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Blockade of Tigit on AML-Derived M2 Macrophages Results in Reprograming into the M1 Phenotype and Enhances CD47-Mediated Phagocytosis


 Background: Bidirectional interactions between the tumor microenvironment (TME) and AML cells lead to disease progression through induction of angiogenesis, migration, cancer stemness and local immunosuppression. Leukemia-associated macrophages (LAM) constitute an important cell population within the TME, but little is known about the phenotype, function, and plasticity of these cells. In the present study we provide an extensive characterization of the macrophage population in patients with AML.
 Methods: The phenotype and expression of co-regulatory receptors was assessed on different bone marrow-derived CD68 +CD14 + LAM populations, in comparison to corresponding CD3 + T-cells and CD117 +CD34 + AML cells (n=35), as well as peripheral blood monocytes from healthy donors (HD, n=16) using multi-parameter flow cytometry. The expression of surface markers and the distribution of LAM subpopulations was correlated with clinical parameters. The effect of a blocking anti-TIGIT antibody on the in vitro plasticity on primary LAMs and monocyte-derived macrophages from healthy donors was investigated. Furthermore, we analyzed if the treatment with blocking anti-TIGIT and anti-CD47 antibodies could increase the anti-leukemic phagocytosis of AML cell lines and in vitro polarized monocyte-derived M2 macrophages.
 Results: Phenotypic analysis of M1 and M2 macrophages in AML and HD revealed that the predominant macrophage population in patients with AML is made up of immunosuppressive alternatively activated M2 LAMs defined by expression of CD163 and CD86 (M1 AML vs. HD p<0.01 and M2 AML vs. HD p=0.02). These M2 LAMs contained significantly higher frequencies of cells expressing the immune checkpoint receptors TIGIT and TIM-3 than M1 LAMs (TIGIT + M2 vs. M1 p<0.01 and TIM-3 + M2 vs. M1 p<0.01, respectively). Regarding co-expression of multiple co-inhibitory receptors, the frequency of macrophages co-expressing TIM-3 or LAG-3 with TIGIT was higher in samples from AML patients in comparison to HDs (p=0.01 and p<0.01, respectively). This difference was caused by the significant up-regulation of TIM-3 and LAG-3 on TIGIT + M2 LAMs in comparison to their corresponding M1 LAMs (p<0.01and p<0.01, respectively).
 Importantly, in vitro blockade of TIGIT in primary LAMs of AML patients or differentiated PB-derived M2 macrophages of HDs resulted in a change in polarization from the M2 towards the M1 phenotype after 24 hours (AML: anti-TIGIT vs. IgG2a p<0.01, n=7 and HD: anti-TIGIT vs. IgG2a p=0.02, n=3).
 Moreover, the additional blockade of TIGIT on PB-derived M2 macrophages augmented the anti-CD47-mediated phagocytosis of the AML cell lines MOLM-13 and MV4-11 after 4 hours (MOLM-13: anti-CD47 vs. IgG1a 31% vs. 10.9%, p=0.04; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 31% vs. 46.4%, p<0.01 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 46.4% vs. 13.6%, p<0.01, n=3 and for MV4-11: anti-CD47 vs. IgG1a 14.4% vs. 7.345%, p=0.03; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 14.4% vs. 28.6%, p=0.03 and combined anti-CD47 + anti-TIGIT vs. IgG1a + IgG2a 28.6% vs. 12.85%, p=0.04, n=2).
 Next, we correlated the phenotypic data with clinical parameters. AML patients of the intermediate risk group according to ELN criteria exhibited a significantly higher frequency of M2 LAMs co-expressing TIGIT and LAG-3 than those in the favorable group (p=0.04 and p=0.01). Moreover, the frequency of TIM-3 + M2 LAMs was significantly increased in patients with adverse and intermediate risk in comparison to those with a favorable risk (p=0.01, p=0.0053). Furthermore, TIGIT + M2 LAMs were significantly more frequent in patients with the FLT3 ITD mutation in comparison with the wilde type (p=0.03).
 Conclusions: Our findings suggest that the proven clinical effect of monoclonal antibodies against TIGIT and TIM-3 in cancer may be due in part to their action on macrophages and depend on macrophage polarization. Our study identifies TIGIT + M2 LAMs co-expressing TIM-3 and LAG-3 as a promising effector population in AML. Further experiments should be conducted to investigate macrophage-mediated cytotoxicity in AML.
 
 
 Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: meeting attendance; Servier: Consultancy, Honoraria, Other: meeting attendance; Jazz Pharmaceuticals: Other: meeting attendance; Novartis: Other: meeting attendance. Bokemeyer: BMS: Honoraria, Other: Travel accomodation, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck Serono: Consultancy, Other: Travel accomodation ; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicine: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Gylcotope GmbH: Research Funding; GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding; Janssen-Cilag: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Merck KGaA: Honoraria; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; Lilly/ImClone: Consultancy; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Fiedler: Celgene: Consultancy; Servier: Consultancy, Other: support for meeting attendance; Abbvie: Consultancy, Honoraria; Morphosys: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Novartis: Consultancy; ARIAD/Incyte: Consultancy; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding.
