high levels of CX3R1 int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools, in early memory. In humans, CX3CR1 int CD8 + T cells were strongly induced following adenovirus-vectored vaccination for hepatitis C virus (HCV) (ChAd3-NSmut) and during natural CMV infection and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1 int cells form an important component of the memory pool in response to persistent viruses and vaccines in both mice and humans., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host's memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words).

Background: Vaccination generating a robust memory population of CD8 + T cells may provide protection against cancer. However, immune therapies for cancer are influenced by the local tumour immune microenvironment. An infiltrate of T cells into tumours of people with colorectal cancer has proven to be a significant indicator of good prognosis., Methods: We used an intracaecal mouse model of cancer to determine whether a protective immune response against a mucosal gut tumour could be generated using a systemic intervention. We investigated the generation of murine memory CD8 + T cells using a sustained antigen release vaccine vehicle (chitosan gel; Gel + OVA) containing the model antigen ovalbumin, chitosan gel alone (Gel) or conventional dendritic cell vaccination (DC + OVA) using the same protein antigen., Results: Following vaccination with Gel + OVA, CD8 + T cell memory populations specific for ovalbumin protein were detected. Only vaccination with Gel + OVA gave decreased tumour burden compared to unvaccinated or DC + OVA-vaccinated mice in the intracaecal cancer challenge model., Conclusion: These results indicate that subcutaneous vaccination with Gel + OVA generates a population of functional CD8 + memory T cells in lymphoid tissue able to protect against intracaecal tumour challenge. Vaccination with chitosan gel may be valuable in anti-cancer treatment at both peripheral and mucosal sites.

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines., Competing Interests: The authors declare that no competing interests exist.

CD8(+) T cells are important in the control of viral infections and cancers because of their cytolytic activity. A vaccine able to generate these cells could be beneficial in the prevention or treatment of these diseases. Chitosan hydrogel is a promising vaccine formulation that has previously been shown to generate effector CD8(+) T cells in a mouse model. This vaccine promotes sustained release of antigen and adjuvant, which generates a robust effector response. For longer lasting immunity, a memory population of these CD8(+) T cells is required to control further disease. We found that vaccination with chitosan hydrogel or dendritic cells using ovalbumin protein as a model antigen and Quil-A adjuvant provided protection in a subcutaneous melanoma challenge 30 days later. Ovalbumin-specific memory CD8(+) T cells were detectable following vaccination with the chitosan hydrogel but not the dendritic cell vaccine and an in vivo cytotoxicity assay demonstrated specific lysis of target cells in chitosan hydrogel vaccinated mice but not those receiving dendritic cell vaccination. These results demonstrate that vaccination with chitosan hydrogel is equally effective as dendritic cell vaccination in tumour protection but has more readily detectable immune correlates of protection. This may be advantageous in predetermining protection in vaccinated individuals.

Rational structure-based design has yielded highly potent inhibitors of cathepsin K (Cat K) with excellent physical properties, selectivity profiles, and pharmacokinetics. Compounds with a 3,4-(CH₃O)₂Ph motif, such as 31, were found to have excellent metabolic stability and absorption profiles. Through metabolite identification studies, a reactive metabolite risk was identified with this motif. Subsequent structure-based design of isoteres culminated in the discovery of an optimized and balanced inhibitor (indazole, 38).

The discovery of nitrile compound 4, a potent inhibitor of Cathepsin K (Cat K) with good bioavailability in dog is described. The compound was used to demonstrate target engagement and inhibition of Cat K in an in vivo dog PD model. The margin to hERG ion channel inhibition was deemed too low for a clinical candidate and an optimisation program to find isosteres or substitutions on benzothiazole group led to the discovery of 20, 24 and 27; all three free from hERG inhibition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Directed screening of nitrile compounds revealed 3 as a highly potent cathepsin K inhibitor but with cathepsin S activity and very poor stability to microsomes. Synthesis of compounds with reduced molecular complexity, such as 7, revealed key SAR and demonstrated that baseline physical properties and in vitro stability were in fact excellent for this series. The tricycle carboline P3 unit was discovered by hypothesis-based design using existing structural information. Optimization using small substituents, knowledge from matched molecular pairs, and control of lipophilicity yielded compounds very close to the desired profile, of which 34 (AZD4996) was selected on the basis of pharmacokinetic profile.