Cytotoxic Phenotype Research Articles

EXTH-11. PEDIATRIC GLIOMA IMMUNE PROFILING IDENTIFIES TIM3 AS A THERAPEUTIC TARGET IN BRAF-FUSION PILOCYTIC ASTROCYTOMA

Abstract Despite being the leading cause of childhood mortality, pediatric gliomas have been relatively understudied. Repurposing of adult brain tumor immunotherapies in pediatric patients has not been successful, highlighting the need for pediatric-specific immunotherapies. Pilocytic astrocytomas (PA) are the most common pediatric glioma. While surgical resection of PA is the first-line treatment, the ten-year progression-free survival rate for patients with residual tumors is <50%. Notably, some PAs undergo spontaneous regression after partial resection, suggesting potential baseline tumor immunoreactivity. Whole transcriptome sequencing performed on a commercial platform of pediatric gliomas (n=250) showed that MAPK-driven gliomas have higher interferon signatures compared to other molecular subgroups. Single-cell sequencing identified a unique activated and cytotoxic microglia population designated MG-Act in BRAF-fused MAPK-activated PA (n=13; p < 0.05), but not in pediatric high-grade gliomas (n=5) or normal brain (n=3). TIM3 was expressed on the luminal side of endothelial cells and MG-Act, but not in the surrounding normal brain based on sequential multiplex imaging. Flow cytometry showed that TIM3 intensity is upregulated on immune cells in the PA tumor microenvironment (TME) relative to matched peripheral blood immune cells (n=6; p < 0.01). Anti-TIM3 reprogrammed fresh ex vivo immune cells from the TME of human PAs (n=3) to a pro-inflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven low-grade gliomas, median survival (MS) was 252 days in mice treated with anti-TIM3 (n=21; p<0.01) relative to IgG control (MS: 110 days; n=20) or anti-PD-1 (MS: 134 days; n=20). The therapeutic activity of anti-TIM3 was abrogated in the CX3CR1 microglia knockout background. ScRNA sequencing data during the therapeutic window of anti-TIM3 in wild-type mice demonstrated enrichment of the MG-Act population within low-grade gliomas at two different longitudinal time points but not in IgG-treated controls. Together, these data indicate that anti-TIM3 should be considered for clinical trials in pediatric low-grade MAPK-driven gliomas.

Multi-omics reveal immune microenvironment alterations in multiple myeloma and its precursor stages

Tumor immune microenvironmental alterations occur early in multiple myeloma (MM) development. In this study, we aim to systematically characterize the tumor immune microenvironment (TME) and the tumor-immune interactions from precursor stages, i.e., monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to newly diagnosed MM, comparing these to healthy donors. Using CIBERSORT, mass cytometry (CyTOF), and single-cell RNA sequencing (scRNA-Seq), we examined innate and adaptive immune changes across these stages. We found a decrease in granulocytes in the TME predicts MM outcomes. HLA-DR is reduced in CD16+ monocytes and plasmacytoid dendritic cells, while myeloid dendritic cells show decreased expression of stress and immune-response genes. NK cells and CD8+ T cells shift from a GZMK+ to a GZMB+ cytotoxic phenotype in the TME, with increased inhibitory markers TIM3 and TIGIT. In paired samples, the proportion and gene expression pattern in patient-specific GZMB+CD8+ T cells remain largely unchanged despite MM progression. Our findings provide a comprehensive immune landscape of MM and its precursors, offering insights into therapeutic strategies. Enhancing neutrophil and NK cell cytotoxicity, tumor antigen presentation, and CD8+ T cell versatility in precursor stages may prevent MM progression.

Acidic polysaccharides from Cistanche deserticola and their effects on the polarization of tumor-associated macrophages

Three purified polysaccharides, CDAP-1, CDAP-2, and CDAP-3, were prepared from the rhizome of Cistanche deserticola and characterized. Structural analysis revealed that CDAP-1 and CDAP-2 are highly branched RG-I-type polysaccharides with side chains, including arabinans, galactans, and/or AGs, whereas CDAP-3 is a typical HG-type polysaccharide. In vivo tests revealed that treatment with the crude polysaccharide fraction (CDCP) significantly prolonged the survival of H22 tumor-bearing mice and exhibited antitumor effects. In vitro experiments demonstrated that all three polysaccharides could polarize M2-like TAMs toward the M1 phenotype. As a major component of CDCP, CDAP-2 could act on M2 macrophages through the TLR4 receptor-mediated NF-κB signaling pathway. An in vitro cell model verified that CDAP-2 could inhibit cell proliferation by reversing the polarization of M2-like TAMs to the cytotoxic M1 phenotype. Overall, we found that CDCP showed a clear antitumor effect and that its major component, CDAP-2, could reverse the suppressive TAM phenotype in the microenvironment, providing a scientific basis for the clinical application and development of C. deserticola.

Hepatocellular carcinoma hosts cholinergic neural cells and tumoral hepatocytes harboring targetable muscarinic receptors

Background & aimsUnexplained interpatient variation and treatment failure in HCC prompt for renewed approaches. Hepatic neurons, belonging to the autonomic nervous system (ANS), mediate liver/whole body cross-talks. Pathological innervation of the ANS have been identified in cancer, nurturing tumor stroma and conferring stronger carcinogenic properties. MethodsWe characterized the innervation of liver tumors from the French Liver Biobank, then applied bioinformatics to the TCGA, several other datasets and a European validation cohort, to re-evaluate patient stratification. Cell biology and pharmacology studies were also done. ResultsDensely packed nucleated DCX+, synaptophysin+, NeuN+, VAChT+, TH-, CD31-, CD45- clusters, to date undetected, were identified in human HCCs, and independently confirmed by scRNA-seq data. Using the new concept of neuronal score, human and rat HCCs displayed tightly netrin-1-associated neural reconfiguration towards cholinergic polarity along with CLD progression, cancer onset and many features of aggressive (proliferative class) HCC, including shortened survival. This score was conditioned by tumoral hepatocytes, and predicted sorafenib efficacy in the STORM HCC phase 3 trial. Conversely, intratumoral adrenergic lymphocytes were enriched in TEMRA and cytotoxic phenotypes. Amongst all cholinergic transcripts, the medically-targeted CHRM3 receptor was enriched in HCC and associated with cells’ and tumors’ pathogenic traits, displayed adverse prognostic value by the log-rank test in HCC stages 1-2, while collapsing upon experimental re-differentiation. Its pharmacological inhibition by low concentrations of anticholinergic drugs, but not cholinomimetics, decreased anchorage-independent and anoikis growth, synergized with sorafenib and lenvatinib in HCC class 1 to 3 lines, yet not in primary human hepatocytes, and preserved mature hepatocytic functions. ConclusionThese data identify cholinergic processes as instrumental in liver carcinogenesis, and support the use of EMA/FDA-approved cholinergic drugs in HCC research. Impact and implicationsHCC care has long been hampered by the enigmatic nature of disease evolution, as well as of response or resistance to treatment. Hepatic neurons are likely the least studied liver cell type and mediate patients singularities to the organ in real-time. Cholinergic inputs identified in this study as pathogenic may be targeted with the well charted pharmacopoeia of neurotropic drugs already available, for basic or clinical research purposes, with an expected high level of safety.

Primary Cutaneous Acral-type CD8-positive T-cell Lymphoproliferations: Widely Divergent Clinical Outcomes Despite Nearly Identical Histomorphology

Abstract Introduction/Objective Non-epidermotropic and acral CD8(+) T-cell proliferations have recently been reclassified as lymphoproliferative disorders (LPDs) due to their indolent course clinical. Here, we describe two cases with similar histomorphology but disparate clinical outcomes, highlighting the diagnostic challenges. Methods/Case Report Case 1: An 89-year-old male developed a 2 cm erythematous papule on the forehead. Biopsy showed a non-epidermotropic pan-dermal lymphocytic infiltrate, a distinct grenz zone, and areas of necrosis. Case 2: A 53-year-old male presented with a rapidly growing, ulcerating 8.9 cm scalp lesion. Biopsy showed a deep dermal lymphoid infiltrate with complete epidermal sparing. In both cases neoplastic cells were positive by immunohistochemistry for CD2, CD3, CD5, CD7 (partial), CD8, granzyme B, TIA-1, and CD68 (perinuclear dot-like pattern). The Ki-67 proliferation index varied modestly between cases (50% in case 1 and 70% in case 2, respectively). Despite treatment, within the span of a few months, case 2 developed adjacent, widely distributed scalp nodules with extensive local tissue destruction. Pathology was consistent with recurrent disease (flow cytometry demonstrating T-cells, co-expressing CD8, CD2, CD3, CD5, CD7 and monotypic TRBC1). Results (if a Case Study enter NA) NA Conclusion CD8+ T-cell lymphoproliferative disorders, specifically primary cutaneous acral CD8+ T-cell lymphoproliferative disorder, is a rare cutaneous T-cell disorder that was re-classified, owing to its indolent clinical course, low risk of systemic dissemination, and favorable prognosis. We report two cases of nearly identical isolated cutaneous lesions that demonstrated a CD8(+)-cytotoxic phenotype with CD68-dot-like positivity. Although all morphologic findings were supportive of the diagnosis, the aggressive clinical progression in case 2 highlights the potential for the disease to defy the classically described indolent behavior. Given that few cases with aggressive clinical courses have been published, we present these cases to underscore their complexity and propose that the classification incorporate clinicopathological correlation to guide management.

Non-homogenous intratumor ionizing radiation doses synergize with PD1 and CXCR2 blockade

The efficacy and side effects of radiotherapy (RT) depend on parameters like dose and the volume of irradiated tissue. RT induces modulations of the tumor immune microenvironment (TIME) that are dependent on the dose. Low dose RT (LDRT, i.e., single doses of 0.5–2 Gy) has been shown to promote immune infiltration into the tumor. Here we hypothesize that partial tumor irradiation combining the immunostimulatory/non-lethal properties of LDRT with cell killing/shrinkage properties of high dose RT (HDRT) within the same tumor mass could enhance anti-tumor responses when combined with immunomodulators. In models of colorectal and breast cancer in immunocompetent female mice, partial irradiation (PI) with millimetric precision to deliver LDRT (2 Gy) and HDRT (16 Gy) within the same tumor induces substantial tumor control when combined with anti-PD1. Using flow cytometry, cytokine profiling and single-cell RNA sequencing, we identify a crosstalk between the TIME of the differentially irradiated tumor volumes. PI reshapes tumor-infiltrating CD8+ T cells into more cytotoxic and interferon-activated phenotypes but also increases the infiltration of pro-tumor neutrophils driven by CXCR2. The combination of the CXCR2 antagonist SB225002 with PD1 blockade and PI improves tumor control and mouse survival. Our results suggest a strategy to reduce RT toxicity and improve the therapeutic index of RT and immune checkpoint combinations.

The Impact of Different Anesthetics on the Distribution and Cytotoxic Function of NK Cell Subpopulations: An In Vitro Study

Only some subpopulations of natural killer (NK) cells have cytotoxic functionality, and the effects of anesthetics on these subpopulations are unknown. This study aimed to evaluate the in vitro effects of various anesthetics, both alone and in combination, on the distribution and cytotoxic function of NK cells and their subpopulations. Peripheral blood mononuclear cells (PBMCs) from eight healthy volunteers were treated for 4 h in vitro with dexmedetomidine, remifentanil, lidocaine, propofol, sevoflurane, and combinations in clinically relevant concentrations or left untreated. Flow cytometry was used to quantify the percentage of sampled NK cells and evaluate their distribution (CD56brightCD16neg, CD56brightCD16dim, CD56dimCD16neg, CD56dimCD16bright, and CD56negCD16bright) and cytotoxicity (Granzyme B (GrzB) and perforin) of NK cell subpopulations. Although the percentage of total NK cells did not change following exposure to anesthesia, the most important cytotoxic subpopulation (CD56dimCD16bright NK cells) decreased after exposure to both propofol (−3.58%, p = 0.045) and sevoflurane (−16.10%, p = 0.008) alone, and most combinations, especially in combination with lidocaine (propofol with lidocaine (−9.66%, p = 0.002) and sevoflurane with lidocaine (−21.90%, p < 0.001)). Dexmedetomidine and remifentanil had no effect on CD56dimCD16bright NK cells. Furthermore, no anesthetic regimen or combination altered the expression of GrzB and perforin in NK cells or NK cell subpopulations. In short, propofol and sevoflurane suppressed the highly cytotoxic phenotype (CD56dimCD16bright) of NK cells, with those exposed to sevoflurane combinations showing greater reductions. Immunosuppression was intensified with the inclusion of lidocaine in the anesthetic regimen.

State of the Field: Cytotoxic Immune Cell Responses in C. neoformans and C. deneoformans Infection.

Cryptococcus neoformans is an environmental pathogen that causes life-threatening disease in immunocompromised persons. The majority of immunological studies have centered on CD4+ T-cell dysfunction and associated cytokine signaling pathways, optimization of phagocytic cell function against fungal cells, and identification of robust antigens for vaccine development. However, a growing body of literature exists regarding cytotoxic cells, specifically CD8+ T-cells, Natural Killer cells, gamma/delta T-cells, NK T-cells, and Cytotoxic CD4+ T-cells, and their role in the innate and adaptive immune response during C. neoformans and C. deneoformans infection. In this review, we (1) provide a comprehensive report of data gathered from mouse and human studies on cytotoxic cell function and phenotype, (2) discuss harmonious and conflicting results on cellular responses in mice models and human infection, (3) identify gaps of knowledge in the field ripe for exploration, and (4) highlight how innovative immunological tools could enhance the study of cytotoxic cells and their potential immunomodulation during cryptococcosis.

Single-cell landscape of bronchoalveolar immune cells in patients with immune checkpoint inhibitor-related pneumonitis

The pathophysiology of immune checkpoint inhibitor-related pneumonitis remains incompletely understood. We conducted single-cell and T-cell receptor transcriptomic sequencing on the bronchoalveolar lavage fluid from five patients with grade ≥2 immune checkpoint inhibitor-related pneumonitis. Our analyses revealed a prominent enrichment of T cells in the bronchoalveolar lavage fluid of patients with immune checkpoint inhibitor-related pneumonitis. Within the CD4 + T cell subset, Tfh-like T cells were highly enriched and exhibited signatures associated with inflammation and clonal expansion. Regulatory T cells were also enriched and displayed enhanced inhibitory functions. Within the CD8 + T-cell subset, effector memory/tissue-resident memory T cells with an elevated cytotoxic phenotype were highly infiltrated. Among myeloid cells, alveolar macrophages were depleted, while pro-inflammatory intermediate monocytes were elevated. Dendritic cells demonstrated enhanced antigen presentation capabilities. Cytokines CXCR4, CXCL13, TNF-α, IFN-α, IFN-γ, and TWEAK were elevated. Through a comprehensive single-cell analysis, we depicted the landscape of immune checkpoint inhibitor-related pneumonitis.

Dynamic Transition of Regulatory T Cells to Cytotoxic Phenotype Amid Systemic Inflammation in Graves' Ophthalmopathy.

Graves' disease (GD) is an autoimmune condition that can progress to Graves' Ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, GD patients without GO, newly diagnosed GO patients, and treated GO patients. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated regulatory T cells (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yinyang1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results revealed the dynamic changes in immune landscape during GO progression and provided novel insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.

Enhancing Selectivity and Inhibitory Effects of Chemotherapy Drugs Against Myelogenous Leukemia Cells with Lippia alba Essential Oil Enriched in Citral.

Acute myelogenous leukemia (AML) is one of the most lethal cancers, lacking a definitive curative therapy due to essential constraints related to the toxicity and efficacy of conventional treatments. This study explores the co-adjuvant potential of Lippia alba essential oils (EO) for enhancing the effectiveness and selectivity of two chemotherapy agents (cytarabine and clofarabine) against AML cells. EO derived from L. alba citral chemotype were produced using optimized and standardized environmental and extraction protocols. Rational fractionation techniques were employed to yield bioactive terpene-enriched fractions, guided by relative chemical composition and cytotoxic analysis. Pharmacological interactions were established between these fractions and cytarabine and clofarabine. The study comprehensively evaluated the cytotoxic, genotoxic, oxidative stress, and cell death phenotypes induced by therapies across AML (DA-3ER/GM/EVI1+) cells. The fraction rich in citral (F2) exhibited synergistic pharmacological interactions with the studied chemotherapies, intensifying their selective cytotoxic, genotoxic, and pro-oxidant effects. This shift favored transitioning from necrosis to a programmed cell death phenotype (apoptotic). The F2-clofarabine combination demonstrated remarkable synergistic anti-leukemic performance while preserving cell integrity in healthy cells. The observed selective antiproliferative effects may be attributed to the potential dual prooxidant/antioxidant behavior of citral in L. alba EO.

Natural killer cells promote neutrophil extracellular traps and restrain macular degeneration in mice.

Neovascular age-related macular degeneration (nvAMD) is the leading cause of blindness in the elderly population. Although it is known that nvAMD is associated with focal inflammation, understanding of the precise immune components governing this process remains limited. Here, we identified natural killer (NK) cells as a prominent lymphocyte population infiltrating the perivascular space of choroidal neovascularization (CNV) lesions in patients with nvAMD and in mouse models. Olink proteomic analysis and single-cell RNA sequencing combined with knockout studies demonstrated the involvement of C-C chemokine receptor 5 (CCR5) in NK cell recruitment and extravasation at the CNV sites of mice. Depletion of NK cells or inhibition of activating receptor NK group 2, member D (NKG2D) inhibited the formation of neutrophil extracellular traps, increased vascular leakage, and exacerbated pathological angiogenesis, indicating that NK cells restrain pathogenesis in this mouse model. Age is the strongest risk factor for AMD, and we show that NK cells from aged human donors exhibited a less cytotoxic phenotype. NK cells from old mice exhibited compromised protective effects in the CNV mouse model. In addition, interleukin-2 complex-mediated expansion of NK cells improved CNV formation in mice. Collectively, our study highlights NK cells as a potential therapeutic target for patients with nvAMD.

Pediatric glioma immune profiling identifies TIM3 as a therapeutic target in BRAF fusion pilocytic astrocytoma.

Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti-PD-1-treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG-KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.

Bis-sulfonamido-2-phenylbenzoxazoles Validate the GroES/EL Chaperone System as a Viable Antibiotic Target.

We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.

Oligoclonal CD4+CXCR5+ T cells with a cytotoxic phenotype appear in tonsils and blood

In clinical situations, peripheral blood accessible CD3+CD4+CXCR5+ T-follicular helper (TFH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of TFH cells in peripheral blood versus tonsils, CD3+CD4+CD45RA–CXCR5+ cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3+CD4+CXCR5+ T-cell subpopulations also appear in tonsils, there with some supplementary TFH characteristics, while peripheral blood-derived TFH cells display markers of proliferation and migration. Three further subsets of TFH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4+CXCR5+ subpopulation presents pronounced cytotoxic properties. Those ‘killer TFH (TFK) cells’ can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4+CXCR5+ T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

Innate and cellular immune response to the Ebola vaccine Ad26.ZEBOV, MVA-BN-Filo: an ancillary study of the EBL2001 phase II trial.

The EBL2001 phase 2 trial tested the two-dose Ad26.ZEBOV, MVA-BN-Filo Ebola vaccine in Europe. Safety and humoral immunogenicity assessments led to EU market authorization in 2020. Complementary analyses of immune responses are warranted to better characterize vaccine effects. We conducted an ancillary study to analyze changes in the serum and cellular responses. Serum biomarkers of activation/inflammation were evaluated using a Luminex assay. Vaccine-elicited T cell responses and functions were evaluated assessing their phenotype, cytokine production, proliferation and cytotoxic potential. Integrated data analysis was performed through correlation and principal component analysis of serum biomarkers and cellular immune responses. Forty-eight volunteers were included. The Ad26.ZEBOV, MVA-BN-Filo vaccine elicited: i) serum increase of inflammatory/activation markers mainly at 1 day after the Ad26.ZEBOV vaccine; ii) durable EBOV-specific T cell proliferation and CD8+ T cells exhibiting a cytotoxic phenotype after Ad26.ZEBOV prime, after MVA-BN-Filo boost and 6 months post vaccination. Integrated analysis revealed correlations between: i) EBOV-specific CD8+ T cell proliferation and cytotoxic phenotype; ii) high EBOV-specific CD8+ T cell cytotoxic phenotype and low inflammatory marker IL-8 at day 1 post vaccination. This study provides unique insights into the in vivo contribution of proliferation/cytotoxic CD8+ T cells and inflammation to the Ad26.ZEBOV, MVA-BN-Filo vaccine-induced potency.

GZMK+CD8+ T cells Target A Specific Acinar Cell Type in Sjögren's Disease.

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2- seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

Terminally differentiated effector memory T cells associate with cognitive and AD-related biomarkers in an aging-based community cohort

Background and purposeThe immune response changes during aging and the progression of Alzheimer’s disease (AD) and related dementia (ADRD). Terminally differentiated effector memory T cells (called TEMRA) are important during aging and AD due to their cytotoxic phenotype and association with cognitive decline. However, it is not clear if the changes seen in TEMRAs are specific to AD-related cognitive decline specifically or are more generally correlated with cognitive decline. This study aimed to examine whether TEMRAs are associated with cognition and plasma biomarkers of AD, neurodegeneration, and neuroinflammation in a community-based cohort of older adults.MethodsStudy participants from a University of Kentucky Alzheimer’s Disease Research Center (UK-ADRC) community-based cohort of aging and dementia were used to test our hypothesis. There were 84 participants, 44 women and 40 men. Participants underwent physical examination, neurological examination, medical history, cognitive testing, and blood collection to determine plasma biomarker levels (Aβ42/Aβ40 ratio, total tau, Neurofilament Light chain (Nf-L), Glial Fibrillary Acidic Protein (GFAP)) and to isolate peripheral blood mononuclear cells (PBMCs). Flow cytometry was used to analyze PBMCs from study participants for effector and memory T cell populations, including CD4+ and CD8+ central memory T cells (TCM), Naïve T cells, effector memory T cells (TEM), and effector memory CD45RA+ T cells (TEMRA) immune cell markers.ResultsCD8+ TEMRAs were positively correlated with Nf-L and GFAP. We found no significant difference in CD8+ TEMRAs based on cognitive scores and no associations between CD8+ TEMRAs and AD-related biomarkers. CD4+ TEMRAs were associated with cognitive impairment on the MMSE. Gender was not associated with TEMRAs, but it did show an association with other T cell populations.ConclusionThese findings suggest that the accumulation of CD8+ TEMRAs may be a response to neuronal injury (Nf-L) and neuroinflammation (GFAP) during aging or the progression of AD and ADRD. As our findings in a community-based cohort were not clinically-defined AD participants but included all ADRDs, this suggests that TEMRAs may be associated with changes in systemic immune T cell subsets associated with the onset of pathology.

IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αβ-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C− cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C− cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C− cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Molecular Pathways and Cellular Subsets Associated with Adverse Clinical Outcomes in Overlapping Immune-Related Myocarditis and Myositis.

Immune checkpoint therapies (ICT) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we performed single-cell RNA sequencing of heart and skeletal muscle biopsies obtained from living patients with cancers treated with ICTs and admitted to the hospital with myocarditis and/or myositis (overlapping myocarditis plus myositis, n = 10; myocarditis-only, n = 1) or ICT-exposed patients ruled out for toxicity utilized as controls (n = 9). All biopsies were obtained within 96 hours of clinical presentation. Analyses of 58,523 cells revealed CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of myeloid cells expressing tissue-resident signatures and FcγRIIIa (CD16a), which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d, which were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results provide insight into the myeloid subsets present in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. See related Spotlight by Fankhauser et al., p. 954.