Biology subjects

Bruns, H.

Publications and source records attributed to Bruns, H..

8 recordsLinked to original sources

bioRxiv2025

TCR activation impairs CAR-T cytotoxicity against separate target cells

Chimeric antigen receptor T cells (CAR-T) are effective therapeutics against cancer and autoimmunity, but whether the endogenous T-cell receptor (TCR) is beneficial, detrimental or irrelevant for CAR-T function and patient outcome remains unclear. We here traced anti-CD19 CAR-T clonotypes in patients with B-cell malignancies pre- and post-infusion using single-cell RNA-, TCR-, and CITE-sequencing. A cytotoxic phenotype, but not CAR-mediated in vitro reactivity to tumor cells, predicted CAR-T persistence. To test the functional impact of endogenous TCR activity on CAR-T behavior, we combined CAR transduction with orthotopic TCR replacement. This revealed that TCR signaling adds to activation of CAR-T cells, but gradually compromises CAR-mediated cytotoxicity, when TCR and CAR antigens are presented by different target cells. Therefore, spatial antigen separation alters TCR/CAR interplay with implications for therapeutic CAR-T design.

immunology↗

bioRxiv2025

Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation

Fate decisions of T helper (Th) cells are tightly linked to their metabolic states, but precise mechanistic links remain unknown, especially in humans. Using in vitro stimulation in combination with gene editing we studied how metabolic regulation shapes human Th1 cell identity and effector function. Differentiated Th1 cells displayed elevated STAT1 phosphorylation at Tyr701 and Ser727 as well as heightened T-bet and IFN{gamma} expression, which were dampened by CRISPR/Cas9-mediated STAT1 deletion. Metabolic profiling revealed enhanced glycolytic activity in Th1 in comparison to Act.T cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, glucose uptake, lactate secretion and NADH abundance. SCENITH analysis demonstrated elevated glycolysis-dependent anabolic activity of Th1 cells. Inhibition of glycolysis reduced IFN{gamma} production and STAT1 phosphorylation independent of JAK1/2 activity, STAT1 abundance or SHP-2 activity, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. Mechanistically, O-Glycosylation, facilitated by O-Glycosyltransferase, emerged as pivotal in modulating STAT1 activity, as evident through immunoprecipitation and Western blot analysis. Pharmaceutical O-Glycosyltransferase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation sites Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFN{gamma} synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-Glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells in antiviral immunity and autoimmunity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/636644v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1ee58aeorg.highwire.dtl.DTLVardef@1001af6org.highwire.dtl.DTLVardef@1ea2ed7org.highwire.dtl.DTLVardef@1a943fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Ubiquitin-specific protease 8 controls B cell proteostasis and cell survival in multiple myeloma

Ubiquitin-specific protease 8 (USP8) is a multifunctional deubiquitinating enzyme that plays a pivotal role in the regulation of endosomal and lysosomal trafficking. Several studies showed that USP8 is critically involved in the pathogenesis of various tumor entities. Recently, USP8 emerged as a vulnerability gene in multiple myeloma (MM), suggesting a functional role in the B- and plasma cell compartment. Here we have comprehensively analyzed mice with deletion of Usp8 at different stages of B-cell development. Furthermore, we evaluated the function of USP8 in proteasome inhibition susceptible and Bortezomib (BTZ) resistant patient derived MM cells using depletion of USP8 and treatment with DUB-IN-2, a widely used inhibitor published to target USP8. Usp8 depletion in Usp8f/fCd19-Cre mice affected B-cell survival and development favoring immature, innate-like B cells, and germinal center and plasma cells, while also elevating immune-responses and causing Roquin depletion. Cells expressing catalytically inactive USP8 accumulated proteins modified with mixed ubiquitin/NEDD8 chains, indicating proteotoxic stress. Moreover, we identified these modifications as preferred substrates of USP8. In MM cells, efficient USP8 knockdown reduced survival by inducing lysosomal dysfunction. In contrast, DUB-IN-2 induced an enhanced ER stress response to treatment with BTZ. Our results highlight the potential of targeting USP8 and the combination of DUB-IN-2 and BTZ in treating BTZ-resistant MM. However, our biochemical and cellular analyses raise fundamental concerns about the function of DUB-IN-2 as a USP8 inhibitor.

molecular biology↗

bioRxiv2024

The integration of network biology and pharmacophore modeling suggests repurposing Clindamycin as an inhibitor of pyroptosis via Caspase-1 blockage in tumor-associated macrophages

BackgroundUveal melanoma (UM) is a highly malignant intraocular tumor with a poor prognosis and response to therapy, including immune checkpoint inhibitors (ICIs), after the onset of liver metastasis. The metastatic microenvironment contains high levels of tumor-associated macrophages (TAMs) that correlate positively with a worse patient prognosis. We hypothesized that one could increase the efficacy of ICIs in UM metastases by immunomodulating UM-associated macrophages. MethodsTo identify potential targets for the immunomodulation, we created a network-based representation of the biology of TAMs and employed (bulk and single-cell) differential gene expression analysis to obtain a regulatory core of UM macrophages-associated genes. We utilized selected targets for pharmacophore-based virtual screening against a library of FDA-approved chemical compounds, followed by refined flexible docking analysis. Finally, we ranked the interactions and selected one novel drug-target combination for in vitro validation. ResultsBased on the generated TAM-specific interaction network (3863 nodes, 9073 edges), we derived a UM macrophages-associated regulatory core (74 nodes, 286 edges). From the regulatory core genes, we selected eight potential targets for pharmacophore-based virtual screening (YBX1, GSTP1, NLRP3, ISG15, MYC, PTGS2, NFKB1, CASP1). Of 266 drug-target interactions screened, we identified the interaction between the antibiotic Clindamycin and Caspase-1 as a priority for experimental validation. Our in vitro validation experiments showed that Clindamycin specifically interferes with activated Caspase-1 and inhibits the secretion of IL-1{beta}, IL-18, and lactate dehydrogenase (LDH) in macrophages after stimulation. Our results suggest that repurposed Clindamycin could reduce pyroptosis in TAMs, a pro-inflammatory form of programmed immune cell death favouring tumor progression. ConclusionWe were able to predict a novel Clindamycin-Caspase-1 interaction that effectively blocks Caspase-1-mediated inflammasome activity and pyroptosis in vitro in macrophages. This interaction is a promising clinical immunomodulator of the tumor microenvironment for improving ICI responsivenss. This work demonstrates the power of combining network-based transcriptomic analysis with pharmacophore-guided screening for de novo drug-target repurposing. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/576201v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@310a9aorg.highwire.dtl.DTLVardef@1af0dcaorg.highwire.dtl.DTLVardef@1b272aborg.highwire.dtl.DTLVardef@86572b_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

Increased pulmonary monocyte infiltration and attenuated phagocytosis defines perinatal dysfunction of innate immunity in Cystic Fibrosis

In Cystic Fibrosis (CF) patients, cycles of infection and inflammation lead to fatal lung damage. While diminished mucus clearance is restored by highly effective CFTR modulator therapy, inflammation and infection persist in treated patients, suggesting alternative mechanisms may contribute to impaired immunity in CF. Here, we made use of a CF pig model to investigate the innate immune system at birth, before the onset of infection. We observed a substantial change in the composition of tissue resident immune cells towards emergency myelopoiesis, accompanied by increased infiltration of monocytes into CF lungs. A more immature status in the transcriptome profile of classical monocytes in CF pigs and preschool children with CF correlated with reduced phagocytic capacity, confirming a congenital and translationally conserved aberration of the immune system in CF. The lack of CFTR expression in circulating monocytes indicates an indirect etiology of these effects and suggests that additional immunological treatments are necessary for CF patients. One Sentence SummaryIncreased infiltration of lung tissue by monomyeloid cells and their impaired phagocytic potential cause dysfunctional imprinting of mucosal immunity in Cystic Fibrosis airways at birth and suggest early and specific treatment of the innate immune system in patients.

immunology↗

bioRxiv2023

An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

The bone marrow permanently harbors high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. We investigated myeloma-associated alterations in marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor-supportive, transcribing increased levels of IL-1{beta}, and myeloma cell survival factor BAFF. Interactions with inflammatory stromal cells can induce neutrophil activation, including BAFF secretion, in a STAT3-dependent manner and once activated, neutrophils gain the ability to reciprocally induce stromal activation. After first-line myeloid-depleting treatment, patient bone marrow retains residual stromal inflammation and newly-formed neutrophils are reactivated. Combined, we identify a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that persists after treatment and warrants novel strategies to target both stromal and immune microenvironments in multiple myeloma.

immunology↗

bioRxiv2021

Reconstitution of T cell immunity against EBV in the immunocompromised host by adoptive transfer of peptide-stimulated T cells after allogeneic stem cell transplantation

Reconstitution of T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by peptide stimulation and adoptively transferred them to a patient with angioimmunoblastic T-cell lymphoma (AITL), who had developed persisting high titers of EBV concomitant to relapse after transplantation. T cell receptor beta (TCR{beta}) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse. Author summaryA characteristic feature of all herpesviruses is their persistence in the hosts body after primary infection. Hence, the hosts immune system is confronted with the problem to control these viruses life-long. Well-known representative of the herpesvirus group are the classic Herpes-Simplex Virus (HSV-1) and Varicella Zoster Virus (VZV, causing chicken pox); a less known representative is Epstein Barr Virus (EBV, causing mononucleosis). When the immune system is severely compromised, for example after stem cell transplantation from a foreign (allogeneic) donor, these viruses can reappear, as they are already in the hosts body. Especially EBV cause life-threatening complications after stem cell transplantation and only reinforcement of the hosts immune system can reestablish viral control. Here we show that ex vivo manufactured EBV-specific T cells can reestablish long-term control of EBV and that these cells persist in the hosts body over months. These results give us a better understanding of viral immune reconstitution post-transplant and of clinically-relevant T cell populations against EBV.

immunology↗

bioRxiv2020

Loss of TP53 mediates suppression of Macrophage Effector Function via Extracellular Vesicles and PDL1 towards Resistance against Chemoimmunotherapy in B-cell malignancies

Chemoimmunotherapy (CIT) is the standard of care in B-cell malignancies. It is relying on synergistic effects of alkylating chemotherapy and monoclonal antibodies via secretory crosstalk with effector macrophages. Here, we observed that loss of p53 function mediates resistance to CIT by suppressing macrophage phagocytic function. Loss of p53 leads to an upregulation of PDL1 and an increased formation of extracellular vesicles (EVs). EVs directly inhibit macrophage phagocytosis by PDL1 surface expression. Suppression of phagocytic function by lymphoma cell-derived EVs could be abrogated by pre-incubation of EVs with anti-PDL1 antibodies, CRISPR-KO of PDL1 and abrogation of EV formation by RAB27A-KO in lymphoma cells. Immune checkpoint inhibition represents a viable strategy to overcome EV-mediated resistance to chemoimmunotherapy in lymphoma. SignificanceLoss of TP53 mediates cell autonomous resistance to genotoxic chemotherapy, moreover non-cell autonomous effects may cause therapy resistance mediated by the tumor microenvironment. We identify a TP53-dependent mechanism that mediates resistance to synergistic chemoimmunotherapy by increasing formation of EVs and expression of the PDL1 immune checkpoint. PDL1 on EVs is directly responsible for macrophage suppression, preventing the exertion of the essential effector function of antibody-dependent cellular phagocytosis. This novel mechanism of resistance is in turn targetable by PDL1 checkpoint inhibition. Enhanced EV-release and immune checkpoint expression in lymphoma are novel mechanisms of macrophage modulation in the lymphoma microenvironment. We provide a novel principle of resistance to chemoimmunotherapy (CIT) representing of immediate relevance to treatment of refractory B-cell lymphoma. HighlightsO_LILoss of TP53 in B-cell lymphoma induces resistance towards chemoimmunotherapy (CIT) by inhibition of macrophage effector function through PDL1 upregulation C_LIO_LILoss of TP53 increases formation of extracellular vesicles (EVs) carrying PDL1 C_LIO_LIEVs inhibit antibody-mediated cellular phagocytosis (ADCP), a key macrophage effector function in CIT C_LIO_LITargeting PDL1 on EVs with immune checkpoint inhibitors overcomes TP53-mediated resistance to CIT C_LI

cell biology↗