A Critical Role for Innate Immunity In Allogeneic Hematopoietic Cell Rejection Via the TLR4/TRIF Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 77-77
Author(s):  
Hong Xu ◽  
Jun Yan ◽  
Ziqiang Zhu ◽  
Yiming Huang ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Innate Immunity ◽  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Innate Immune System ◽  
Critical Role ◽  
Innate Immune ◽  
Wild Type

Abstract Abstract 77 Adaptive immunity, especially T cells, has long been believed to be the dominant immune barrier in allogeneic transplantation. Targeting host T cells significantly reduces conditioning for bone marrow cell (BMC) engraftment. Innate immunity has been recently shown to pose a significant barrier in solid organ transplantation, but has not been addressed in bone marrow transplantation (BMT). Using T cell deficient (TCR-β/δ−/−) or T and B cell deficient (Rag−/−) mice, we found that allogeneic BMC rejection occurred early before the time required for T cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. Therefore, we hypothesized that by controlling both innate and adaptive immunity, the donor BMC would have a window of advantage to engraft. Survival of BMC in vivo was significantly improved by depleting recipient macrophages and/or NK cells, but not neutrophils. Moreover, depletion of macrophages and NK cells in combination with co-stimulatory blockade with anti-CD154 and rapamycin as a novel form of conditioning resulted in 100% allogeneic engraftment without any irradiation and T cell depletion. Donor chimerism remained stable and durable up to 6 months. Moreover, specific Vβ5½ and Vβ11 clonal deletion was detected in host CD4+ T cells in chimeras, indicating central tolerance to donor alloantigens. Whether and how the innate immune system recognizes or responds to allogeneic BMCs remains unknown. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors. The adaptor MyD88 transmits signals emanating from all TLR, except TLR3 while TRIF specifically mediates TLR3 and TLR4 signaling via type 1 IFN. To further determine the innate signaling pathways in allogeneic BMC rejection, B6 background (H2b) MyD88−/− and TRIF−/− mice were conditioned with anti-CD154/rapamycin plus 100 cGy total body irradiation and transplanted with 15 × 106 BALB/c (H2d) BMC. Only 33.3% of MyD88−/− recipients engrafted at 1 month, resembling outcomes for wild-type B6 mice. In contrast, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month, significantly higher than in MyD88−/− and wild-type B6 controls (P < 0.005). To determine the mechanism of innate signaling in BMC rejection, we examined whether TRIF linked TLR3 or TLR4 is the key pattern recognition receptor involved in BMC recognition. To this end, TLR3−/− and TLR4−/− mice were transplanted with BALB/c BMC with same conditioning. None of the TLR3−/− mice engrafted. In contrast, engraftment was achieved in 100% of TLR4−/− mice up to 6 months follow up. Taken together, these results suggest that rejection of allogeneic BMC is uniquely dependent on the TLR4/TRIF signaling pathway. Thus, our results clearly demonstrate a previously unappreciated role for innate immunity in allogeneic BMC rejection. Our current findings are distinct from prior reports demonstrating a critical role of MyD88 in rejection of allogeneic skin grafts and lung, and may reflect unique features related to BMC. The findings of the role of innate immunity in BMC rejection would lead to revolutionary changes in our understanding and management of BMT. This would be informative in design of more specific innate immune targeted conditioning proposals in BMT to avoid the toxicity. Disclosures: Bozulic: Regenerex LLC: Employment. Ildstad:Regenerex LLC: Equity Ownership.

Innate Immunity Is Responsible for Early Allorejection of Donor Bone Marrow Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3523-3523
Author(s):  
Hong Xu ◽  
Jun Yan ◽  
Ziqiang Zhu ◽  
Lala-Rukh Hussain ◽  
Yiming Huang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Innate Immunity ◽  
Immune System ◽  
T Cell ◽  
Innate Immune System ◽  
Immune Cell ◽  
Marrow Cell ◽  
Donor Cell ◽  
Innate Immune ◽  
Kinetics Of

Abstract Abstract 3523 Poster Board III-460 Adaptive immunity, especially T cells, has long been believed to be the dominant immune response in allogeneic transplantation. However, innate immunity has been recently shown to pose a significant barrier to the induction of tolerance to solid organ transplants. The role of the innate immune system in bone marrow cell alloreactivity has not been addressed. The innate immune system provides the first line of defense in the removal of pathogens because of the delay in generation of adaptive immune responses. Our present findings show that innate immunity is a significant first line barrier in bone marrow cell (BMC) rejection. To determine the effect of innate immune cell populations on rejection of donor BMCs, T cell deficient mice (TCR β/δ−/−) were used as BMC recipients in in vivo cytotoxicity assays (Figure A). TCRβ/δ−/− mice have normal innate immune cell populations but do not initiate adaptive cell-mediated cytotoxic or humoral responses. 20 × 106 CFSE labeled donor (high CFSE fluorescence intensity) and recipient control splenocytes (low CFSE fluorescence intensity) were injected into TCRβ/δ−/− and wildtype control B6 recipient mice. Donor cell survival was compared over time. Donor BALB/c splenocytes were eliminated in wildtype B6, with rejection complete by day 3. The kinetics of elimination of donor cells in TCRβ/δ−/− mice was similar to that for wildtype B6 controls, with donor cells eliminated by day 3. These results indicate that early rejection of the splenocytes in wildtype mice was T cell-independent. The acute rejection of BMC in wildtype B6 recipients occurred within 3 days, which is prior to the time required for T cell activation. Thus the effectors mediating BMC rejection would be the innate immune cells: macrophages, neutrophils, or NK cells. To rule out potential involvement of natural Abs in the cytotoxicity we observed in the TCRβ/δ−/− mice, Rag−/− mice were used as recipients (Figure B). Rag−/− mice do not produce mature T cells or B cells. 20 × 106 CFSE labeled donor (high CFSE intensity) and recipient control BM cells (low CFSE intensity) were injected into Rag−/−mice. Rag−/− and wildtype B6 control mice exhibited similar kinetics of donor cell cytolysis. The rapid elimination of allogeneic cells from immunocompetent mice is comparable with that observed in T cell- (TCRβ/δ−/−) or T and B cell- (Rag−/−) deficient mice indicates that allogeneic cells are subject to T cell-independent rejection at the early time period after cell infusion (≤ 3 days). As the kinetics of cytotoxicity were similar in experiments using either splenocytes or BMCs as target cells in our later experiments, our data suggest that the innate immune system is responsible for early allorejection of donor BMC at the early inductive period for adaptive immunity. These findings may have significant impact on the development of immune-based nonmyeloablative conditioning strategies and show for the first time that a dominant factor in BMC rejection is contributed by innate immune responses. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals Toll-like receptor-mediated innate immunity against herpesviridae infection: a current perspective on viral infection signaling pathways

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Wenjin Zheng ◽  
Qing Xu ◽  
Yiyuan Zhang ◽  
Xiaofei E ◽  
Wei Gao ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Immune System ◽  
Signaling Pathways ◽  
Critical Role ◽  
Innate Immune ◽  
Host Cells ◽  
Main Body ◽  
Current Perspective ◽  
Viral Components

Abstract Background In the past decades, researchers have demonstrated the critical role of Toll-like receptors (TLRs) in the innate immune system. They recognize viral components and trigger immune signal cascades to subsequently promote the activation of the immune system. Main body Herpesviridae family members trigger TLRs to elicit cytokines in the process of infection to activate antiviral innate immune responses in host cells. This review aims to clarify the role of TLRs in the innate immunity defense against herpesviridae, and systematically describes the processes of TLR actions and herpesviridae recognition as well as the signal transduction pathways involved. Conclusions Future studies of the interactions between TLRs and herpesviridae infections, especially the subsequent signaling pathways, will not only contribute to the planning of effective antiviral therapies but also provide new molecular targets for the development of antiviral drugs.

scholarly journals Effector CD8+ T Lymphocytes against Liver Stages of Plasmodium yoelii Do Not Require Gamma Interferon for Antiparasite Activity

2008 ◽  
Vol 76 (8) ◽  
pp. 3628-3631 ◽  
Author(s):  
Sumana Chakravarty ◽  
G. Christian Baldeviano ◽  
Michael G. Overstreet ◽  
Fidel Zavala
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protective Immunity ◽  
Critical Role ◽  
Plasmodium Yoelii ◽  
Gamma Interferon ◽  
Parasite Development ◽  
Wild Type ◽  
Ifn Γ

ABSTRACT The protective immune response against liver stages of the malaria parasite critically requires CD8+ T cells. Although the nature of the effector mechanism utilized by these cells to repress parasite development remains unclear, a critical role for gamma interferon (IFN-γ) has been widely assumed based on circumstantial evidence. However, the requirement for CD8+ T-cell-mediated IFN-γ production in protective immunity to this pathogen has not been directly tested. In this report, we use an adoptive transfer strategy with circumsporozoite (CS) protein-specific transgenic T cells to examine the role of CD8+ T-cell-derived IFN-γ production in Plasmodium yoelii-infected mice. We show that despite a marginal reduction in the expansion of naive IFN-γ-deficient CS-specific transgenic T cells, their antiparasite activity remains intact. Further, adoptively transferred IFN-γ-deficient CD8+ T cells were as efficient as their wild-type counterparts in limiting parasite growth in naive mice. Taken together, these studies demonstrate that IFN-γ secretion by CS-specific CD8+ T cells is not essential to protect mice against live sporozoite challenge.

scholarly journals Role of Bim in Regulating CD8+ T-Cell Responses during Chronic Viral Infection

2006 ◽  
Vol 80 (17) ◽  
pp. 8627-8638 ◽  
Author(s):  
Jason M. Grayson ◽  
Ashley E. Weant ◽  
Beth C. Holbrook ◽  
David Hildeman
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Viral Infection ◽  
T Cell Responses ◽  
Mutant Mice ◽  
Chronic Viral Infection ◽  
Wild Type ◽  
Cell Responses

ABSTRACT Apoptosis is critical for the development and maintenance of the immune system. The proapoptotic Bcl-2 family member Bim is important for normal immune system homeostasis. Although previous experiments have shown that Bim is critical for the apoptosis of antigen-specific CD8+ T cells during acute viral infection, the role of Bim during chronic viral infection is unclear. Using lymphocytic choriomeningitis virus clone 13 infection of mice, we demonstrate a role for Bim in CD8+ T-cell apoptosis during chronic viral infection. Enumeration of antigen-specific CD8+ T cells by major histocompatibility complex class I tetramer staining revealed that CD8+ DbNP396-404+ T cells, which undergo extensive deletion in wild-type mice, exhibited almost no decrease in Bim mutant mice. This contrasts with CD8+ DbGP33-41+ and CD8+ DbGP276-286+ T cells that underwent similar decreases in numbers in both Bim mutant and wild-type mice. Increased numbers of CD8+ DbNP396-404+ T cells in Bim mutant mice were due to lack of apoptosis and could not be explained by altered proliferation, differential homing to tissues, or increased help from CD4+ T cells. When viral titers were examined, high levels were initially observed in both groups, but in Bim mutant mice, clearance from the spleen and sera was slightly accelerated. These experiments demonstrate the critical role of Bim during chronic viral infection to down-regulate CD8+ T-cell responses and have implications for designing strategies for optimizing immunotherapies during situations where antigen persists, such as chronic infection, autoimmune syndromes, and cancer.

Critical Role of Hem1 in Hematopoietic Cell Chemotaxis and Innate Immunity to S. Pneumoniae

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1630-1630
Author(s):  
Robert B. Lorsbach ◽  
Yoon-Sang Kim ◽  
Jennifer Moore ◽  
Hope Smith-Sielicki ◽  
Tiffany M. Jones ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Innate Immunity ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Critical Role ◽  
Hematopoietic Cell ◽  
Wild Type ◽  
Regulatory Complex

Abstract Hem1 is a hematopoietic specific member of the HEM family proteins, which have been identified as components of the WAVE regulatory complex. To further characterize the role of Hem1 in hematopoietic cell function, we have generated mice deficient in Hem1 using gene targeting methodology. Hem1-deficient mice manifest several phenotypic abnormalities, including peripheral blood lymphopenia and neutrophilia, splenomegaly, premature mortality, and with variable penetrance thymic hypoplasia and alopecia. Flow cytometric analysis of peripheral blood and spleen demonstrated that Hem1−/ − mice have a marked reduction in peripheral B cells, changes that were evident in both adult and neonatal mice; the splenomegaly in Hem1−/ − mice was attributable to increased extramedullary hematopoiesis. The frequency of bone marrow B cell progenitors was also markedly reduced in Hem1−/ − mice. To assess the role of Hem1 in hematopoietic stem cell (HSC) function, competitive bone marrow transplantation assays were performed. In contrast to wild-type HSCs, Hem1-deficient HSCs had poor competitive repopulating activity in irradiated recipient mice. KSL cell analysis demonstrated no significant difference in the frequency of lin-c-kit+Sca1+ HSCs between wild-type and Hem1−/ − bone marrow, suggesting that the defective competitive repopulating activity of Hem1−/ − HSCs is attributable to defective bone marrow homing or stem cell niche interaction. Given the biochemical evidence implicating the HEM proteins as a component of the WAVE regulatory complex, we also assessed the ability of Hem1-deficient leukocytes to undergo cytoskeletal remodeling in vitro. Using a transwell assay, Hem1−/ − bone marrow storage pool neutrophils demonstrated markedly blunted chemotactic responses to formylated peptide which was attributable to defective f-actin formation. Hem1−/ − peripheral CD4+ T cells similarly manifested chemotactic defects in response to SDF-1, and showed blunted proliferation when stimulated with antibodies against CD3 and CD28. Finally, a model of Streptococcus pneumoniae infection was employed to test the role of Hem1 in the in vivo function of neutrophils. Hem1−/ − mice were dramatically more sensitive to S. pneumoniae than wild-type littermates, as manifested by the inability to eliminate S. pneumoniae organisms in vivo and higher mortality. In summary, Hem1 deficiency results in deficiencies and functional defects in multiple hematopoietic lineages due to defective signaling to the actin cytoskeleton, and importantly, Hem1 plays a critical role in innate immunity to S. pneumoniae in vivo.

MerTK Receptor Tyrosine Kinase Inhibition As a Potential Strategy to Augment Immune-Mediated Clearance of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4044-4044
Author(s):  
Lauren Shelby Page ◽  
Xiaodong Wang ◽  
H. Shelton Earp ◽  
Stephen Frye ◽  
Craig T Jordan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Receptor Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Wild Type ◽  
Vehicle Treatment ◽  
Equity Ownership

Abstract The MerTK receptor tyrosine kinase is aberrantly expressed in 80-100% of acute myeloid leukemia (AML), and inhibition of MerTK prolongs leukemia-free survival in vivo, making it an appealing therapeutic target. MerTK is normally expressed on macrophages (MMs) and dendritic cells (DCs) and facilitates engulfment of apoptotic debris. Through this process, MerTK promotes tolerogenicity and anti-inflammatory cytokine production. We hypothesized that inhibition of MerTK will also mediate anti-leukemia effects through relief of microenvironment immunosuppression and stimulation of innate MM and DC activity leading to enhanced T-cell immunity. To test this hypothesis, we transplanted wild-type C57BL/6 mice with syngeneic murine MLL-ENL fusion AML or primary mouse MLL-AF9 AML. We evaluated the effects of MerTK inhibition using MRX-2843, a MerTK kinase inhibitor. Mice were treated daily and survival was monitored. Treatment with MRX-2843 prolonged median survival (43 days) compared to vehicle treatment (17 days) in mice transplanted with MLL-ENL AML (p<0.0001), and MLL-AF9 primary murine AML (42 days with MRX-2843, 30 days with vehicle; p<0.05). To explore the immune effects of MerTK inhibition in AML, we evaluated in vivoMMs and DCs by flow cytometry. Compared to non-leukemic controls, vehicle-treated leukemic mice demonstrated a significant infiltration of MMs and DCs into the primary sites of leukemic burden (spleen, bone marrow). These MMs and DCs expressed high levels of co-inhibitory ligands PD-L1 and PD-L2. Treatment with MRX-2843 significantly decreased PD-L1 and PD-L2 expressing MM and DCs in the spleen (p<0.01) and bone marrow (p<0.01) down to non-leukemic levels. Similar results were not obtained when mice were treated with the non-MerTK targeted FLT3 inhibitor AC220, suggesting that these effects are likely specific to MerTK inhibition. We next evaluated effects of MerTK inhibition on the leukemia microenvironment. We confirmed that T-cells do not express MerTK in-vivowith or without AML, nor +/- MRX-2843. Compared to non-leukemic mice, leukemic mice significantly upregulated co-inhibitory receptors PD-1 and Tim-3 on CD4 and CD-8 T-cells in the spleen and bone marrow. Treatment with MRX-2843 in leukemic mice significantly decreased CD4 and CD8 T-cell PD-1 and Tim-3 expression in the spleen (p<0.05) and bone marrow (p<0.05) down to non-leukemic levels. We next evaluated the role of functional T-cells in the anti-leukemia effects of MerTK inhibition. Wild-type mice and mice harboring a T-cell receptor mutation (TCRa-/-) were transplanted with MLL-ENL AML, treated with MRX-2843 or vehicle, and monitored for symptoms of leukemia. Median survival of all vehicle-treated mice with leukemia was 20-22 days. Median survival of TCRa-/- mice treated with MRX-2843 was 30 days, whereas median survival of MRX-2843 treated wild-type mice was significantly extended to 40 days (p<0.01). To confirm these findings, leukemic wild-type mice were CD8 depleted and treated as above. Median survival of CD8-depleted MRX-2843 treated mice was significantly diminished (30 days) compared to isotype (control) + MRX-2843 treatment (40 days, p<0.01). These data suggest an additional non-cell autonomous role of T-cell anti-tumor immunity in mice treated with a MerTK inhibitor. We next evaluated peripheral serum cytokines using Luminex magnetic bead analysis. Treatment with MerTK inhibition significantly increased serum [IL-18] (p<0.01) and [MCP-3/CCL7] (p<0.05) by 2-fold compared to vehicle-treated mice. Additionally, there was no correlation between co-inhibitor expression and serum [IFN-g]. These changes appeared to be mediated through MerTK and not solely attributable to tumor burden given that MLL-ENL cells in culture did not exhibit these changes +/-MRX-2843. However, MLL-ENL cells did secrete significantly less IL-4 (p<0.01) and LIF (p<0.05) with MerTK inhibition in vitrocompared to vehicle treatment. In conclusion, inhibition of MerTK decreased infiltration of immunosuppressive co-inhibitory ligand-expressing MMs and DCs into primary sites of leukemic engraftment. Furthermore, inhibition of MerTK altered the leukemia microenvironment to decrease T-cell co-inhibitory receptor expression and improve anti-leukemia effects. These preliminary studies demonstrate the potential of MerTK inhibition as an immunotherapeutic strategy in AML. Disclosures Wang: Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx: Employment, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843.

scholarly journals Innate Pathways of Immune Activation in Transplantation

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Todd V. Brennan ◽  
Keri E. Lunsford ◽  
Paul C. Kuo
Keyword(s):  
Immune System ◽  
Innate Immune System ◽  
Critical Role ◽  
Adaptive Immune System ◽  
Innate Immune ◽  
Microbial Pathogens ◽  
Immune Recognition ◽  
First Line ◽  
Adaptive Immune

Studies of the immune mechanisms of allograft rejection have predominantly focused on the adaptive immune system that includes T cells and B cells. Recent investigations into the innate immune system, which recognizes foreign antigens through more evolutionarily primitive pathways, have demonstrated a critical role of the innate immune system in the regulation of the adaptive immune system. Innate immunity has been extensively studied in its role as the host's first-line defense against microbial pathogens; however, it is becoming increasingly recognized for its ability to also recognize host-derived molecules that result from tissue damage. The capacity of endogenous damage signals acting through the innate immune system to lower immune thresholds and promote immune recognition and rejection of transplant grafts is only beginning to be appreciated. An improved understanding of these pathways may reveal novel therapeutic targets to decrease graft alloreactivity and increase graft longevity.

216 Anti-tumor activity of iosH2 by blocking LILRB2 receptor signalling

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A235-A235
Author(s):  
Osiris Marroquin Belaunzaran ◽  
Anahita Rafiei ◽  
Anil Kumar ◽  
Julia Kolibaba ◽  
Lorenz Vogt ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Innate Immune System ◽  
Cell Activation ◽  
Innate Immune ◽  
High Affinity ◽  
Anti Tumor Activity

BackgroundThe human leukocyte immunoglobulin-like receptor family B (LILR B) acts as check point blockade of the innate immune system by inhibiting leukocyte activation through SHP phosphatase recruitment. Some of the physiological ligands include classical HLA class I molecules, including beta-2-microglobulin (B2M) free open conformers (OC). Natural HLA-OC expression is known from autoimmune disease leading to immune activation by pleiotropic effects since they bind to LILRB and KIR family members reducing Treg and MDSC numbers and increased effector T-cell and NK-cell activation, respectively. We have generated an IgG4-HLA-57 open conformer (OC) molecule (iosH2) with high affinity for LILRB molecules and demonstrate its anti-cancer activity in vitro and in vivo.Methods iosH2 was produced by transient gene expression in CHO cells and purified by standard chromatography. Affinity of iosH2 binding was quantified by ELISA and SPR analysis. HLA-G mediated signaling and competition was assessed using functional cell lines. Effect of iosH2 on activation of SHP1/2 was assessed using Western Blot. Functional assays including in vitro polarization and phagocytosis potential of primary macrophages was assessed by flow cytometry in the presence of iosH2 or isotype control. Effect of iosH2 on T cell activation was evaluated in co-cultures of cancer and T cells. Mouse models were used to assess in vivo activity.Results iosH2 binds to LILRB2 with high affinity and blocks the activation of HLA-G. In addition, iosH2 blocks receptor-mediated activation of SHP1/2. iosH2 promotes a shift from M2 to M1 macrophages with enhanced tumor cell phagocytosis in vitro. iosH2 enhances activation and killing potential of T cells in cancer cells and T cells co-culture assay. iosH2 exerts therapeutic efficacy in mouse transgenic (melanoma) and different syngeneic tumor models (e.g. pancreatic, colon and breast cancer) as monotherapy. Moreover, it acts synergistically in vivo with PD1 blocking antibodies achieving long-term tumor control. Ex vivo tumor sample analysis demonstrates a significant reduction of MDSC and Tregs and a shift towards an activated inflammatory M1 macrophage phenotype. Loss of MDSC functionality was paralleled by enhanced CD8+ T cell expansion and activity.Conclusions iosH2 binds to LILRB2 with high affinity, restores immune cell function in vitro and demonstrates anti-tumor activity in different in vivo mouse models. In addition, it acts synergistically in vivo with PD1. iosH2 is a first-in-class OC therapeutic with robust anti-tumor activity by promoting key components of the innate immune system. Clinical development is under way and phase I trial in preparation.

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