IFN-γ Production Research Articles

Electroacupuncture suppresses NK cell infiltration and activation in the ischemic mouse brain through STAT3 inhibition.

Electroacupuncture (EA) at Shuigou (GV26) and Baihui (GV20) has shown therapeutic benefits for stroke patients. Given that natural killer (NK) cell infiltration into the brain significantly contributes to the exacerbation of cerebral ischemic injury, this study investigated the impact of EA at Shuigou (GV26) and Baihui (GV20) on post-ischemic brain infiltration and activation of NK cells. Neurological deficit score, rotarod test, adhesive removal test, and TTC staining were used to evaluate the beneficial effects of EA in middle cerebral artery occlusion (MCAO) mice. The inhibitory effect of EA on STAT3 activation was assessed using Western blot. Flow cytometry was used to explore the impact of EA on post-ischemic brain infiltration of NK cells, as well as the activating receptor NKG2D expression and interferon-γ (IFN-γ) production by these infiltrated NK cells. EA significantly alleviated neurological functional deficits and reduced brain infarction in MCAO mice. Abundant NK cells infiltrated into the ischemic hemisphere, but this infiltration was significantly suppressed by EA. Furthermore, EA attenuated NKG2D levels and reduced production of IFN-γ by NK cells in the ischemic brain. Notably, EA's inhibitory effect on post-ischemic NK cell brain infiltration and activation was comparable to that of STAT3 inhibition. The combination of EA and STAT3 inhibition did not result in further enhancement of the inhibitory effect. Moreover, the protective effects of EA against MCAO injury were abolished when STAT3 was activated. Our findings suggest that EA at Shuigou (GV26) and Baihui (GV20) inhibits the post-ischemic brain infiltration and activation of NK cells through STAT3 inhibition, significantly contributing to its therapeutic effects against brain ischemia.

Nutrient transporter pattern in CD56dim NK cells: CD16 (FcγRIIIA)-dependent modulation and association with memory NK cell functional profile

BackgroundHuman memory NK cells represent a heterogeneous CD56dim population that expands and persists in human cytomegalovirus (HCMV)-seropositive healthy individuals. They are characterized by the preferential, not fully overlapping, expression of NKG2C (activating receptor for HLA-E) and CD57 maturation marker, and by the lack of FcεRIγ adaptor chain. Hyperresponsiveness to Fcγ receptor IIIA (CD16) engagement represents the distinctive functional signature of memory NK cells. Although CD16 engagement was shown to acutely enhance glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet.ResultsHere, we describe the peculiar nutrient transporter expression pattern of FcεRIγ- memory NK cells, characterized by higher levels of CD98 neutral amino acid antiporter and CD71 transferrin receptor, and lower expression of GLUT1 glucose transporter, with respect to FcεRIγ+ conventional NK cells. Although CD16 engagement acutely enhances glycolytic and oxidative pathways, its capability to induce a persisting metabolic reprogramming of human NK cells is poorly understood yet. Our results firstly show that sustained CD16 engagement by contact with IgG-opsonized target cells induces the mTORC1-dependent upregulation of CD98 and CD71 nutrient receptors on CD56dim NK cells, in a transporter-specific fashion, that is finely tuned by cell-dependent (grade of functional maturation, and memory or conventional lineage) and stimulus-dependent (time length and cooperation with cytokines) factors. We also demonstrate that CD98 antiporter function is required for CD16-dependent IFN-γ production, and that enhanced CD98-mediated neutral amino acid uptake associates with heightened memory NK cell functional response.ConclusionCollectively, our work documents that CD16 engagement leads to a metabolic rewiring of human NK cells and suggests that a distinct nutrient transporter expression pattern may contribute to memory NK cell peculiar functional features.

In utero human cytomegalovirus infection expands NK-like FcγRIII+ CD8+ T cells that mediate Fc antibody functions.

Human cytomegalovirus (HCMV) profoundly impacts host T and natural killer (NK) cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells following HCMV exposure in utero. Most FcγRIII+ CD8+ T cells express the canonical αβ T cell receptor (TCR) but a proportion express non-canonical γδ TCR. FcγRIII+ CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+ CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+ CD8+ T cells mediate antibody-dependent IFNγ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+ CD8+ T cell Fc effector functions were further enhanced by interleukin-15 (IL-15), as has been observed in neonatal NK cells. Our study reveals that FcγRIII+ CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

Malnutrition disrupts adaptive immunity during visceral leishmaniasis by enhancing IL-10 production.

Protein-energy malnutrition (PEM) is a risk factor for developing visceral leishmaniasis (VL). While nutrient deficiency can impair immunity, its mechanistic impact on protective adaptive immune responses following Leishmania infection remains unknown. To determine the potential negative impacts of malnutrition on anti-parasitic responses in chronic VL, we provided mice with a polynutrient-deficient diet (deficient protein, energy, zinc, and iron) that mimics moderate human malnutrition. The polynutrient-deficient diet resulted in growth stunting and reduced mass of visceral organs and following infection with Leishmania infantum, malnourished-mice harbored more parasites in the spleen and liver. Malnourished and infected mice also had fewer T lymphocytes, with reduced T cell production of IFN-γ required for parasite clearance and enhanced production of the immunosuppressive cytokine, IL-10. To determine if IL-10 was causative in disease progression in the malnourished mice, we treated infected mice with monoclonal antibody α-IL-10R. α-IL-10R treatment reduced the parasite number in malnourished mice, restored the number of T cells producing IFN-γ, and enhanced hepatic granuloma formation. Our results indicate that malnutrition increases VL susceptibility due to defective IFN-γ-mediated immunity attributable to increased IL-10 production.

TMIC-41. RBBP4/P300 COMPLEX CONTROLS GENES MODULATING ANGIOGENESIS AND IMMUNOSUPPRESSION IN GLIOBLASTOMA MICROENVIRONMENT

Abstract The RBBP4/p300 complex was recently shown to control genes critical for DNA damage repair and may play a role in glioblastoma (GBM) therapy responsiveness, especially to temozolomide (TMZ). However, the role of this complex in the modulation of the GBM tumor microenvironment (GBM-TME) remains elusive. Because pro-tumorigenic GBM-TME is primarily due to angiogenesis and immune tolerance, we evaluated whether the RBBP4/p300 complex may control these critical processes. To that end, we used shRNA to silence each RBBP4/p300 complex member in GBM43 cells and RNAseq to identify angiogenesis and immune genes regulated by this complex. Our data demonstrated that silencing RBBP4 or p300 suppressed the expression of HIF1α and several other hypoxia response genes, including VEGFA and DDIT4, suggesting that RBBP4/p300 complex may regulate angiogenesis. Consistent with a role in regulating angiogenesis, culture medium conditioned by GBM43 cells expressing the control non-targeting shRNA (shNT) significantly induced angiogenic tube formation by human umbilical vein endothelial cells (HUVEC). In contrast, this effect was blocked by the medium conditioned by the shRBBP4 or shp300 expressing cells. Both shRBBP4 and shp300 expressing cells failed to activate HIF1α accumulation and other signaling in response to hypoxia. Additionally, the control shNT-conditioned medium inhibited IFN-γ production while the medium conditioned by the shRBBP4 or shp300 expressing cells supported IFN-ɣ production from CD8+ T cells. Intriguingly, silencing RBBP4 or p300 also suppressed the expression of CCL2 and PD-L1 (CD274). CCL2 protein is a chemokine that promotes TME infiltration by myeloid-derived suppressor cells (MDSCs), while PD-L1 interacts with PD1 to suppress cytotoxic CD8+ T cell activation. Although this is an ongoing investigation, we conclude that RBBP4/p300 complex mediates the expression of hypoxia response genes, including HIF1α, and immunosuppressive genes, including PD-L1, and this activity may enhance angiogenic and immunosuppressed GBM-TME, making this complex a potential target for GBM therapy.

Homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung natural killer cells

Natural killer (NK) cells are important innate immune effector cells for controlling tumor growth and metastasis. Differentiated mature NK cells preferentially reside in the peripheral tissues and express higher levels of self-major histocompatibility complex class I (MHC-I)-recognizing inhibitory receptors. MHC-I recognition by NK cells are known to be important for their development and maturation processes, however, the role of homeostatic MHC-I recognition in maintaining effector functions of mature NK cells in the peripheral tissues needs to be elucidated. In this study, we utilized a pan anti-MHC-I blocking monoclonal antibody (anti-MHC-I) to examine the role of homeostatic MHC-I recognition in the response of pulmonary mature NK cells in an experimental lung metastasis model of B16F10 melanoma. Anti-MHC-I treatment showed significant inhibition of the lung metastasis of B16F10 melanoma in NK cell- and IFN-γ-dependent mechanisms. The blockade of homeostatic MHC-I recognition increased mature lung NK cell responsiveness, such as direct cytotoxicity and IFN-γ production, rather than the number of lung NK cells. Mechanistically, the gene expression of activating receptors including DNAX accessory molecule-1 (DNAM-1) was upregulated in NK cells treated with anti-MHC-I, and further the enhanced NK cell cytotoxicity against B16F10 cells was DNAM-1-dependent. Collectively, homeostatic self-MHC-I recognition regulates anti-metastatic function of mature lung NK cells by restraining the expression of activating receptors.

Age-specific dynamics of neutralizing antibodies, cytokines, and chemokines in response to La Crosse virus infection in mice.

La Crosse virus (LACV) is a primary cause of pediatric arboviral encephalitis in the United States, particularly affecting children aged 16 years or younger. This age-related susceptibility extends to murine models, where weanling mice (3 weeks old) succumb to LACV infection, while adults (≥6 weeks old) demonstrate resistance. Despite its clinical relevance, the host immune response to LACV is not fully understood. In this study, we investigated the roles of neutralizing antibodies (nAbs), cytokines, and chemokines in weanling and adult mice following infection with 5 × 105 plaque-forming units (PFU) of LACV. Weanling mice demonstrated early disease onset with elevated peripheral viremia, but passive transfer of adult serum, confirmed to have nAbs, to naïve weanlings prior to infection completely rescued them from death. Moreover, adult mice had increased Th1 cytokines, Th9/Th17/Th22/Treg cytokines, and many chemokines. In contrast, weanlings had higher Th2 cytokines, correlating with symptoms onset. Flow cytometry and intracellular cytokine staining further demonstrated that weanling mice produced higher levels of IL-4 by CD4+ and CD8+ T cells compared to adults, regardless of infection status. Conversely, LACV-infected adult mice had increased IFN-γ production by CD8+ T cells compared to uninfected controls. Finally, the adoptive transfer of splenocytes from immune adult mice to naïve weanlings delayed neurological symptoms and improved survival. In conclusion, this study links nAbs and cytokine and chemokine responses to protective immunity in adult mice, contrasting with the pathogenesis seen in weanlings. These findings underscore the importance of further research into innate and adaptive immune mechanisms during LACV infection.IMPORTANCELa Crosse virus (LACV) is a primary cause of pediatric encephalitis in the United States, with an impact on children aged 16 years or younger. This age-related susceptibility is recapitulated in mouse models, where young mice succumb to LACV-induced disease, while adults demonstrate resistance. Our understanding of host responses to LACV remains underexplored. This study sheds light on the dynamics of neutralizing antibodies (nAbs), cytokines, and chemokines following LACV infection in both adult and weanling mice. Our study reveals age-specific variations in viremia, neutralizing antibody titers, survivability, and levels of cytokines and chemokines. Adult mice exhibit significantly elevated levels of Th1 cytokines, contrasting with elevated levels of Th2 cytokines observed in weanling mice, often coinciding with the onset of symptoms. These data reveal age-specific dynamics in cytokines and chemokines associated with protective versus pathogenic immunity, emphasizing the need for further studies on innate and adaptive immunity.

The role of mitochondrial transfer in the suppression of CD8+ T cell responses by Mesenchymal stem cells

Background. CD8+ Cytotoxic T lymphocytes play a key role in the pathogenesis of autoimmune diseases and clinical conditions such as graft versus host disease and graft rejection. Mesenchymal Stromal Cells (MSCs) are multipotent cells with tissue repair and immunomodulatory capabilities. Since they are able to suppress multiple pathogenic immune responses, MSCs have been proposed as a cellular therapy for the treatment of immune-mediated diseases. However, the mechanisms underlying their immunosuppressive properties are not yet fully understood. MSCs have the remarkable ability to sense tissue injury and inflammation and respond by donating their own mitochondria to neighboring cells. Whether mitochondrial transfer has any role in the repression of CD8+ responses is unknown.Methods and results. We have utilized CD8+ T cells from Clone 4 TCR transgenic mice that differentiate into effector cells upon activation in vitro and in vivo to address this question. Allogeneic bone marrow derived MSCs, co-cultured with activated Clone 4 CD8+ T cells, decreased their expansion, the production of the effector cytokine IFNγ and their diabetogenic potential in vivo. Notably, we found that during this interaction leading to suppression, MSCs transferred mitochondria to CD8+ T cells as evidenced by FACS and confocal microscopy. Transfer of MSC mitochondria to Clone 4 CD8+ T cells also resulted in decreased expansion and production of IFNγ upon activation. These effects overlapped and were additive with those of prostaglandin E2 secreted by MSCs. Furthermore, preventing mitochondrial transfer in co-cultures diminished the ability of MSCs to inhibit IFNγ production. Finally, we demonstrated that both MSCs and MSC mitochondria downregulated T-bet and Eomes expression, key transcription factors for CTL differentiation, on activated CD8+ T cells.Conclusion. In this report we showed that MSCs are able to interact with CD8+ T cells and transfer them their mitochondria. Mitochondrial transfer contributed to the global suppressive effect of MSCs on CD8+ T cell activation by downregulating T-bet and Eomes expression resulting in impaired IFNγ production of activated CD8+ T cells.

Piezo1 restrains proinflammatory response but is essential in T cell-mediated immunopathology.

Piezo1 is a mechanosensitive, nonselective Ca2+ channel which is broadly expressed in CD4+ T cells. Using lineage-specific Piezo1 knockout mice (Piezo1cKO), we show that loss of Piezo1 in CD4+ T cells significantly increased IFNγ and IL-17 production in vitro under TH1 and TH17 polarizing conditions, respectively. Despite their intrinsic proinflammatory phenotype, Piezo1cKO T cells are incapable of establishing disease in vivo in three separate adoptive transfer (AT) T cell-mediated inflammatory mouse models, including experimental autoimmune encephalomyelitis, inflammatory bowel disease (IBD), and graft versus-host disease. These phenomena coincided with a decreased effector memory (CD44hiCD62Llo) CD4+ T cell pool derived from donor Piezo1cKO T cells, an observation that is related to intrinsic T-cell fitness, as co-transfer IBD mouse model revealed a deficiency in the CD4+ effector memory population derived only from the naïve Piezo1cKO but not co-infused Piezo1WT CD4+ T cell source. Taken together, our results support Piezo1 as restraining proinflammatory T cell differentiation while contributing to the generation and persistence of the effector memory pool during CD4+ T cell-mediated immunopathology.

Sea Buckthorn Flavonoid Extracted with High Hydrostatic Pressure Alleviated Shrimp Allergy in Mice through the Microbiota and Metabolism.

Sea buckthorn (Hippophaë rhamnoides L.) known as the deciduous shrub has been reported to have effects of antioxidant, anti-inflammatory, and immunomodulatory activities. Tropomyosin (TM) induced a regulatory immune response associated with food allergy. In this study, a mouse model of food allergy sensitized to tropomyosin (TM) was established to assess the antiallergic properties of sea buckthorn flavonoid extract (SBF). SBF alleviated mice's allergic symptoms and exhibited a significant reduction in the levels of IgE and histamine. Meanwhile, SBF repaired the allergic Th2 cell overpolarization generated by TM, via downregulating the IL-4 production and upregulating IFN-γ production to restore the balance of Th1/Th2 cells. Furthermore, the 16S RNA analysis showed that SBF primarily restored the gut microbiota via increasing the abundance in Chitinophilidae and decreasing in Burkholderiaceae, Pneumatobacteriaceae, and Sphingomonadaceae. Gut metabolomes determined by liquid chromatography-mass spectrometry (LC-MS) suggested that TM upregulated PE (14:0/22:1(13Z)) and SBF decreased formimino-l-glutamic acid and urocanic acid levels. According to the KEGG pathway analysis, SBF treatment has been shown to modulate glycerophospholipid and histidine metabolism to improve allergic reactions. SBF holds great promise as a novel potential agent for the treatment of food allergies.

The Antigen-Specific Response of NK Cells to SARS-CoV-2 Correlates With KIR2DS4 Expression.

Natural killer (NK) cells play a pivotal role in the immune response against viral infections, including SARS-CoV-2. However, our understanding of memory NK cell responses in the context of SARS-CoV-2 remains limited. To address this, we investigated the memory-like response of NK cells to SARS-CoV-2 peptides, presented by autologous cells. Blood samples from 45 donors underwent analysis for SARS-CoV-2 IgG antibodies, categorizing them into four groups based on the antibody kind and level. NK cells from SARS-CoV-2-experienced donors demonstrated enhanced degranulation and activation levels, IFNγ production and proliferative potential in response to SARS-CoV-2 peptides. Investigation of highly proliferating NK cells demonstrated the formation of distinct clusters depending on the SARS-CoV-2 peptide supplementation and the donor group. RNA sequencing revealed differential gene expression patterns, highlighting metabolism, protein transport, and immune response genes. Notably, KIR2DS4 expression correlated with enhanced IFNγ production, degranulation and proliferation levels, suggesting a role in SARS-CoV-2 recognition. Collectively, these findings provide detailed insights into antigen-specific NK cell responses to SARS-CoV-2 peptides, indicating potential mechanisms underlying NK cell activation in antiviral immunity.

Interleukin-12 decorated nanosized semiflexible Immunofilaments enable directed targeting and augmented IFNγ responses of Natural Killer Cells

Immunotherapies are a powerful strategy to treat cancer by modulating the immune system to raise an anti-tumor immune response. A prime example of immunotherapies are cytokines - small immunomodulatory molecules that are widely used to stimulate immune cells. Undirected administration of cytokines, however, can cause severe side effects, preventing the use of potent cytokines, such as Interleukin (IL)-12, which induces IFNγ responses by cytotoxic effector lymphocytes, including NK cells. Biomaterials, like nanoparticles, can encapsulate IL-12 and accumulate at the tumor site to alleviate side effects. Yet, the released IL-12 might not be directly targeted to extracellular IL-12 receptors on the specific effector cells, thereby potentially compromising the cytokine's therapeutic efficacy. Here, we develop a polymer-based platform to target NK cells, which we call immunofilaments. Immunofilaments are nanosized linear polymers that present an anti-CD16 antibody and IL-12 effectively to NK cells and lead to synergistic NK cell activation as highlighted by an increase in TNFα and IFNγ production and upregulation of multiple activation markers, including CD25, CD69, and degranulation marker CD107a. NK cell proliferation is enhanced in the presence of both anti-CD16 antibody and IL-12 compared to giving IL-12 separately. Finally, we demonstrate that the IF platform is suitable for in vivo applications, as immunofilaments readily activate human NK cells upon administration to mice. STATEMENT OF SIGNIFICANCE: IL-12 is a potent cytokine that stimulates IFNγ responses in NK cells, which supports an anti-tumor immune response. Due to its high potency, the delivery of IL-12 needs to be highly controlled to prevent severe adverse side effects, which can be achieved by using biomaterials. This study shows that nanosized polymers termed Immunofilaments can be used to immobilize IL-12 and effectively target and activate NK cells by co-conjugation of anti-CD16 antibodies. This work is a prime example of careful engineering of innovative biomaterials to improve immunotherapy.

In situ endoscopic photodynamic therapy combined with immature DC vaccination induces a robust T cell response against peritoneal carcinomatosis.

Immunogenic cell death (ICD) and ferroptosis have recently emerged as key factors in the anticancer immune response. Among the treatments able to induce ICD and the associated release of danger signals is photodynamic therapy (PDT). Ferroptosis for its part results from lipid peroxidation and is induced by CD8+ T cells to kill nearby cancer cells on IFN-γ production. We aimed to combine the two concepts, that is, to evaluate whether the strong pro-oxidant effects of PDT may promote ferroptosis and antigen release and to develop a procedure for in situ PDT to prepare the soil for highly endocytotic immature dendritic cell (iDC) adoptive transfer. This approach was implemented for managing peritoneal carcinomatosis, a lesion often associated with poor outcomes. We used three-dimensional (3D) heterotypic spheroids made of cancer cells, exposed them to a white light-activated OR141 photosensitizer (PS), and subsequently complexified them by adding iDC and naive lymphocytes. We next used a model of mouse peritoneal carcinomatosis and administered PDT using laparoscopy to locally induce photoactivation using the endoscope light. The immune response following adoptive transfer of iDC was tracked both in vivo and ex vivo using isolated immune cells from in situ vaccinated mice. Cancer cells undergoing PDT-induced cell death significantly increased ICD markers and the infiltration of iDCs in spheroids, relying on ferroptosis. These actions induced the sequential activation of CD8+ and CD4+ T cells as revealed by a significant spheroid 3D structure deterioration and, remarkably, were not recapitulated by conventional ferroptosis inducer RSL3. Using LED light from an endoscope for in situ photoactivation of PS enabled us to apply the vaccination modality in mice with peritoneal tumors. Consecutive intraperitoneal injection of iDCs resulted in delayed tumor growth, increased survival rates, and prevented tumor relapse on rechallenge. CD8+ T cell response was supported by depletion experiments, nodal detection of early activated T cells, and ex vivo T cell-induced cytotoxicity toward spheroids. The combination of in situ PDT locally delivered by an endoscope light and iDC administration induces a durable memory immune response against peritoneal carcinomatosis thereby opening new perspectives for the treatment of a life-threatening condition.

Persistence of activated anti-mesothelin hYP218 chimeric antigen receptor T cells in the tumour is associated with efficacy in gastric and colorectal carcinomas.

Patients with advanced gastric and colorectal cancers have limited treatment options. Since mesothelin is highly expressed in these tumour types, we evaluated the therapeutic benefits of anti-mesothelin hYP218 CAR T cells alone, and in combination with anti-PD1 antibody, pembrolizumab. GEPIA analysis was performed using human gastric (n=408) and colon cancer tumours (n=275) in TCGA database, to evaluate mRNA expression of mesothelin, compared to normal tissues. Mesothelin expression in gastric and colorectal cancer cell-lines (n=5) was analysed using flow cytometry. In vitro efficacy by hYP218 CAR T cells was tested by cytotoxicity and cytokine release assays. In vivo anti-tumour efficacy of hYP218 CAR T cells alone, and in combination with pembrolizumab, was evaluated in NSG mice bearing human gastric (HGC27) and colorectal (SW48) tumour xenografts. Additionally, hYP218 CAR-T cell persistence, activation and exhaustion marker-expression were studied. Mesothelin expression was significantly higher in gastric and colon cancer biopsies compared to normal tissues (p<.005). Mesothelin expression in gastric and colon cancer cell lines ranged from 10000 to 70000 molecules per cell. hYP218 CAR T cells demonstrated strong cytotoxic activity at low effector to target ratio, ranging from 0.24 to 1.0. In NSG mouse-models, hYP218 CAR T cells demonstrated anti-tumour efficacy and persisted in the tumour microenvironment in a functional state at day 40 posttreatment with expression of activation markers CD39 and CD69, increased production of IFN-γ and TNF-α and ability to kill tumour cells in vitro when isolated from tumours. There was increased PD1 expression. In combination with pembrolizumab, hYP218 CAR T cells led to slower tumour growth in NSG mice bearing large but not small HGC27 tumours. Anti-tumour efficacy of hYP218 CAR T cells is due to increased accumulation of activated CAR T cells in the tumour and combination with pembrolizumab resulted in improvement in anti-tumour activity of large established tumours. HIGHLIGHTS: Mesothelin expression is significantly higher in gastric and colorectal cancers than normal tissues. hYP218 CAR T cells demonstrate strong anti-tumour activity against mesothelin-positive gastric and colorectal carcinomas. Activated hYP218 CAR T cells persist in the tumour microenvironment and retain their cytotoxic activity. Addition of pembrolizumab in larger tumours enhance CAR T cell efficacy.

HIF1α-regulated glycolysis promotes activation-induced cell death and IFN-γ induction in hypoxic T cells

Hypoxia is a common feature in various pathophysiological contexts, including tumor microenvironment, and IFN-γ is instrumental for anti-tumor immunity. HIF1α has long been known as a primary regulator of cellular adaptive responses to hypoxia, but its role in IFN-γ induction in hypoxic T cells is unknown. Here, we show that the HIF1α-glycolysis axis controls IFN-γ induction in both human and mouse T cells, activated under hypoxia. Specific deletion of HIF1α in T cells (Hif1α–/–) and glycolytic inhibition suppresses IFN-γ induction. Conversely, HIF1α stabilization by hypoxia and VHL deletion in T cells (Vhl–/–) increases IFN-γ production. Hypoxic Hif1α–/– T cells are less able to kill tumor cells in vitro, and tumor-bearing Hif1α–/– mice are not responsive to immune checkpoint blockade (ICB) therapy in vivo. Mechanistically, loss of HIF1α greatly diminishes glycolytic activity in hypoxic T cells, resulting in depleted intracellular acetyl-CoA and attenuated activation-induced cell death (AICD). Restoration of intracellular acetyl-CoA by acetate supplementation re-engages AICD, rescuing IFN-γ production in hypoxic Hif1α–/– T cells and re-sensitizing Hif1α–/– tumor-bearing mice to ICB. In summary, we identify HIF1α-regulated glycolysis as a key metabolic control of IFN-γ production in hypoxic T cells and ICB response.

Performance of a Global Functional Assay Based on Interferon-γ Release to Predict Infectious Complications and Cancer After Kidney Transplantation.

The QuantiFERON-Monitor assay (QTF-Monitor) is intended to assess innate and adaptive immune responses by quantifying interferon (IFN)-γ release upon whole blood stimulation with a TLR7/8 agonist and an anti-CD3 antibody. We performed the QTF-Monitor in 126 kidney transplant recipients (KTRs) at different points during the first 6 post-transplant months. The primary outcome was overall infection, whereas secondary outcomes included bacterial infection, opportunistic infection and de novo cancer. The association between IFN-γ production and outcomes was analyzed as "low" immune responses (<15IU/mL) and as a continuous variable to explore alternative thresholds. There were no significant differences in the occurrence of overall infection according to the QTF-Monitor at any monitoring point. Regarding secondary outcomes, KTRs with a low response at week 2 experienced a higher incidence of bacterial infection (50.8% versus 24.4%; P-value = 0.006). Low response at month 1 was also associated with opportunistic infection (31.6% versus 14.3%; P-value = 0.033). The discriminative capacity of IFN-γ levels was poor (areas under the ROC curve: 0.677 and 0.659, respectively). No differences were observed for the remaining points or post-transplant cancer. In conclusion, the QTF-Monitor may have a role to predict bacterial and opportunistic infection in KTRs when performed early after transplantation.

Glycolysis regulated exosomal LINC01214 inhibited CD8+ T cell function and induced anti-PD1 resistance in melanoma via modulating miR-4492/PPP1R11 axis

BackgroundLong non-coding RNAs (lncRNAs) can be incorporated into exosomes to mediate the intercellular communication, regulating the occurrence, development, and immunosuppression of cancers. T cell dysfunction has been a hallmark of many cancers, including melanoma, which enables cancer cells escape from host immune surveillance. However, the molecular mechanism of exosome-transmitted lncRNAs in CD8+ T cell dysfunction in melanoma remains largely unclear. MethodThe expression of circulating LINC01214 (cirLINC01214) was detected by quantitative real-time polymerase chain reaction (RT-qPCR). Exosomes were isolation from the culture medium and plasma of melanoma patients via ultracentrifugation and characterized by transmission electronic microscopy. The regulation of exosomal LINC01214 on CD8+ T cell function was determined by ELISA. The molecular mechanism of exosomal LINC01214 in CD8+ T cells were assessed by the RNA immunoprecipitation and pull-down assay. A mouse model with reconstituted human immune system was used to explore the role of exosomal LINC01214 in the resistance to anti-PD1 therapy. ResultsLINC01214 was highly expressed in melanoma tissues compared with matched adjacent normal tissues. Increased levels of circulating LINC01214 (cirLINC01214) was observed in melanoma patient plasma and correlated with poor PD-1 immunotherapy response. The cirLINC01214 was predominantly released by melanoma cells in an exosome manner. Melanoma cell-derived exosomal LINC01214 inhibits the production of IFN-γ, TNF-α, Granzyme-B and Perforin by CD8+ T cells. Further mechanism study found that cirLINC01214 delivered by exosomes suppressed CD8+ T cell function by up-regulating the expression of Protein Phosphatase 1 Regulatory Inhibitor Subunit 11 (PPP1R11) through sponging miR-4492. CirLINC01214 conferred resistance to PD-1 immunotherapy in melanoma xenograft mouse model. Melanoma patients with poor prognosis after PD-1 treatment carried high levels of exosomal LINC01214. Additionally, the secretion of exosomal cirLINC01214 was enhanced by the Warburg effect, which was consistent with the reprogrammed glucose metabolism of melanoma. ConclusionsOur results demonstrated that exosomal LINC01214 released by melanoma cells promoted immunotherapy resistance by inducing CD8+ T cell dysfunction via the miR-4492/PPP1R11 regulatory loop. Targeting cirLINC01214 might be a potential therapeutic strategy to enhance the outcome of immunotherapy in melanoma.

HIV-1 latency reversal agent boosting is not limited by opioid use.

Opioid use may impact the HIV-1 reservoir and its reversal from latency. We studied forty-seven virally suppressed people with HIV (PWH) and observed that lower concentration of HIV-1 latency reversal agents (LRA), used in combination with small molecules that did not reverse latency, synergistically increased the magnitude of HIV-1 re-activation ex vivo, regardless of opioid use. This LRA boosting, which combined a Smac mimetic or low-dose protein kinase C agonist with histone deacetylase inhibitors, generated significantly more unspliced HIV-1 transcription than phorbol 12-myristate 13-acetate (PMA) with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation, modulated surface activation-induced markers, and altered T cell production of TNFα, IL-2, and IFNγ. HIV-1 reservoirs in PWH contained unspliced and polyadenylated (polyA) virus mRNA, the ratios of which were greater in resting than total CD4+ T cells and correct to 1:1 with PMAi exposure. We characterized treated suppressed HIV-1 infection as a period of inefficient, not absent, virus transcription. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent post-transcriptional block. LRA boosting can be leveraged to probe mechanisms of an effective cellular HIV-1 latency reversal program.

Antiviral immune response against HTLV-1 invalidates T-SPOT.TB® results in patients with HTLV-1-positive rheumatic diseases.

T-SPOT.TB®, one of the screening tests for latent tuberculosis infection (LTBI), yields invalid results in human T-cell leukemia virus type 1 (HTLV-1)-positive patients with rheumatoid arthritis. However, the detailed mechanisms behind this invalidation are unclear. Additionally, it remains unclear whether T-SPOT.TB® or QuantiFERON-TB (QFT) is more useful in HTLV-1-positive patients with rheumatic disease (RD). Among all of the HTLV-1-positive RD patients who visited our department between August 2012 and December 2022, 44 patients who were screened using T-SPOT.TB® were included in the analysis. QFT testing was performed in 33 of the 44 patients, and the results were compared with that of T-SPOT.TB®. Furthermore, we performed a culture experiment mimicking T-SPOT.TB® using peripheral blood mononuclear cells (PBMCs) obtained from HTLV-1-positive patients with RD. Additionally, T-cell subsets with autonomous product IFN-γ were analyzed using a flow cytometer. Of the included patients, 13 (29.5%) were invalid for T-SPOT.TB® because of the increased number of negative control spots. The median HTLV-1 proviral load in the invalid group was higher than that in the valid group (2.45 vs. 0.49 copies/100 PBMCs, respectively, p = 0.002). QFT was performed in all 33 patients, including 13 patients who were invalid in T-SPOT.TB®. The main source of IFN-γ production was CD8+ T-cells in the T-SPOT.TB® mimic experiment. Furthermore, Tax-expressing CD4+ T-cells and Tax-specific cytotoxic CD8+ T-cells were more frequently observed in patients with invalid results than in patients with valid results. CD4+ T-cell depletion in the T-SPOT.TB® mimic experiment reduced the population of IFN-γ producing CD8+ T cells. T-SPOT.TB® may be invalidated by the interaction between Tax-expressing CD4+ T-cells and cytotoxic CD8+ T-cells. Moreover, HTLV-1-associated immune reactions due to contact between these cells may be unlikely to occur in QFT using whole blood. Therefore, our results reveal the superiority of QFT over T-SPOT.TB® as a screening test for LTBI in HTLV-1-positive patients with RD.

Proteomic approach to identify host cell attachment proteins provides protective Pseudomonas aeruginosa vaccine antigen FtsZ

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes severe nosocomial infections in susceptible individuals due to the emergence of multidrug-resistant strains. There are no approved vaccines against P. aeruginosa infections nor candidates in active clinical development, highlighting the need for novel candidates and strategies. Using a cell-blot proteomic approach, we reproducibly identified 49 proteins involved in interactions with human lung epithelial cells across four P. aeruginosa strains. Among these were cell division protein FtsZ and outer membrane protein OpmH. Escherichia coli BL21 cells overexpressing recombinant FtsZ or rOpmH showed a 66- and 15-fold increased ability to attach to 16HBE14o− cells, further supporting their involvement in host cell attachment. Both antigens led to proliferation of NK and CD8+ cytotoxic T cells, significant increases in the production of IFN-γ, IL-17A, TNF and IL-4 in immunised mice and elicited strong antigen-specific serological IgG1 and IgG2c responses. Immunisation with FtsZ significantly reduced bacterial burden in the lungs by 1.9-log CFU and dissemination to spleen by 1.8-log CFU. The protective antigen candidate, FtsZ, would not have been identified by traditional approaches relying on either virulence mechanisms or sequence-based predictions, opening new avenues in the development of an anti-P. aeruginosa vaccine.