CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

The Transcription Factor Runx3 Establishes Chromatin Accessibility of cis-Regulatory Landscapes that Drive Memory Cytotoxic T Lymphocyte Formation.

T cell receptor (TCR) stimulation of naive CD8+ T cells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.

CD4+ T Cell Help Confers a Cytotoxic T Cell Effector Program Including Coinhibitory Receptor Downregulation and Increased Tissue Invasiveness.

CD4+ T cells optimize the cytotoxic T cell (CTL) response in magnitude and quality, by unknown molecular mechanisms. We here present the transcriptomic changes in CTLs resulting from CD4+ T cell help after anti-cancer vaccination or virus infection. The gene expression signatures revealed that CD4+ T cell help during priming optimized CTLs in expression of cytotoxic effector molecules and many other functions that ensured efficacy of CTLs throughout their life cycle. Key features included downregulation of PD-1 and other coinhibitory receptors that impede CTL activity, and increased motility and migration capacities. "Helped" CTLs acquired chemokine receptors that helped them reach their tumor target tissue and metalloprotease activity that enabled them to invade into tumor tissue. A very large part of the "help" program was instilled in CD8+ T cells via CD27 costimulation. The help program thus enhances specific CTL effector functions in response to vaccination or a virus infection.

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo.

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B-cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL-tumor contacts in vivo. In the therapeutic settings of anti-CD19 CAR T cells, the majority of CAR T cell-tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti-tumor responses in vivo.

The frequency and function of nucleoprotein-specific CD8+ T cells are critical for heterosubtypic immunity against influenza virus infection.

Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.

Higher HIV-1 evolutionary rate is associated with cytotoxic T lymphocyte escape mutations in infants.

Escape from cytotoxic T lymphocyte (CTL) responses toward HIV-1 Gag and Nef has been associated with reduced control of HIV-1 replication in adults. However, less is known about CTL-driven immune selection in infants as longitudinal studies of infants are limited. Here, 1,210 gag and 1,264 nef sequences longitudinally collected within 15 months after birth from 14 HIV-1 perinatally infected infants and their mothers were analyzed. The number of transmitted founder (T/F) viruses and associations between virus evolution, selection, CTL escape, and disease progression were determined. The analyses indicated that a paraphyletic-monophyletic relationship between the mother-infant sequences was common (80%), and that the HIV-1 infection was established by a single T/F virus in 10 of the 12 analyzed infants (83%). Furthermore, most HIV-1 CTL escape mutations among infants were transmitted from the mothers and did not revert during the first year of infection. Still, immune-driven selection was observed at approximately 3 months after HIV-1 infection in infants. Moreover, virus populations with CTL escape mutations in gag evolved faster than those without, independently of disease progression rate. These findings expand the current knowledge of HIV-1 transmission, evolution, and CTL escape in infant HIV-1 infection and are relevant for the development of immune-directed interventions in infants.IMPORTANCEDespite increased coverage in antiretroviral therapy for the prevention of perinatal transmission, paediatric HIV-1 infection remains a significant public health concern, especially in areas of high HIV-1 prevalence. Understanding HIV-1 transmission and the subsequent virus adaptation from the mother to the infant's host environment, as well as the viral factors that affect disease outcome, is important for the development of early immune-directed interventions for infants. This study advances our understanding of vertical HIV-1 transmission, and how infant immune selection pressure is shaping the intra-host evolutionary dynamics of HIV-1.

Recognition of HIV-1-infected fibrocytes lacking Nef-mediated HLA-B downregulation by HIV-1-specific T cells.

Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE: Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.

Time required for commitment to T cell proliferation depends on TCR affinity and cytokine response.

T cell activation and effector functions are determined by the affinity of the interaction between T cell receptor (TCR) and its antigenic peptide MHC (pMHC) ligand. A better understanding of the quantitative aspects of TCR-pMHC affinity-dependent T cell activation is critical for the development of new immunotherapeutic strategies. However, the role of TCR-pMHC affinity in regulating the kinetics of CD8+ T cell commitment to proliferation and differentiation is unknown. Here, we show that the stronger the TCR-pMHC affinity, the shorter the time of T cell-APC co-culture required to commit CD8+ T cells to proliferation. The time threshold for T cell cytokine production is much lower than that for cell proliferation. There is a strong correlation between affinity-dependent differences in AKT phosphorylation and T cell proliferation. The cytokine IL-15 increases the poor proliferation of T cells stimulated with low affinity pMHC, suggesting that pro-inflammatory cytokines can override the affinity-dependent features of T cell proliferation.

K33 only mutant ubiquitin augments bone marrow-derived dendritic cell-mediated CTL priming via PI3K-Akt pathway.

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.

A case of response to combination treatment with TSA-DC-CTL immunotherapy and osimertinib in EGFR mutated advanced lung adenocarcinoma.

BACKGROUND: This study details a case of a patient with advanced lung adenocarcinoma harboring an exon 19 deletion in the EGFR gene. METHOD: A 46-year-old female patient was diagnosed with stage IVb left lung adenocarcinoma, with multiple bone and lymph node metastases. Following the identification of tumor-specific antigen peptides, the patient received a combination treatment of immunotherapy (TSA-DC-CTL) and oral osimertinib. Peripheral blood circulating immune cells and circulating tumor cells (CTCs) were monitored before and after treatment. PET-CT and CT scans were used to assess the tumor response to treatment. RESULTS: A significant increase in total lymphocyte percentage and decrease in the number of CTCs in the patient was observed. Imaging studies showed a notable reduction in tumor metastases. CONCLUSION: This report demonstrates the safety and efficacy of TSA-DC-CTL cell immunotherapy combined with osimertinib in the treatment of a patient with advanced lung adenocarcinoma with an EGFR exon 19 deletions. This study describes a promising new treatment option for patients with advanced lung cancer with EGFR mutations.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Single-cell landscape of functionally cured chronic hepatitis B patients reveals activation of innate and altered CD4-CTL-driven adaptive immunity.

BACKGROUND & AIMS: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC) is considered the optimal therapeutic outcome for patients with chronic hepatitis B (CHB). However, the immune-pathological biomarkers and underlying mechanisms of FC remain unclear. In this study we comprehensively interrogate disease-associated cell states identified within intrahepatic tissue and matched PBMCs (peripheral blood mononuclear cells) from patients with CHB or after FC, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. METHODS: We combined single-cell transcriptomics (single-cell RNA sequencing) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs. FC. RESULTS: We found that the intrahepatic environment in CHB and FC displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with the emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes, and an activated innate response represented by liver-resident natural killer cells, specific Kupffer cell subtypes and marginated neutrophils. Surprisingly, we found MHC class II-expressing hepatocytes in patients achieving FC, as well as low but persistent levels of covalently closed circular DNA and pregenomic RNA, which may play an important role in FC. CONCLUSIONS: Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic development. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis. IMPACT AND IMPLICATIONS: This study dissects the immuno-pathological cell states associated with functionally cured chronic hepatitis B (defined by the loss of HBV surface antigen or HBsAg). We identified the sustained presence of very low viral load, accessory antigen-presenting hepatocytes, adaptive-memory-like natural killer cells, and the emergence of helper CD4 T cells with cytotoxic or effector-like signatures associated with functional cure, suggesting previously unsuspected alterations in the adaptive immune response, as well as a key role for the innate immune response in achieving or maintaining functional cure. Overall, the insights generated from this study may provide new avenues for the development of alternative therapies as well as patient surveillance for better clinical management of chronic hepatitis B.

USP47 deficiency in mice modulates tumor infiltrating immune cells and enhances antitumor immune responses in prostate cancer.

This study investigates the role of USP47, a deubiquitinating enzyme, in the tumor microenvironment and its impact on antitumor immune responses. Analysis of TCGA database revealed distinct expression patterns of USP47 in various tumor tissues and normal tissues. Prostate adenocarcinoma showed significant downregulation of USP47 compared to normal tissue. Correlation analysis demonstrated a positive association between USP47 expression levels and infiltrating CD8+ T cells, neutrophils, and macrophages, while showing a negative correlation with NKT cells. Furthermore, using Usp47 knockout mice, we observed a slower tumor growth rate and reduced tumor burden. The absence of USP47 led to increased infiltration of immune cells, including neutrophils, macrophages, NK cells, NKT cells, and T cells. Additionally, USP47 deficiency resulted in enhanced activation of cytotoxic T lymphocytes (CTLs) and altered T cell subsets within the tumor microenvironment. These findings suggest that USP47 plays a critical role in modulating the tumor microenvironment and promoting antitumor immune responses, highlighting its potential as a therapeutic target in prostate cancer.

Deubiquitinating enzymes: potential regulators of the tumor microenvironment and implications for immune evasion.

Ubiquitination and deubiquitination are important forms of posttranslational modification that govern protein homeostasis. Deubiquitinating enzymes (DUBs), a protein superfamily consisting of more than 100 members, deconjugate ubiquitin chains from client proteins to regulate cellular homeostasis. However, the dysregulation of DUBs is reportedly associated with several diseases, including cancer. The tumor microenvironment (TME) is a highly complex entity comprising diverse noncancerous cells (e.g., immune cells and stromal cells) and the extracellular matrix (ECM). Since TME heterogeneity is closely related to tumorigenesis and immune evasion, targeting TME components has recently been considered an attractive therapeutic strategy for restoring antitumor immunity. Emerging studies have revealed the involvement of DUBs in immune modulation within the TME, including the regulation of immune checkpoints and immunocyte infiltration and function, which renders DUBs promising for potent cancer immunotherapy. Nevertheless, the roles of DUBs in the crosstalk between tumors and their surrounding components have not been comprehensively reviewed. In this review, we discuss the involvement of DUBs in the dynamic interplay between tumors, immune cells, and stromal cells and illustrate how dysregulated DUBs facilitate immune evasion and promote tumor progression. We also summarize potential small molecules that target DUBs to alleviate immunosuppression and suppress tumorigenesis. Finally, we discuss the prospects and challenges regarding the targeting of DUBs in cancer immunotherapeutics and several urgent problems that warrant further investigation.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Hypoxia and the Hypoxia-Inducible Factors in Lymphocyte Differentiation and Function.

Molecular oxygen doubles as a biomolecular building block and an element required for energy generation and metabolism in aerobic organisms. A variety of systems in mammalian cells sense the concentration of oxygen to which they are exposed and are tuned to the range present in our blood and tissues. The ability to respond to insufficient O2 in tissues is central to regulation of erythroid lineage cells, but challenges also are posed for immune cells by a need to adjust to very different oxygen concentrations. Hypoxia-inducible factors (HIFs) provide a major means of making such adjustments. For adaptive immunity, lymphoid lineages are initially defined in bone marrow niches; T lineage cells arise in the thymus, and B cells complete maturation in the spleen. Lymphocytes move from these first stops into microenvironments (bloodstream, lymphatics, and tissues) with distinct oxygenation in each. Herein, evidence pertaining to functions of the HIF transcription factors (TFs) in lymphocyte differentiation and function is reviewed. For the CD4+ and CD8+ subsets of T cells, the case is very strong that hypoxia and HIFs regulate important differentiation events and functions after the naïve lymphocytes emerge from the thymus. In the B lineage, the data indicate that HIF1 contributes to a balanced regulation of B-cell fates after antigen (Ag) activation during immunity. A model synthesized from the aggregate literature is that HIF in lymphocytes generally serves to modulate function in a manner dependent on the molecular context framed by other TFs and signals.

Murine Models of Familial Cytokine Storm Syndromes.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disease caused by mutations in effectors and regulators of cytotoxicity in cytotoxic T cells (CTL) and natural killer (NK) cells. The complexity of the immune system means that in vivo models are needed to efficiently study diseases like HLH. Mice with defects in the genes known to cause primary HLH (pHLH) are available. However, these mice only develop the characteristic features of HLH after the induction of an immune response (typically through infection with lymphocytic choriomeningitis virus). Nevertheless, murine models have been invaluable for understanding the mechanisms that lead to HLH. For example, the cytotoxic machinery (e.g., the transport of cytotoxic vesicles and the release of granzymes and perforin after membrane fusion) was first characterized in the mouse. Experiments in murine models of pHLH have emphasized the importance of cytotoxic cells, antigen-presenting cells (APC), and cytokines in hyperinflammatory positive feedback loops (e.g., cytokine storms). This knowledge has facilitated the development of treatments for human HLH, some of which are now being tested in the clinic.

TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Immunoinformatic prediction of potential immunodominant epitopes from cagW in order to investigate protection against Helicobacter pylori infection based on experimental consequences.

Helicobacter pylori is a leading cause of stomach cancer and peptic ulcers. Thus, identifying epitopes in H. pylori antigens is important for disease etiology, immunological surveillance, enhancing early detection tests, and developing optimal epitope-based vaccines. We used immunoinformatic and computational methods to create a potential CagW epitope candidate for H. pylori protection. The cagW gene of H. pylori was amplified and cloned into pcDNA3.1 (+) for injection into the muscles of healthy BALB/c mice to assess the impact of the DNA vaccine on interleukin levels. The results will be compared to a control group of mice that received PBS or cagW-pcDNA3.1 (+) vaccinations. An analysis of CagW protein antigens revealed 8 CTL and 7 HTL epitopes linked with AYY and GPGPG, which were enhanced by adding B-defensins to the N-terminus. The vaccine's immunogenicity, allergenicity, and physiochemistry were validated, and its strong activation of TLRs (1, 2, 3, 4, and 10) suggests it is antigenic. An in-silico cloning and immune response model confirmed the vaccine's expression efficiency and predicted its impact on the immune system. An immunofluorescence experiment showed stable and bioactive cagW gene expression in HDF cells after cloning the whole genome into pcDNA3.1 (+). In vivo vaccination showed that pcDNA3.1 (+)-cagW-immunized mice had stronger immune responses and a longer plasmid DNA release window than control-plasmid-immunized mice. After that, bioinformatics methods predicted, developed, and validated the three-dimensional structure. Many online services docked it with Toll-like receptors. The vaccine was refined using allergenicity, antigenicity, solubility, physicochemical properties, and molecular docking scores. Virtual-reality immune system simulations showed an impressive reaction. Codon optimization and in-silico cloning produced E. coli-expressed vaccines. This study suggests a CagW epitopes-protected H. pylori infection. These studies show that the proposed immunization may elicit particular immune responses against H. pylori, but laboratory confirmation is needed to verify its safety and immunogenicity.