InVivoPure pH 6.5 Dilution Buffer

In mBio on 31 October 2023 by Esher, S. K., Harriett, A. J., et al.

PubMed

Polymicrobial intra-abdominal infections are serious clinical infections that can lead to life-threatening sepsis, which is difficult to treat in part due to the complex and dynamic inflammatory responses involved. Our prior studies demonstrated that immunization with low-virulence Candida species can provide strong protection against lethal polymicrobial sepsis challenge in mice. This long-lived protection was found to be mediated by trained Gr-1+ polymorphonuclear leukocytes with features resembling myeloid-derived suppressor cells (MDSCs). Here we definitively characterize these cells as MDSCs and demonstrate that their mechanism of protection involves the abrogation of lethal inflammation, in part through the action of the anti-inflammatory cytokine interleukin (IL)-10. These studies highlight the role of MDSCs and IL-10 in controlling acute lethal inflammation and give support for the utility of trained tolerogenic immune responses in the clinical treatment of sepsis.

• Cancer Research
,

In Clinical Cancer Research on 1 September 2023 by Kohlmeyer, J. L., Lingo, J. J., et al.

PubMed

Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes. ©2023 American Association for Cancer Research.

• Cancer Research
,
• Immunology and Microbiology

In Cancers on 16 August 2023 by Gerton, T. J., Green, A., et al.

PubMed

High-grade serous ovarian cancer (HGSOC) is responsible for the majority of gynecology cancer-related deaths. Patients in remission often relapse with more aggressive forms of disease within 2 years post-treatment. Alternative immuno-oncology (IO) strategies, such as immune checkpoint blockade (ICB) targeting the PD-(L)1 signaling axis, have proven inefficient so far. Our aim is to utilize epigenetic modulators to maximize the benefit of personalized IO combinations in ex vivo 3D patient-derived platforms and in vivo syngeneic models. Using patient-derived tumor ascites, we optimized an ex vivo 3D screening platform (PDOTS), which employs autologous immune cells and circulating ascites-derived tumor cells, to rapidly test personalized IO combinations. Most importantly, patient responses to platinum chemotherapy and poly-ADP ribose polymerase inhibitors in 3D platforms recapitulate clinical responses. Furthermore, similar to clinical trial results, responses to ICB in PDOTS tend to be low and positively correlated with the frequency of CD3+ immune cells and EPCAM+/PD-L1+ tumor cells. Thus, the greatest response observed with anti-PD-1/anti-PD-L1 immunotherapy alone is seen in patient-derived HGSOC ascites, which present with high levels of systemic CD3+ and PD-L1+ expression in immune and tumor cells, respectively. In addition, priming with epigenetic adjuvants greatly potentiates ICB in ex vivo 3D testing platforms and in vivo tumor models. We further find that epigenetic priming induces increased tumor secretion of several key cytokines known to augment T and NK cell activation and cytotoxicity, including IL-6, IP-10 (CXCL10), KC (CXCL1), and RANTES (CCL5). Moreover, epigenetic priming alone and in combination with ICB immunotherapy in patient-derived PDOTS induces rapid upregulation of CD69, a reliable early activation of immune markers in both CD4+ and CD8+ T cells. Consequently, this functional precision medicine approach could rapidly identify personalized therapeutic combinations able to potentiate ICB, which is a great advantage, especially given the current clinical difficulty of testing a high number of potential combinations in patients.

• Immunology and Microbiology

In ACS Chemical Biology on 16 June 2023 by Griffin, M. E., Tsukidate, T., et al.

PubMed

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific and effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

• Cancer Research
,
• Immunology and Microbiology

Preprint on BioRxiv : the Preprint Server for Biology on 8 June 2023 by DuCote, T. J., Song, X., et al.

PubMed

ABSTRACT Two important factors that contribute to resistance to immune checkpoint inhibitors (ICIs) are an immune-suppressive microenvironment and limited antigen presentation by tumor cells. In this study, we examine if inhibition of the methyltransferase EZH2 can increase ICI response in lung squamous cell carcinomas (LSCCs). Our in vitro experiments using 2D human cancer cell lines as well as 3D murine and patient derived organoids treated with two inhibitors of the EZH2 plus interferon-γ (IFNγ) showed that EZH2 inhibition leads to expression of both major histocompatibility complex class I and II (MHCI/II) expression at both the mRNA and protein levels. ChIP-sequencing confirmed loss of EZH2-mediated histone marks and gain of activating histone marks at key loci. Further, we demonstrate strong tumor control in models of both autochthonous and syngeneic LSCC treated with anti-PD1 immunotherapy with EZH2 inhibition. Single-cell RNA sequencing and immune cell profiling demonstrated phenotypic changes towards more tumor suppressive phenotypes in EZH2 inhibitor treated tumors. These results indicate that this therapeutic modality could increase ICI responses in patients undergoing treatment for LSCC.

• Mus musculus (House mouse)
,
• Cardiovascular biology
,
• Immunology and Microbiology
,
• Neuroscience

Preprint on BioRxiv : the Preprint Server for Biology on 12 March 2023 by Wayne, C. R., Bremner, L., et al.

PubMed

SUMMARY Group A Streptococcus (GAS) infections can cause neuropsychiatric sequelae in children due to post-infectious encephalitis. Multiple GAS infections induce migration of Th17 lymphocytes from the nose into the brain, which are critical for microglial activation, blood-brain barrier (BBB) and neural circuit impairment in a mouse disease model. How endothelial cells (ECs) and microglia respond to GAS infections, and which Th17-derived cytokines are essential for these responses are unknown. Using single-cell RNA sequencing and spatial transcriptomics, we found that ECs downregulate BBB genes and microglia upregulate interferon-response, chemokine and antigen-presentation genes after GAS infections. Several microglial-derived chemokines were elevated in patient sera. Administration of a neutralizing antibody against interleukin-17A (IL-17A), but not ablation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, partially rescued BBB dysfunction and microglial expression of chemokine genes. Thus, IL-17A is critical for neuropsychiatric sequelae of GAS infections and may be targeted to treat these disorders.

• Cancer Research

In International Journal of Radiation Oncology, Biology, Physics on 1 March 2023 by Mare, S. D., Nishri, Y., et al.

PubMed

Diffusing alpha-emitters Radiation Therapy (DaRT) releases alpha-emitting atoms into the tumor microenvironment. The treatment effectively ablates human and mice xenografts and shows 100% response rates in skin or head and neck squamous cell carcinoma patients. DaRT induces specific and systemic antitumor immune activation and synergizes with immune stimulation and modulation in mice. Here, the transcriptional profile activated by DaRT, and its potential to enhance responsiveness to immune checkpoint inhibition by programmed cell death protein 1 (PD-1) blockade were studied. Squamous cell carcinoma tumor- bearing BALB/C mice were treated with DaRT or inert seeds in combination with anti-PD-1 (aPD-1) or IgG control antibody. Sixteen days after seed insertion, tumors and spleens were subjected to immunophenotyping and immunohistochemical staining. Combination of DaRT and aPD-1 was tested for efficacy. Gene expression analysis was performed on mRNA extracted from tumors 7 days after DaRT or inert insertion using Nanostring PanCancer-IO-360 panel, and tumors and spleens were subjected to flow cytometry analysis. DaRT in combination with aPD-1 delayed tumor development, induced CD3 and CD8 lymphocytes infiltration more efficiently than either monotherapy. The combined treatment reduced splenic polymorphonuclear myeloid derived suppressor cells more than aPD-1 therapy or control. Granzyme B release in the tumor was increased only in the combinational treatment and was correlated with T-lymphocyte infiltration. Gene expression and gene set enrichment analysis of mRNA levels 7 days after DaRT insertion indicated that DaRT upregulated apoptosis, p53 signaling, G1/S-related arrest, interferon signaling and myeloid related transcription, while downregulating DNA repair, cell proliferation, and notch-related transcription. Flow cytometry showed that DaRT increased dendritic cells activation and led to changes in MDSCs distribution. DaRT promotes a "hot" tumor microenvironment and changes in immune suppression that lead to a potentiation of aPD-1 blockade induced effector T cell function and improved treatment efficacy. This study provides rationale for investigating DaRT and aPD-1 combination in patients with squamous cell carcinoma. Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

• IHC
,
• Mus musculus (House mouse)
,
• Cancer Research
,
• Immunology and Microbiology

Preprint on BioRxiv : the Preprint Server for Biology on 23 February 2023 by Nguyen, L. L., Watson, Z. L., et al.

PubMed

ABSTRACT Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

• Cardiovascular biology

In Pharmaceutics on 30 January 2023 by Ahmed, M., Hernández-Verdin, I., et al.

PubMed

Therapeutic antibodies targeting immune checkpoints have shown limited efficacy in clinical trials in glioblastoma (GBM) patients. Ultrasound-mediated blood-brain barrier opening (UMBO) using low-intensity pulsed ultrasound improved drug delivery to the brain. We explored the safety and the efficacy of UMBO plus immune checkpoint inhibitors in preclinical models of GBM. A blood-brain barrier (BBB) opening was performed using a 1 MHz preclinical ultrasound system in combination with 10 µL/g microbubbles. Brain penetration of immune checkpoint inhibitors was determined, and immune cell populations were evaluated using flow cytometry. The impact of repeated treatments on survival was determined. In syngeneic GL261-bearing immunocompetent mice, we showed that UMBO safely and repeatedly opened the BBB. BBB opening was confirmed visually and microscopically using Evans blue dye and magnetic resonance imaging. UMBO plus anti-PDL-1 was associated with a significant improvement of overall survival compared to anti-PD-L1 alone. Using mass spectroscopy, we showed that the penetration of therapeutic antibodies can be increased when delivered intravenously compared to non-sonicated brains. Furthermore, we observed an enhancement of activated microglia percentage when combined with anti-PD-L1. Here, we report that the combination of UMBO and anti-PD-L1 dramatically increases GL261-bearing mice's survival compared to their counterparts treated with anti-PD-L1 alone. Our study highlights the BBB as a limitation to overcome in order to increase the efficacy of anti-PD-L1 in GBM and supports clinical trials combining UMBO and in GBM patients.

• Immunology and Microbiology

Preprint on BioRxiv : the Preprint Server for Biology on 27 January 2023 by Griffin, M. E., Tsukidate, T., et al.

PubMed

ABSTRACT The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N -arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N -arylpyrazole NOD2 agonist is enantiomer-specific, effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo . Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

• Cancer Research
,
• Immunology and Microbiology

In Journal for Immunotherapy of Cancer on 1 December 2022 by Qiao, M., Zhou, F., et al.

PubMed

Anti-PD-1(L1) therapies are less efficacious in patients with EGFR-mutated non-small-cell lung cancer. However, the underlying mechanism is poorly understood. The characteristics of T cells in EGFR-mutated and wild-type tumors were analyzed based on The Cancer Genome Atlas database and clinical samples. Plasma levels of 8 T-cell-related cytokines were evaluated and its association with immunotherapy efficacy were explored. Association between EGFR signaling pathway and IL-10 was examined through tumor cell lines and clinical tumor samples. In vitro restimulation model of human CD8+T cells isolated from peripheral blood was used to analyze the impact of IL-10 on T cells. Doxycycline-inducible transgenic EGFRL858R mouse models were used to investigate the efficacy of combining recombinant mouse IL-10 protein and PD-1 blockade and its underlying mechanism in vivo. EGFR-mutated tumors showed a lack of CD8+T cell infiltration and impaired CD8+T cell cytotoxic function. The incompetent CD8+T cells in EGFR-mutated tumors were characterized as absence of CD39 expression, which defined hallmarks of cytotoxic and exhausted features and could not be reinvigorated by anti-PD-1(L1) treatment. Instead, CD39 expression defined functional states of CD8+T cells and was associated with the therapeutic response of anti-PD-1(L1) therapies. Mechanically, IL-10 upregulated CD39 expression and was limited in EGFR-mutated tumors. IL-10 induced hallmarks of CD8+T cells immunity in CD39-dependent manner. Using autochthonous EGFR L858R-driven lung cancer mouse models, combining recombinant mouse IL-10 protein and PD-1 blockade optimized antitumor effects in EGFR-mutated lung tumors. Our study suggested that owing to low level of IL-10 to induce the expression of CD39 on CD8+T cells, fewer phenotypically cytotoxic and exhausted CD39+CD8+T cells in EGFR-mutated tumors could be potentially reinvigorated by anti-PD-1(L1) treatment. Hence, IL-10 could potentially serve as a cytokine-based strategy to enhance efficacy of anti-PD-1(L1) treatment in EGFR-mutated tumors. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

• Cancer Research
,
• Immunology and Microbiology

In Cancer Cell on 14 November 2022 by Zhou, Z., Chen, M. M., et al.

PubMed

Checkpoint inhibition immunotherapy has revolutionized cancer treatment, but many patients show resistance. Here we perform integrative transcriptomic and proteomic analyses on emerging immuno-oncology targets across multiple clinical cohorts of melanoma under anti-PD-1 treatment, on both bulk and single-cell levels. We reveal a surprising role of tumor-intrinsic SIRPA in enhancing antitumor immunity, in contrast to its well-established role as a major inhibitory immune modulator in macrophages. The loss of SIRPA expression is a marker of melanoma dedifferentiation, a key phenotype linked to immunotherapy efficacy. Inhibition of SIRPA in melanoma cells abrogates tumor killing by activated CD8+ T cells in a co-culture system. Mice bearing SIRPA-deficient melanoma tumors show no response to anti-PD-L1 treatment, whereas melanoma-specific SIRPA overexpression significantly enhances immunotherapy response. Mechanistically, SIRPA is regulated by its pseudogene, SIRPAP1. Our results suggest a complicated role of SIRPA in the tumor ecosystem, highlighting cell-type-dependent antagonistic effects of the same target on immunotherapy. Copyright © 2022 Elsevier Inc. All rights reserved.

• Immunology and Microbiology
,
• In Vivo
,
• Mus musculus (House mouse)

In Cellular and Molecular Gastroenterology and Hepatology on 25 April 2021 by Go, D. M., Lee, S. H., et al.

PubMed

Helicobacter pylori has been reported to modulate local immune responses to colonize persistently in gastric mucosa. Although the induced expression of programmed cell death ligand 1 (PD-L1) has been suggested as an immune modulatory mechanism for persistent infection of H pylori, the main immune cells expressing PD-L1 and their functions in Helicobacter-induced gastritis still remain to be elucidated. The blockades of PD-L1 with antibody or PD-L1-deficient bone marrow transplantation were performed in Helicobacter-infected mice. The main immune cells expressing PD-L1 in Helicobacter-infected stomach were determined by flow cytometry and immunofluorescence staining. Helicobacter felis or H pylori-infected dendritic cell (DC)-deficient mouse models including Flt3-/-, Zbtb46-diphtheria toxin receptor, and BDCA2-diphtheria toxin receptor mice were analyzed for pathologic changes and colonization levels. Finally, the location of PD-L1-expressing DCs and the correlation with H pylori infection were analyzed in human gastric tissues using multiplexed immunohistochemistry. Genetic or antibody-mediated blockade of PD-L1 aggravated Helicobacter-induced gastritis with mucosal metaplasia. Gastric classical DCs expressed considerably higher levels of PD-L1 than other immune cells and co-localized with T cells in gastritis lesions from Helicobacter-infected mice and human beings. H felis- or H pylori-infected Flt3-/- or classical DC-depleted mice showed aggravated gastritis with severe T-cell and neutrophil accumulation with low bacterial loads compared with that in control mice. Finally, PD-L1-expressing DCs were co-localized with T cells and showed a positive correlation with H pylori infection in human subjects. The PD-1/PD-L1 pathway may be responsible for the immune modulatory function of gastric DCs that protects the gastric mucosa from Helicobacter-induced inflammation, but allows persistent Helicobacter colonization. Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

• Cancer Research
,
• Immunology and Microbiology

In Gut on 1 January 2021 by Knudsen, E. S., Kumarasamy, V., et al.

PubMed

This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer. Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment. We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors. Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy. © Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.

• Cancer Research
,
• Immunology and Microbiology

In Journal for Immunotherapy of Cancer on 1 October 2020 by He, X., Du, Y., et al.

PubMed

The survival benefits of combining chemotherapy (at the maximum tolerated dose, MTD) with concurrent immunotherapy, collectively referred to as chemoimmunotherapy, for the treatment of squamous cell lung carcinoma (SQCLC) have been confirmed in recent clinical trials. Nevertheless, optimization of chemoimmunotherapy in order to enhance the efficacy of immune checkpoint inhibitors (ICIs) in SQCLC remains to be explored. Cell lines, syngeneic immunocompetent mouse models, and patients' peripheral blood mononuclear cells were used in order to comprehensively explore how to enhance ectopic lymphoid-like structures (ELSs) and upregulate the therapeutic targets of anti-programmed death 1 (PD-1)/anti-PD-1 ligand (PD-L1) monoclonal antibodies (mAbs), thus rendering SQCLC more sensitive to ICIs. In addition, molecular mechanisms underlying optimization were characterized. Low-dose chemotherapy contributed to an enhanced antigen exposure via the phosphatidylinositol 3-kinase/Akt/transcription factor nuclear factor kappa B signaling pathway. Improved antigen uptake and presentation by activated dendritic cells (DCs) was observed, thus invoking specific T cell responses leading to systemic immune responses and immunological memory. In turn, enhanced antitumor ELSs and PD-1/PD-L1 expression was observed in vivo. Moreover, upfront metronomic (low-dose and frequent administration) chemotherapy extended the time window of the immunostimulatory effect and effectively synergized with anti-PD-1/PD-L1 mAbs. A possible mechanism underlying this synergy is the increase of activated type I macrophages, DCs, and cytotoxic CD8+ T cells, as well as the maintenance of intestinal gut microbiota diversity and composition. In contrast, when combining routine MTD chemotherapy with ICIs, the effects appeared to be additive rather than synergistic. We first attempted to optimize chemoimmunotherapy for SQCLC by investigating different combinatorial modes. Compared with the MTD chemotherapy used in current clinical practice, upfront metronomic chemotherapy performed better with subsequent anti-PD-1/PD-L1 mAb treatment. This combination approach is worth investigating in other types of tumors, followed by translation into the clinic in the future. © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

• Cardiovascular biology
,
• Immunology and Microbiology

In Cell Reports Medicine on 22 September 2020 by Marinaik, C. B., Kingstad-Bakke, B., et al.

PubMed

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2. © 2020 The Author(s).