Regulation of the hematopoietic stem cell pool by C-Kit-associated trogocytosis.

Hematopoietic stem cells (HSCs) are routinely mobilized from the bone marrow (BM) to the blood circulation for clinical transplantation. However, the precise mechanisms by which individual stem cells exit the marrow are not understood. This study identified cell-extrinsic and molecular determinants of a mobilizable pool of blood-forming stem cells. We found that a subset of HSCs displays macrophage-associated markers on their cell surface. Although fully functional, these HSCs are selectively niche-retained as opposed to stem cells lacking macrophage markers, which exit the BM upon forced mobilization. Macrophage markers on HSCs could be acquired through direct transfer by trogocytosis, regulated by receptor tyrosine-protein kinase C-Kit (CD117), from BM-resident macrophages in mouse and human settings. Our study provides proof of concept that adult stem cells utilize trogocytosis to rapidly establish and activate function-modulating molecular mechanisms.

Human Tumor-Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis.

The clinical benefits of tumor-targeting antibodies (tAb) are modest in solid human tumors. The efficacy of many tAbs is dependent on Fc receptor (FcR)-expressing leukocytes that bind Fc fragments of tAb. Tumor-associated macrophages (TAM) and neutrophils (TAN) represent the majority of FcR+ effectors in solid tumors. A better understanding of the mechanisms by which TAMs and TANs regulate tAb response could help improve the efficacy of cancer treatments. Here, we found that myeloid effectors interacting with tAb-opsonized lung cancer cells used antibody-dependent trogocytosis (ADT) but not antibody-dependent phagocytosis. During this process, myeloid cells "nibbled off" tumor cell fragments containing tAb/targeted antigen (tAg) complexes. ADT was only tumoricidal when the tumor cells expressed high levels of tAg and the effectors were present at high effector-to-tumor ratios. If either of these conditions were not met, which is typical for solid tumors, ADT was sublethal. Sublethal ADT, mainly mediated by CD32hiCD64hi TAM, led to two outcomes: (i) removal of surface tAg/tAb complexes from the tumor that facilitated tumor cell escape from the tumoricidal effects of tAb; and (ii) acquisition of bystander tAgs by TAM with subsequent cross-presentation and stimulation of tumor-specific T-cell responses. CD89hiCD32loCD64lo peripheral blood neutrophils (PBN) and TAN stimulated tumor cell growth in the presence of the IgG1 anti-EGFR Ab cetuximab; however, IgA anti-EGFR Abs triggered the tumoricidal activity of PBN and negated the stimulatory effect of TAN. Overall, this study provides insights into the mechanisms by which myeloid effectors mediate tumor cell killing or resistance during tAb therapy. SIGNIFICANCE: The elucidation of the conditions and mechanisms by which human FcR+ myeloid effectors mediate cancer cell resistance and killing during antibody treatment could help develop improved strategies for treating solid tumors.

The transplant rejection response involves neutrophil and macrophage adhesion-mediated trogocytosis and is regulated by NFATc3.

The anti-foreign tissue (transplant rejection) response, mediated by the immune system, has been the biggest obstacle to successful organ transplantation. There are still many enigmas regarding this process and some aspects of the underlying mechanisms driving the immune response against foreign tissues remain poorly understood. Here, we found that a large number of neutrophils and macrophages were attached to the graft during skin transplantation. Furthermore, both types of cells could autonomously adhere to and damage neonatal rat cardiomyocyte mass (NRCM) in vitro. We have demonstrated that Complement C3 and the receptor CR3 participated in neutrophils/macrophages-mediated adhesion and damage this foreign tissue (NRCM or skin grafts). We have provided direct evidence that the damage to these tissues occurs by a process referred to as trogocytosis, a damage mode that has never previously been reported to directly destroy grafts. We further demonstrated that this process can be regulated by NFAT, in particular, NFATc3. This study not only enriches an understanding of host-donor interaction in transplant rejection, but also provides new avenues for exploring the development of novel immunosuppressive drugs which prevent rejection during transplant therapy.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Trogocytosis of CAR molecule regulates CAR-T cell dysfunction and tumor antigen escape.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical response in treating both hematologic malignancies and solid tumors. Although instances of rapid tumor remissions have been observed in animal models and clinical trials, tumor relapses occur with multiple therapeutic resistance mechanisms. Furthermore, while the mechanisms underlying the long-term therapeutic resistance are well-known, short-term adaptation remains less understood. However, more views shed light on short-term adaptation and hold that it provides an opportunity window for long-term resistance. In this study, we explore a previously unreported mechanism in which tumor cells employ trogocytosis to acquire CAR molecules from CAR-T cells, a reversal of previously documented processes. This mechanism results in the depletion of CAR molecules and subsequent CAR-T cell dysfunction, also leading to short-term antigen loss and antigen masking. Such type of intercellular communication is independent of CAR downstream signaling, CAR-T cell condition, target antigen, and tumor cell type. However, it is mainly dependent on antigen density and CAR sensitivity, and is associated with tumor cell cholesterol metabolism. Partial mitigation of this trogocytosis-induced CAR molecule transfer can be achieved by adaptively administering CAR-T cells with antigen density-individualized CAR sensitivities. Together, our study reveals a dynamic process of CAR molecule transfer and refining the framework of clinical CAR-T therapy for solid tumors.

Outsmarting trogocytosis to boost CAR NK/T cell therapy.

Chimeric antigen receptor (CAR) NK and T cell therapy are promising immunotherapeutic approaches for the treatment of cancer. However, the efficacy of CAR NK/T cell therapy is often hindered by various factors, including the phenomenon of trogocytosis, which involves the bidirectional exchange of membrane fragments between cells. In this review, we explore the role of trogocytosis in CAR NK/T cell therapy and highlight potential strategies for its modulation to improve therapeutic efficacy. We provide an in-depth analysis of trogocytosis as it relates to the fate and function of NK and T cells, focusing on its effects on cell activation, cytotoxicity, and antigen presentation. We discuss how trogocytosis can mediate transient antigen loss on cancer cells, thereby negatively affecting the effector function of CAR NK/T cells. Additionally, we address the phenomenon of fratricide and trogocytosis-associated exhaustion, which can limit the persistence and effectiveness of CAR-expressing cells. Furthermore, we explore how trogocytosis can impact CAR NK/T cell functionality, including the acquisition of target molecules and the modulation of signaling pathways. To overcome the negative effects of trogocytosis on cellular immunotherapy, we propose innovative approaches to modulate trogocytosis and augment CAR NK/T cell therapy. These strategies encompass targeting trogocytosis-related molecules, engineering CAR NK/T cells to resist trogocytosis-induced exhaustion and leveraging trogocytosis to enhance the function of CAR-expressing cells. By overcoming the limitations imposed by trogocytosis, it may be possible to unleash the full potential of CAR NK/T therapy against cancer. The knowledge and strategies presented in this review will guide future research and development, leading to improved therapeutic outcomes in the field of immunotherapy.

Measurement of Trogocytosis: Quantitative Analyses Validated with Rigorous Controls.

Trogocytosis is a process in which receptors on acceptor cells remove and internalize cognate ligands from donor cells. Trogocytosis has a profound and negative impact on mAb-based cancer immunotherapy, as seen in the treatment of chronic lymphocytic leukemia (CLL) with CD20 mAbs, such as rituximab (RTX) and ofatumumab (OFA). Our clinical observations of RTX/OFA-mediated loss of the CD20 target from circulating CLL cells have been replicated in our in vitro studies. Here we describe flow cytometry and fluorescence microscopy experiments, which demonstrate that acceptor cells, such as monocytes/macrophages that express FcγR, remove and internalize both antigen and donor cell-bound cognate IgG mAbs for several different mAb-donor cell pairs. Fluorescent mAbs and portions of the plasma cell membrane are transferred from donor cells to acceptor cells, which include the THP-1 monocytic cell line as well as freshly isolated monocytes. We describe rigorous controls to validate the reactions and eliminate dissociation or internalization as alternative mechanisms. Trogocytosis is likely to contribute to neutropenia, thrombocytopenia, and liver damage associated with use of antibody-drug conjugates. The methods we have described should allow for examination of strategies focused on blocking trogocytosis and its adverse effects. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Trogocytosis of mAb-opsonized donor cells mediated by adherent THP-1 cells Alternate Protocol: Application of fluorescence microscopy to examine THP-1 cell-mediated trogocytosis Support Protocol 1: Alexa labeling of mAbs and determination of F/P ratios Support Protocol 2: Standard washing procedure Support Protocol 3: Labeling and opsonization of cells Basic Protocol 2: Trogocytosis mediated by human monocytes as acceptor cells Support Protocol 4: Isolation of human monocytes Basic Protocol 3: Trogocytosis mediated by THP-1 cells in solution Support Protocol 5: Retinoic acid treatment of THP-1 cells Support Protocol 6: Culturing of SCC-25, BT-474, MOLT-4 and THP-1 cell lines.

Settling the uncertainty about unconventional circulating tumor cells: Epithelial-to-mesenchymal transition, cell fusion and trogocytosis.

Circulating tumor cells (CTCs) were first described 150 years ago. The so-called "classical" CTC populations (EpCAM+/CK+/CD45-) have been fully characterized and proposed as the most representative CTC subset, with clinical relevance. Nonetheless, other "atypical" or "unconventional" CTCs have also been identified, and their critical role in metastasis formation was demonstrated. In this chapter we illustrate the studies that led to the discovery of unconventional CTCs, defined as CTCs that display both epithelial and mesenchymal markers, or both cancer and immune markers, also in the form of hybrid cancer-immune cells. We also present biological explanations for the origin of these unconventional CTCs: epithelial to mesenchymal transition, cell-cell fusion and trogocytosis. We believe that a deeper knowledge on the biology of CTCs is needed to fully elucidate their role in cancer progression and their use as cancer biomarkers.

Both APRIL and antibody-fragment-based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here, we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs, and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL, single-chain-variable-fragment, heavy-chain-only). METHODS: Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo, for their polyfunctionality, immune synapse formation, memory, exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms, we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS: APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BBζ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BBζ CAR T cells. After CAR T cell-myeloma cell contact, BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis, and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse, we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway, we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION: Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect, but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies.

ATF3 and CH25H regulate effector trogocytosis and anti-tumor activities of endogenous and immunotherapeutic cytotoxic T lymphocytes.

Effector trogocytosis between malignant cells and tumor-specific cytotoxic T lymphocytes (CTLs) contributes to immune evasion through antigen loss on target cells and fratricide of antigen-experienced CTLs by other CTLs. The mechanisms regulating these events in tumors remain poorly understood. Here, we demonstrate that tumor-derived factors (TDFs) stimulated effector trogocytosis and restricted CTLs' tumoricidal activity and viability in vitro. TDFs robustly altered the CTL's lipid profile, including depletion of 25-hydroxycholesterol (25HC). 25HC inhibited trogocytosis and prevented CTL's inactivation and fratricide. Mechanistically, TDFs induced ATF3 transcription factor that suppressed the expression of 25HC-regulating gene-cholesterol 25-hydroxylase (CH25H). Stimulation of trogocytosis in the intratumoral CTL by the ATF3-CH25H axis attenuated anti-tumor immunity, stimulated tumor growth, and impeded the efficacy of chimeric antigen receptor (CAR) T cell adoptive therapy. Through use of armored CAR constructs or pharmacologic agents restoring CH25H expression, we reversed these phenotypes and increased the efficacy of immunotherapies.

KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape.

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.

Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality.

Successful translation of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors has proved to be troublesome, mainly due to the complex tumor microenvironment promoting T cell dysfunction and antigen heterogeneity. Mesothelin (MSLN) has emerged as an attractive target for CAR T cell therapy of several solid malignancies, including ovarian cancer. To improve clinical response rates with MSLN-CAR T cells, a better understanding of the mechanisms impacting CAR T cell functionality in vitro is crucial. Here, we demonstrated superior cytolytic capacity of CD28-costimulated MSLN-CAR T cells (M28z) relative to 4-1BB-costimulated MSLN-CAR T cells (MBBz). Furthermore, CD28-costimulated MSLN CAR T cells displayed enhanced cytolytic capacity against tumor spheroids with heterogeneous MSLN expression compared to MBBz CAR T cells. In this study, we identified CAR-mediated trogocytosis as a potential impeding factor for successful MSLN-CAR T cell therapy due to fratricide killing and contributing to tumor antigen heterogeneity. Moreover, we link antigen-dependent upregulation of LAG-3 with reduced CAR T cell functionality. Taken together, our study highlights the therapeutic potential and bottlenecks of MSLN-CAR T cells, providing a rationale for combinatorial treatment strategies.

Blockade of CD47 enhances the antitumor effect of macrophages in renal cell carcinoma through trogocytosis.

Immune checkpoint inhibitors and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR TKIs) are mainstream treatments for renal cell carcinoma (RCC). Both T cells and macrophages infiltrate the tumor microenvironment of RCC. CD47, an immune checkpoint of macrophages, transmits the "don't eat me" signal to macrophages. We propose a novel therapeutic strategy that activates the antitumor effect of macrophages. We found that CD47 was expressed in patients with RCC, and high CD47 expression was indicative of worse overall survival in datasets from The Cancer Genome Atlas. We observed that CD47-blocking antibodies enhanced the antitumor effect of macrophages against human RCC cell lines. Trogocytosis, rather than phagocytosis, occurred and was promoted by increased cell-to-cell contact between macrophages and RCC cells. Trogocytosis induced by CD47 blockade occurred in the presence of CD11b integrin signaling in macrophages and was augmented when RCC cells were exposed to VEGFR TKIs, except for sunitinib. In conclusion, this study presents evidence that anti-CD47 blocking antibodies improve the antitumor effect of macrophages in RCC. In combination with VEGFR TKIs, CD47 blockade is a potential therapeutic strategy for patients with RCC.

CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation.

Immunotherapies targeting the "don't eat me" myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Balamuthia mandrillaris trophozoites ingest human neuronal cells via a trogocytosis-independent mechanism.

BACKGROUND: Environmental protozoa need an adaptation mechanism to survive drastic changes in niches in the human body. In the brain parenchyma, Balamuthia mandrillaris trophozoites, which are causative agents of fatal brain damage, must acquire nutrients through the ingestion of surrounding cells. However, the mechanism deployed by the trophozoites for cellular uptake remains unknown. METHODS: Amoebic ingestion of human neural cell components was investigated using a coculture system of clinically isolated B. mandrillaris trophozoites and human neuroblastoma SH-SY5Y cells. Cell-to-cell interactions were visualized in a three-dimensional manner using confocal and holotomographic microscopes. RESULTS: The B. mandrillaris trophozoites first attached themselves to human neuroblastoma SH-SY5Y cells and then twisted themselves around the cytoplasmic bridge. Based on fluorescence-based cell tracking, the B. mandrillaris trophozoites then inserted invadopodia into the cytoplasm of the human cells. Subsequently, the human protein-enriched components were internalized into the trophozoites in the form of nonmembranous granules, whereas the human lipids were dispersed in the cytoplasm. Intervention of trogocytosis, a process involving nibbling on parts of the target cells, failed to inhibit this cellular uptake. CONCLUSIONS: Human cell ingestion by B. mandrillaris trophozoites likely differs from trogocytosis, suggesting that a pathogen-specific strategy can be used to ameliorate brain damage.

Measuring trogocytosis between ovarian tumor and natural killer cells.

Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021).

Tim-3 mediates T cell trogocytosis to limit antitumor immunity.

T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human programmed cell death 1-positive (PD-1+) Tim-3+CD8+ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen-presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3+ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen-specific CD8+ T cells and PD-1+Tim-3+ CD8+ TILs isolated from patients with melanoma. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8+ TILs in 2 melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not in CD8+ T cells impeded the trogocytosis of CD8+ TILs in vivo. Trogocytosed CD8+ T cells presented tumor peptide-major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism Tim-3 uses to limit antitumor immunity.

Amivantamab (JNJ-61186372) induces clinical, biochemical, molecular, and radiographic response in a treatment-refractory NSCLC patient harboring amplified triple EGFR mutations (L858R/ T790M/G796S) in cis.

The sequential use of 1st-/2nd-generation to 3rd-generation epidermal growth factor (EGFR) tyrosine kinase inhibitors (TKIs) has led to the emergence of triple EGFR mutations generally consisting of the founder mutation (del 19 or L858R), gatekeeper mutation (T790M) and mutation (C797S) that abolishes the covalent binding of osimertinib to the EGFR protein (i.e., del 19 or L858R/T790M/C797S). Besides C797S, other tertiary mutations confer structural steric hindrance to osimertinib rather than preventing its covalent binding to the EGFR kinase domain such as solvent front mutation (G796S) or others such as L792F/H mutation. "Fourth-generation" EGFR TKIs are being developed to inhibit these triple mutations, in particular, in the background of compound T790M/C797S mutations but they are still in early clinical stages of development. Amivantamab, a bi-specific EGFR/MET monoclonal antibody that can affect Fc mediated trogocytosis of the EGFR protein has been approved for the treatment of EGFR exon20 insertion mutations and has demonstrated activity against a myriad of compound EGFR mutations. Here we report amivantamab monotherapy induced symptomatic, biochemical, molecular, and radiographic responses in a NSCLC patient with triple EGFR mutations in cis in the background of EGFR amplification.