Impaired T-cell Research Articles

P-2346. Mpox-Specific Cellular and Humoral immunity in Mpox-Survivors Living With HIV

Abstract Background Mpox is an orthopoxvirus with a rodent reservoir that causes a disease similar to mild smallpox. In 2022, a sexually-transmitted global mpox outbreak infected over 90,000 individuals. Some immunocompromised patients experienced prolonged, severe mpox resulting in significant morbidity, amputations, and death. People living with HIV (PLWH) were at particularly high risk due to impaired cell-mediated immunity, as CD4 and CD8 T-cells help eradicate the virus from infected lesions. Mpox virion total binding titer in PLWH with or without history of mpox infection Methods We compared immune responses to mpox in PLWH who were either mpox-naïve (n = 10) versus mpox-survivors (n = 15). We measured total mpox antibody levels with a novel ELISA assay using lysed mpox virions. We characterized mpox and orthopoxvirus-specific cell-mediated immunity using intracellular cytokine staining with an enhanced protocol to allow detection of low-frequency cellular populations. Poxvirus-specific T-cell responses in PLWH as determined by intracellular cytokine stimulation using live vaccinia virus (MVA), conserved orthopoxvirus peptide pools (VCD4 and VCD8), and mpox-specific peptide pools (MCD4 and MCD8). Pools provided by Alba Grifoni and Alessandro Sette (La Jolla Institute). Interferon-gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin 2 (IL-2) were the predominant cytokines detected. Responders defined as having any CD4 or CD8 response for any of the 9 stimulations above the 90th percentile of the control group. Results Our assays differentiated well between the mpox-naïve and mpox-survivors. All mpox-survivors had binding titers of 10 to 1,000 times background with no antibodies detected in mpox-uninfected PLWH. Mpox-specific CD4 and CD8 responses of 0.01 to 0.3% were present in all infected individuals, comparable to immunocompetent mpox-survivors. The magnitude of mpox-specific T-cell responses correlated well with vaccinia-specific T-cell responses, demonstrating induction of broad anti-orthopoxvirus cell-mediated immunity. CD4 T-cell responses were TH1-type, with higher polyfunctionality in mpox-specific T-cells compared to HIV and CMV-specific T-cells. The magnitude of the IFNγ and TNFα CD4 and CD8 responses correlated with mpox-binding antibody titers, but did not predict cross-neutralization titers from vaccinia, a closely related virus that is used to vaccinate for smallpox and mpox. Host CD4 count did not correlate with the magnitude of mpox-specific cellular or humoral immunity. Conclusion These new assays effectively quantify and characterize mpox-specific immunity, with strong induction of orthopoxvirus-specific humoral and cellular immunity in immunocompromised hosts with HIV. Although PLWH are at increased risk for complications from mpox, their potent immune responses post-infection suggest that most are at reduced risk for severe complications from re-infection. Disclosures Samuel D. Stampfer, MD/PhD, Gilead: Stocks/Bonds (Public Company) Colleen F. Kelley, MD, MPH, Gilead: Grant/Research Support|Humanigen: Grant/Research Support|Moderna: Grant/Research Support|Novavax: Grant/Research Support|ViiV: Grant/Research Support

P-1864. Early Combination Therapy with Isavuconazole and Immune Checkpoint Inhibitors Improves Infection Outcomes and Alleviates Paralyzed Antifungal Host Immunity in Neutropenic Mice with Invasive Pulmonary Mucormycosis

Abstract Background Invasive pulmonary mucormycosis (IPM) is a severe opportunistic mold infection whose outcome is predominantly host-driven. Preclinical studies and several clinical case reports suggested a benefit of adjunct immunotherapy with immune checkpoint inhibitors (ICIs). However, the mechanisms of IPM-associated pulmonary immune paralysis and its mitigation by ICIs require further study. Timeline of experimental interventions. Abbreviations: CPM = cyclophosphamide, i.p. = intra-peritoneally, ISAV = isavuconazonium sulfate, IT = immunotherapy, PD-L1 = Programmed Death Ligand 1, p.o. = per os (oral gavage). Methods Eight-week-old cyclophosphamide-immunosuppressed BALB/c mice were infected intranasally with 50,000 Rhizopus arrhizus spores. The Mucorales-active triazole isavuconazonium sulfate (ISAV) was initiated on day (d) 3 post-infection (2×64 mg/kg/d by oral gavage). Additionally, mice received 2×0.25 mg/kg anti (α)-PD-L1 or a non-targeting isotype antibody either on d3+5 (early) or d6+8 (late) (Fig. 1). Infection severity was scored using the murine sepsis score (MSS; 0 = healthy – 3 = moribund, 4 = dead). nCounter-based transcriptomics and Ingenuity pathway enrichment analysis were performed on lung tissue homogenates. Murine sepsis scores (MSS, 0 = healthy – 3 = moribund, 4 = dead) of immunosuppressed mice with invasive pulmonary mucormycosis according to the treatment arm. Kruskal-Wallis test with Dunn’s post-test. * p<0.05, *** p<0.001. Abbreviations: ISAV = isavuconazonium sulfate, IT = immunotherapy, ns = not significant, PD-L1 = Programmed Death Ligand 1. Results Untreated neutropenic mice developed severe IPM (median day-10 MSS: 2.9, Fig. 2). Pulmonary transcriptomics at d4 revealed strong innate immune activation and induction of type-1 (Th1) and type-17 (Th17) T-helper-cell responses. However, by d7, proinflammatory signals had markedly decreased and nCounter analysis revealed impaired intercellular crosstalk and T-cell signaling (Fig. 3). Compared to ISAV + isotype (median d10 MSS, 2.0), early and late adjunct α-PD-L1 therapy improved median d10 MSS to 1.0 (p < 0.001) and 1.4 (p = 0.01), respectively (Fig. 2). While ISAV delayed the tipping point from immune activation to full-blown immune paralysis, early adjunct α-PD-L1 therapy elicited significantly stronger dendritic cell and classical (M1) macrophage signaling, activation of key cytokine pathways (e.g., IL-1, IL-2, IL-12), reinvigoration of Th1 and Th17 signaling, and suppression of IPM-induced co-inhibitory checkpoint pathways than both ISAV + isotype and ISAV + late α-PD-L1 (Fig. 3). Enrichment of selected immune-related pathways on day 10 after IPM infection (7 days after initiation of therapy) depending on the treatment arm and comparison with surrogates of immune paralysis in untreated infected mice. All differentially induced pathways were statistically significant (Benjamini-Hochberg adjusted p-values <0.05). Abbreviations: IPM = invasive pulmonary mucormycosis, ISAV = isavuconazonium sulfate, PD-L1 = Programmed Death Ligand 1. Conclusion IPM is associated with rapidly emerging immune paralysis. Combined antifungal and early immunomodulatory (α-PD-L1) therapy was the most effective strategy for early interception of immune paralysis and improvement of morbidity/mortality in our IPM model. Disclosures Sebastian Wurster, MD, MSc, Astellas Pharma: Grant/Research Support|Gilead Sciences: Grant/Research Support Dimitrios P. Kontoyiannis, MD, AbbVie: Advisor/Consultant|Astellas Pharma: Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Gilead Sciences: Honoraria|Knight: Advisor/Consultant|Merck: Advisor/Consultant|Scynexis: Advisor/Consultant

Is Kaposi sarcoma a novel comorbidity of cutaneous lymphoma? A systematic review of the literature.

Patients with cutaneous lymphomas (CL) are at an increased risk of developing secondary malignancies. This study aimed to assess the frequency of association between CL and Kaposi sarcoma (KS) and to identify factors that may promote the co-occurrence of these two diseases. On January 25, 2024, we conducted a systematic search of four electronic medical databases to identify all published cases of KS associated with CL. The clinical course and outcomes of these patients were summarized. For critical appraisal, we applied the JBI Checklist for Case Reports. The study was registered in the PROSPERO database (CRD42022313204). A total of 40 articles reporting on 45 patients were assessed for eligibility. We included 27 cases in the final analysis (26 cutaneous T-cell lymphomas, 1 cutaneous B-cell lymphoma). In 71% of cases, the diagnosis of CL preceded KS. Nearly half (48%) of the patients had erythrodermic mycosis fungoides or Sézary syndrome. KS lesions were predominantly limited to the skin, with complete remission achieved in 53% of cases. The association between KS and CL is rare, limiting our study due to the small sample size and potential reporting bias. Skin-targeted therapies, a restricted T-cell repertoire, and impaired T-cell responses in erythrodermic CTCL patients may contribute to the development of KS.

Nivolumab to restore T-cell fitness in CAR-T refractory multiple myeloma.

Efficacy and durability remain central shortcomings of T-cell based therapies in multiple myeloma (MM). Here, we employ blood-based transcriptional T-cell profiling to define impaired T-cell fitness as putative biomarker associated with sensitivity to PD1 inhibition in CAR-T refractory MM patients.

Redirecting glucose flux during invitro expansion generates epigenetically and metabolically superior Tcells for cancer immunotherapy.

Cellular therapies are living drugs whose efficacy depends on persistence and survival. Expansion of therapeutic Tcells employs hypermetabolic culture conditions to promote Tcell expansion. We show that typical invitro expansion conditions generate metabolically and functionally impaired Tcells more reliant on aerobic glycolysis than those expanding invivo. We used dichloroacetate (DCA) to modulate glycolytic metabolism during expansion, resulting in elevated mitochondrial capacity, stemness, and improved antitumor efficacy in murine Tcell receptor (TCR)-Tg and human CAR-T cells. DCA-conditioned Tcells surprisingly show no elevated intratumoral effector function but rather have improved engraftment. DCA conditioning decreases reliance on glucose, promoting usage of serum-prevalent physiologic carbon sources. Further, DCA conditioning promotes metabolic flux from mitochondria to chromatin, resulting in increased histone acetylation at key longevity genes. Thus, hyperglycemic culture conditions promote expansion at the expense of metabolic flexibility and suggest pharmacologic metabolic rewiring as a beneficial strategy for improvement of cellular immunotherapies.

MYO1F regulates T-cell activation and glycolytic metabolism by promoting the acetylation of GAPDH.

Proper cellular metabolism in T cells is critical for a productive immune response. However, when dysregulated by intrinsic or extrinsic metabolic factors, T cells may contribute to a wide spectrum of diseases, such as cancers and autoimmune diseases. However, the metabolic regulation of T cells remains incompletely understood. Here, we show that MYO1F is required for human and mouse T-cell activation after TCR stimulation and that T-cell-specific Myo1f knockout mice exhibit an increased tumor burden and attenuated EAE severity due to impaired T-cell activation in vivo. Mechanistically, after TCR stimulation, MYO1F is phosphorylated by LCK at tyrosines 607 and 634, which is critical for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acetylation at Lys84, 86 and 227 mediated by α-TAT1, which is an acetyltransferase, and these processes are important for its activation, cellular glycolysis and thus the effector function of T cells. Importantly, we show that a fusion protein of VAV1-MYO1F, a recurrent peripheral T-cell lymphoma (PTCL)-associated oncogenic protein, promotes hyperacetylation of GAPDH and its activation, which leads to aberrant glycolysis and T-cell proliferation, and that inhibition of the activity of GAPDH significantly limits T-cell activation and proliferation and extends the survival of hVAV1-MYO1F knock-in mice. Moreover, hyperacetylation of GAPDH was confirmed in human PTCL patient samples containing the VAV1-MYO1F gene fusion. Overall, this study revealed not only the mechanisms by which MYO1F regulates T-cell metabolism and VAV1-MYO1F fusion-induced PTCL but also promising therapeutic targets for the treatment of PTCL.

Booster COVID-19 mRNA vaccination ameliorates impaired B-cell but not T-cell responses in older adults

Age-associated differences in the effect of repetitive vaccination, particularly on memory T-cell and B-cell responses, remain unclear. While older adults (aged ≥65 years) exhibited enhanced IgG responses following COVID-19 mRNA booster vaccination, they produced fewer spike-specific circulating follicular helper T cells-1 than younger adults. Similarly, the cytotoxic CD8+ T-cell response remained diminished with reduced PD-1 expression even after booster vaccination compared with that in younger adults, suggesting impaired memory T-cell activation in older adults. In contrast, although B-cell responses in older adults were weaker than those in younger adults in the primary response, the responses were significantly enhanced upon booster vaccination, reaching levels comparable with that observed in younger adults. Therefore, while booster vaccination ameliorates impaired humoral immunity in older adults by efficiently stimulating memory B-cell responses, it may less effectively enhance T-cell-mediated cellular immunity. Our study provides insights for the development of effective therapeutic and vaccine strategies for the most vulnerable older population.

JAML overexpressed in colorectal cancer promotes tumour proliferation by activating the PI3K-AKT-mTOR signalling pathway

The expression and biological function of junctional adhesion molecule-like protein (JAML) in colorectal cancer (CRC) remain unclear. Paraffin tissue samples from 50 cases of CRC were collected to determine the expression of JAML. JAML was overexpressed or knock-down in CRC cells to evaluated the proliferation, migration and invasion in vitro and in vivo. Western-blot and others were applied to explore the mechanisms. The study showed that JAML was highly expressed within cancer tissues in 50% (25/50) of patients with CRC, and was correlated with higher TNM stage (p < 0.05). Patients of JAML-high group had poorer overall survival compared to JAML-low group (p = 0.0362, HR = 0.4295, 95% CI of 0.1908–0.9667). The tumour infiltrating lymphocytes (TILs) was lower in the JAML-high group than in the JAML-low group (p < 0.05). Overexpression of JAML promoted the proliferation, migration, and invasion of CRC by activating the PI3K-AKT-mTOR signalling pathway both in vitro and in vivo. TILs were reduced in JAML-high tumour tissues by decreasing chemokines such as CCL20 and CXCL9/10/11. Our study identified JAML, a potentially ideal target that is specifically highly expressed in CRC tissues, which promoted tumour proliferation, impaired T-lymphocytes infiltration, provided a promising therapeutic strategy for patients with CRC.

Depletion of monocytic myeloid-derived suppressor cells in LP-BM5 murine retroviral infection has a positive impact on virus-induced host immunodeficiency

We have shown the induction of CD11b+Ly6C+ monocytic myeloid-derived suppressor cells (M-MDSCs) during infection of B6 mice by LP-BM5 immunodeficiency-inducing retrovirus. We published that the molecular mechanisms of these M-MDSCs vary, and depend on the cell type targeted by the suppression –defined by use of biochemical inhibitors, mouse M-MDSCs knock-out strains and blocking antibodies. These M-MDSCs suppressed proliferation and function of T cells, via nitric oxide synthase/nitric oxide; and that of B cells, ∼50% via INOS/NO along with the negative checkpoint regulator VISTA, reactive nitrogen and oxygen species, and other soluble mediators. Here, LP-BM5 infected mice were treated weekly with 5-Fluorouracil (5-FU), resulting in depletion of peripheral blood and splenic M-MDSCs, reduced MDSC activity, and significantly decreased standard disease parameters of: splenomegaly, impaired B-and T-cell ex vivo polyclonal responses, and viral load. In addition, 5-FU treatment significantly increased percentages of CD4+ and CD8+ T cells.

Global awareness of antigenic peptides - Unraveling the impact on sickle cell anemia patients in the presence of pathogens: A narrative review.

Sickle cell anemia (SCA) is a genetic blood disorder characterized by the production of abnormal hemoglobin S (HbS), leading to sickle-shaped red blood cells and various complications, including increased susceptibility to infections. The presence of antigenic peptides, short amino acid sequences derived from pathogens or altered self-proteins, plays a crucial role in immune responses. This review explores the global awareness of antigenic peptides, their role in immune responses in SCA patients, and the challenges and opportunities in managing infections within this vulnerable population. Antigenic peptides are central to the adaptive immune response, facilitating the recognition and elimination of pathogens by T-cells. In SCA, altered antigen presentation and impaired T-cell responses due to chronic inflammation, functional asplenia, and ongoing hemolysis contribute to increased susceptibility to infections. Pathogens such as Streptococcus pneumoniae and Haemophilus influenzae pose significant risks to SCA patients, highlighting the importance of robust immune responses mediated by antigenic peptides. Strategies such as vaccination and immunotherapy aim to enhance immune function by targeting specific antigenic peptides, thereby reducing infection rates and improving patient outcomes. Advances in genomics and proteomics offer insights into individual variations in antigen presentation and immune responses, guiding the development of tailored therapeutic interventions. Global collaborations are essential to address disparities in healthcare access and implement effective preventive measures, ensuring equitable outcomes for SCA patients worldwide.

Management of Refractory/Resistant Herpes Simplex Virus Infections in Haematopoietic Stem Cell Transplantation Recipients: A Literature Review.

Herpes simplex virus (HSV) infections in allogeneic haematopoietic stem cell transplantation (HSCT) recipients pose significant challenges, with higher incidence, severity, and risk of emergence of resistance to antivirals due to impaired T-cell mediated immunity. This literature review focuses on acyclovir-refractory/resistant HSV infections in HSCT recipients. The review addresses the efficacy of antiviral prophylaxis, the incidence of acyclovir-refractory/resistant HSV infections, and the identification of risk factors and potential prognostic impact associated with those infections. Additionally, alternative therapeutic options are discussed. While acyclovir prophylaxis demonstrates a significant benefit in reducing HSV infections in HSCT recipients and, in some cases, overall mortality, concerns arise about the emergence of drug-resistant HSV strains. Our systematic review reports a median incidence of acyclovir-resistant HSV infections of 16.1%, with an increasing trend in recent years. Despite limitations in available studies, potential risk factors of emergence of HSV resistance to acyclovir include human leucocyte antigen (HLA) mismatches, myeloid neoplasms and acute leukaemias, and graft-versus-host disease (GVHD). Limited evidences suggest a potentially poorer prognosis for allogeneic HSCT recipients with acyclovir-refractory/resistant HSV infection. Alternative therapeutic approaches, such as foscarnet, cidofovir, topical cidofovir, optimised acyclovir dosing, and helicase-primase inhibitors offer promising options but require further investigations. Overall, larger studies are needed to refine preventive and therapeutic strategies for acyclovir-refractory/resistant HSV infections in allogeneic HSCT recipients and to identify those at higher risk.

Comprehensive exploration of FCHO1 mutations: Clinical manifestations and implications across disorders.

FCH domain only 1 (FCHO1) is a key player in clathrin-mediated endocytosis, vital for various cellular processes, including immune regulation and cancer progression. However, the clinical implications of FCHO1 mutations, particularly in combined immunodeficiency, remain unclear. This systematic review aims to provide an objective analysis of the molecular genetics, clinical manifestations, and potential therapeutic targets associated with FCHO1 mutations. A systematic search following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was conducted across electronic databases up to March 25, 2024, to identify studies investigating the relationship between FCHO1 and different clinical manifestations. Eligibility criteria were applied to screen studies, and data extraction included study characteristics, reported symptoms, genetic variants, and primary outcomes. In silico analyses were performed to assess protein-protein interactions and gene expression patterns. Five studies were included, offering insights into the molecular genetics, T-cell deficiency mechanisms, clinical manifestations, and potential therapeutic targets associated with FCHO1 mutations. Molecular analyses identified specific mutations disrupting FCHO1 function, leading to impaired T-cell proliferation, cytokine production, and susceptibility to infections. Clinically, patients exhibited recurrent infections, lymphopenia, and malignancies, with allogeneic hematopoietic stem cell transplantation emerging as a therapeutic option. In silico analyses revealed potential interactions and co-expression between FCHO1 and genes involved in cancer progression and immune signaling pathways. This systematic review objectively elucidates the multifaceted role of FCHO1 in immune regulation and disease pathogenesis. Understanding the molecular mechanisms underlying FCHO1 mutations and their impact on disease manifestations is crucial for guiding clinical management and developing targeted therapeutic strategies.

Integrative transcriptome analysis reveals the molecular events underlying impaired T-cell responses in EGFR-mutant lung cancer

EGFR mutations are critical oncogenic drivers in lung adenocarcinoma (LUAD). However, the mechanisms by which they impact the tumor microenvironment (TME) and tumor immunity are unclear. Furthermore, the reasons underlying the poor response of EGFR-mutant (EGFR-MU) LUADs to immunotherapy with PD-1/PD-L1 inhibitors are unknown. Utilizing single-cell RNA (sc-RNA) and bulk RNA sequencing datasets, we conducted high-dimensional weighted gene coexpression network analysis to identify key genes and immune-related pathways contributing to the immunosuppressive TME. EGFR-MU cancer cells downregulated MHC class I genes to evade CD8+ cytotoxic T cells, expressed substantial levels of MHC class II molecules, and engaged with CD4+ regulatory T cells (Tregs). EGFR-MU tumors may recruit Tregs primarily through the CCL17/CCL22/CCR4 axis, leading to a Treg-enriched TME. High levels of MHC class II-positive cancer-associated fibroblasts and tumor endothelial cells were found within EGFR-MU tumors. Owing to the absence of costimulatory factors, they may inhibit rather than activate the tumor antigen-specific CD4+ T-cell response, contributing further to immune suppression. Multiplex immunohistochemistry analyses in a LUAD cohort confirmed increased expression of MHC class II molecules in cancer cells and fibroblasts in EGFR-MU tumors. Our research elucidates the highly immunosuppressive TME in EGFR-MU LUAD and suggests potential targets for effective immunotherapy.

Novel Mutation in the Moesin (MSN) Gene Leads to Immunodeficiency with Epstein-Barr Virus (EBV) Infection and Dermatomyositis-Like Symptoms.

Moesin (MSN) deficiency is a recently reported combined immunodeficiency, and few cases have been reported to date. We describe a Chinese patient with a novel mutation causing MSN deficiency and a novel phenotype. Clinical and immunological data were collected. Whole-exome sequencing was performed to identify gene mutations. MSN protein expression and T cell proliferation and activation were determined by flow cytometry. Cell migration was confirmed with a Transwell assay. Autoantibody levels were analyzed using antigen microarrays. The patient was a 10-year-old boy who presented with recurrent fever, oral ulcers and dermatomyositis-like symptoms, such as periorbital edema, facial swelling, elevated creatine kinase levels, and abnormal electromyography and muscle biopsy results. Epstein-Barr virus (EBV) DNA was detected in the serum, cells and tissues of this patient. He further developed nasal-type NK/T-cell lymphoma. A novel hemizygous mutation (c.68A > G, p.N23S) in the MSN gene was found. The immunological phenotype of this patient included persistent decreases in T and B lymphocyte counts but normal immunoglobulin IgG levels. The patient had attenuated MSN protein expression and impaired T-cell proliferation and migration. The proportions of Tfh cells and CD21low B cells in the patient were higher than those in the controls. Moreover, 82 IgG and 102 IgM autoantibodies were more abundant in the patient than in the healthy controls. The novel mutation N23S is pathogenic and leads to a severe clinical phenotype. EBV infection, tumor, and dermatomyositis-like autoimmune symptoms may be associated with MSN deficiency, further expanding the understanding of the disease.

Clonal hematopoiesis driven by mutated DNMT3A promotes inflammatory bone loss

Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory Tcell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.

Local delivery of accutox® synergises with immune-checkpoint inhibitors at disrupting tumor growth

BackgroundThe Accum® platform was initially designed to accumulate biomedicines in target cells by inducing endosomal-to-cytosol escape. Interestingly however, the use of unconjugated Accum® was observed to trigger cell death in a variety of cancer cell lines; a property further exploited in the development of Accum®-based anti-cancer therapies. Despite the impressive pro-killing abilities of the parent molecule, some cancer cell lines exhibited resistance. This prompted us to test additional Accum® variants, which led to the identification of the AccuTOX® molecule.MethodsA series of flow-cytometry and cell-based assays were used to assess the pro-killing properties of AccuTOX® along with its ability to trigger the production of reactive oxygen species (ROS), endosomal breaks and antigen presentation. RNA-seq was also conducted to pinpoint the most prominent processes modulated by AccuTOX® treatment in EL4 T-cell lymphoma. Finally, the therapeutic potency of intratumorally-injected AccuTOX® was evaluated in three different murine solid tumor models (EL4, E0771 and B16) both as a monotherapy or in combination with three immune-checkpoint inhibitors (ICI).ResultsIn total, 7 Accum® variants were screened for their ability to induce complete cell death in 3 murine (EL4, B16 and E0771) and 3 human (MBA-MD-468, A549, and H460) cancer cell lines of different origins. The selected compound (hereafter refereed to as AccuTOX®) displayed an improved killing efficiency (~ 5.5 fold compared to the parental Accum®), while retaining its ability to trigger immunogenic cell death, ROS production, and endosomal breaks. Moreover, transcriptomic analysis revealed that low dose AccuTOX® enhances H2-Kb cell surface expression as well as antigen presentation in cancer cells. The net outcome culminates in impaired T-cell lymphoma, breast cancer and melanoma growth in vivo especially when combined with anti-CD47, anti-CTLA-4 or anti-PD-1 depending on the animal model.ConclusionsAccuTOX® exhibits enhanced cancer killing properties, retains all the innate characteristics displayed by the parental Accum® molecule, and synergizes with various ICI in controlling tumor growth. These observations will certainly pave the path to continue the clinical development of this lead compound against multiple solid tumor indications.Graphical

Exploring T-cell exhaustion features in Acute myocardial infarction for a Novel Diagnostic model and new therapeutic targets by bio-informatics and machine learning

BackgroundT-cell exhaustion (TEX), a condition characterized by impaired T-cell function, has been implicated in numerous pathological conditions, but its role in acute myocardial Infarction (AMI) remains largely unexplored. This research aims to identify and characterize all TEX-related genes for AMI diagnosis.MethodsBy integrating gene expression profiles, differential expression analysis, gene set enrichment analysis, protein-protein interaction networks, and machine learning algorithms, we were able to decipher the molecular mechanisms underlying TEX and its significant association with AMI. In addition, we investigated the diagnostic validity of the leading TEX-related genes and their interactions with immune cell profiles. Different types of candidate small molecule compounds were ultimately matched with TEX-featured genes in the “DrugBank” database to serve as potential therapeutic medications for future TEX-AMI basic research.ResultsWe screened 1725 differentially expressed genes (DEGs) from 80 AMI samples and 71 control samples, identifying 39 differential TEX-related transcripts in total. Functional enrichment analysis identified potential biological functions and signaling pathways associated with the aforementioned genes. We constructed a TEX signature containing five hub genes with favorable prognostic performance using machine learning algorithms. In addition, the prognostic performance of the nomogram of these five hub genes was adequate (AUC between 0.815 and 0.995). Several dysregulated immune cells were also observed. Finally, six small molecule compounds which could be the future therapeutic for TEX in AMI were discovered.ConclusionFive TEX diagnostic feature genes, CD48, CD247, FCER1G, TNFAIP3, and FCGRA, were screened in AMI. Combining these genes may aid in the early diagnosis and risk prediction of AMI, as well as the evaluation of immune cell infiltration and the discovery of new therapeutics.

Glucose-driven histone lactylation promotes the immunosuppressive activity of monocyte-derived macrophages in glioblastoma

Immunosuppressive macrophages restrict anti-cancer immunity in glioblastoma (GBM). Here, we studied the contribution of microglia (MGs) and monocyte-derived macrophages (MDMs) to immunosuppression and mechanisms underlying their regulatory function. MDMs outnumbered MGs at late tumor stages and suppressed Tcell activity. Molecular and functional analysis identified a population of glycolytic MDM expressing GLUT1 with potent immunosuppressive activity. GBM-derived factors promoted high glycolysis, lactate, and interleukin-10 (IL-10) production in MDMs. Inhibition of glycolysis or lactate production in MDMs impaired IL-10 expression and Tcell suppression. Mechanistically, intracellular lactate-driven histone lactylation promoted IL-10 expression, which was required to suppress Tcell activity. GLUT1 expression on MDMs was induced downstream of tumor-derived factors that activated the PERK-ATF4 axis. PERK deletion in MDM abrogated histone lactylation, led to the accumulation of intratumoral Tcells and tumor growth delay, and, in combination with immunotherapy, blocked GBM progression. Thus, PERK-driven glucose metabolism promotes MDM immunosuppressive activity via histone lactylation.

Ifit2 interacts with murine-β-coronavirus RSA59 nucleocapsid protein to restrict its replication and spread

Abstract The antiviral efficacy of Ifit2, an interferon-induced protein with tetratricopeptide repeats 2, is known for its ability to restrict the neurotropic murine-β-coronavirus, RSA59 infection. Intracranial injection of RSA59 in Ifit2 deficient (-/-) compared to wild-type (WT) mice results in impaired microglial activation and peripheral lymphocyte infiltration into the brain with severe morbidity and mortality in the acute phase of the disease. RSA59-infected Ifit2-/- mice also showed extensive viral replication, spreading throughout the spinal cord with impaired microglial activation and T-cell infiltration. Cervical-lymph-nodes of RSA59-infected Ifit2-/- mice exhibited reduced activation of CD4+ T cells and impaired IFNγ expression while preserving the blood-brain barrier integrity. To understand Ifit2's impact on the chronic demyelination phase, Ifit2-/- and Ifit2+/+ (WT) mice were observed for neuropathological outcomes upon RSA59 infection. The chronic disease phase in RSA59-infected Ifit2-/- mice demonstrated severe demyelination and persistent viral load. Furthermore, understanding molecular interactions in a reductionist approach using co-immunoprecipitation showed that upregulated Ifit2 in RSA59-infected primary astrocytes can interact with viral nucleocapsid protein. Thus, this study highlights that Ifit2's direct interaction with viral nucleocapsid protein can restrict RSA59 spread throughout the CNS in the acute phase, thus limiting severe chronic demyelination.

SARS-CoV-2 infection induces thymic atrophy mediated by IFN-γ in hACE2 transgenic mice.

Pathogenic infections cause thymic atrophy, perturb thymic T-cell development, and alter immunological response. Previous studies reported dysregulated T-cell function and lymphopenia in coronavirus disease-19 (COVID-19). However, immunopathological changes in the thymus associated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection have not been elucidated. Here, we report that SARS-CoV-2 infects thymocytes, and induces CD4+CD8+ (double positive; DP) T-cell apoptosis leading to thymic atrophy and loss of peripheral TCR repertoire in K18-hACE2 transgenic mice. Infected thymus led to increased CD44+CD25- T-cells, indicating an early arrest in the T-cell maturation pathway. Thymic atrophy was notably higher in male hACE2-Tg mice than in females and involved an upregulated de-novo synthesis pathway of thymic glucocorticoid. Further, IFN-γ was crucial for thymic atrophy, as anti-IFN-γ -antibody neutralization bluntedthymic involution. Therapeutic use of Remdesivir also rescued thymic atrophy. While the Omicron variant and its sub-lineage BA.5 variant caused marginal thymic atrophy, the delta variant of SARS-CoV-2 exhibited severe thymic atrophy characterized by severely depleted DP T-cells. Recently characterized broadly SARS-CoV-2 neutralizing monoclonal antibody P4A2 was able to rescue thymic atrophy and restore the thymic maturation pathway of T-cells. Together, we report SARS-CoV-2-associated thymic atrophy resulting from impaired T-cell maturation pathway which may contribute to dyregulated T cell response during COVID-19.