Human CD4 Research Articles

CX 3 CR1 + macrophages interact with HSCs to promote HCC through CD8 + T-cell suppression.

HSCs contribute to HCC progression by regulating multiple factors. However, the entire immunoregulatory functions of HSCs are still obscure. Here, we aim to investigate whether HSCs impose CX 3 CR1 + macrophages to protumorigenic properties in the peritumoral area. In single-cell RNA-sequencing analysis of patients with HCC, a subpopulation of macrophages specifically expressed Arg1 and Cx3cr1 in the peritumoral area and were highly enriched with retinol metabolism-related genes. Flow cytometry analysis showed significantly increased frequencies of CD14 + CD11b + HLA-DR - macrophages with CX 3 CR1 in the HCC adjacent region where α-smooth muscle actin-expressing activated hepatic stellate cells (aHSCs) showed colocalized expression of CX 3 CL1. Accordingly, in tumor-bearing mice, Cx3cl1 mRNA expression was notably increased in aHSCs within the adjacent HCC, where infiltration of CX 3 CR1 + Ly6C + macrophages was mostly observed with decreased CD8 + T cells. In adoptive transfer and in vitro coculture of myeloid cells, we demonstrated that CX 3 CR1 + Ly6C + macrophages migrated and highly expressed arginase-1 by interacting with retinoid-enriched aHSCs in the adjacent HCC. Direct treatment of retinoids or coculturing with retinol-storing mouse aHSCs or human LX-2 cells significantly increased arginase-1 expression in CX 3 CR1 + Ly6C + macrophages and human blood CD14 + cells, leading to the suppression of CD8 + T-cell proliferation. Moreover, genetic deficiency of CX 3 CR1 in myeloid cells or pharmacological inhibition of retinol metabolism remarkably attenuated HCC development. We showed that CX 3 CR1 + Ly6C + macrophages migrate and interact with aHSCs in the peritumoral region where retinoids induce arginase-1 expression in CX 3 CR1 + Ly6C + macrophages, subsequently depriving CD8 + T cells of arginine and promoting HCC.

CD74 is a functional MIF receptor on activated CD4+ T cells

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22(TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

An atlas of transcribed enhancers across helper T cell diversity for decoding human diseases.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro granzyme-mediated target cell killing by CD8 T or NK cells.

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

Psoriatic arthritis subtypes are phenocopied in humanized mice.

Psoriatic arthritis (PsA) is a complex inflammatory disease that challenges diagnosis and complicates the rational selection of effective therapies. Although T cells are considered active effectors in psoriasis and PsA, the role of CD8+ T cells in pathogenesis is not well understood. We selected the humanized mouse model NSG-SGM3 transgenic strain to examine psoriasis and PsA endotypes. Injection of PBMCs and sera from patients with psoriasis and PsA generated parallel skin and joint phenotypes in the recipient mouse. The transfer of human circulating memory T cells was followed by migration and accumulation in the skin and synovia of these immunodeficient mice. Unexpectedly, immunoglobulins were required for recapitulation of the clinical phenotype of psoriasiform lesions and PsA domains (dactylitis, enthesitis, bone erosion). Human CD8+ T cells expressing T-bet, IL-32 and CXCL14 were detected by spatial transcriptomics in murine synovia and by immunofluorescence in the human PsA synovia. Importantly, depletion of human CD8+ T cells prevented skin and synovial inflammation in mice humanized with PsA peripheral blood cells. The humanized model of psoriasis and PsA represents a valid platform for accelerating the understanding of disease pathogenesis, improving the design of personalized therapies, and revealing psoriatic disease targets.

Expanded Antigen-Specific Elimination Assay to Measure Human CD8+ T Cell Cytolytic Potential.

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.

GABA-mediated inhibition of human CD4+ T cell functions is enhanced by insulin but impaired by high glucose levels

γ-aminobutyric acid (GABA), known as the main inhibitory neurotransmitter in the brain, exerts immunomodulatory functions by interaction with immune cells, including T cells. Metabolic programs of T cells are closely linked to their effector functions including proliferation, differentiation, and cytokine production. The physiological molecules glucose and insulin may provide environmental cues and guidance, but whether they coordinateto regulate GABA-mediated T cell immunomodulation is still being examined. CD4+ T cells that were isolated from blood samples from healthy individuals and from patients with type 1 diabetes (T1D) were activated invitro. We carried out metabolic assays, multiple proximity extension assay (PEA), ELISA, qPCR, immunoblotting, immunofluorescence staining, flow cytometry analysis, MS-based proteomics, as well as electrophysiology and live-cell Ca2+ imaging. We demonstrate that GABA-mediated reduction of metabolic activity and the release of inflammatory proteins, including IFNγ and IL-10, were abolished in human CD4+ T cells from healthy individuals and patients with T1D when the glucose concentration was elevated above levels typically observed in healthy people. Insulin increased GABAA receptor-subunit ρ2 expression, enhanced the GABAA receptors-mediated currents and Ca2+ influx. GABA decreased, whereas insulin sustained, hexokinase activity and glycolysis in a glucose concentration-dependent manner. These findings support that metabolic factors, such as glucose and insulin, influence the GABA-mediated immunomodulation of human primary T cells effector functions. The Swedish Children's Diabetes Foundation, The Swedish Diabetes Foundation, The Swedish Research Council 2018-02952, EXODIAB, The Ernfors Foundation, The Thurings Foundation and the Science for Life Laboratory.

IRF4 impedes human CD8 T cell function and promotes cell proliferation and PD-1 expression

Human CD8 tumor-infiltrating lymphocytes (TILs) with impaired effector functions and PD-1 expression are categorized as exhausted. However, the exhaustion-like features reported in TILs might stem from their activation rather than the consequence of Tcell exhaustion itself. Using CRISPR-Cas9 and lentiviral overexpression in CD8 Tcells from non-cancerous donors, we show that the Tcell receptor (TCR)-induced transcription factor interferon regulatory factor 4 (IRF4) promotes cell proliferation and PD-1 expression and hampers effector functions and expression of nuclear factor κB (NF-κB)-regulated genes. While CD8 TILs with impaired interferon γ (IFNγ) production exhibit activation markers IRF4 and CD137 and exhaustion markers thymocyte selection associated high mobility group box (TOX) and PD-1, activated Tcells in patients with COVID-19 do not demonstrate elevated levels of TOX and PD-1. These results confirm that IRF4+ TILs are exhausted rather than solely activated. Our study indicates, however, that PD-1 expression, low IFNγ production, and active cycling in TILs are all influenced by IRF4 upregulation after Tcell activation.

Targeting a disintegrin and metalloprotease (ADAM) 17-CD122 axis enhances CD8+ T cell effector differentiation and anti-tumor immunity

CD8+ T cell immune responses are regulated by multi-layer networks, while the post-translational regulation remains largely unknown. Transmembrane ectodomain shedding is an important post-translational process orchestrating receptor expression and signal transduction through proteolytic cleavage of membrane proteins. Here, by targeting the sheddase A Disintegrin and Metalloprotease (ADAM)17, we defined a post-translational regulatory mechanism mediated by the ectodomain shedding in CD8+ T cells. Transcriptomic and proteomic analysis revealed the involvement of post-translational regulation in CD8+ T cells. T cell-specific deletion of ADAM17 led to a dramatic increase in effector CD8+ T cell differentiation and enhanced cytolytic effects to eliminate pathogens and tumors. Mechanistically, ADAM17 regulated CD8+ T cells through cleavage of membrane CD122. ADAM17 inhibition led to elevated CD122 expression and enhanced response to IL-2 and IL-15 stimulation in both mouse and human CD8+ T cells. Intriguingly, inhibition of ADAM17 in CD8+ T cells improved the efficacy of chimeric antigen receptor (CAR) T cells in solid tumors. Our findings reveal a critical post-translational regulation in CD8+ T cells, providing a potential therapeutic strategy of targeting ADAM17 for effective anti-tumor immunity.

Spontaneous human CD8 T cell and autoimmune encephalomyelitis-induced CD4/CD8 T cell lesions in the brain and spinal cord of HLA-DRB1*15-positive multiple sclerosis humanized immune system mice.

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein-Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.

Spontaneous human CD8 T cell and autoimmune encephalomyelitis-induced CD4/CD8 T cell lesions in the brain and spinal cord of HLA-DRB1*15-positive multiple sclerosis humanized immune system mice

Autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are only partially represented in current experimental models and the development of humanized immune mice is crucial for better understanding of immunopathogenesis and testing of therapeutics. We describe a humanized mouse model with several key features of MS. Severely immunodeficient B2m-NOG mice were transplanted with peripheral blood mononuclear cells (PBMCs) from HLA-DRB1-typed MS and healthy (HI) donors and showed rapid engraftment by human T and B lymphocytes. Mice receiving cells from MS patients with recent/ongoing Epstein–Barr virus reactivation showed high B cell engraftment capacity. Both HLA-DRB1*15 (DR15) MS and DR15 HI mice, not HLA-DRB1*13 MS mice, developed human T cell infiltration of CNS borders and parenchyma. DR15 MS mice uniquely developed inflammatory lesions in brain and spinal cord gray matter, with spontaneous, hCD8 T cell lesions, and mixed hCD8/hCD4 T cell lesions in EAE immunized mice, with variation in localization and severity between different patient donors. Main limitations of this model for further development are poor monocyte engraftment and lack of demyelination, lymph node organization, and IgG responses. These results show that PBMC humanized mice represent promising research tools for investigating MS immunopathology in a patient-specific approach.

CBP/P300 Inhibition Impairs CD4+ T Cell Activation: Implications for Autoimmune Disorders.

T cell activation is critical for an effective immune response against pathogens. However, dysregulation contributes to the pathogenesis of autoimmune diseases, including Juvenile Idiopathic Arthritis (JIA). The molecular mechanisms underlying T cell activation are still incompletely understood. T cell activation promotes the acetylation of histone 3 at Lysine 27 (H3K27ac) at enhancer and promoter regions of proinflammatory cytokines, thereby increasing the expression of these genes which is essential for T cell function. Co-activators E1A binding protein P300 (P300) and CREB binding protein (CBP), collectively known as P300/CBP, are essential to facilitate H3K27 acetylation. Presently, the role of P300/CBP in human CD4+ T cells activation remains incompletely understood. To assess the function of P300/CBP in T cell activation and autoimmune disease, we utilized iCBP112, a selective inhibitor of P300/CBP, in T cells obtained from healthy controls and JIA patients. Treatment with iCBP112 suppressed T cell activation and cytokine signaling pathways, leading to reduced expression of many proinflammatory cytokines, including IL-2, IFN-γ, IL-4, and IL-17A. Moreover, P300/CBP inhibition in T cells derived from the inflamed synovium of JIA patients resulted in decreased expression of similar pathways and preferentially suppressed the expression of disease-associated genes. This study underscores the regulatory role of P300/CBP in regulating gene expression during T cell activation while offering potential insights into the pathogenesis of autoimmune diseases. Our findings indicate that P300/CBP inhibition could potentially be leveraged for the treatment of autoimmune diseases such as JIA in the future.

Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages.

Adeno-associated virus (AAV) serotypes from primates are being developed and clinically used as vectors for human gene therapy. However, the evolutionary mechanism of AAV variants is far from being understood, except that genetic recombination plays an important role. Furthermore, little is known about the interaction between AAV and its natural hosts, human and nonhuman primates. In this study, natural AAV capsid genes were subjected to systemic evolutionary analysis with a focus on selection drives during the diversification of AAV lineages. A number of positively selected sites were identified from these AAV lineages with functional relevance implied by their localization on the AAV structures. The selection drives of the two AAV2 capsid sites were further investigated in a series of biological experiments. These observations did not support the evolution of the site 410 of the AAV2 capsid driven by selection pressure from the human CD4+ T-cell response. However, positive selection on site 548 of the AAV2 capsid was directly related to host humoral immunity because of the profound effects of mutations at this site on the immune evasion of AAV variants from human neutralizing antibodies at both the individual and population levels. Overall, this work provides a novel interpretation of the genetic diversity and evolution of AAV lineages in their natural hosts, which may contribute to their further engineering and application in human gene therapy.

Polyomavirus BK-Specific CD4+ T Cells Response to VP1 Stimulation in Kidney Transplant Recipients.

Reactivation of Polyomavirus BK (BKPyV) is related to reduction of T cells response in kidney transplant recipients (KTRs). Here, we examined the differentiation of CD4+ T cells subsets in response to BKPyV KTRs, using the BKPyV VP1 (viral capsid protein 1) as a stimulator. We categorized our samples into three distinct groups: 1. Reactive BKPyV (BKPyV+), 2. non-reactive (BKPyV-) KTRs and 3. Healthy controls. BKPyV- KTRs and healthy controls stimulated with VP1 and BKPyV+ unstimulated with VP1. The human CD4+ T cells was stimulation with VP1-Ag. The proportion of CD4+ T lymphocytes and their various subsets, including naive T cells, central memory T cells (TCM), and effector memory T cells (TEM) was measured using flowcytometry. BKPyV- KTRs VP1+ indicated significantly lower TCM CD4+ T cells in contrast with both BKPyV+ KTRs VP1-, and healthy controls VP1+. This indicates that VP1 stimulation may reduce TCM cell levels in these patients. The percentage of TEM in the BKPyV- KTRs VP1+ group was significantly less prevalent than the BKPyV+ KTRs VP1- group. The percentage of TEM cells in BKPyV+ KTRs VP1- was significantly lower than the healthy controls VP1+. Stimulation with VP1 protein significantly increased the frequency of cytotoxic CD4+ T cells in BKPyV- KTRs VP1+ compared to BKPyV+ KTRs VP1-. The present research has shown that the VP1 stimulation of CD4+ T cells can induce cytotoxic CD4+ T cells responses that may help overcome BKPyV infection in KTRs. However, VP1 stimulation may also differentially affect TCM and TEM CD4+ T cells subsets.

In vitro IL-15-activated human naïve CD8+ T cells down-modulate the CD8β chain and become CD8αα T cells.

Antigen-driven human effector-memory CD8+ T cells expressing low levels of the CD8β chain have been previously described. However, little is known on a possible antigen-independent trigger. We have examined the impact that IL-15 has on the expression of CD8β on purified human naïve CD8+ T cells after CFSE labeling and culture with IL-15. As expected, IL-15 induced naïve CD8+ T cells to proliferate and differentiate. Remarkably, the process was associated with a cell-cycle dependent down-modulation of CD8β from the cell surface, leading to the generation of CD8αβlow and CD8αβ- (i.e., CD8αα) T cells. In contrast, expression of the CD8α chain remained steady or even increased. Neither IL-2 nor IL-7 reproduced the effect of IL-15. Determination of mRNA levels for CD8α and CD8β isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8β M-4 isoform, while levels of the M-1/M-2 isoforms and of CD8α increased. Noteworthy, CD8+ T cell blasts obtained after culture of CD8+ T cells with IL-15 showed a cell-cycle dependent increase in the level of the tyrosine kinase Lck, when compared to CD8+ T cells at day 0. This study has shown for the first time that IL-15 generates CD8αα+αβlow and CD8αα+αβ- T cells containing high levels of Lck, suggesting that they may be endowed with unique functional features.

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications

BackgroundMacrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ.Aim of the studyTo scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype.MethodsDifferentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling.ResultsMreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased.ConclusionExposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Impact of urolithin A supplementation, a mitophagy activator on mitochondrial health of immune cells (MitoIMMUNE): A randomized, double-blind, placebo-controlled trial in healthy adults.

e14562 Background: Mitochondrial dysfunction and stem cell exhaustion are hallmarks of aging, driving inflammation and limiting immune function. Likewise, cancer is characterized by repression of mitochondrial dynamics in tumor-infiltrating leukocytes that fosters terminal T cell exhaustion. Yet, interventions to reliably improve immune function are still lacking. Urolithin A (UA) is a postbiotic known to improve muscle function in aged adults upon oral intake. We have recently shown that UA induces mitophagy in human CD8+ cells to induce formation of T memory stem cells, while sensitizing murine cancer to immunotherapy. Methods: In this double-blind, prospective, placebo-controlled trial (NCT05735886) we sought to characterize for the first time the effects of UA on the human immune system. Fifty healthy participants aged 45-70 years were randomized (1:1) to an intervention of single-dose (1000mg) UA or placebo per day for a study duration of 28 days. Randomization was performed based on age, gender and BMI. Exclusion criteria were prior medical conditions, BMI &gt;35 kg/m2, smoking, and intake of prescription medication or other dietary supplements. The primary end points of the study were changes in peripheral CD3+ cells, as well as alterations in mitochondrial activity in select immune populations of the peripheral blood as assessed by flow cytometry. Results: Intake of UA was safe and well-tolerated in the intervention cohort of 25 participants. After the study period, total peripheral lymphocytes and circulating NK cells were expanded in the UA group. Performing a complete immunophenotyping via spectral flow cytometry, we found that UA elicits immune remodeling characterized by broad changes of immune surface markers and mitochondrial measurements. CD8+ cells of participants in the intervention arm displayed greater mitochondrial mass, while preferably taking on a naive phenotype and expressing more Ki67. In addition, circulating monocytes exhibited a less inflammatory signature. UA intake reduced plasma levels of several proinflammatory cytokines. Conclusions: Collectively, our study constitutes an unprecedented intervention-based assessment of immune aging that globally characterizes the effect of UA on the immune system, proposing validation in future confirmatory studies and potential combination with cancer immunotherapy. Clinical trial information: NCT05735886 .

Histone Lactylation Drives CD8 T Cell Metabolism and Function.

The activation and functional differentiation of CD8 T cells are linked to metabolic pathways that result in the production of lactate. Lactylation is a lactate-derived histone post-translational modification (hPTM); however, the relevance of histone lactylation in the context of CD8 T cell activation and function is not known. Here, we show the enrichment of H3K18-lactylation (H3K18la) and H3K9-lactylation (H3K9la) in human and murine CD8 T cells which act as transcription initiators of key genes regulating CD8 T cell phenotype and function. Further, we note distinct impacts of H3K18la and H3K9la on CD8 T cell subsets linked to their specific metabolic profiles. Importantly, we demonstrate that modulation of H3K18la and H3K9la by targeting metabolic and epigenetic pathways regulates CD8 T cell effector function including anti-tumor immunity in preclinical models. Overall, our study uncovers the unique contributions of H3K18la and H3K9la in modulating CD8 T cell phenotype and function intricately associated with metabolic state.

A novel B7-H3 x CD3 bispecific T-cell engager for the treatment of B7-H3+ tumors.

e14509 Background: B7-H3 (CD276) is a transmembrane protein highly expressed in solid tumors but with limited expression in normal tissues. Its presence is strongly associated with the inhibition of T-cell activity, cancer progression, and invasion. Furthermore, B7-H3 is strongly associated with poor prognosis and reduced patient survival. Given its crucial role in tumor biology, B7-H3 has emerged as an attractive therapeutic target for drug development. We utilized ExMab heterodimer platform and common light chain technology to develop EX105, a next-generation B7-H3×CD3 bispecific antibody. This innovative T-cell engager exhibits excellent anti-tumor activity in mouse models and good safety profile in NHP tox study. Methods: EX105 was generated using ExMab heterodimer platform and common light chain technology to form a heterodimeric IgG1 structure. Preclinical pharmacology studies, efficacy, and safety were evaluated. Anti-tumor activity study of EX105 includes in vitro T-cell cytotoxicity assays and in vivo PBMC reconstituted xenograft mice models. Preclinical safety evaluation of EX105 was performed through GLP single-dose and repeat-dose tox studies in non-human primates (NHPs). Results: Based on ELISA and FACS analyses, EX105 can bind to both human and cynomolgus monkey B7-H3 and CD3 antigens. Furthermore, SPR data suggest that EX105 exhibits a high affinity to B7-H3 and a much lower affinity to CD3. In consistent with that, EX105 only induced relatively low cytokine release. Nevertheless, EX105 showed a potent in vitro T cell cytotoxicity against B7-H3+ tumor cell lines. The in vivo anti-tumor activity of EX105 was further determined in several tumor xenograft models. Treatment with EX105 resulted in remarkable tumor regression in both “hot” tumor model and “cold” tumor model, indicating its effectiveness across a range of tumor types. Tox study in NHPs showed minimal adverse effects and no liver impairment after EX105 administration, even at high doses. The cytokine release data observed in this study suggests a good safety profile of EX105, with much lower cytokine release compared to other T cell engagers. Conclusions: These findings collectively indicate that EX105 has the potential to become a promising therapeutic agent for the treatment of various B7-H3 positive cancers. The first-in-human clinical trial for patients with advanced B7-H3 positive cancers is due to start.