PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.

Forimtamig, a novel GPRC5D-targeting T-cell bispecific antibody with a 2+1 format, for the treatment of multiple myeloma.

Despite several approved therapies, multiple myeloma (MM) remains an incurable disease with high unmet medical need. "Off-the-shelf" T-cell bispecific antibodies (TCBs) targeting BCMA and GPRC5D have demonstrated high objective response rates (ORR) in heavily pre-treated MM patients, however, primary resistance, short duration of response and relapse driven by antigen shift frequently occurs. Although GPRC5D represents the most selective target in MM, recent findings indicate antigen loss occurs more frequently than with BCMA. Thus, anti-GPRC5D immunotherapies must hit hard during a short period of time to kill as many myeloma cells as possible. Here, we characterize forimtamig, a novel GPRC5D-targeting TCB with 2+1 format, using preclinical models of MM. Bivalent binding of forimtamig to the N-terminus of GPRC5D confers higher affinity as compared to classical 1+1 TCB formats correlating with formation of more stable immunological synapses and higher potency in tumor cell killing and T cell activation. Using an orthotopic mouse model of MM, forimtamig recruited T effector cells to the bone marrow and induced rapid tumor killing even after the introduction of step-up dosing to mitigate cytokine release. Combination of forimtamig with standard-of-care (SoC) agents including anti-CD38 antibodies, immunomodulatory drugs and proteasome inhibitors improved depth and duration of response. The combination of forimtamig with novel therapeutic agents including BCMA-TCB and Cereblon E3 Ligase Modulatory Drugs (CELMoDs) was potent and prevented occurrence of GPRC5D-negative tumor relapse. Forimtamig is currently being evaluated in Phase 1 clinical trials in relapsed and refractory myeloma (RRMM) patients for monotherapy and in combination treatments. NCT04557150.

B- and T-cell acute lymphoblastic leukemias evade chemotherapy at distinct sites in the bone marrow.

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support escape from treatment. Using three-dimensional fluorescence imaging of ten primary ALL xenografts we identified sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detected B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon shortterm chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment.

Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations.

The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

Multicolor quantitative confocal imaging cytometry.

Multicolor 3D quantitative imaging of large tissue volumes is necessary to understand tissue development and organization as well as interactions between distinct cell types in situ. However, tissue imaging remains technically challenging, particularly imaging of bone and marrow. Here, we describe a pipeline to reproducibly generate high-dimensional quantitative data from bone and bone marrow that may be extended to any tissue. We generate thick bone sections from adult mouse femurs with preserved tissue microarchitecture and demonstrate eight-color imaging using confocal microscopy without linear unmixing. We introduce XiT, an open-access software for fast and easy data curation, exploration and quantification of large imaging data sets with single-cell resolution. We describe how XiT can be used to correct for potential artifacts in quantitative 3D imaging, and we use the pipeline to measure the spatial relationship between hematopoietic cells, bone matrix and marrow Schwann cells.

Ubiquitous overexpression of CXCL12 confers radiation protection and enhances mobilization of hematopoietic stem and progenitor cells.

C-X-C motif chemokine ligand 12 (CXCL12; aka SDF1α) is a major regulator of a number of cellular systems, including hematopoiesis, where it influences hematopoietic cell trafficking, proliferation, and survival during homeostasis and upon stress and disease. A variety of constitutive, temporal, ubiquitous, and cell-specific loss-of-function models have documented the functional consequences on hematopoiesis upon deletion of Cxcl12. Here, in contrast to loss-of-function experiments, we implemented a gain-of-function approach by generating a doxycycline-inducible transgenic mouse model that enables spatial and temporal overexpression of Cxcl12. We demonstrated that ubiquitous CXCL12 overexpression led to an increase in multipotent progenitors in the bone marrow and spleen. The CXCL12+ mice displayed reduced reconstitution potential as either donors or recipients in transplantation experiments. Additionally, we discovered that Cxcl12 overexpression improved hematopoietic stem and progenitor cell mobilization into the blood, and conferred radioprotection by promoting quiescence. Thus, this new CXCL12+ mouse model provided new insights into major facets of hematopoiesis and serves as a versatile resource for studying CXCL12 function in a variety of contexts.

In vitro biomimetic engineering of a human hematopoietic niche with functional properties.

In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs' behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis.

An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.

Multicolor 3D Confocal Imaging of Thick Tissue Sections.

In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700 µm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.

Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated With Anti-Tumor Immunity.

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1-PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

Fibroblast Activation Protein α-Targeted CD40 Agonism Abrogates Systemic Toxicity and Enables Administration of High Doses to Induce Effective Antitumor Immunity.

PURPOSE: CD40 agonists hold great promise for cancer immunotherapy (CIT) as they enhance dendritic cell (DC) activation and concomitant tumor-specific T-cell priming. However, the broad expression of CD40 accounts for sink and side effects, hampering the efficacy of anti-CD40 antibodies. We hypothesized that these limitations can be overcome by selectively targeting CD40 agonism to the tumor. Therefore, we developed a bispecific FAP-CD40 antibody, which induces CD40 stimulation solely in presence of fibroblast activation protein α (FAP), a protease specifically expressed in the tumor stroma. EXPERIMENTAL DESIGN: FAP-CD40's in vitro activity and FAP specificity were validated by antigen-presenting cell (APC) activation and T-cell priming assays. In addition, FAP-CD40 was tested in subcutaneous MC38-FAP and KPC-4662-huCEA murine tumor models. RESULTS: FAP-CD40 triggered a potent, strictly FAP-dependent CD40 stimulation in vitro. In vivo, FAP-CD40 strongly enhanced T-cell inflammation and growth inhibition of KPC-4662-huCEA tumors. Unlike nontargeted CD40 agonists, FAP-CD40 mediated complete regression of MC38-FAP tumors, entailing long-term protection. A high dose of FAP-CD40 was indispensable for these effects. While nontargeted CD40 agonists induced substantial side effects, highly dosed FAP-CD40 was well tolerated. FAP-CD40 preferentially accumulated in the tumor, inducing predominantly intratumoral immune activation, whereas nontargeted CD40 agonists displayed strong systemic but limited intratumoral effects. CONCLUSIONS: FAP-CD40 abrogates the systemic toxicity associated with nontargeted CD40 agonists. This enables administration of high doses, essential for overcoming CD40 sink effects and inducing antitumor immunity. Consequently, FAP-targeted CD40 agonism represents a promising strategy to exploit the full potential of CD40 signaling for CIT.

Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF.

Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.

A Novel GATA2 Protein Reporter Mouse Reveals Hematopoietic Progenitor Cell Types.

The transcription factor (TF) GATA2 plays a key role in organ development and cell fate control in the central nervous, urogenital, respiratory, and reproductive systems, and in primitive and definitive hematopoiesis. Here, we generate a knockin protein reporter mouse line expressing a GATA2VENUS fusion from the endogenous Gata2 genomic locus, with correct expression and localization of GATA2VENUS in different organs. GATA2VENUS expression is heterogeneous in different hematopoietic stem and progenitor cell populations (HSPCs), identifies functionally distinct subsets, and suggests a novel monocyte and mast cell lineage bifurcation point. GATA2 levels further correlate with proliferation and lineage outcome of hematopoietic progenitors. The GATA2VENUS mouse line improves the identification of specific live cell types during embryonic and adult development and will be crucial for analyzing GATA2 protein dynamics in TF networks.

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells.

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

A 3D Tissue-wide Digital Imaging Pipeline for Quantitation of Secreted Molecules Shows Absence of CXCL12 Gradients in Bone Marrow.

Technological limitations have hampered understanding of how individual molecules, including putative stem cell regulators, are distributed throughout tissues and stem cell niches. Here, we report adaptation of the proximity ligation assay (PLA) for large-volume, in situ imaging of individual proteins with multiple additional fluorescent channels with integrated 3D quantification strategies and software. Using this platform, we quantified the bone marrow (BM) distribution of individual CXCL12 chemokine proteins, both before and after their depletion by granulocyte-colony stimulating factor (G-CSF) treatment. We found ubiquitous CXCL12 distributions with local enrichments but no long-range gradients, in contrast to current assumptions about how CXCL12 controls migration of hematopoietic stem and progenitor cells (HSPCs) within BM. This pipeline for discrete digital quantitative, large-volume, multicolor imaging, with up to single-molecule sensitivity, may be broadly applied to any antibody epitope and tissue, enabling further insights into molecular organization of tissues and cellular interactions.

Fate Distribution and Regulatory Role of Human Mesenchymal Stromal Cells in Engineered Hematopoietic Bone Organs.

The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs' fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments.

Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules.

The bone marrow (BM) microenvironment contains many types of cells and molecules with roles in hematopoiesis, osteogenesis, angiogenesis and metabolism. The spatial distribution of the different bone and BM cell types remains elusive, owing to technical challenges associated with bone imaging. To map nonhematopoietic cells and structures in bone and BM, we performed multicolor 3D imaging of osteoblastic, vascular, perivascular, neuronal and marrow stromal cells, and extracellular-matrix proteins in whole mouse femurs. We analyzed potential interactions between cells and molecules on the basis of colocalization of markers. Our results shed light on the markers expressed by different osteolineage cell types; the heterogeneity of vascular and perivascular cells; the neural subtypes innervating marrow and bone; the diversity of stromal cells; and the distribution of extracellular-matrix components. Our complete imaging data set is available for download and can be used in research in bone biology, hematology, vascular biology, neuroscience and extracellular-matrix biology.

Muscular coordination of biceps brachii and brachioradialis in elbow flexion with respect to hand position.

Contribution of synergistic muscles toward specific movements over multi joint systems may change with varying position of distal or proximal joints. Purpose of this study is to reveal the relationship of muscular coordination of brachioradialis and biceps brachii during elbow flexion with respect to hand position and biomechanical advantages and disadvantages of biceps brachii. A group of 16 healthy subjects has been advised to perform 20 repetitions of single elbow flexion movements in different hand positions (pronated, neutral, and supinated). With a speed of 20°/s, simultaneously sEMG of biceps brachii and brachioradialis and kinematics of the movement were recorded in a motion analysis laboratory. Normalized to MVC the sEMG amplitudes of both muscles contributing to elbow flexion movements were compared in pronated, supinated, and neutral hand position over elbow joint angle. Significant differences in the contribution of brachioradialis were found in pronated hand position compared to supinated and neutral hand position while the muscular activity of biceps brachii shows no significant changes in any hand position. In conclusion, a statistical significant dependency of the inter-muscular coordination between biceps brachii and brachioradialis during elbow flexion with respect to hand position has been observed depending on a biomechanical disadvantage of biceps brachii.