CD94 deficiency or blockade unleashes the anti-tumor immunity in mice and humanized murine models.

NKG2 family members have emerged as promising targets in tumor immunotherapy. CD94 can dimerize with both inhibitory and activating NKG2 proteins, while the overall effect and value of targeting CD94 on anti-tumor immunity are unclear. Here, it is shown that the expression of CD94 is upregulated on tumor-infiltrating natural killer (NK) cells and CD8+ T cells, and is related to their exhausted characteristics. Tumor-bearing CD94 knockout (CD94-KO) mice exhibit delayed tumor growth, decreased lung metastases, and prolonged survival. Single cell RNA-seq reveals a remodeled tumor microenvironment in CD94-KO mice, with a reduction in immunosuppressive cells and an increase in anti-tumor immune cells. Moreover, NK cells and CD8+ T cells become proliferative and strongly tumoricidal in CD94-KO mice, thus contributing to the tumor inhibition effect of CD94 deficiency. Treatment with a humanized anti-CD94 blocking antibody (h15C10) alone, in tumor-bearing humanized mouse, delays tumor progression, and improves the therapeutic efficacy of PD-L1 blockade through combination therapy. Our study indicates that CD94 may work as a candidate target in checkpoint immunotherapy.

Multimodal probing of T-cell recognition with hexapod heterostructures.

Studies using antigen-presenting systems at the single-cell and ensemble levels can provide complementary insights into T-cell signaling and activation. Although crucial for advancing basic immunology and immunotherapy, there is a notable absence of synthetic material toolkits that examine T cells at both levels, and especially those capable of single-molecule-level manipulation. Here we devise a biomimetic antigen-presenting system (bAPS) for single-cell stimulation and ensemble modulation of T-cell recognition. Our bAPS uses hexapod heterostructures composed of a submicrometer cubic hematite core (α-Fe2O3) and nanostructured silica branches with diverse surface modifications. At single-molecule resolution, we show T-cell activation by a single agonist peptide-loaded major histocompatibility complex; distinct T-cell receptor (TCR) responses to structurally similar peptides that differ by only one amino acid; and the superior antigen recognition sensitivity of TCRs compared with that of chimeric antigen receptors (CARs). We also demonstrate how the magnetic field-induced rotation of hexapods amplifies the immune responses in suspended T and CAR-T cells. In addition, we establish our bAPS as a precise and scalable method for identifying stimulatory antigen-specific TCRs at the single-cell level. Thus, our multimodal bAPS represents a unique biointerface tool for investigating T-cell recognition, signaling and function.

A single-cell atlas reveals shared and distinct immune responses and metabolic profiles in SARS-CoV-2 and HIV-1 infections.

Introduction: Within the inflammatory immune response to viral infection, the distribution and cell type-specific profiles of immune cell populations and the immune-mediated viral clearance pathways vary according to the specific virus. Uncovering the immunological similarities and differences between viral infections is critical to understanding disease progression and developing effective vaccines and therapies. Insight into COVID-19 disease progression has been bolstered by the integration of single-cell (sc)RNA-seq data from COVID-19 patients with data from related viruses to compare immune responses. Expanding this concept, we propose that a high-resolution, systematic comparison between immune cells from SARS-CoV-2 infection and an inflammatory infectious disease with a different pathophysiology will provide a more comprehensive picture of the viral clearance pathways that underscore immunological and clinical differences between infections. Methods: Using a novel consensus single-cell annotation method, we integrate previously published scRNA-seq data from 111,566 single PBMCs from 7 COVID-19, 10 HIV-1+, and 3 healthy patients into a unified cellular atlas. We compare in detail the phenotypic features and regulatory pathways in the major immune cell clusters. Results: While immune cells in both COVID-19 and HIV-1+ cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activity, and downregulated mitophagy. Discussion: Our results indicate that differential IFN-I signaling regulates the distinct immune responses in the two diseases, revealing insight into fundamental disease biology and potential therapeutic candidates.

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.

BACKGROUND: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. METHODS: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. RESULTS: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. CONCLUSION: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

The use of supercytokines, immunocytokines, engager cytokines, and other synthetic cytokines in immunotherapy.

Cytokines exert powerful immunomodulatory effects that are critical to physiology and pathology in humans. The application of natural cytokines in clinical studies has not been clearly established, and there are often problems associated with toxicity or lack of efficacy. The key reasons can be attributed to the pleiotropy of cytokine receptors and undesired activation of off-target cells. With a deeper understanding of the structural principles and functional signals of cytokine-receptor interactions, artificial modification of cytokine signaling through protein engineering and synthetic immunology has become an increasingly feasible and powerful approach. Engineered cytokines are designed to selectively target cells. Herein, the theoretical and experimental evidence of cytokine engineering is reviewed, and the "supercytokines" resulting from structural enhancement and the "immunocytokines" generated by antibody fusion are described. Finally, the "engager cytokines" formed by the crosslinking of cytokines and bispecific immune engagers and other synthetic cytokines formed by nonnatural analogs are also discussed.

A single-cell atlas reveals shared and distinct immune responses and metabolism during SARS-CoV-2 and HIV-1 infections.

SARS-CoV-2 and HIV-1 are RNA viruses that have killed millions of people worldwide. Understanding the similarities and differences between these two infections is critical for understanding disease progression and for developing effective vaccines and therapies, particularly for 38 million HIV-1 + individuals who are vulnerable to SARS-CoV-2 co-infection. Here, we utilized single-cell transcriptomics to perform a systematic comparison of 94,442 PBMCs from 7 COVID-19 and 9 HIV-1 + patients in an integrated immune atlas, in which 27 different cell types were identified using an accurate consensus single-cell annotation method. While immune cells in both cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activities, and downregulated mitophagy. Our results elucidate transcriptional signatures associated with COVID-19 and HIV-1 that may reveal insights into fundamental disease biology and potential therapeutic targets to treat these viral infections. HIGHLIGHTS: COVID-19 and HIV-1 + patients show disease-specific inflammatory immune signatures COVID-19 patients show more productive humoral responses than HIV-1 + patients SARS-CoV-2 elicits more enriched IFN-I signaling relative to HIV-IDivergent, impaired metabolic programs distinguish SARS-CoV-2 and HIV-1 infections.

The Role of Oxidative Stress in Intervertebral Disc Degeneration.

Intervertebral disc degeneration is a very common type of degenerative disease causing severe socioeconomic impact, as well as a major cause of discogenic low back pain and herniated discs, placing a heavy burden on patients and the clinicians who treat them. IDD is known to be associating with a complex process involving in extracellular matrix and cellular damage, and in recent years, there is increasing evidence that oxidative stress is an important activation mechanism of IDD and that reactive oxygen and reactive nitrogen species regulate matrix metabolism, proinflammatory phenotype, autophagy and senescence in intervertebral disc cells, apoptosis, autophagy, and senescence. Despite the tremendous efforts of researchers within the field of IDD pathogenesis, the proven strategies to prevent and treat this disease are still very limited. Up to now, several antioxidants have been proved to be effective for alleviating IDD. In this article, we discussed that oxidative stress accelerates disc degeneration by influencing aging, inflammation, autophagy, and DNA methylation, and summarize some antioxidant therapeutic measures for IDD, indicating that antioxidant therapy for disc degeneration holds excellent promise.

Antigen-Multimers: Specific, Sensitive, Precise, and Multifunctional High-Avidity CAR-Staining Reagents.

Although chimeric antigen receptor (CAR) T-cell therapy has transformed cancer treatment, high-quality and universal CAR-staining reagents are urgently required to manufacture CAR T cells, predict therapy response, decipher CAR biology, and engineer new CARs. Here, we developed tetrameric and dodecameric forms of a multifunctional and extensible category of high-avidity CAR-staining reagents: antigen-multimers. Antigen-multimers detected CARs against CD19, HER2, and Tn-glycoside with significantly higher specificity, sensitivity, and precision than existing reagents. In addition to accurate CAR T-cell detection by flow cytometry, antigen-multimers also enabled ≥100-fold magnetic enrichment of rare CAR T cells, selective CAR T-cell stimulation, and high-dimensional CAR T-cell profiling by single-cell multi-omics analyses. Finally, antigen-multimers accurately captured clinical anti-CD19 CAR T cells from patients' cellular infusion products, post-infusion peripheral blood, and tumor biopsies. Antigen-multimers can be readily extended to other CAR systems by switching its antigen ligand. As such, antigen-multimers have broad clinical and research applications.

All-trans retinoic acid induces leukemia resistance to NK cell cytotoxicity by down-regulating B7-H6 expression via c-Myc signaling.

BACKGROUND: The interaction between activating receptor NKp30 and its major tumor ligand B7-H6 is important for NK cell-mediated tumor rejection. However, the regulation of B7-H6 by tumor therapeutics remains largely unknown. In this study, we investigated the regulation of B7-H6 by all-trans retinoic acid (atRA), a terminal differentiation inducer of tumor cells that is extensively used for clinical leukemia therapy. METHODS: We investigated the role of NKp30:B7-H6 axis in NK cell-mediated tumor lysis against leukemia cells and the influence of atRA treatment on the cytotoxicity of NK cells using NK cell lines (NK92 and NKG) and leukemia cell lines (U-937 and THP-1). We evaluated the effect of atRA treatment on the expression of B7-H6 using real-time PCR, flow cytometry and western blotting. We used CRISPR/Cas9 to knockdown B7-H6 expression and siRNA to knockdown c-Myc in U-937 cells to evaluate the role of B7-H6 and c-Myc in atRA-induced tumor resistance against NK cells. RESULTS: NK cell-mediated U-937 cell lysis was mainly dependent on NKp30/B7-H6 interaction. Blockade of B7-H6 by monoclonal antibody significantly impaired NK cytotoxicity. atRA treatment induced U-937 resistance to NK cell cytotoxicity by reducing B7-H6 expression, and showed no effect on NK cytotoxicity against B7-H6 knockdown U-937 cells. Epigenetic modifications, such as DNA methylation and histone deacetylase (HDAC), were not responsible for atRA-mediated B7-H6 down-regulation as inhibitors of these pathways could not restore B7-H6 mRNA expression. On the other hand, atRA treatment reduced c-Myc expression, which in turn inhibited the transcription of B7-H6 on leukemia cells. CONCLUSION: atRA treatment promotes tumor cell resistance against NK cell-mediated lysis by down-regulating B7-H6 expression via the c-Myc signaling pathway, suggesting that more attention needs to be paid to the immunological adverse effects in the clinical use of atRA treatment.

Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors.

BACKGROUND: Although checkpoint-based immunotherapy has shown exciting results in the treatment of tumors, around 70% of patients have experienced unresponsiveness. PVRIG is a recently identified immune checkpoint receptor and blockade of which could reverse T cell exhaustion to treat murine tumor; however, its therapeutic potential via NK cells in mice and human remains seldom reported. METHODS: In this study, we used patient paraffin-embedded colon adenocarcinoma sections, various murine tumor models (MC38 colon cancer, MCA205 fibrosarcoma and LLC lung cancer), and human NK cell- or PBMC-reconstituted xenograft models (SW620 colon cancer) to investigate the effect of PVRIG on tumor progression. RESULTS: We found that PVRIG was highly expressed on tumor-infiltrating NK cells with exhausted phenotype. Furthermore, either PVRIG deficiency, early blockade or late blockade of PVRIG slowed tumor growth and prolonged survival of tumor-bearing mice by inhibiting exhaustion of NK cells as well as CD8+ T cells. Combined blockade of PVRIG and PD-L1 showed better effect in controlling tumor growth than using either one alone. Depletion of NK or/and CD8+ T cells in vivo showed that both cell types contributed to the anti-tumor efficacy of PVRIG blockade. By using Rag1-/- mice, we demonstrated that PVRIG blockade could provide therapeutic effect in the absence of adaptive immunity. Further, blockade of human PVRIG with monoclonal antibody enhanced human NK cell function and inhibited human tumor growth in NK cell- or PBMC-reconstituted xenograft mice. CONCLUSIONS: Our results reveal the importance of NK cells and provide novel knowledge for clinical application of PVRIG-targeted drugs in future.

Lattice Light-Sheet Microscopy Multi-dimensional Analyses (LaMDA) of T-Cell Receptor Dynamics Predict T-Cell Signaling States.

Lattice light-sheet microscopy provides large amounts of high-dimensional, high-spatiotemporal resolution imaging data of cell surface receptors across the 3D surface of live cells, but user-friendly analysis pipelines are lacking. Here, we introduce lattice light-sheet microscopy multi-dimensional analyses (LaMDA), an end-to-end pipeline comprised of publicly available software packages that combines machine learning, dimensionality reduction, and diffusion maps to analyze surface receptor dynamics and classify cellular signaling states without the need for complex biochemical measurements or other prior information. We use LaMDA to analyze images of T-cell receptor (TCR) microclusters on the surface of live primary T cells under resting and stimulated conditions. We observe global spatial and temporal changes of TCRs across the 3D cell surface, accurately differentiate stimulated cells from unstimulated cells, precisely predict attenuated T-cell signaling after CD4 and CD28 receptor blockades, and reliably discriminate between structurally similar TCR ligands. All instructions needed to implement LaMDA are included in this paper.

The histone methyltransferase EZH2 primes the early differentiation of follicular helper T cells during acute viral infection.

Epigenetic modifications to histones dictate the differentiation of naïve CD4+ T cells into different subsets of effector T helper (TH) cells. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been implicated in the mechanism regulating the differentiation of TH1, TH2 and regulatory T (Treg) cells. However, whether and how EZH2 regulates follicular helper T (TFH) cell differentiation remain unknown. Using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, we observed abundant EZH2 expression and associated H3K27me3 modifications preferentially in the early committed virus-specific TFH cells compared to those in TH1 cells. Ablation of EZH2 in LCMV-specific CD4+ T cells leads to a selective impairment of early TFH cell fate commitment, but not late TFH differentiation or memory TFH maintenance. Mechanistically, EZH2 specifically stabilizes the chromatin accessibility of a cluster of genes that are important for TFH fate commitment, particularly B cell lymphoma 6 (Bcl6), and thus directs TFH cell commitment. Therefore, we identified the chromatin-modifying enzyme EZH2 as a novel regulator of early TFH differentiation during acute viral infection.

TCR-pMHC bond conformation controls TCR ligand discrimination.

A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR-pMHC bonds and the conformations of individual TCR-CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR-pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR-pMHC bond conformation.

Tumor Therapeutics Work as Stress Inducers to Enhance Tumor Sensitivity to Natural Killer (NK) Cell Cytolysis by Up-regulating NKp30 Ligand B7-H6.

Immune cells are believed to participate in initiating anti-tumor effects during regular tumor therapy such as chemotherapy, radiation, hyperthermia, and cytokine injection. One of the mechanisms underlying this process is the expression of so-called stress-inducible immunostimulating ligands. Although the activating receptor NKG2D has been proven to play roles in tumor therapy through targeting its ligands, the role of NKp30, another key activating receptor, is seldom addressed. In this study, we found that the NKp30 ligand B7-H6 was widely expressed in tumor cells and closely correlated to their susceptibility to NK cell lysis. Further studies showed that treatment of tumor cells with almost all standard tumor therapeutics, including chemotherapy (cisplatin, 5-fluorouracil), radiation therapy, non-lethal heat shock, and cytokine therapy (TNF-α), could up-regulate the expression of B7-H6 in tumor cells and enhance tumor sensitivity to NK cell cytolysis. B7-H6 shRNA treatment effectively dampened sensitization of tumor cells to NK-mediated lysis. Our study not only reveals the possibility that tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis but also suggests that B7-H6 could be a potential target for tumor therapy in the future.

Revealing Invisible Photonic Inscriptions: Images from Strain.

Photonic structural materials have received intensive interest and have been strongly developed over the past few years for image displays, sensing, and anticounterfeit materials. Their "smartness" arises from their color responsivity to changes of environment, strain, or external fields. Here, we introduce a novel invisible photonic system that reveals encrypted images or characters by simply stretching, or immersing in solvents. This type of intriguing photonic material is composed of regularly arranged core-shell particles that are selectively cross-linked by UV irradiation, giving different strain response compared to un-cross-linked regions. The images reversibly appear and disappear when cycling the strain and releasing it. The unique advantages of this soft polymer opal system compared with other types of photonic gels are that it can be produced in roll to roll quantities, can be vigorously deformed to achieve strong color changes, and has no solvent evaporation issues because it is a photonic rubber system. We demonstrate potential applications together with a fabrication procedure which is straightforward and scalable, vital for user take-up. Our work deepens understanding of this rubbery photonic system based on core-shell nanospheres.

CRACC-CRACC interaction between Kupffer and NK cells contributes to poly I:C/D-GalN induced hepatitis.

CD2-like receptor activating cytotoxic cells (CRACC) is known as a critical activating receptor of natural killer (NK) cells. We have previously reported that NK cells contribute to Poly I:C/D-galactosamine (D-GalN)-induced fulminant hepatitis. Since natural killer group 2, member D (NKG2D) is considered critical but not the only activating receptor for NK cells, we investigated the role of CRACC in this model. We found that CRACC was abundant on hepatic NK cells but with low expression levels on Kupffer cells under normal conditions. Expression of CRACC on NK cells and Kupffer cells was remarkably upregulated after poly I:C injection. Hepatic CRACC mRNA levels were also upregulated in Poly I:C/D-GalN-treated mice, and correlated positively with the serum alanine aminotransferase (ALT) levels. CRACC expression on Kupffer cells was specifically silenced by nano-particle encapsulated siRNA in vivo, which significantly reduced Poly I:C/D-GalN-induced liver injury. In co-culture experiments, it was further verified that silencing CRACC expression or blockade of CRACC activation by mAb reduced the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Collectively, our findings suggest that CRACC-CRACC interaction between NK cells and resident Kupffer cells contributes to Poly I:C/D-GalN-induced fulminant hepatitis.