Transsynaptic nanocolumns are nanoscale alignments of pre- and postsynaptic proteins that ensure efficient synaptic transmission. Autoantibodies against the transsynaptic protein LGI1, implicated in LGI1 autoimmune encephalitis, are known to disrupt synaptic function, but their impact on nanocolumn architecture remains unclear. To investigate this, we employed a multi-modal super-resolution imaging strategy combining post-gelation immunolabeling, expansion microscopy with Airyscan super-resolution imaging and compared the results with another advanced super-resolution microscopy technique – d STORM. By physically expanding hippocampal neuron cultures 10.3-fold, our approach enabled decrowding of dense synaptic regions and improved epitope accessibility as well as labeling efficiency. Post-expansion immunolabeling followed by multicolor Airyscan imaging achieved 20–30 nm resolution, allowing detailed visualization of transsynaptic nanocolumns. With this approach we observed LGI1 autoantibody-induced sharpening of Munc13-1 – GluA1 alignment and a shift in AMPA receptor positioning. These findings highlight how advanced expansion-based imaging enables quantitative analysis of nanoscale synaptic alterations in disease contexts.

Super-resolution microscopy in combination with genetic labeling methods allows imaging of single proteins in cells. However, visualizing endogenous proteins on primary cells remains challenging due to the use of sterically demanding antibodies for labeling. Here, we demonstrate how immunolabeling conditions and antibody cross-linking influence the quantification and identification of membrane receptor stoichiometry on cells using single-molecule localization microscopy. We developed an optimized immunolabeling and analysis protocol and demonstrate the performance of the approach by resolving the molecular organization of endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the usefulness of the method for immunotherapy applications, we investigated the interaction of primary multiple myeloma cells with the therapeutic monoclonal antibodies daratumumab and isatuximab and a polyclonal anti-CD38 antibody. Our approach might lay the foundation for improved personalized diagnostics and treatment with therapeutic antibodies.

This chapter presents a step-by-step protocol to resolve the organization of the nuclear pore complex (NPC) in HeLa-NUP107-GFP cells by eight-fold expansion microscopy (ExM). To enhance the efficiency of the procedure, we introduce an accelerated expansion protocol designed to allow rapid iteration and improve experimental throughput. This protocol involves both pre-expansion staining with wheat germ agglutinin (WGA) and post-digestion staining with nanobodies against GFP to visualize the NPC. The subsequent use of confocal microscopy enables multicolor super-resolution imaging with approximately 30 nm spatial resolution.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) has become a widely adopted single-molecule localization microscopy (SMLM) technique owing to its high spatial resolution, versatile labeling strategies, and theoretically unlimited multiplexing capability. Recent developments in repetitive docking strand designs have enabled faster image acquisition by increasing the number of potential binding motifs per target. However, the effect of such architectural modifications on effective spatial resolution remains largely unexplored. Here, we systematically quantify how repetitive docking strands influence localization distributions and effective resolution using the well-defined geometry of the trimeric proliferating cell nuclear antigen (PCNA) as a model system. Whereas classical single-motif docking strands resolve the expected ∼6 nm spacing between PCNA subunits with high precision, repetitive docking motifs produce broadened localization distributions, despite comparable localization precision. Our results suggest that spatial blurring arises from a combination of variable binding site geometry, rotational flexibility of elongated multivalent DNA docking sequences, as well as the dynamic behavior of imager strands. This study provides a quantitative framework for understanding how docking strand architecture determines resolution limits in DNA-PAINT and underscores the need to balance multiplexing and imaging speed with structural fidelity. Our results thus offer guidance for the rational design of docking strands for high-precision DNA-PAINT imaging of protein complexes.

ABSTRACT Understanding the complex interplay between therapeutic monoclonal antibodies (mAbs) and target receptors within the plasma membrane is essential for improving immunotherapy efficacy. However, direct visualization of lipid nanodomains remains challenging due to their nanoscale dimensions and dynamic behavior. Here, we combine expansion microscopy (ExM) with a fluorescent perfringolysin O domain‐4 probe to map cholesterol‐rich nanodomains in the membrane of whole intact cells with a spatial resolution approaching ∼40 nm on a confocal setup. We demonstrate that cholesterol‐rich domains predominantly localize to actin‐supported membrane protrusions in COS‐7 cells. We directly visualize how the binding of therapeutic mAbs rituximab (RTX) and daratumumab (DARA) redistributes CD20 and CD38 on lymphoma and multiple myeloma cells, respectively, into cholesterol‐rich nanodomains. Accumulation of receptor/mAb complexes within lipid nanodomains creates Fc fragment‐dense regions that enhance signal transduction, apoptotic signaling, and complement‐dependent cytotoxicity. Furthermore, RTX binding induces pronounced B‐cell polarization and accumulation of CD20/RTX complexes in cholesterol‐rich membrane nanodomains, whereas CD38/DARA complexes exhibit a more homogeneous membrane distribution indicating that mAb binding‐induced receptor reorganization occurs in a cell‐type‐ and receptor‐dependent manner at the nanoscale. Our results show how ExM can be used advantageously to improve our understanding of the complex interplay of mAbs and lipid nanodomains.

Abstract Structural characterization of macromolecular assemblies within intact cells remains one of the central challenges in cell biology. While cryo-electron tomography provides unparalleled structural information, its applicability is limited by sample thickness, imaging throughput, and accessibility. Fluorescence microscopy offers molecular specificity and compatibility with intact biological specimens but has so far lacked the spatial resolution required to visualize cellular ultrastructure. Here we introduce Mega-expansion microscopy (Mega-ExM), a fluorescence imaging approach that enables structural visualization of whole cells using conventional confocal microscopes. Mega-ExM combines iterative hydrogel expansion with whole-proteome NHS-dye labeling and post-expansion immunostaining to achieve tunable expansion factors of up to ∼1,500-fold while preserving ultrastructure. At expansion factors of 40-260x, Mega-ExM resolves centrioles, mitochondrial cristae, protein-dense domains within mitochondrial cristae consistent with respiratory-chain supercomplexes, the synaptonemal complex, and nuclear pore complexes (NPCs) with high fidelity. Particle averaging of ∼200x expanded NPCs yields reconstructions with a structural resolution of ∼35 Å, approaching what cryo-electron tomography has achieved for selected protein assemblies. By combining molecular specificity, large imaging volumes, and nanoscale structural resolution on conventional fluorescence microscopes, Mega-ExM establishes a broadly accessible platform for in situ structural biology.

Immunotherapies targeting surface antigens have transformed the treatment landscape of multiple myeloma (MM), with GPRC5D emerging as a promising therapeutic target. Monoallelic loss of GPRC5D is frequently observed in newly diagnosed MM patients, and the incidence of acquired GPRC5D alterations increases following exposure to GPRC5D-directed therapies. However, the functional consequences of both baseline monoallelic and therapy-induced biallelic GPRC5D alterations remain poorly understood. In this study, we modeled monoallelic versus biallelic loss of GPRC5D to investigate their impact on MM cell biology and responsiveness to GPRC5D-targeted immunotherapies. Our results demonstrate that monoallelic GPRC5D loss in OPM-2 cells reduces surface expression of the antigen and confers resistance to GPRC5D-directed therapies. Complete loss of GPRC5D alters the transcriptional state of MM cells and promotes reprogramming of the phosphoproteomic circuitry ultimately resulting in a pro-proliferative chemokine environment. As a result, GPRC5D deficiency increases the basal proliferation rate of MM cells thereby providing a competitive advantage which may further be amplified by selecting these aggressive phenotypes during ongoing treatment with anti-GPRC5D immunotherapies.

Super-resolution fluorescence microscopy enables the visualization of protein structures at nanometer resolution, providing insights into receptor organization on the plasma membrane that are essential for the development and optimization of immunotherapies. In this context, monoclonal antibodies are employed, which typically bind only a subset of available membrane receptors, due to steric hindrance or otherwise limited epitope accessibility, to quantify the accessible targets. These accessible targets, rather than the total receptor density, are critical for determining therapeutic efficacy. Here, we present a simplified, robust protocol to quantify antibody-accessible endogenous receptors using monoclonal antibodies directly labeled with fluorescent dyes in combination with total internal reflection fluorescence (TIRF) direct stochastic optical reconstruction microscopy (dSTORM). The method employs optimized labeling and fixation conditions to preserve the native receptor distribution, enabling precise quantification of accessible receptors and their stoichiometry at single-molecule resolution. Omitting secondary antibodies and minimizing fixation-induced artifacts prevents artificial clustering and maintains the physiological binding pattern of therapeutic antibodies. The standardized workflow delivers therapy-relevant information about receptor accessibility and organization underlying therapeutic antibody binding, thereby advancing the mechanistic understanding of immunotherapy resistance and personalized treatment strategies across diverse membrane protein targets. Key features • Influence of fixation conditions on receptor epitope accessibility. • Use of therapeutic antibodies for quantitative estimation of receptor availability relevant to immunotherapy. • Fluorophore localizations provide information on antibody binding events below the optical resolution limit.

Efficient protein labeling with minimal linkage error is a key requirement for super-resolution fluorescence microscopy. Many commonly used labeling strategies, including antibodies, fluorescent proteins, and self-labeling enzymes are limited by steric hindrance, and therefore they limit labeling density or constrain labeling to protein termini, thereby restricting achievable resolution at the molecular scale. Here, we describe a practical and broadly applicable protocol for site-specific protein labeling based on genetic code expansion and bioorthogonal inverse electron-demand Diels-Alder click chemistry. The method relies on the incorporation of a strained alkene-modified noncanonical amino acid at a defined position within a protein of interest, followed by rapid and selective covalent labeling with tetrazine-conjugated organic fluorophores. This approach enables the attachment of small, bright dyes with minimal linkage error and is compatible with both live-cell and fixed-cell imaging. The protocol provides detailed guidance on the design of suitable click sites, expression of amber mutants in mammalian cells, selection of appropriate tetrazine dyes, and optimization of labeling conditions for super-resolution microscopy, including single-molecule localization microscopy. Critical parameters, common pitfalls, and limitations are discussed to facilitate robust implementation across different protein classes and experimental systems. This workflow supports high-density, stoichiometric labeling and enables molecular-scale imaging of proteins in their native cellular context.

Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable ~15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration–dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens.

Introduction CD20-targeting monoclonal antibodies (mAbs) are in clinical use and standard of care in the treatment of hematological malignancies and autoimmune diseases, including rituximab (RTX), obinutuzumab (OBZ) or ofatumumab (OFA). These agents differ in their structure, epitope specificity and modes of action (MoA), yet the precise molecular basis for their divergent clinical profiles remains incompletely understood. Historically, MoAs have been inferred using biochemical complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays, flow cytometry for B cell depletion kinetics and confocal microscopy to determine cell viability and apoptosis. However, these approaches lack the combined spatial and temporal resolution needed to resolve real-time dynamics at the single cell or molecular level. Here, lattice light-sheet (LLS) microscopy can be used advantageously to visualize in real-time interactions of tumor cells with mAbs reaching high spatiotemporal resolution giving novel insights into binding kinetics, receptor cluster formation and complement deposition. Such dynamic high-resolution insights into antibody-antigen-engagement and downstream immune synapse formation are not achievable with conventional flow cytometry or confocal microscopy, offering a powerful new platform to elucidate and potentially optimize the molecular mechanisms that underlie CD20-directed therapies. Methods To acquire an interaction profile of cells with components of the immune system, fluorescently labeled mAbs RTX (5µg), OFA (10µg), OBZ (20µg) and 2H7 (10µg, serving as control mAb) were individually applied to Raji cells and primary B cells isolated from anonymized CLL patient samples. Cells were co-incubated with 40µg of the prelabeled complement component 1q (C1q) for 1 hour at 37 °C in RPMI medium, while imaging with an LLS microscope. In a second set of experiments, SiR actin (1:1000) was utilized to visualize mAb-binding induced cytoskeletal changes of B cells. Colocalization of C1q with the respective mAbs and accumulation on the surface of tumor cells was determined over time. To investigate cytoskeletal remodeling, the lengths of membrane protrusions and the degree of actin polarization were measured dynamically during the imaging period. Results Raji cells co-incubated with OFA and C1q showed a significantly reduced total amount of complex binding compared to RTX and 2H7. The C1q binding to OBZ was also significantly reduced but remained higher than observed with OFA. In actin-stained cells, the total amount of bound complex was reduced for all mAbs, but still highest for RTX. During image acquisition OFA-treated cells maintained stable protrusion lengths, whereas for RTX-treated Raji cells the lengths reduced significantly. This reduction was accompanied by a higher polarization degree observable for all mAbs except OFA. Notably, in our first experiments the total amount of bound complex for each tested mAb showed heterogenic distribution across individual cells and patient samples: In a therapy-naïve CLL patient OFA induced the highest C1q binding events, while OBZ showed the lowest. In contrast, a second therapy-naïve CLL patient displayed the opposite pattern with OBZ inducing strongest C1q binding. In a third patient, who was in first relapse 10 years after RTX-bendamustine therapy, the highest binding was observed for 2H7 whereas OBZ again showed the lowest activity. Until now, clear cytoskeletal changes such as protrusions and polarization have been less pronounced in primary CLL samples compared to Raji cells. For each condition 20-30 cells were imaged and analyzed. Conclusion LLS microscopy enables real-time visualization of live-cell interactions between patient derived B cells, therapeutic mAbs and the C1q protein, offering novel insights into inter-individual variability in treatment response. The observed differences in C1q binding and cytoskeletal remodeling suggest that these responses are patient specific and may be influenced by underlying factors such as disease genetics or prior therapies. LLS microscopy holds significant potential to support personalization of immunotherapies in hematologic malignancies by identifying the most effective mAb for each individual patient. When integrated with genetic and molecular profiling, LLS microscopy will further contribute to a broader understanding of underlying mechanisms of organism-treatment interactions.

The treatment landscape in Multiple Myeloma (MM) shifts towards immunotherapies with the G protein-coupled receptor class C group 5 member D (GPRC5D) as a promising antigen for T cell engagers (TCE) and investigational CART constructs. Monoallelic genetic alterations in GPRC5D are present in up to 15% of MM patients prior GPRC5D directed therapy. The incidence of acquired biallelic GPRC5D events following treatment has to be determined. Recently, biallelic loss of GPRC5D was identified as the underlying mechanism for acquired resistance toward talquetamab, the first approved GPRC5D directed TCE. In this study, we modeled GPRC5D genetic alterations and studied their impact on the biology of the disease and responsiveness to anti-MM immunotherapies. Mono- and bi-allelic GPRC5D knock-out (KO) cell models were created using the MM cell line OPM-2 via CRISPR-Cas9 technology. Surface receptor distribution and epitope quantification of common immunotherapeutic targets was performed via Direct stochastic optical reconstruction microscopy (dSTORM). MM cells were engineered to perform cytotoxicity, functional and clonal competition assays using immunotherapeutic agents in presence of effector T cells from healthy donors. dSTORM revealed a 2-fold reduction in GPRC5D surface expression on GPRC5DWt/Del (0, 16±0, 02 clusters/µm2) vs. WT cells (0, 32±0, 10 clusters/µm2). GPRC5DDel/Del had barely detectable GPRC5D expression with 0, 03±0, 07 clusters/µm2. CD38 expression was unexpectedly diminished in GPRC5D Wt/Del (5, 74±0.43 CD38 clusters/µm2) compared to WT parental cells (9, 59±0, 71 clusters/µm2, p=0.04), with an even greater reduction detected in GPRC5D Del/Del clones (2, 22±0.203 clusters/µm2, p<0, 0001). GPRC5D-altered cells with reduced CD38 expression depicted a significant resistance towards CD38 targeting antibodies (8.01% specific lysis in GPRC5DWt/Del cells, 6.38 % in GPRC5DDel/Del compared to 31.50% in WT cells, p <0.0001). GPRC5DWt/Delcells depicted a significant resistance towards Talquetamab. Additionally, in presence of talquetamab GPRC5DDel/Del models showed a proliferation gain (2 fold, p<0, 0001). High content chemokine profiling using the scioCyto microarray platform revealed significantly altered chemokine profiles, including growth and proliferation promoting cytokines. In addition, upregulation of MAPK and PI3K pathways involved in growth proliferation and downregulation of the RAP1 signaling pathway involved in cell adhesion was observed in GPRC5D altered cells via bulk RNA seq. To decipher potential mechanism of growth induction and a link to CD38, scRNA seq and phospho-proteomic based analysis are ongoing. Our work suggest a link between CD38 and GPRC5D in MM and supports an anti-proliferative function of GPRC5D suggesting treatment over progression with talquetamab may favor tumor growth in GPRC5D deficient cells. Umair Munawar, Seungbin Han, Elena Gerhard-Hartmann, Cornelia Vogt, Silvia Nerreter, Shilpa Kurian, Thomas Nerreter, Johanna Thurner, Julia Weingart, Patrick Eiring, Xiang Zhou, Nina Rein, Johanna Lehmann, Max Koeppel, Andreas Rosenwald, Ondrej Slaby, Michael Hudecek, Hermann Einsele, Leo Rasche, Markus Sauer, Johannes Waldschmidt, Bernhard Kuester, Klaus Martin Kortuem. GPRC5D alterations impact CD38 expression and provide proliferative advantage in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4602.

Visualizing the ultrastructure of mitotic spindles, the macromolecular machines that segregate chromosomes during mitosis, by fluorescence imaging remains challenging. Here we introduce an azido- and amino-functionalized docetaxel probe, which upon labeling of microtubules can be fixed, click-labeled and linked into hydrogels. The new probe is particularly useful for super-resolution imaging of dense microtubule structures in mitotic spindles. Multicolor expansion microscopy of mitotic cells allowed us to visualize the different phases of mitosis with unprecedented spatial resolution.

Introduction Immunotherapies have shown exceptional potential in achieving deep and durable remissions in Multiple Myeloma (MM). However, the complex interactions between immunoreceptors on MM cells and their surrounding microenvironment are not yet fully understood, presenting a major hurdle in optimizing these therapies. The advent of super-resolution imaging technologies, such as the single-molecule-sensitive methoddirect Stochastic Optical Reconstruction Microscopy (dSTORM) and lattice light-sheet microscopy (LLS), enable precise visualization of receptor organization, clustering, and interaction dynamics. Leveraging these tools, we aim to decode the spatial organization and functional dynamics of immunoreceptors in MM, using CD38 as a model receptor. Methods We applied dSTORM to map CD38 expression in Daratumumab (Dara) resistant (n=22) and Dara naïve (n=30) MM patients, detecting residual CD38 in CD38 negative patients (n=3) using the multi-epitope (ME) antibody CD38ME. As two-color dSTORM imaging and subsequent colocalization analysis revealed evidence of epitope shielding by therapeutic Dara, we tested whether Isatuximab (Isa) can overcome this effect. We compared the detectable receptors by Dara, Isa or a 1 to 1 mixture, and performed colocalization analysis in AMO1 cells. Additionally, LLS was employed to visualize the CD38 distribution in RPMI8226 cells for 1 hour following therapeutic antibody addition. Results Using dSTORM we found a significant reduction of detectable CD38 receptors in resistant versus naïve patients (21.7±3.8 vs. 32.1±2.4 clusters/µm2, p=0.004). We identified 3 patients as CD38-negative by flow cytometry (FC), but found residual CD38 expression using dSTORM. As the interval between sampling and last Dara treatment was 14 to 24 days, we hypothesized the cause could be epitope shielding by therapeutic Dara still bound to CD38 receptors. Two-color dSTORM images revealed barely any colocalization ranging from 0.6% to 1.3% between CD38ME and Dara. Much more colocalization (7.5% and 7.2%) was found in 2 patients who had last been treated 91 and 468 days before sampling. Together this data confirms epitope shielding by therapeutic Dara for at least 24 days post-treatment. To determine whether this effect can be overcome by treatment with Isa, AMO1 cells stained with Dara, Isa or a 1 to 1 mixture of both were imaged. We detected more CD38 receptors with Dara compared to Isa (44.0±4.7 vs 26.2±3.1 clusters/µm2, p=0.03), but their combination yielded no significant difference compared to Dara alone. Colocalization analysis of two-color dSTORM images revealed only 2% of colocalization between Dara and Isa, suggesting that Dara and Isa compete for binding to CD38. Applying LLS to investigate the CD38 distribution upon therapeutic antibody engagement, we found that CD38 polarization occurred only in 4% of Dara-treated cells within the first 10 minutes, whereas Isa induced polarization more slowly but also more efficiently, reaching 24% after about 25 minutes. We observed that the majority of CD38 clusters localized near the microtubule-organizing center, but no consistent spatial pattern could be identified. Similar results were observed when Dara and Isa were co-incubated. The mechanisms behind these observations remain unclear and need further study. Conclusion Our study demonstrates the value of dSTORM in overcoming detection limits. We identified residual CD38 expression in patients deemed CD38-negative by FC using CD38ME. Minimal colocalization between Dara and CD38ME was found up to 24 days post-treatment, in contrast with greater colocalization at later time points, suggesting epitope shielding by therapeutic Dara for up to 24 days post-treatment. Additionally, our findings show competitive binding to CD38 by Dara and Isa, hence Isa staining and/or treatment cannot overcome the issue of epitope shielding. By LLS we showed that Dara and Isa affect CD38 spatial distribution similarly, with both antibodies inducing CD38 polarization. However, Isa induces slower but more sustained CD38 polarization compared to Dara, highlighting potential differences in their mechanisms of action. These two imaging approaches combined can provide critical insights into the mechanisms of immune cell-mediated killing as well as mechanisms underlying therapeutic response and resistance, thereby improving diagnostics and enabling personalized treatment strategies for MM patients.

Background: Treatment paradigm in Multiple Myeloma (MM) has shifted towards immunotherapies such as monoclonal antibodies,T cell engagers (TCE) and CART cells targeting surface antigens. While G protein-coupled receptor class C group 5 member D (GPRC5D) has emerged as a promising antigen for TCE, newly acquired GPRC5D alterations have been reported to convey resistance to the anti-GPRC5D-CD3 TCE talquetamab. Since surface epitome expression in MM cells is highly impacted by secondary genetic events, we investigated the role of GPRC5D alterations on the surface expression of common immunotherapy targets (BCMA, CD38, CD138) in MM. Methods: CRISPR-Cas9 technology was utilized to create GPRC5D knock out (KO) cell models using the MM cell line OPM-2. Direct stochastic optical reconstruction microscopy (dSTORM) was used for high-resolution receptor quantification and distribution estimation. Gene expression profiling at transcriptomic and protein level was performed utilizing Real time PCR (RT-PCR), RNA sequencing and Western blotting. MM cells were engineered with Sleeping Beauty transposons system for stable expression of luciferase to perform cytotoxicity, functional and clonal competition assays using immunotherapeutic agents in presence of effector T cells from healthy donors. Results: Digital droplet PCR (ddPCR) and Sanger sequencing were used to verify the generation of GPRC5D mono- and bi-allelic KO cells. Ultra-high resolution surfaceome screening via dSTORM confirmed a significant reduction of surface GPRC5D expression in KO cell models (0.032±0.007 clusters/µm2 vs 0.323±0.102 clusters/µm2 in WT cells, p=0.002). Surprisingly, GPRC5D altered cells also showed a significant reduction in CD38 surface expression: GPRC5DWt/Del cells had 5.745±0.432 CD38 clusters/µm2 compared to WT parental cells with 9.594±0.717 clusters/µm2, (p=0.04). CD38 cluster density was further reduced in GPRC5DDel/Del clones (2.226±0.203 clusters/µm2, p<0,0001). Of note, no changes in other immunotherapeutic surface antigens (BCMA, SLAMF7, CD138) were observed, suggesting an exclusive impact of GPRC5D alterations on CD38 expression. Western blotting confirmed a reduction in CD38 expression at protein level. No changes at RNA levels were observed via bulk RNA sequencing and RT-PCR implying a post-transcriptional link between GPRC5D and CD38. To understand the functional impact of CD38 reduction in GPRC5D models, cells were treated with the CD38 targeting immunotherapeutic agents daratumumab (Dara) and isatuximab (Isa). GPRC5DWt/Del cells with reduced CD38 expression depicted a significant resistance towards CD38 targeting antibodies (8.01% specific lysis compared to 31.50% in WT cells, p <0.0001). This resistance was further enhanced in GPRC5DDel/Del clones (6.387 % specific lysis, p <0.0001), whereas no differences in sensitivity towards genotoxic drugs, proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) were seen. Exogenous overexpression of GPRC5D in WT and KO cells did not impact CD38 at transcript level but an increase in CD38 protein level was observed via Western blotting. Patients with GPRC5D alterations are being screened for daratumumab sensitivity, notably one patient from our institution with biallelic GPRC5D loss did not respond to subsequent Dara-PACE therapy. Summary: This study suggests that GPRC5D alterations are associated with downregulation of CD38 at post transcriptomic level. Co-localization and clonal competition experiments are ongoing along with clinical analysis of anti-GPRC5D treated patients.

Background: G protein-coupled receptor class C group 5 member D (GPRC5D) has recently emerged as a promising immunotherapy target in Multiple Myeloma (MM) although its function remains unknown. Mono-allelic GPRC5D alterations are found in 15% patients with newly diagnosed MM and these numbers are likely to increase in relapsed / refractory MM where most patients have been exposed to the anti-GPRC5D-CD3 bispecific antibody talquetamab. The functional impact of such baseline mono-allelic and acquired bi-allelic alterations of the GPRC5D gene remains to be elucidated. In this study, we modeled various GPRC5D genetic alterations and studied their impact on the biology of the disease and treatment responsiveness to anti-MM immunotherapies. Methods: Mono- and bi-allelic GPRC5D knock-out (KO) cell models were created using the MM cell line OPM-2 via CRISPR-Cas9 technology. GPRC5D allelic status in the cells was confirmed using digital droplet PCR (ddPCR) and Sanger sequencing. Direct stochastic optical reconstruction microscopy (dSTORM) was utilized for estimation of receptor distribution and epitope quantification of common immunotherapeutic targets such as GPRC5D, CD38, BCMA and SLAMF7. Functional assays were conducted using healthy effector T cells and treatment efficacy was tested for various immunotherapies and conventional anti-MMdrugs. Results: Gene expression profiling with RT-PCR confirmed that GPRC5DWt/Del cells had a 50% reduction in expression of GPRC5D mRNA (2 folds decrease in 2-ΔΔct) compared to WT cells. Whereas, GPRC5DDel/Del cells had 98% reduction in GPRC5D expression at mRNA level. These transcriptomic results were confirmed by ultra-high resolution microscopy (dSTORM). There was a 2 folds reduction in GPRC5D surface expression on GPRC5DWt/Del cells (0.1624±0.0222 clusters/µm2) compared to WT cells (0.3237±0.1024 clusters/µm2). GPRC5DDel/Del had barely detectable GPRC5D expression with 0.0322±0.007 clusters/µm2. These results were also confirmed at protein level with Western blots. When exposed to the anti-GPRC5D-CD3 bispecific antibody talquetamab, GPRC5DWt/Del cells depicted a significant resistance which was further enhanced in GPRC5DDel/Del cells. Surprisingly, in the presence of talquetamab GPRC5D deficient models showed a proliferation gain (0.5 fold, p=0.016) compared to the WT parental cells . Notably, this was most pronounced in the biallelelicly inactivated cells (2 folds increase, p<0.0001). In absence of talquetamab, the baseline cell proliferation rate did not differ between the WT cells and our models. No proliferation changes were seen in cell models treated with talquetamab in absence of effector T cells. High content Cyto/Chemokine profiling on the GPRC5D coculture experiments was perfomed using the scioCyto microarray platform which revealed significantly altered cyto/chemokine profiles, including growth and proliferation promoting cytokines such as CCL28, IL34, HGF and CXCL9. In addition, differential gene expression analysis from Bulk RNA sequencing of the GPRC5D WT and KO cells confirmed upregulation of MAPK and PI3K pathways involved in growth proliferation and downregulation of the RAP1 signaling pathway involved in cell adhesion. Unspecific or alternate binding of talquetamab to the GPRC5DDel/Del cell surface was not detectable using flow cytometry, to decipher potential mechanism of growth induction via non-specific binding of talquetamab, pulldown assays combined with proteomic based analysis are ongoing. Conclusion Here we provide first data that monoallelic GPRC5D inactivation impairs talquetamab efficiency. Our work supports an anti-proliferative function of GPRC5D and suggests that treatment over progression with talquetamab may favor tumor growth in GPRC5D deficient cells.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Background: Multiple myeloma (MM) is characterized by complex genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus and secondary mutations involving key oncogenic drivers. This temporal complexity represents a constant challenge for the clinical management of MM, but at the same time may provide new avenues for immunotherapy-based approaches including CAR-T cells and T-cell engaging antibodies. In this study, we investigated the impact of secondary genetic events on the surface expression of common targets for immunotherapy in MM. Methods: Cell models harboring two hotspot activating mutations in KRAS (G12A, A146T) and TP53 (R282W) were created using the MM cell lines OPM-2 and AMO1 via CRISPR-Cas9 technology. Direct stochastic optical reconstruction microscopy ( dSTORM) was used for high-resolution receptor density estimation and epitope quantification of common immunotherapeutic targets such as CD38, BCMA and SLAMF7. Results: dSTORM imaging of cell line models carrying patient-derived secondary genetic alterations revealed a distinct pattern linked to each mutation respectively. In KRAS G12A, we observed a reduction in BCMA density by 2-fold as compared to wild type cells (5.9 ± 0.3 vs. 10.8 ± 1.2 localization clusters/µm 2, p=0005). This reduction in BCMA surface expression was accompanied by a decrease in other key immunotargets, including SLAMF7 (6.0 ± 0.8 vs 11.8 ± 0.8 localization clusters/µm 2, p <0,0001) and CD38 (12.2 ± 1.5 vs 17.9 ± 1.6 localization clusters/µm 2, p=0,012). In KRAS A146, CD38 density increased, whereas mean BCMA and SLAMF7 localizations were consistently reduced as compared to wild type control. To ensure that BCMA downregulation is a consistent feature in genomic high-risk disease, we next simulated a TP53 double-hit situation by introducing an inactivating hotspot mutation R282W in a TP53 deletion background. Surfaceome profiling by dSTORM revealed a minor increase in CD38 levels for TP53R282W/Del as compared to wild type control (51.5 ± 5.7 vs. 36.4 ± 4.2 localization clusters/µm 2, p=0.06). Interestingly, the expression level of BCMA, known to be comparably low on WT tumor cells, further decreased in TP53 double-hit cells (0.6 ± 0.1 vs 1.1 ± 0.1 localization clusters/µm 2, p=0.002), whereas no major differences in the cluster density of SLAMF7 were observed. This leaves room for speculation whether these effects on biallelic TP53 inactivated tumor cells may underlie reduced susceptibility towards BCMA-targeting therapy. Of note, we also observed altered cell morphology in the TP53 double-hit model, characterized by elongated stress fibers enriched with CD138, potentially interfering with receptor accessibility of surface receptors (Figure 1). Recently, secondary genetic events have been reported in BCMA and GPRC5D following targeted immunotherapy inducing acquired drug resistance. We created cell models representing homo- and heterozygous genetic alterations in BCMA and GPRC5D, studies to assess their effects on the surfaceome are ongoing and will be presented at the meeting. Summary: Our data suggest that surface epitope expression in MM cells seems to be less agnostic to secondary genomic events than expected. These findings may provide a direct link between genetic high-risk disease and inferior efficacy of anti-BCMA immunotherapies in the context of e.g. TP53 double-hit MM.