Macrophage-mediated Phagocytosis Research Articles

A Sampling of Highlights from the Literature

Inflamed tumor (by Y. Rosen via Wikimedia Commons)The use of 300 patient samples across 22 cancer types from PD-1 blockade trials revealed that inflammatory gene expression profiling and the tumor mutational burden are independent biomarkers for objective response rates to anti–PD-1. This finding was validated in a set of more than 6,000 tumors from TCGA database, in which biomarker-defined patterns identified potential targets to overcome resistance to therapy.Cristescu R, …, Kaufman D. Science 2018 Oct 12;362:eaar3593.Clustered macrophages from hyperprogression (from Fig. 2 of Lo Russo et al., Clin Cancer Res 2018)Blockade of immune checkpoints in some NSCLC patients causes a hyperprogression that correlates with the presence of myeloperoxidase+ myeloid cells and CD163+CD33+PD-L1+ macrophages. When mice with patient-derived xenografts are treated with anti–PD-1, tumors hyperprogress and increase their numbers of M2 macrophages. Treatment with anti–PD-1 F(ab)2 fragments, however, does not induce hyperprogression. Thus, stimulation of innate cell Fc receptors within tumors can sustain hyperprogression.Lo Russo G, …, Garassino MC. Clin Cancer Res 2018 Sep 11. DOI: 10.1158/1078-0432.CCR-18-1390.Blocking phagocytosis inhibition (from Ahkâm via Free Icons PNG)Tumor-promoting macrophages can express CD47, which inhibits phagocytosis. A phase Ib trial tested blockage of the CD47-SIRPα interaction, combined with rituxibab (anti-CD20), in 22 patients with relapsed/refractory non-Hodgkin lymphoma. Refractory lymphoma cells are eliminated via macrophage-mediated phagocytosis. Half of patients achieved objective responses, with manageable grade 1 or 2 adverse effects. This combination boosts antitumor immunity and shows promise for patients with aggressive lymphomas.Advani R, …, Smith SM. N Engl J Med 2018 Nov 1;379:1711–21.Macrophage-surrounded tumor (by R. Weissleder via NCI).Complement is key to inflammation and may promote protumoral microenvironments. Urokinase+ macrophages in squamous carcinomas produce C5a, which attracts other phagocytes and induces their degranulation while promoting secretion of proangiogenic and tissue remodeling cytokines. PMX-53 blocks C5a from activating its receptor and causes an increase in M1 macrophages, setting up chemokine and cytokine conditions that, with chemotherapy, recruit CD8+ T cells and inhibit tumor growth.Medler TR, …, Coussens LM. Cancer Cell 2018 Oct 8;34:561–78.Identifying standout epitopes (by Trajaner via Pixabay)Identification of immunodominant epitopes in disease can be accomplished with a deep neural network for genome-scale predictions. The described "bacteria originated T cell antigen" (BOTA) predictor identifies MHC class II–restricted epitopes from model pathogens. The CD4+ T-cell epitopes were validated through high-throughput screening that could identify reactive TCRs based on the genomic sequence of bacterial epitopes. Application of this technique to cancer may help identify immunodominant epitopes in tumors.Graham DB, …, Xavier RJ. Nat Med 2018 Oct 22;24:1762–72.Protein barcodes for CRISPR (by by Marius Walter via Wikimedia Commons)CRISPR can be used to produce unique nucleotide sequences (barcodes), and usually these are “read” on the DNA level. A barcoding system at the protein level called Pro-codes has been developed. Synthesized sequences encoding 3 combinations of 14 different linear epitopes, results in more than 300 Pro-codes. Each Pro-code was paired to CRISPR single-guide RNA and used to screen for genes affecting breast cancer sensitivity to antigen-specific T-cell responses. Identification of Rtp4, Psmb8, and Socs1 indicates this method can identify mechanisms of immune evasion.Wroblewska A, …, Brown BD. Cell 2018 Nov 1;175:1141–55.

Interleukin 4 Induces Macrophage-Mediated Phagocytosis of Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive disease associated with poor prognosis, hence, new therapeutic strategies are needed. The AML cells reside in the bone marrow niche and depend on external signals such as cytokines for their growth and survival. In contrast to providing a supporting role, cytokines can act as negative regulators of AML cells. We previously identified that interleukin 4 (IL-4) induces apoptosis of AML cells in a Stat6-dependent manner and demonstrated strong anti-leukemic activity of IL-4 in a murine AML model driven by MLL-AF9 (Peña-Martínez et al., Leukemia 2017). Ectopic expression of IL-4 in AML cells resulted in prolonged survival in C57BL/6 mice, with mice almost devoid of leukemia cells in bone marrow and spleen upon the time of sacrifice. However, the IL-4-induced apoptosis of AML cells did not fully explain the strong antileukemic activity of IL-4 in vivo.To investigate whether the anti-leukemic effect of ectopic IL-4 expression in vivo was microenvironment dependent, we used IL-2 receptor gamma(-/-) immunodeficient mice, which lack a functional IL-4 receptor type I complex, as recipients for transplantation of murine MLL-AF9 AML cells. Notably, there was no antileukemic activity of IL-4 in these mice, suggesting that the in vivo anti-leukemic effect of IL-4 was predominantly microenvironment-dependent. To identify the cell population that was responsible for the antileukemic effect of IL-4 stimulation, we performed flow cytometry analysis of bone marrow and spleen cells in mice exposed to high IL-4 levels. We found that the anti-leukemic activity of IL-4 was associated with a significant expansion of F4/80+ macrophages in both the bone marrow and spleen, which was confirmed by immunohistochemistry.To determine whether macrophages were responsible for the anti-leukemic effect of IL-4, we depleted macrophages with clodronate liposomes in mice receiving IL-4-secreting AML cells. Depletion of macrophages partially rescued the anti-leukemic effect of IL-4, indicating that macrophages stimulated with IL-4 were responsible for the antileukemic activity. Consistent with these findings, in macrophage differentiation assays with either murine or human monocytes, IL-4 enhanced the phagocytosis of leukemia cells.To characterize the IL-4-induced macrophages, we performed RNA sequencing of F4/80+ macrophages from animals exposed to high IL-4 levels. Gene expression analysis revealed IL-4-induced M2 polarization of the macrophages, as key markers of M2 activation such as Arg1, Retnla and Chil3 were upregulated. Moreover, consistent with the in vitro findings, gene set enrichment analysis revealed an enrichment of phagocytosis signatures in the macrophages stimulated with IL-4.Strikingly, we found that exposure of AML cells to IL-4 resulted in strong upregulation of CD47, a ‘don't eat me signal’, which protects cells from macrophage-mediated phagocytosis. By CRISPR/Cas9-mediated disruption of Stat6 in IL-4 treated AML cells, we found that the CD47 upregulation was Stat6 dependent, recognizing a previously unknown link between IL-4 signalling and CD47 upregulation. Consistent with this finding, combined antibody blockade of CD47 on leukemia cells and IL-4 stimulation of macrophages resulted in enhanced phagocytosis of AML cells.Taken together, this study shows that IL-4 has anti-leukemic activity by activating macrophages, findings that might translate into new immunotherapeutic opportunities in AML and possibly other malignancies. DisclosuresNo relevant conflicts of interest to declare.

CD47 Expression Defines the Efficacy of Rituximab in Non-Germinal Center B-Cell (non-GCB) Diffuse Large B-Cell Lymphoma (DLBCL)

▪CD47 is an immune-checkpoint protein that binds SIRPa on immune cells to deliver an inhibitory “don't eat me” signal. CD47 is a prominent new target in B-cell malignancies, in which CD47 antibody in combination with the CD20 antibody rituximab (R) is explored in clinical trials. Addition of R to CHOP chemotherapy significantly improves survival of patients with DLBCL, but how CD47 expression contributes to this positive impact of R on patient outcome is currently unclear. Especially in the context of the different cell-of-origin DLBCL subtypes (Germinal Center B-cell (GCB) vs non-GCB DLBCL).We therefore investigated the impact of CD47 expression on rituximab efficacy. We first studied a clinically well annotated transcriptome cohort of 939 DLBCL patients treated with either CHOP or R-CHOP, and we validated these findings in functional in vitro assays. Our analyses showed that overall survival (OS) for patients with high CD47 expression (i.e. above median) is worse compared to patients with low CD47 expression (i.e. below median) after R-CHOP (p=0.001), but not after CHOP treatment (p=0.645). Correspondingly, patients with low CD47 expression benefited most from addition of R to CHOP (HR=0.32, CI:[0.21-0.50], p<0.0001). Non–GCB patients showed a worse survival compared to GCB DLBCL patients after R-CHOP treatment (p<0.0001). Interestingly, with respect to COO classification, high expression of CD47 impacted the OS in non-GCB R-CHOP-treated patients (HR=1.9, CI:[1.14-3.26], p=0.013) but not in GCB DLBCL patients (HR=1.16, CI:[0.68-1.99], p=0.58). Importantly, high CD47 expression was found to determine the worse overall survival of non-GCB versus GCB patients (p=0.0006). No difference in OS was observed between GCB and non-GCB-patients with low CD47 expression (p=0.7662). Pro-phagocytic receptors expressed on DLBCL, i.e. SLAMF7, did not determine outcome upon R-CHOP treatment. In multivariate analysis, high CD47 expression remained significantly associated with poor survival only in non-GCB-patients.We further investigated this differential impact of CD47 in non-GCB and GCB subtypes in vitro. Non-GCB and GCB DLBCL cell lines (n=6) were mixed with allogeneic M1 macrophages and treated with a CD47 blocking antibody, which comprises human IgG4. Results confirmed that macrophage-mediated phagocytosis by R was augmented with CD47-blocking antibody only in non-GCB DLBCL cell lines, and not in GCB DLBCL cell lines. Also, CD47 blocking co-treatment also increased the number of tumor cells ingested per macrophage, with a clear significant increase in phagocytic index in non-GCB cells, but not in GCB DLBCL cells (p=0.001).In conclusion, the patient benefit of addition of R to CHOP is limited by CD47 specifically in non-GCB patients. In addition, we confirmed that CD47-blocking only augmented rituximab-mediated phagocytosis in non-GCB cell-lines. Together, these data suggest that especially non-GCB DLBCL patients with a currently worse prognosis may benefit from CD47-targeted therapy combined with rituximab. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single-Arm Phase II Study of MOR208 Combined with Lenalidomide in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: L-Mind

Single-Arm Phase II Study of MOR208 Combined with Lenalidomide in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma: L-Mind

Attenuation of CD47-SIRPα Signal in Cholangiocarcinoma Potentiates Tumor-Associated Macrophage-Mediated Phagocytosis and Suppresses Intrahepatic Metastasis

The involvement of chronic inflammation in cholangiocarcinoma (CCA) progression is well established. Cluster of differentiation 47 (CD47) is mutually expressed in various cancers and serves as a protective signal for phagocytic elimination. CD47 signaling blockage is a recent treatment strategy; however, little is known regarding CD47 in CCA. Therefore, the potential use of CD47 targeting in CCA was focused. CD47 was highly expressed in CCA compared to hepatocellular carcinoma (HCC). Disturbance of CD47-signal regulatory protein-α (SIRPα) interaction by blocking antibodies promoted the macrophage phagocytosis. The therapeutic potential of anti-CD47 therapy was demonstrated in liver metastatic model; alleviation of cancer colonization together with dense macrophage infiltrations was observed. The usefulness of anti-CD47 was emphasized by its universal facilitating macrophage activities. Moreover, increased production of inflammatory cytokines, such as IL-6 and IL-10, in macrophage exposed to CCA-conditioned media suggested that CCA alters macrophages toward cancer promotion. Taken together, interfering of CD47-SIRPα interaction promotes macrophage phagocytosis in all macrophage subtypes and consequently suppresses CCA growth and metastasis. The unique overexpression of CD47 in CCA but not HCC offers an exceptional opportunity for a targeted therapy. CD47 is therefore a novel target for CCA treatment.

CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin’s Lymphoma

BackgroundThe Hu5F9-G4 (hereafter, 5F9) antibody is a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis. 5F9 synergizes with rituximab to eliminate B-cell non-Hodgkin’s lymphoma cells by enhancing macrophage-mediated antibody-dependent cellular phagocytosis. This combination was evaluated clinically.MethodsWe conducted a phase 1b study involving patients with relapsed or refractory non-Hodgkin’s lymphoma. Patients may have had diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 5F9 (at a priming dose of 1 mg per kilogram of body weight, administered intravenously, with weekly maintenance doses of 10 to 30 mg per kilogram) was given with rituximab to determine safety and efficacy and to suggest a phase 2 dose.ResultsA total of 22 patients (15 with DLBCL and 7 with follicular lymphoma) were enrolled. Patients had received a median of 4 (range, 2 to 10) previous therapies, and 95% of the patients had disease that was refractory to rituximab. Adverse events were predominantly of grade 1 or 2. The most common adverse events were anemia and infusion-related reactions. Anemia (an expected on-target effect) was mitigated by the strategy of 5F9 prime and maintenance dosing. Dose-limiting side effects were rare. A selected phase 2 dose of 30 mg of 5F9 per kilogram led to an approximate 100% CD47-receptor occupancy on circulating white and red cells. A total of 50% of the patients had an objective (i.e., complete or partial) response, with 36% having a complete response. The rates of objective response and complete response were 40% and 33%, respectively, among patients with DLBCL and 71% and 43%, respectively, among those with follicular lymphoma. At a median follow-up of 6.2 months among patients with DLBCL and 8.1 months among those with follicular lymphoma, 91% of the responses were ongoing.ConclusionsThe macrophage checkpoint inhibitor 5F9 combined with rituximab showed promising activity in patients with aggressive and indolent lymphoma. No clinically significant safety events were observed in this initial study. (Funded by Forty Seven and the Leukemia and Lymphoma Society; ClinicalTrials.gov number, NCT02953509.)

Unveiling the fate of adhering bacteria to antimicrobial surfaces: expression of resistance-associated genes and macrophage-mediated phagocytosis

Since most antibacterial coatings reported to fight biomaterial-associated infections (BAI) fail in completely preventing bacterial colonization, it is crucial to know the impact of that small fraction of adhered bacteria in BAI recrudescence. This study aims to understand the fate of Staphylococcus aureus able to adhere to an antimicrobial coating previously developed, in terms of potential development of bacterial resistance and their macrophage-mediated phagocytosis. Antimicrobial coating comprised the co-immobilization of Palm peptide and DNase I onto polydimethylsiloxane. Expression of genes associated to resistance and virulence mechanisms showed that cells in contact with antimicrobial surfaces for a long period of 30 days, exhibit genes equally or less expressed, as compared to cells recovered from control surfaces. Recovered cells also exhibit the same susceptibility patterns, which strengthens the evidence of no resistance development. Remarkably, cells adhered to modified surfaces shows a reduced metabolic activity upon vancomycin treatment unlike the cells found on control surfaces, which can be identified as a clinical opportunity for prophylactically administration after implant surgery. Furthermore, results highlight that functionalization of PDMS with Palm and DNase I should not compromise the action of host immune cells. The overall results reinforce the potential of this antimicrobial strategy to fight BAI.

IFN-γ protects from apoptotic neutrophil-mediated tissue injury during acute Listeria monocytogenes infection.

Listeria monocytogenes (LM) is a foodborne Gram-positive intracellular pathogen that can cause listeriosis in humans and animals. Although phagocytes are known to be involved in the response to this infection, the role of neutrophils is not entirely clear. Here, we have demonstrated that soon after LM infection, a large number of IFN-γ-producing neutrophils quickly accumulated in the spleen, blood, and peritoneal cavity. Both in vivo and in vitro experiments demonstrated that neutrophils were an important source of IFN-γ. IFN-γ played a critical protective role against acute LM infection, as demonstrated by the poor survival of Ifng-/- mice. Moreover, IFN-γ promoted bacterial clearance by the neutrophils, thereby inhibiting LM-induced neutrophil apoptosis and spleen damage. In addition to this, IFN-γ could effectively drive macrophage-mediated phagocytosis of apoptotic neutrophils, which was accompanied with TGF-β secretion and was involved in protection against tissue injury. Importantly, by phagocytizing apoptotic neutrophils, macrophages obtained myeloperoxidase, an important bactericidal molecule only produced by neutrophils, which further promoted the antibacterial activity of macrophages. These findings demonstrate that neutrophils are an important source of IFN-γ at the early stage of LM infection, which is characterized by both LM elimination and tissue-protective effects.

Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Like conventional transplants, immunosuppression is required to facilitate survival and function of human embryonic stem cell (hESC) derivatives after implantation into xenogeneic recipients. We have previously reported that T cells alone are sufficient to reject allogeneic murine ESC derivatives; and strategies that inhibit T-cell activation, including coreceptor and costimulation blockade, prevent hESC derivatives from being rejected. This study aimed to investigate, in addition to T cells, whether macrophages contribute to transplant rejection of hESC xenografts with nonobese diabetic (NOD)/SCID mice that lack functional T and B cells but have macrophages. We show that acute rejection against hESC-derived endothelial cells (hESC-ECs) was mediated, to some degree, by infiltrating macrophages that phagocytosed them. Transgenic expression of murine CD47 on cell surface of hESC-ECs mitigates macrophage-mediated phagocytosis and improves their survival after transplantation. Our results highlight that innate immune cells, such as macrophages, can reject hESC derivatives, raising concern against the use of NOD/SCID as transplant recipients for testing in vivo function of hESC-derived tissues. Augmenting CD47 signaling promotes survival and function of hESC derivatives after xenogeneic transplantation.-Leung, C. S., Li, J., Xu, F., Wong, A. S. L., Lui, K. O. Ectopic expression of recipient CD47 inhibits mouse macrophage-mediated immune rejection against human stem cell transplants.

Inhibition of autophagy potentiated the anti-tumor effects of VEGF and CD47 bispecific therapy in glioblastoma.

Glioblastoma, characterized by extensive microvascular proliferation and invasive tumor growth, is one of the most common and lethal malignancies in adults. Benefits of the conventional anti-angiogenic therapy were only observed in a subset of patients and limited by diverse relapse mechanism. Fortunately, recent advances in cancer immunotherapy have offered new hope for patients with glioblastoma. Herein, we reported a novel dual-targeting therapy for glioblastoma through simultaneous blockade of VEGF and CD47 signaling. Our results showed that VEGFR1D2-SIRPαD1, a VEGF and CD47 bispecific fusion protein, exerted potent anti-tumor effects via suppressing VEGF-induced angiogenesis and activating macrophage-mediated phagocytosis. Meanwhile, autophagy was activated by VEGFR1D2-SIRPαD1 through inactivating Akt/mTOR and Erk pathways in glioblastoma cells. Importantly, autophagy inhibitor or knockdown of autophagy-related protein 5 potentiated VEGFR1D2-SIRPαD1-induced macrophage phagocytosis and cytotoxicity against glioblastoma cells. Moreover, suppression of autophagy led to increased macrophage infiltration, angiogenesis inhibition, and tumor cell apoptosis triggered by VEGF and CD47 dual-targeting therapy, thus eliciting enhanced anti-tumor effects in glioblastoma. Our data revealed that VEGFR1D2-SIRPαD1 alone or in combination with autophagy inhibitor could effectively elicit potent anti-tumor effects, highlighting potential therapeutic strategies for glioblastoma through disrupting angiogenetic axis and CD47-SIRPα anti-phagocytic axis alone or in combination with autophagy inhibition.

Generation, safety and immunogenicity of an Actinobacillus pleuropneumoniae quintuple deletion mutant SLW07 (ΔapxICΔapxIICΔorf1ΔcpxARΔarcA)

We inactivated a virulence determinant, ArcA, in an Actinobacillus pleuropneumoniae quadruple deletion mutant SLW06 (ΔapxICΔapxIICΔorf1ΔcpxAR, serovar 1), and a quintuple deletion mutant SLW07 was generated. SLW07 showed decreased adherence to and invasion of host cells, compared to its parent strain SLW06. SLW07 was more sensitive in RAW264.7 macrophage-mediated phagocytosis and clearance. SLW07 was less virulent in mice. An immunization assay indicated that both SLW07 and SLW06 preferentially stimulated T helper cell type 2 response in mice. Live vaccines induced the production of interleukin-6 and tumor necrosis factor-α by splenic lymphocytes. Furthermore, the protective immunity of SLW07 was not affected after ArcA mutation. Immunization with SLW07 could provide a complete protection following virulent A. pleuropneumoniae challenge in mice. Our results suggest that SLW07 is a promising live vaccine candidate, which is further attenuated from and shares similar protective efficacy with its quadruple deletion parent SLW06.

Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

Beta-defensins 2 and 3 (BD2 and BD3) are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA). Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1) and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.

Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice.

Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage's ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Alleviates Atherosclerosis via Enhancing Efferocytosis and Facilitating a Shift in the Th1/Th2 Balance Toward Th2 Polarization

Background/Aims: We and other groups have demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (ALA-SDT) induces macrophage and foam cell apoptosis and stabilizes atherosclerosis (AS) plaques in animal models. Lymphocytes also play vital roles in the development of AS. The primary purpose of the present study was to investigate the effects of ALA-SDT on T helper (Th) cell fate and function, Th subset differentiation, and atherosclerotic lesion stability. Methods: We utilized ALA-SDT on Western diet-fed apoE^-/-mice in vivo and human Jurkat cells in vitro. Hematoxylin and eosin staining and TUNEL assays were used to evaluate the atherosclerotic plaque size and apoptosis within the atheroma. ALA induced cytotoxicity on cultured Jurkat cells was determined with CCK-8 assay. To address the mechanisms, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (MPTP) opening were evaluated by staining with fluorescent probes. Western blot analysis and confocal microscopy were used to analyze the protein levels of caspases, Bax and cytochrome c and the release of cytochrome c. Cell apoptosis and necrosis and phagocytosis were examined by flow cytometry. ELISAs and immunofluorescent staining were used to assess the corresponding cytokine levels and Th subset cell numbers within the atheroma. Results: Our studies revealed that ALA-SDT significantly enhanced CD4⁺ cell apoptosis and macrophage-mediated phagocytosis and hence reduced the necrotic core size. ALA-SDT activated the mitochondrial apoptotic signaling pathway with minimal necrosis in Jurkat cells. ALA-SDT inhibited the Th1 response and enhanced the Th2 response. These effects of ALA-SDT were mediated primarily through the generation of ROS. Conclusion: ALA-SDT alleviates AS by enhancing cytotoxic effects on Th cells, subsequently stimulating efferocytosis and facilitating a shift in the Th1/Th2 balance toward Th2 cells, a discovery that might help elucidate the mechanism underlying SDT as a potential treatment to prevent atherothrombotic events.

Abstract B002: CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells

Abstract Most human cancers express CD47 to escape the macrophage surveillance. To reinstate macrophage functionality in clearing tumor cells, targeting CD47 has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines of both solid and blood tumors that are frequently used in cancer drug research and in Mixeno® animal study in Crown Bioscience for CD47. Despite its being reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47 despite gene expression data, such as RNA-seq data, suggesting that they do. To establish a robust and reproducible in vitro assay platform to support anti-CD47 drug research, we applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve &amp;gt; 80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was cocultured with target tumor cells labeled for flow cytometry analysis. In the presence of anti-CD47 antibody, steady phagocytosis effect was observed with aCD47 BRIC126 antibody and target tumor cells expressing high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. To prove the finding, we constructed isogenic cell models from the same parental cells in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems are also subject to the CD47 biology studies and can be served as positive and negative controls in phagocytosis assays. Detailed data will be discussed. Citation Format: Huajun Yang, Zhongliang Li, Beibei Tang, Frank Xing, Qian Shi. CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B002.

Abstract B007: Potent antitumor activity of a novel and orally available small-molecule antagonist targeting the CD47/SIRPα pathway

Abstract Antibodies targeting PD-1 and PD-L1 towards stimulation of T cells have revolutionized cancer therapy because of the highly durable response in multiple cancer indications. Although macrophages and other myeloid immune cells offer much promise as effectors of cancer immunotherapy, efforts to modulate them for therapeutic benefit have gained momentum only in the last few years. The CD47/signal regulatory protein alpha (SIRPα) axis is a critical regulator of myeloid cell activation and serves as an immune checkpoint for macrophage-mediated phagocytosis. Because of its frequent upregulation in several cancers, CD47 contributes to immune evasion and cancer progression. Disruption of CD47-SIRPα interaction is now being evaluated as a therapeutic strategy for cancer with the use of monoclonal antibodies targeting CD47 or SIRPα, and engineered receptor decoys. In view of the requirement for the intravenous dosing for all reported CD47 targeting agents, we sought to discover and develop an orally available small-molecule CD47 antagonist. An oral CD47 agent potentially offers the convenience, flexibility to adjust the dose and schedule to address any emergent adverse events, and ease of combination therapy. Through rational design based on the crystal structure of CD47/SIRPα interacting interface, we generated a series of peptides capable of disrupting CD47-SIRPα interactions. Relative binding affinities of the peptides were determined in a newly established cellular assay that utilizes a SIRPα-derived peptide as the probe. The high-affinity peptides were further truncated to identify the shortest peptide pharmacophore. The elements of this pharmacophore were incorporated into nonpeptidic small-molecule scaffolds, resulting in lead compounds disrupting CD47/SIRPα interaction. The lead CD47 antagonists induced phagocytototic activity of human macrophages to a similar extent as commercially available anti-CD47 antibodies. Further optimization of these leads resulted in compounds with desirable physicochemical properties and good oral bioavailability. An advanced lead CD47 antagonist inhibited primary tumor growth (~90%TGI) in a mouse syngeneic model of B-cell lymphoma upon twice-a-day oral dosing. Biomarker characterization and efficacy studies in additional tumor models are ongoing. These findings support further development of these orally bioavailable agents for use in the clinic. Citation Format: Pottayil G. Sasikumar, Chennakrishnareddy Gundala, Wesley R. Balasubramanian, Sudarshan S. Naremaddepalli, Archana Bhumireddy, Sandeep S. Patil, Amit A. Dhudashiya, Vijaysai Rayavarapu, Anirudha Lakshminarasimhan, Dodheri S. Samiulla, Kiran Aithal, Girish Daginakatte, Murali Ramachandra. Potent antitumor activity of a novel and orally available small-molecule antagonist targeting the CD47/SIRPα pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B007.

Correction to: Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

In the original publication, the fifth author name was erroneously published as "Patmika Jiaravuthiasan". The correct author name should read as, "Patmika Jiaravuthisan". The original article was corrected.

Abstract 2653: The anti-myeloma activity of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, is enhanced when combined with a proteasome inhibitor

Abstract CD47 is transmembrane glycoprotein that delivers an anti-phagocytic (“do not eat”) signal by binding to its receptor, signal-regulatory protein α (SIRPα), on the surface of macrophages. Many tumors, including multiple myeloma (MM), express high levels of CD47 as a means to exploit the CD47-SIRPα pathway and escape macrophage-mediated immune surveillance. TTI-621 (SIRPαFc) is a soluble recombinant fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG1. It is designed to promote anti-tumor responses by blocking the CD47 “do not eat” signal and engaging activating Fcγ receptors on macrophages. We have previously shown that TTI-621 enhances phagocytosis of malignant cells in vitro and exhibits anti-tumor activity in acute myeloid leukemia and B lymphoma xenograft models. In this study we investigated the anti-myeloma activity of TTI-621 as both a single agent and in combination with bortezomib or carfilzomib, two proteasome inhibitors that are approved for use in MM patients. The ability of TTI-621 to trigger macrophage phagocytosis of MM cells was assessed using a flow cytometry-based phagocytosis assay. Blockade of CD47 using TTI-621 effectively triggered macrophage-mediated phagocytosis of MM cells, and this anti-tumor effect was significantly enhanced by pre-treatment of MM cells with bortezomib or carfilzomib. In order to understand the molecular mechanism behind this enhanced phagocytic effect, we performed immunophenotyping of the tumor cells and observed an upregulation of pro-phagocytic “eat” signals on the tumor cell surface that potentially augment the phagocytic response. To investigate whether CD47 blockade in the context of proteasome inhibition translates to enhanced anti-tumor activity in vivo, we employed a MM xenograft model. NOD.SCID mice were subcutaneously engrafted with human MM cells followed by treatment with TTI-621, in the absence or presence of concurrent administration of a proteasome inhibitor. As monotherapies, both TTI-621 and the proteasome inhibitor reduced tumor burden relative to vehicle controls. Moreover, the combination of CD47 blockade and proteasome inhibition resulted in a greater reduction in tumor growth as compared to the two drugs alone. In conclusion, these data demonstrate that TTI-621 exhibits anti-myeloma activity that is further enhanced by combination with bortezomib or carfilzomib. TTI-621 monotherapy is currently being evaluated in a Phase 1b study in patients with MM and other hematological malignancies (NCT02663518). These data provide a rationale to evaluate a combination study of TTI-621 and a proteasome inhibitor in MM patients. Citation Format: Emma Linderoth, Simone Helke, Vivian Lee, Tapfuma Mutukura, Mark Wong, Gloria H. Lin, Lisa D. Johnson, Xinli Pang, Jeff Winston, Penka S. Petrova, Robert A. Uger, Natasja N. Viller. The anti-myeloma activity of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, is enhanced when combined with a proteasome inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2017-2653

SIRPα-CD47 Blockade–Mediated Tumor Cell Phagocytosis Requires SLAMF7

Abstract SLAMF7 expression is required for macrophage-mediated phagocytosis of hematopoietic tumor cells.

Human CD200 suppresses macrophage-mediated xenogeneic cytotoxicity and phagocytosis.

Various strategies, such as the generation of alpha-1,3-galactosyltransferase knocked-out pigs and CD55 transgenic pigs, have been investigated to inhibit pig to human xenogeneic rejection. Our aim is to develop strategies to overcome the hurdle of not only hyper acute rejection, but also that of cellular xenogeneic rejection (CXR). Although macrophages have been well known to play a critical role in CXR, monocyte/macrophage-mediated xenogeneic rejection has not been well studied. In this study, we evaluated the effect of CD200 in xenogeneic rejection by macrophages. Naïve swine endothelial cells (SEC) and SEC/CD200 were co-cultured with M0 macrophages and the cytotoxicity was measured by a WST-8 assay. The phagocytosis of SEC and SEC/CD200 by macrophages was analyzed by flow cytometry. While CD200 failed to suppress a significant amount of cytotoxicity against SEC by monocytes, M0 macrophage-mediated cytotoxicity was significantly suppressed by human CD200. The phagocytosis by M0 macrophages was also tested. The phagocytosis assay revealed that human CD200 suppresses M0 macrophage-mediated phagocytosis. Our findings indicate that human CD200 suppresses the xenogeneic rejection by CD200R+ macrophages and that the generation of hCD200 transgenic pigs for use in xenografts is very attractive for preventing the macrophage-mediated rejection.