T cell-intrinsic PKD3 fine-tunes differentiation into CD8+ central memory T cells and CD8 single positive thymocyte development.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.

T cell-intrinsic protein kinase D3 is dispensable for the cells' activation.

Protein kinases D (PKDs) are implicated in T cell receptor (TCR) signaling. Of the two T cell-expressed isoforms PKD2 and PKD3, however, only the former one is rather well understood in this immune cell type. Recently, we have observed a putative hyper-phenotype of T cells from conventional PKD3-knockout mice, which we explained as a secondary effect due to a skewed T cell compartment from naïve towards effector/memory T cells already under steady state conditions. Nonetheless, to this end it is not clear whether these aberrations are mediated by a T cell-intrinsic or -extrinsic function of PKD3. To address this question, we have investigated mice lacking PKD3 specifically in the T cell compartment. We could show that T cells from CD4-Cre-driven conditional knockout mice did not phenocopy the ones from conventional PKD3-knockout mice. In brief, no skewing in the T cell compartment of peripheral lymphoid organs, no hyper-activation upon stimulation in vitro or in vivo as well as no aberrations in follicular helper T cells (TFH) upon immunization were observed. Hence, although PKD3 is strongly regulated upon TCR stimulation, in T cells this kinase seems to be dispensable for their activation. The described skewing in the T cell compartment of conventional PKD3-deficient mice seems to be mediated by T cell-extrinsic mechanisms, thus once more emphasizing the importance of cell type-specific mouse models.

A MLR-Based Approach to Analyze Regulators of T Lymphocyte Activation In Vivo.

Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.1/2 and cell proliferation dye-labeling. This setup allows us to track adoptively transferred allogenic CD4+ and CD8+ T lymphocytes and analyze their phenotype as well as the proliferation by flow cytometry in the blood and spleen. We could show hypo-reactive responses of T lymphocytes isolated from knockout mice with a known defect in T lymphocyte activation. Thus, this MLR-based in vivo model provides the opportunity to analyze positive regulators of T cell responses under physiological conditions of polyclonal T lymphocyte activation in vivo.

Addressing the role of PKD3 in the T cell compartment with knockout mice.

BACKGROUND: The Protein kinase D3 (PKD3) has been implicated in signal transduction downstream of the T cell receptor (TCR). However, its role for the activation of primary T lymphocytes has not been elucidated so far. METHODS: Expression of PKD isoforms in primary murine T cells was determined by RT-PCR and SDS-Page. A germline PKD3-knockout mouse line was analyzed for its immune response to OVA/alum intraperitoneal immunization. Phenotyping of the T cell compartment ex vivo as well as upon stimulation in vitro was performed by flow cytometry. Additionally, cytokine expression was assessed by flow cytometry, RT-PCR and Luminex technology. RESULTS: PKD expression in T cells is modulated by TCR stimulation, leading to a rapid down-regulation on mRNA and on protein level. PKD3-deficient mice respond to immunization with enhanced T follicular helper cell generation. Furthermore, peripheral PKD3-deficient CD4+ T cells express more interleukin-2 than wild type CD4+ T cells upon TCR stimulation ex vivo. However, purified naïve CD4+ T cells do not differ in their phenotype upon differentiation in vitro from wild type T cells. Moreover, we observed a shift towards an effector/memory phenotype of splenic T cells at steady state, which might explain the contradictory results obtained with pan-T cells ex vivo and naïve-sorted T cells. CONCLUSION: While PKD3-deficiency in vivo in mice leads to a skewing of the T cell compartment towards a more activated phenotype, this kinase seems to be dispensable for naïve CD4+ T cell differentiation in vitro. Video Abstract.

Emerging Next-Generation Target for Cancer Immunotherapy Research: The Orphan Nuclear Receptor NR2F6.

Additional therapeutic targets suitable for boosting anti-tumor effector responses have been found inside effector CD4+ and CD8+ T cells. It is likely that future treatment options will combine surface receptor and intracellular protein targets. Utilizing germline gene ablation as well as CRISPR/Cas9-mediated acute gene mutagenesis, the nuclear receptor NR2F6 (nuclear receptor subfamily 2 group F member 6, also called Ear-2) has been firmly characterized as such an intracellular immune checkpoint in effector T cells. Targeting this receptor appears to be a strategy for improving anti-tumor immunotherapy responses, especially in combination with CTLA-4 and PD-1. Current preclinical experimental knowledge firmly validates the immune checkpoint function of NR2F6 in murine tumor models, which provides a promising perspective for immunotherapy regimens in humans in the near future. While the clinical focus remains on the B7/CD28 family members, protein candidate targets such as NR2F6 are now being investigated in laboratories around the world and in R&D companies. Such an alternative therapeutic approach, if demonstrated to be successful, could supplement the existing therapeutic models and significantly increase response rates of cancer patients and/or expand the reach of immune therapy regimens to include a wider range of cancer entities. In this perspective review, the role of NR2F6 as an emerging and druggable target in immuno-oncology research will be discussed, with special emphasis on the unique potential of NR2F6 and its critical and non-redundant role in both immune and tumor cells.

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127-KLRG1+) or memory precursors cells (MPECs, CD127+KLRG1-) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8+ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6-/- OT-I T-cells showed that the augmented memory formation is CD8+ T-cell intrinsic. Although the relative difference between the Nr2f6+/+ and Nr2f6-/- OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8+ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8+ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8+ T cells in vivo.

Chemically modified mRNA nucleofection of primary human T cells.

Here we show that an approach of in-vitro transcribed mRNA nucleofection expands the range of transfection of primary human T cells. It represents a reproducible and time-efficient technology, and is thus an ideal tool in basic research involving highly controlled in-vitro experiments with a gene of interest aiming at identifying its biological human T cell function.

Targeting NAD immunometabolism limits severe graft-versus-host disease and has potent antileukemic activity.

Acute graft-versus-host disease (aGVHD) and tumor relapse remain major complications after allogeneic hematopoietic stem cell transplantation. Alloreactive T cells and cancer cells share a similar metabolic phenotype to meet the bioenergetic demands necessary for cellular proliferation and effector functions. Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in energy metabolism and is constantly replenished by nicotinamide phosphoribosyl-transferase (Nampt), the rate-limiting enzyme in the NAD salvage pathway. Here we show, that Nampt blockage strongly ameliorates aGVHD and limits leukemic expansion. Nampt was highly elevated in serum of patients with gastrointestinal GVHD and was particularly abundant in human and mouse intestinal T cells. Therapeutic application of the Nampt small-molecule inhibitor, Fk866, strongly attenuated experimental GVHD and caused NAD depletion in T-cell subsets, which displayed differential susceptibility to NAD shortage. Fk866 robustly inhibited expansion of alloreactive but not memory T cells and promoted FoxP3-mediated lineage stability in regulatory T cells. Furthermore, Fk866 strongly reduced the tumor burden in mouse leukemia and graft-versus-leukemia models. Ex vivo studies using lymphocytes from GVHD patients demonstrated potent antiproliferative properties of Fk866, suggesting potential clinical utility. Thus, targeting NAD immunometabolism represents a novel approach to selectively inhibit alloreactive T cells during aGVHD with additional antileukemic efficacy.

Loss-of-function phenotype of a PKCθT219A knockin mouse strain.

BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4+ T lymphocyte activation.

BACKGROUND: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. METHODS: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. RESULTS: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. CONCLUSION: Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity.

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts.

Protein kinase C theta is dispensable for suppression mediated by CD25+CD4+ regulatory T cells.

The activation of conventional T cells upon T cell receptor stimulation critically depends on protein kinase C theta (PKCθ). However, its role in regulatory T (Treg) cell function has yet to be fully elucidated. Using siRNA or the potent and PKC family-selective pharmacological inhibitor AEB071, we could show that murine Treg-mediated suppression in vitro is independent of PKCθ function. Likewise, Treg cells of PKCθ-deficient mice were fully functional, showing a similar suppressive activity as wild-type CD25+CD4+ T cells in an in vitro suppression assay. Furthermore, in vitro-differentiated wild-type and PKCθ-deficient iTreg cells showed comparable Foxp3 expression as well as suppressive activity. However, we observed a reduced percentage of Foxp3+CD25+ CD4+ T cells in the lymphatic organs of PKCθ-deficient mice. Taken together, our results suggest that while PKCθ is involved in Treg cell differentiation in vivo, it is dispensable for Treg-mediated suppression.

Role for coronin 1 in mouse NK cell function.

Coronin 1, a member of the evolutionary conserved WD repeat protein family of coronin proteins is expressed in all leukocytes, but a role for coronin 1 in natural killer (NK) cell homeostasis and function remains unclear. Here, we have analyzed the number and functionality of NK cells in the presence and absence of coronin 1. In coronin 1-deficient mice, absolute NK cell numbers and phenotype were comparable to wild type mice in blood, spleen and liver. Following in vitro stimulation of the activating NK cell receptors NK1.1, NKp46, Ly49D and NKG2D, coronin 1-deficient NK cells were functional with respect to interferon-γ production, degranulation and intracellular Ca2+ mobilization. Also, both wild type as well as coronin 1-deficient NK cells showed comparable cytotoxic activity. Furthermore, activation and functionality of NK cells following Vesicular Stomatitis Virus (VSV) infection was similar between wild type and coronin 1-deficient mice. Taken together these data suggest that coronin 1 is dispensable for mouse NK cell homeostasis and function.

Proof of Principle for a T Lymphocyte Intrinsic Function of Coronin 1A.

Coronins are evolutionarily conserved proteins that were originally identified as modulators of actin-dependent processes. Studies analyzing complete Coronin 1a knock-out mice have shown that this molecule is an important regulator of naive T cell homeostasis and it has been linked to immune deficiencies as well as autoimmune disorders. Nevertheless, because Coronin 1A is strongly expressed in all leukocyte subsets, it is not conclusive whether or not this phenotype is attributed to a T cell-intrinsic function of Coronin 1A. To address this research question, we have generated a T cell-specific Coronin 1a knock-out mouse (Coro1afl/fl × Cd4[Cre]). Deletion of Coronin 1A specifically in T cells led to a strong reduction in T cell number and a shift toward the effector/memory phenotype in peripheral lymphoid organs when compared with Cd4[Cre] mice expressing wild-type Coronin 1A. In contrast to peripheral lymphoid tissue, thymocyte number and subsets were not affected by the deletion of Coronin 1a Furthermore, T cell-specific Coronin 1a knock-out mice were largely resistant to the induction of autoimmunity when tested in the myelin oligoglycoprotein-induced EAE mouse model of multiple sclerosis. Thus, the phenotype of T cell-specific Coronin 1a deletion resembles the phenotype observed with conventional (whole body) Coronin 1a knock-out mice. In summary, our findings provide formal proof of the predominant T cell-intrinsic role of Coronin 1A.

Role of PKCtheta in macrophage-mediated immune response to Salmonella typhimurium infection in mice.

BACKGROUND: The serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta (-/-)) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections. RESULTS: Our results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta (-/-) mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages. CONCLUSIONS: Taken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.

The Nuclear Orphan Receptor NR2F6 Is a Central Checkpoint for Cancer Immune Surveillance.

Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is an orphan member of the nuclear receptor superfamily. Here, we show that genetic ablation of Nr2f6 significantly improves survival in the murine transgenic TRAMP prostate cancer model. Furthermore, Nr2f6(-/-) mice spontaneously reject implanted tumors and develop host-protective immunological memory against tumor rechallenge. This is paralleled by increased frequencies of both CD4(+) and CD8(+) T cells and higher expression levels of interleukin 2 and interferon γ at the tumor site. Mechanistically, CD4(+) and CD8(+) T cell-intrinsic NR2F6 acts as a direct repressor of the NFAT/AP-1 complex on both the interleukin 2 and the interferon γ cytokine promoters, attenuating their transcriptional thresholds. Adoptive transfer of Nr2f6-deficient T cells into tumor-bearing immunocompetent mice is sufficient to delay tumor outgrowth. Altogether, this defines NR2F6 as an intracellular immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity.

Novel protein kinase C θ: coronin 1A complex in T lymphocytes.

BACKGROUND: Protein kinase C-θ (PKCθ) plays an important role in signal transduction down-stream of the T cell receptor and T cells deficient of PKCθ show impaired NF-κB as well as NFAT/AP-1 activation resulting in strongly decreased IL-2 expression and proliferation. However, it is not yet entirely clear, how the function of PKCθ - upon T cell activation - is regulated on a molecular level. FINDINGS: Employing a yeast two-hybrid screen and co-immunoprecipitation analyses, we here identify coronin 1A (Coro1A) as a novel PKCθ-interacting protein. We show that the NH2-terminal WD40 domains of Coro1A and the C2-like domain of PKCθ are sufficient for the interaction. Furthermore, we confirm a physical interaction by GST-Coro1A mediated pull-down of endogenous PKCθ protein. Functionally, wild-type but not Coro1A lacking its actin-binding domain negatively interferes with PKCθ-dependent NF-κB, Cyclin D1 and IL-2 transactivation when analysed with luciferase promoter activation assays in Jurkat T cells. This could be phenocopied by pharmacological inhibitors of actin polymerization and PKC, respectively. Mechanistically, Coro1A overexpression attenuates both lipid raft and plasma membrane recruitment of PKCθ in CD3/CD28-activated T cells. Using primary CD3(+) T cells, we observed that (opposite to PKCθ) Coro1A does not localize preferentially to the immunological synapse. In addition, we show that CD3(+) T cells isolated from Coro1A-deficient mice show impaired IKK/NF-κB transactivation. CONCLUSIONS: Together, these findings both in Jurkat T cells as well as in primary T cells indicate a regulatory role of Coro1A on PKCθ recruitment and function downstream of the TCR leading to NF-κB transactivation.

Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

Protein kinase C θ (PKCθ) is involved in signaling downstream of the T cell antigen receptor (TCR) and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures) and in vivo (experimental autoimmune encephalomyelitis model, EAE) techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt), accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ) and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ-/- CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

Macrophages eliminate circulating tumor cells after monoclonal antibody therapy.

The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (FcγRI) and the low-affinity IgG-binding Fc receptor (FcγRIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity.