Stimulation Of Immune Cells Research Articles

Mannosylated imiquimod-terbinafine co-loaded transethosomes for cutaneous leishmaniasis; assessment of its anti-leishmanial potential, in vivo safety and immune response modulation

Current treatment options for cutaneous leishmaniasis are associated with myriad limiting factors including low penetration, poor efficacy, and drug toxicities. Herein, we reported imiquimod and terbinafine co-loaded mannosylated transethosomes (IMQ-TER-MTES) with enhanced cutaneous retention, macrophage targeting, anti-leishmanial potential, and dermal immunomodulation. IMQ-TER-MTES were optimized using Design Expert® followed by their loading into chitosan gel. Moreover, the antileishmanial response against amastigotes-infected macrophages and Leishmania-infected BALB/c mice was evaluated. Finally, the safety and immunomodulation activity of IMQ-TER-MTES gel was performed using BALB/c mice. Optimized IMQ-TER-MTES showed nano-sized particles with low poly-dispersibility index (PDI) and high drug entrapment. Mannosylation has augmented macrophage targeting and the internalization capability of TES. IMQ-TER-MTES showed significantly reduced IC50 value (19.56 ± 3.62 μg/ml), higher selectivity index (29.24), and synergism against Leishmania major (L. major) amastigotes. In L. major infected BALB/c mice, the cutaneous lesion healing potential of IMQ-TER-MTES was also elevated with reduced lesion size (1.52 ± 0.43 mm). Superior safety of IMQ-TER-MTES was observed in BALB/c mice along with adequate stimulation of dermal immune cells, in contrast to the ALDARA®. Moreover, incremented Nuclear factor Kappa-β (NF-κβ) and nitric oxide (NO) biosynthesis were observed with IMQ-TER-MTES.

Cancer secretome: finding out hidden messages in extracellular secretions.

Secretome analysis has gained popularity recently as a very well-designed proteomic approach that is being used to study various interactions and their effects on cellular activity. This analysis is especially helpful while studying the effects of the cells on their microenvironment, paracrine and autocrine processes, their therapeutic purposes, and as a new diagnostic perspective. Cancer is a condition rather than a specific type of disease and is still yet to be fully understood. Cancer secretome is a fairly new concept that is being implemented to examine the interactions taking place in the tumor microenvironment and can help to understand the phenomena like induction of tumorigenesis, stimulation of immune cells, etc. The secretome analysis helps to gain a different perspective on the existing knowledge on cancer and its effects. The recent advances in secretome studies are directed toward secreted components as drug targets, biomarkers, and companion tools for diagnostic and prognostic purposes in cancer. This review aims to find the interactors in different types of cancer and understand the existing unstructured secretome data and its application in prognosis, diagnosis, and in biomarker study.

Sustained delivery of CpG oligodeoxynucleotide by acetalated dextran microparticles augments effector response to Computationally Optimized Broadly Reactive Antigen (COBRA) influenza hemagglutinin

A subunit or protein-based influenza vaccine can be a safer alternative to live attenuated vaccine (Flumist) and require fewer boosts than an inactivated vaccine (e.g. Fluzone). However, to form an effective subunit vaccine, an adjuvant is often needed. In this work we used electrospray to encapsulate the hydrophilic adjuvant CpG into microparticles made from the hydrophobic biodegradable polymer acetalated dextran. To understand the rate of particle degradation on CpG release, polymer that was slow (21 h at phagosomal pH 5) and fast (0.25 h at pH 5) degrading was used to encapsulate the adjuvant. The slow-degrading particles exhibited the greatest degree of innate immune stimulation of antigen-presenting cells in vitro. In mice, the broadly acting Computationally Optimized Broadly Reactive Antigen (COBRA) Y2 influenza hemagglutinin (HA) antigen was used with CpG particles, soluble CpG, or MF-59 like adjuvant Addavax. Particles and soluble CpG elicited similar induction of anti-HA antibodies and protection against lethal influenza challenge, but the sustained release particles elicited the highest levels antibody effector functions. These results demonstrate a suitable method for encapsulation of CpG oligonucleotide in a hydrophobic particle matrix, and suggest that sustained release of CpG from Ace-DEX microparticles could potentially be used to induce potent antibody effector functions.

Modakafusp Alfa (TAK-573), a Novel CD38-Targeting Attenuated Interferon-Alpha Immunocytokine, Kills MM Cells Via NK Cell Activation

Background Despite recent success of monoclonal antibodies (mAb) and T cell redirecting therapies, relapsed/refractory (RR) multiple myeloma (MM) patients remain in urgent need of new therapies with distinct mechanisms of action (MoA). Modakafusp alfa, previously known as TAK-573, is a novel immune-targeted attenuated cytokine specifically designed to deliver interferon-alpha to CD38-expressing cells via a CD38-targeting IgG4 mAb. Preliminary results of a first in human phase 1 trial with modakafusp alfa monotherapy (1.5mg/kg, every four weeks) in RRMM patients (90% anti-CD38 mAb refractory; 37% anti-BCMA agent exposed) indicate a promising 40% overall response with manageable toxicity (Kaufman et al. EHA 2022 abstract S181). Prior studies suggest a unique and multimodal MoA of modakafusp alfa, including anti-proliferative effects on tumor cells as well as stimulation of CD38-positive immune effector cells. Towards a thorough understanding of these immunomodulatory effects, specifically regarding the activation of innate immunity, we here studied the effects of modakafusp alfa on the anti-MM activity of NK cells, which highly express CD38. Aims We assessed the ex vivo activity of modakafusp alfa on NK cell activation, degranulation, and antibody-independent cytotoxicity. In addition, we investigated the impact of modakafusp alfa on NK cell-mediated, antibody-dependent cellular cytotoxicity (ADCC) against MM cells using daratumumab (anti-CD38) or elotuzumab (anti-SLAMF7). Methods NK cell activation was explored by flow cytometry after 24-hour incubation of peripheral blood mononuclear cells (PBMC) from healthy donors with modakafusp alfa or negative/specificity controls. These pre-incubated PBMCs were also used to examine NK cell degranulation after subsequent 4 hour stimulation with K562 or PMA/Ionomycin, or in a 16-hour cytotoxicity assay against K562. In addition, modakafusp alfa pre-incubated PBMCs or enriched NK cells were assessed for antibody-independent cytotoxicity or daratumumab/elotuzumab-mediated ADCC in 24-48 hour assays against MM cell lines RPMI-8226, MM.1S or UM9. Alternatively, modakafusp alfa was directly added to the assays without pre-incubation steps. Results We found that incubation of PBMCs with modakafusp alfa for 24 hours resulted in upregulation of activation markers CD38 and CD69 on the surface of NK cells. In addition, modakafusp alfa pre-incubation significantly improved the degranulation of NK cells after 4-hour stimulation with K562 or PMA/Ionomycin. Importantly, overnight pre-incubation of PBMCs or enriched NK cells with modakafusp alfa showed enhanced antibody-independent cytotoxicity against K562 tumor cells and MM cell lines RPMI-8226, MM.1S or UM9. Consequently, this also resulted in augmented killing of MM cells in the presence of daratumumab or elotuzumab. We observed similar effects when modakafusp alfa was directly added to the cytotoxicity assays without pre-incubation of immune effector cells. Conclusions Modakafusp alfa rapidly activates NK cells, and improves their degranulation and anti-MM effectivity. This important MoA also results in improved efficacy of daratumumab or elotuzumab in short-term cytotoxicity assays. Our findings warrant clinical investigation of combining modakafusp alfa with NK cell-exploiting immunotherapies.

NEO-TRA1: A CD25-Targeted De Novo non-Alpha Agonist of the IL-2 Receptor Selectively Expands Regulatory T Cells

Introduction: Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and their dysfunction contributes to inflammatory and autoimmune conditions, such as graft versus host disease (GVHD). IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low dose IL-2 therapy effectively expands Tregs, this intervention is limited by coincident stimulation of conventional T cells and natural killer cells. Investigational therapeutics derived from directed mutagenesis of native IL-2 sequences have resulted in some improvements in Treg selectivity. To optimize the magnitude and selectivity of Treg expansion, we utilized de novo protein design technology to fine-tune the individual receptor interfaces of IL-2 mimetic proteins targeted to Tregs with CD25 antibodies. We found decoupling receptor agonism and targeting is an effective means of improving Treg stimulation and corresponding therapeutic candidates elicit robust expansion of Treg populations in vivo without detectable stimulation of inflammatory immune cells. Methods: We designed a series of de novo IL-2Ra independent agonists of the IL-2 receptor that demonstrate reduced binding affinity to CD122 and CD132, the beta and gamma chains of the IL-2 receptor complex, by up to 5 logs when compared to IL-2. These non-alpha IL-2 mimetic variants were fused to a humanized anti-CD25 mAb that lacks effector function for cis-targeting to Tregs. Candidate molecules were screened on primary human T cells to assess pSTAT5 signaling. Treg-specific molecules were administered to humanized NSG mice weekly (four times) and peripheral blood was collected for immune cell profiling via flow cytometry. Results: Screening of TRA candidates in primary human T cells identified several highly selective Treg agonists, including NEO-TRA1 (Fig1). At all concentrations, CD8+ and CD4+ non-Tregs showed low or negligible levels of pSTAT5 signaling upon treatment with NEO-TRA1. Nevertheless, CD4+CD25highCD127low Tregs show increasing pSTAT5 signaling at a wide range of concentrations starting at 20pM, and demonstrate preferential signaling in Tregs by NEO-TRA1. To establish the ability of our TRA to expand Treg in vivo, NSG mice engrafted with human hematopoietic stem cells to create a humanized immune system received weekly injections of NEO-TRA1, vehicle control, or a previously described Treg selective IL-2 mutein (IL-2v-Fc). Characterization of Day 13 CD4+CD25+Foxp3+ Treg percentages in peripheral blood mononuclear cells show that the Treg population expanded to ~30% of total CD4+ cells in mice receiving NEO-TRA1, while Treg percentages reached ~22% in mice receiving IL-2v-Fc (Fig2). Despite a slight decrease at Day 20, the Treg percentages remained higher in NEO-TRA1-treated cohorts than in control groups throughout the study. Conclusions: NEO-TRA1 exhibits potent expansion of Treg populations within primary human samples and in humanized mouse models. The observed profile of STAT5 phosphorylation demonstrates selective activation of Tregs over other lymphocyte populations. NEO-TRA1 is the first Treg agonist based on de novo protein design. NEO-TRA1 is both specific and potent and has high therapeutic potential for the treatment of GVHD and other autoimmune or inflammation diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessment of cell-mediated immune responses to SARS-CoV-2 in Syrian hamsters

BACKGROUND: The pandemic caused by the SARS-CoV-2 virus at the end of 2019 remains to be a serious healthcare problem. Constant antigenic drift of the pathogen led to a decrease of licensed COVID-19 vaccines effectiveness. And the development of broad-spectrum vaccines with high effectiveness rate against evolutionarily divergent SARS-CoV-2 variants remains an urgent issue. Unlike virus-specific antibodies with limited spectrum of action, T-cell immunity has a wider cross-protective potential. Syrian hamsters are the most appropriate model for preclinical evaluation of new vaccine candidates, since these animals are susceptible to SARS-CoV-2 infection and show clinical symptoms of the disease. However, study of T-cell vaccine response in hamsters is complicated by the lack of available reagents and test systems for adequate assessment of the virus-specific cellular immunity levels after vaccination.
 AIM: In this work, we report an optimized protocol of stimulation of Syrian hamsters immune cells with a live SARS-CoV-2 virus to assess virus-specific T-cell responses.
 MATERIALS AND METHODS: Intranasal infection of animals with SARS-CoV-2 virus followed by stimulation of immune cells with different doses of whole live coronavirus and counting of IFN-producing cells by ELISpot method.
 RESULTS: Stimulation of spleen and lung cells with SARS-CoV-2 at a dose 0.1 TCID50/cell is the most optimal viral concentration for detecting maximum of cytokine-producing cells in SARS-CoV-2-infected animals. Stimulation of cells with whole virus revealed greater number of virus-specific cells compared to a stimulation with pools of SARS-CoV-2 lyophilized peptides (S and N proteins).
 CONCLUSIONS: Overall, the new methodology allows assessment of the immunogenicity of COVID-19 T-cell vaccines more accurately in preclinical studies using the Syrian hamsters model.

Isolating and targeting a highly active, stochastic dendritic cell subpopulation for improved immune responses.

Dendritic cell (DC) activation via pathogen-associated molecular patterns (PAMPs) is critical for antigen presentation and development of adaptive immune responses, but the stochastic distribution of DC responses to PAMP signaling, especially during the initial stages of immune activation, is poorly understood. In this study, we isolate a unique DC subpopulation via preferential phagocytosis of microparticles (MPs) and characterize this subpopulation of "first responders" (FRs). We present results that show these cells (1) can be isolated and studied via both increased accumulation of the micron-sized particles and combinations of cell surface markers, (2) show increased responses to PAMPs, (3) facilitate adaptive immune responses by providing the initial paracrine signaling, and (4) can be selectively targeted by vaccines to modulate both antibody and Tcell responses invivo. This study presents insights into a temporally controlled, distinctive cell population that influences downstream immune responses. Furthermore, it demonstrates potential for improving vaccine designs via FR targeting.

114 (PB-114) Poster - Exosomes from mistletoe treated tumor cells for the stimulation of immune cells

Background: Breast carcinoma is the most common and one of the most malignant tumors in women. Multimodal standard therapy currently increases the 5-year survival rate to over 80%. For some years now, complementary medicine has also been playing an increasingly role in the field of cancer therapy. In particular, mistletoe extract is used in breast cancer therapy to reduce the side effects of tumor therapy and to improve patients’ quality of life. Different approaches exist to stimulate the immune system.

Prognostic significance of immunoscore related markers in bladder cancer

BackgroundThe significance of total and specific subpopulations of tumor-infiltrating lymphocytes (TILs) in cancer is now well-documented. In the present study, we investigated the relevance of CD3+, CD8 +, CD45RO +, and FOXP3 + TILs to the prognosis and survival of patients with bladder cancer and the disease's clinical-pathological parameters.MethodsInfiltration of each subset was immunohistochemically evaluated in both stromal and intratumoral regions of tumor tissues from 85 patients with urothelial cell carcinoma of the bladder, with known survival.ResultsOur results indicated that intratumoral CD45RO+ lymphocytes were significantly higher in high-grade tumors than in low-grade ones (P = 0.028). The frequencies of intratumoral CD3+ (P = 0.002), CD8 + (P = 0.008), intratumoral (P = 0.002), and stromal (P = 0.017) CD45RO+ lymphocytes were also higher in patients with muscular invasion than those without invasion. The frequencies of intratumoral CD3+ (P = 0.043), CD8+ (P = 0.003), CD45RO+ (P = 0.023), and total CD45RO+ (P = 0.015), showed variation in patients with different T-stage, as well; mostly increased in T2 versus Ta and T1. Comparing patients in different stages revealed an increase in the frequencies of total CD3+ (P = 0.011), intratumoral CD3+ (P = 0.006), total CD8+ (P = 0.012), intratumoral CD8+ (P = 0.009) and stromal CD8+ (P = 0.034), as well as total and stromal CD45RO+ lymphocytes (P = 0.01 and P = 0.034, respectively) in stage II comparing to stage I, while the frequencies of stromal CD3+ (P = 0.077) and CD8+ (P = 0.053) cells tended to be decreased in stage III compared to stage II.ConclusionsWe collectively observed that the frequency of immune cells, especially CD45RO+, CD3+, and CD8+ lymphocytes, were significantly higher in early-progressed tumors. This observation could be explained by continuous and prolonged stimulation of immune cells with tumor antigens during tumor progression or an increase in the recruiting factors, especially in the early stages, to eliminate tumor cells. However, with tumor progression to the late stages, the inhibitory microenvironment provided by tumor cells suppresses or changes the functionality of the effector and memory immune cells to help tumor growth. However, more functional studies with larger sample sizes are needed to reveal the real status of the immune system in patients with bladder cancer.

Immune Memory After Respiratory Infection With Streptococcus pneumoniae Is Revealed by in vitro Stimulation of Murine Splenocytes With Inactivated Pneumococcal Whole Cells: Evidence of Early Recall Responses by Transcriptomic Analysis.

The in vitro stimulation of immune system cells with live or killed bacteria is essential for understanding the host response to pathogens. In the present study, we propose a model combining transcriptomic and cytokine assays on murine splenocytes to describe the immune recall in the days following pneumococcal lung infection. Mice were sacrificed at days 1, 2, 4, and 7 after Streptococcus pneumoniae (TIGR4 serotype 4) intranasal infection and splenocytes were cultured in the presence or absence of the same inactivated bacterial strain to access the transcriptomic and cytokine profiles. The stimulation of splenocytes from infected mice led to a higher number of differentially expressed genes than the infection or stimulation alone, resulting in the enrichment of 40 unique blood transcription modules, including many pathways related to adaptive immunity and cytokines. Together with transcriptomic data, cytokines levels suggested the presence of a recall immune response promoting both innate and adaptive immunity, stronger from the fourth day after infection. Dimensionality reduction and feature selection identified key variables of this recall response and the genes associated with the increase in cytokine concentrations. This model could study the immune responses involved in pneumococcal infection and possibly monitor vaccine immune response and experimental therapies efficacy in future studies.

Abstract 1986: Differential T cell response within the tumor microenvironment observed across different indications

Abstract Cytotoxic T-cells (CTLs) in the Tumor immune Microenvironment (TiME) play an important role in mounting anti-tumor immune response. Immunotherapeutic (I/O) agents like checkpoint inhibitor aim to prevent anergy whilst T cell agonists co-stimulate CTLs to reinvigorate them and thus leading to tumor cytotoxicity. In this study we investigate the abundance and functionality of the intratumoral CTLs post stimulation, across different tumor indications, to understand the differential response across samples and indications to immune cell stimulation. These include Head and Neck Squamous cell carcinoma (HNSCC), Renal Cell Carcinoma (RCC), stomach adenocarcinoma (ca-Stomach) and Triple Negative Breast Cancer (TNBC). Using the Farcast࣪ TiME tumor histo-culture platform, that preserves both tumor and immune components in culture, we stimulated tumor explants with anti-CD3 (10ng/ml) + IL2 (100units/ml) for T-cell activation. Supernatants were collected pre-treatment and at 24-hour intervals for evaluation of cytokine release. At termination the explants were either fixed in formalin to generate paraffin blocks or dissociated into single cells for flowcytometry. 3-5 samples from each tumor indication were included in this study. Flowcytometry based immunophenotyping indicated presence of 48.6% ±5.4 live CD3+ T-cells and 20.6% ± 3.1 live CD3+CD8+ CTLs across all indications in the absence of any stimulation. HNSCC, RCC and ca-Stomach had similar proportion of CTLs, while TNBC had much lower proportions of T cell and CTLs. HNSCC and TNBC had a very low proportion of functional Granzyme-B+ CTLs (0.29%±0.08) whereas ca-Stomach and RCC had relatively higher proportion of these cells (6.9%±2.5). The proliferating Ki67+ CTLs sub-population was low across all indications (0.37%±0.2). Upon stimulation there was a significant increase (>2.5-fold, p<0.05) in the proliferating CTLs sub-population across all indications though the cytokine release pattern varied. HNSCC showed a consistent increase (7.4-fold) in functional CTLs in all samples accompanied by a significant increase in INFɣ, Granzyme B and Perforin secretion. RCC and TNBC showed minimal change in functional CTLs but showed a significant increase in secretion of these cytokines. In ca-Stomach there was an increase in INFɣ secretion, but not Granzyme B and Perforin. In summary, while CTLs were present in TiME, the proportions of activated and proliferating sub-populations differed across indications. The presence of active CTLs did not always translate to tumor cytotoxicity. This could be due to other pro-tumor cells and factors that offset the anti-tumor activity of T cells. The TiME platform thus provides a unique platform to study complex interactions between tumor and intratumoral immune cell populations that could be further probed to understand differential response in patients to immunotherapy mediated tumor cytotoxicity. Citation Format: Nandini P. Basak, Vasanth K, Kowshik Jaganathan, Gowri Shankar K, Manimaran A, Rajashekar M, MS Ganesh, Amritha Prabha, Prakash BV, C Jaya Prakash, Sindhu Govindan, Ritu Malhotra, Pradeep Kar, Oliyarasi M, Anirudh Suri, Rachita Rao, Satish Sankaran. Differential T cell response within the tumor microenvironment observed across different indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1986.

Abstract CT249: First-in-human study of TAK-500, a novel STING agonist immune stimulating antibody conjugate (ISAC), alone and in combination with pembrolizumab in patients with select advanced solid tumors

Abstract Background: Although immune checkpoint inhibitors (CPIs) have revolutionized cancer treatment, resistance remains a challenge. Reduced interferon (IFN) signaling, immune escape and immunosuppressive tumor phenotypes have been proposed as resistance mechanisms, suggesting innate immune cell stimulation in the tumor microenvironment as a potential strategy to overcome resistance. Stimulator of Interferon Genes (STING) is a cytosolic protein critical for induction of type 1 IFN-dependent innate immunity. Cysteine-cysteine chemokine receptor type 2 (CCR2) is expressed by tumor-infiltrating myeloid cells, including tumor associated macrophages (TAM), and promotes immune escape by limiting CD8+ T-cell infiltration. TAK-500 is an ISAC that consists of three parts: a STING agonist payload based on TAK-676 (currently under phase 1 clinical evaluation [NCT04420884, NCT04879849]), the IgG1 anti-CCR2 antibody (previously evaluated in early phase studies), and a self-immolating maleimide-containing protease-cleavable peptide linker. By targeting STING to CCR2 expressing myeloid cells, TAK-500 has three possible mechanisms of action: activation of IFN response, reprogramming of suppressive intratumoral CCR2+ cells to an inflammatory phenotype, and blockade of suppressive TAM recruitment. TAK-500 thus has the potential to overcome resistance to CPIs in both CPI refractory and immunologically excluded or deserted tumors. Methods: This phase 1a/1b open-label study (NCT05070247) will evaluate the safety, tolerability, antitumor activity, pharmacokinetics and pharmacodynamics of TAK-500 in patients aged ≥18 years with gastroesophageal adenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, non-squamous non-small cell lung cancer, squamous cell carcinoma of the head and neck, mesothelioma or triple-negative breast cancer. Patients must have had progressive disease or intolerance to all standard therapy. In the initial dose escalation phase, patients will receive single-agent intravenous (IV) TAK-500 administered once every 3 weeks (Q3W) in 21-day cycles to determine the pharmacologically active dose (PAD) range. An additional escalation cohort will receive TAK-500 in combination with IV pembrolizumab 200 mg Q3W in 21-day cycles, with the initial TAK-500 dose level beginning 1-2 dose levels below the predicted single agent PAD range. Dose escalation in both single agent and combination cohorts will be guided by the Bayesian Optimal Interval design. A subsequent dose expansion phase will evaluate TAK-500 in combination with pembrolizumab. Planned enrollment for both escalation and expansion cohorts is ~106 patients. Citation Format: Jennifer R. Diamond, Jason T. Henry, Gerald S. Falchook, Anthony J. Olszanski, Harshabad Singh, E. Jane Leonard, Richard C. Gregory, Vicky A. Appleman, John P. Gibbs, Carole E. Harbison, Cong Li, Jessica M. Sapiro, Tomoki Yoneyama, Alexander Parent, Vincent Chung. First-in-human study of TAK-500, a novel STING agonist immune stimulating antibody conjugate (ISAC), alone and in combination with pembrolizumab in patients with select advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT249.

Phase 1a/1b study design of the novel STING agonist, immune-stimulating antibody-conjugate (ISAC) TAK-500, with or without pembrolizumab in patients with advanced solid tumors.

TPS2690 Background: Tumor resistance to immune checkpoint inhibitors (CPIs), including pembrolizumab, is common. Suggested mechanisms of resistance include reduced interferon (IFN) signaling, immune escape, and immunosuppressive tumor phenotypes. Innate immune cell stimulation in the tumor microenvironment may be a potential pathway to overcome this resistance. Stimulator of interferon genes (STING) is a cytosolic protein critical for initiation of type-1 IFN-dependent innate immunity. TAK-500 is a novel ISAC comprising a STING agonist (based on TAK-676, which is currently in phase 1 clinical trials [NCT04420884, NCT04879849]), an IgG1 anti-cysteine-cysteine chemokine receptor 2 (CCR2) antibody, and a self-immolating maleimide-containing protease-cleavable peptide linker. CCR2-expressing myeloid cells, including tumor associated macrophages (TAMs), promote immune evasion in part by reducing infiltration of CD8+ T cells into the tumor microenvironment. As such, TAK-500 has the potential to mitigate CPI resistance in solid tumors via targeted STING activation of tumor-infiltrating CCR2-expressing myeloid cells, thus leading to stimulation of innate and adaptive immunity within the tumor microenvironment through three potential mechanisms of action: activation of IFN signaling, reprogramming of CCR2-expressing myeloid cells to an inflammatory phenotype, and blockade of suppressive TAM recruitment. Methods: Adult patients with a diagnosis of locally advanced or metastatic gastroesophageal adenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, non-squamous non-small cell lung cancer, squamous cell carcinoma of the head and neck, mesothelioma or triple-negative breast cancer are eligible. All patients must have progressive disease while on, or be intolerant to, all current standard therapies. Approximately 106 patients in total will be enrolled to the dose escalation and expansion cohorts. In the dose escalation phase, intravenous (IV) TAK-500 will be administered over the range 8–480 µg/kg on day 1 of every 21-day cycle (Q3W) to establish the pharmacologically active dose (PAD) range. An additional escalation cohort of patients will receive TAK-500 + IV pembrolizumab 200 mg Q3W, starting at a dose of TAK-500 that is 1–2 dose levels below the lowest PAD range established in the single agent cohort. Dose escalation in both cohorts will be guided by Bayesian Optimal Interval design. In the dose expansion phase, only the combination of TAK-500 + pembrolizumab will be evaluated. Primary objectives of this study are safety and tolerability; secondary objectives include determination of the PAD range, the recommended phase 2 dose, pharmacokinetics, pharmacodynamics, and antitumor activity of TAK-500 as a single agent and in combination with pembrolizumab. Clinical trial information: NCT05070247.

Biological Consequences of HIV‐1 Interactions with Bacteria

Microbiota dysbiosis due to bacterial vaginosis and other venereal diseases has been associated with an increased risk of HIV‐1 transmission and acquisition. The prevalent idea is that microbial perturbations compromise the mucosal integrity and create inflammatory conditions in the mucosa which results in the recruitment and stimulation of immune cells that harbor or are targeted by HIV‐1. However, it is also possible that HIV‐1 virions bind bacteria or bacterial products in a manner that impacts virus infectivity and transmissibility. This study aims to examine direct interactions between HIV‐1 and bacteria and to better understand the biological consequences of such interactions. Specifically, we focus on HIV‐1 interactions via glycan‐binding proteins, or lectins, expressed on bacterial surfaces. We investigated multiple lectins expressed by bacteria: FimH from uropathogenic Escherichia coli, MsL from vaginal and oral non‐pathogenic Lactobacillus plantarum, and three Siglec‐like adhesins (SLBR‐B, SLBR‐H, and SLBR‐N) from oral Streptococcus gordonii. FimH and MsL bind high mannose N‐glycans while the SLBRs bind different α‐2,3 sialylated O‐glycan structures. N‐ and O‐glycans have been identified on the surface of HIV‐1 on Env or host‐derived glycoproteins. Both FimH and MsL had no impact on HIV‐1 infectivity, while SLBR‐N increased HIV‐1 infection by up to 3‐fold. The observed effects of SLBR‐N were recapitulated by plant lectins specific for similar glycan epitopes, further validating these findings. We hypothesize that this enhancement effect could be due to accumulation of viral particles and/or bridging effects between the cell surface and HIV‐1 virions. Studies are in progress to decipher the molecular mechanisms for this enhancement effect. This data will help us better understand the importance of direct interactions between viral particles and bacteria in HIV‐1 acquisition.

Acoustofluidic Stimulation of Functional Immune Cells in a Microreactor (Adv. Sci. 16/2022)

Acoustofluidic Stimulation of Functional Immune Cells In article number 2105809, Jinsoo Park, Hyung Jin Sung, Jessie S. Jeon, and co-workers employ surface acoustic waves to agitate fluid in a microreactor capable of dynamic cell culture in a controllable and contact-free manner. This microreactor prevents cellular damages that can be seen in the acoustic vortex by adjusting the duty cycle and applied power of the acoustical actuation. Using the microreactor, the functional changes of natural killer cells are observed upon the acoustical stimulation, namely, Ca2+ influx to the cells and their enhanced cell-mediated cytotoxicity to the tumor cells in the copresence of protein kinase C activator.

Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques

Abstract TRM constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. Experiments in mice demonstrated that TRM reactivation triggers antiviral responses in neighboring cells by promoting local stimulation of innate and adaptive immune cells, and by recruiting immune effectors. Collectively this is referred to as ‘sensing and alarm’ function. We wished to broadly identify TRM functions in non-human primates (NHP). Here we applied a prime-boost vaccine modality in rhesus macaques to generate abundant SIVgag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLT. To assess sensing and alarm function, macaques were challenged intravaginally with CM9 peptide from the SIV gag protein and necropsied 24 or 48 hours later. CD8 TRM reactivation increased expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the FRT at 24h, in situ proliferation (Ki67 expression) by 48h, and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells by 24h. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. Mucosal CD4 T cells expressed various HIV restriction factors, and had reduced expression of the HIV-coreceptor CCR5. Increased numbers of vaginal T and B cells coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T cells, B cells and SIV-env-specific B cells. These data demonstrate that vaccine-elicited CD8 TRM rapidly trigger local activation and the recruitment of innate, cellular, and humoral immune responses to the site of antigen exposure.

Abstract 245: Antibody Responses To ApoA-I Rely On An Interplay Between B And T Cells

Apolipoprotein A-I (ApoA-I), the major protein of high-density lipoprotein (HDL), has been investigated as the target and regulatory element of immune responses in both patients and mice susceptible to atherosclerosis. Previous work by our lab has focused on investigating the immune responses to ApoA-I in wild-type mice as a strategy to understand their role in models of atherosclerosis and other inflammatory conditions. We hypothesize that peripheral immunologic tolerance is responsible for regulating the dynamic immune response to ApoA-I. To examine this, we first evaluated antibody profiles to ApoA-I in both wild-type C57BL/6 and LDL receptor knockout mice and found that anti-ApoA-I antibodies naturally develop to the protein. To determine whether this response was T cell dependent or independent, we examined whether nu/nu mice on a BALB/c background, and the parent strain, displayed the phenotype. These studies showed that both nu/nu, or wild type BALB/c mice fail to mount antibody responses to ApoA-I suggesting a regulatory mechanism might be responsible for suppressing this antibody response. Ex vivo stimulation of immune cells with ApoA-I provide evidence of both B cell and T cell activation, suggesting that peripheral tolerance mechanisms may be responsible for regulating the humoral and cellular immune response to ApoA-I. These findings agree with previous studies where ApoA-I has been shown to regulate T cell activity in the context of atherosclerosis by inducing the development of regulatory T cells, which assist in suppressing the development of plaque formation; as well as studies showing a higher propensity toward Treg development in BALB/c mice. Ongoing experiments, which more clearly characterize the role of B cells and T cells in response to ApoA-I, will be presented with the goal of clarifying the immune mechanisms responsible for anti-ApoA-I autoimmunity. These data will help guide continued understanding of the role of immune responses to ApoA-I in atherosclerotic disease progression.

Acoustofluidic Stimulation of Functional Immune Cells in a Microreactor.

The cytotoxic response of natural killer (NK) cells in a microreactor to surface acoustic waves (SAWs) is investigated, where the SAWs produce an acoustic streaming flow. The Rayleigh‐type SAWs form by an interdigital transducer propagated along the surface of a piezoelectric substrate in order to allow the dynamic stimulation of functional immune cells in a noncontact and rotor‐free manner. The developed acoustofluidic microreactor enables a dynamic cell culture to be set up in a miniaturized system while maintaining the performance of agitating media. The present SAW system creates acoustic streaming flow in the cylindrical microreactor and applies flow‐induced shear stress to the cells. The suspended NK cells are found to not be damaged by the SAW operation of the adjusted experimental setup. Suspended NK cell aggregates subjected to an SAW treatment show increased intracellular Ca2+ concentrations. Simultaneously treating the NK cells with SAWs and protein kinase C activator enhances the lysosomal protein expressions of the cells and the cell‐mediated cytotoxicity against target tumor cells. These have important implications by showing that acoustically actuated system allows dynamic cell culture without cell damages and further alters cytotoxicity‐related cellular activities.

Mechanical Activation of Immune T Cells via a Water Driven Nanomotor.

As a key step during immune response, antigen recognition requires direct mechanical interaction between T cells and antigen presenting cells. Upon subjection to mechanical forces, mechanotransduction is triggered. In this study, the mechanical forces generated by water driven synthetic Au-Zn nanomotors are used to activate mechanosensitive Jurkat T cells. The triggering and activation of the cellular Ca2+ channel is observed. It is revealed that the mechanosensitive cells experience different degrees of activation upon receiving different mechanical input signals and demonstrate that external mechanical forces can optimize T cell activation. Compared with T cell activation with cytokines which can lead to the risky widespread activation of T cells and systemic immune storm, nanomotors can present mechanical force and achieve localized immune cell stimulation. It is expected that mechano nanomotors will contribute to the emerging T cell immunology field and facilitate more comprehensive understanding of the T cell mechanical response and function.

Molecular phylogenetic analysis of amylase trypsin inhibitors (ATIs) from a selection of ancient and modern wheat

Wheat amylase-trypsin inhibitors (ATIs) are a family of wheat proteins, which play an important role in plant defence against pest attacks. Recently, ATIs have been identified as major stimulators of human innate immune cells leading to cause Non-celiac Wheat Sensitivity. Information about ATI sequence differences among wheat species is scarce, especially considering ancient wheat genotypes. In this study, ten selected wheat accessions with different ploidy level and year of release were used for gene sequencing of four representative alpha-amylase/trypsin inhibitor genes (WMAI, WDAI, WTAI-CM3 and CMx genes). The phylogenetic analysis and the PCA analysis performed on the deduced amino acid sequences of ATI genes evidenced that the ten wheat genotypes can be differentiated on the basis of their ploidy level, but not with respect to ancient or recently developed wheat genotypes. The haplotype analysis based on Nei's genetic distances, beside confirming these results, also allowed the separation of the hulled from the naked wheat genotypes. The genetic sequence differences highlighted in this study among the ten genotypes can be the basis for further studies aimed at identifying proinflammatory sequences in ATI genes. This is the first study analyzing the ATI genetic sequences of a set of ancient and modern wheat genotypes with a different ploidy level.