Activating Receptors 2B4 Research Articles

The Oncogenic Fusion Protein CBFB-SMMHC Downregulates CD48 to Evade NK Cell Recognition

Chromosomal translocations are a common cytogenetic aberration in acute leukemia, which frequently generate fusion proteins with oncogenic properties. We have previously studied the immune evasion mechanisms of two common oncogenic fusion proteins in acute myeloid leukemia (AML), PML-RARA and AML1-ETO. We investigated, in particular, the activity of natural killer (NK) cells, which are part of the innate immune system and play a significant role in eliminating leukemic cells. We found that both oncogenic fusion proteins downregulate the expression of CD48, a ligand of the NK cell activating receptor 2B4, to escape NK cell-mediated recognition of the tumor cells.In the current study we tested the possibility that other oncogenic fusion proteins are also able to evade NK cell recognition. To this aim we cloned several leukemic fusion proteins into lentiviral vectors and expressed them in U937 cells, which are commonly used to express leukemic fusion proteins. The leukemic fusion proteins included MLL-AF4, NUP98-HOXA9, DEK-NUP98 and CBFB-SMMHC. By using flow cytometry, we examined the effect of these fusion proteins on the expression of several NK cell ligands. CBFB-SMMHC was the only fusion protein which downregulated the expression of CD48.Next, we tested the functional significance of the downregulation of CD48 by CBFB-SMMHC by performing cytotoxicity assays with NK cells. We first used the NK cell line YTS eco since the cytotoxicity of these cells is mainly dependent on the 2B4-CD48 interaction. We found that cells which express CBFB-SMMHC were killed significantly less than the control cells, and that this reduced killing was due to CD48 downregulation. We also tested the killing of CBFB-SMMHC expressing cells by primary bulk NK cells and found that cells which express this fusion protein were killed significantly less.Mechanistically we show that overexpression of CBFB-SMMHC reduces the mRNA levels of CD48. Additionally, we tested whether treatment with HDAC inhibitors (HDACi) can reverse the downregulation of CD48 by CBFB-SMMHC. We treated cells which express CBFB-SMMHC with various HDACi and examined the effect of this treatment on the expression of CD48. Two specific class I HDACi, mocetinostat and entinostat, upregulated the expression of CD48 in cells which express this fusion protein.Taken together we show that modulation of CD48 expression is an immune evasion mechanism, which is specific to core binding factor (CBF) proteins (AML1-ETO and CBFB-SMMHC) and PML-RARA. Although the three fusion proteins which we found to down-regulate the expression of CD48 are associated with better prognosis in AML, about 30% of CBF-AML patients eventually relapse. Based on our findings we suggest that down-regulation of CD48 by these fusion proteins, which leads to NK cell immune evasion, contributes to the persistence of residual disease in these subtypes of leukemia and eventually to a clinical relapse. Finally, we propose that NK cell based therapies (i.e., NK cell infusion) or class I HDACi could be potential adjunctive therapies for CBF-AML or APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TGF-β regulated leukemia cell susceptibility against NK targeting through the down-regulation of the CD48 expression

Transforming growth factor-β (TGF-β) is known to function as a dual role regulatory cytokine for being either a suppresser or promoter during tumor initiation and progression. In solid tumors, TGF-β secreted from tumor microenvironment acts as a suppresser against host immunity, like natural killer (NK) cells, to favor tumor evasion. However, besides solid tumors, the underlying mechanism of how TGF-β regulates leukemogenesis, tumor progression, immunoediting, and NK function is still not clear in detail. In this study, we found that TGF-β induced leukemia MEG-01 and U937 cells to become less sensitive to NK-92MI targeting by down-regulating CD48, a ligand for NK activating receptor 2B4, but not down-regulating other tumor-associated carbohydrate antigens (TACAs). In CD48-knockdown cells, cells responding to NK-92MI targeting displayed a phenotype of less NK susceptibility and cell conjugation. On the other hand, when NK cells were treated with TGF-β, TGF-β suppressed NK recognition, degranulation, and killing activity in time-dependent manner by regulating ICAM-1 binding capacity instead of affecting expressions of activating and inhibitory receptors. Taken together, both leukemia cells and immune NK cells could be regulated by TGF-β through suppressing leukemia cell surface CD48 to escape from host surveillance and down-regulating NK cell surface ICAM-1 binding activity to impair NK functions, respectively. Our results suggested that TGF-β had effect in leukemia similar to that observed in solid tumors but through different regulatory mechanism.

2B4 costimulatory domain enhancing cytotoxic ability of anti-CD5 chimeric antigen receptor engineered natural killer cells against T cell malignancies

BackgroundChimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering.MethodsTwo second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo.ResultsBoth BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK.ConclusionsAnti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

Immune evasion by oncogenic proteins of acute myeloid leukemia

AbstractPML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immuoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.

The activating receptors 2B4 and NTB-A, but not CRACC are subject to ligand-induced down-regulation on human natural killer cells

Activation of natural killer cells can be mediated by different receptors. Stimulation of the receptors 2B4, NTB-A and CRACC, members of the SLAM-related receptor family, induces cytotoxicity and cytokine production. The surface expression of 2B4 and other activating natural killer cell receptors is down-modulated after receptor engagement, which results in a weaker response to consecutive stimulation. We tested whether this regulatory mechanism applies to all SLAM-related receptors expressed by primary human natural killer cells. After co-culture with target cells expressing the respective ligands different effects on receptor surface expression were observed. While 2B4 ex-pression was strongly reduced, NTB-A showed less prominent down-modulation and the expression level of CRACC remained unchanged. The expression levels of the receptor-proximal signaling molecules SAP, EAT-2 and FynT did not change after receptor engagement. Co-culture with target cells expressing the ligands for NTB-A or CRACC had no impact on subsequent NTB-A or CRACC-mediated NK cell activation.

The activating receptors 2B4 and NTB-A, but not CRACC are subject to ligand-induced down-regulation on human natural killer cells

AbstractActivation of natural killer cells can be mediated by different receptors. Stimulation of the receptors 2B4, NTB-A and CRACC, members of the SLAM-related receptor family, induces cytotoxicity and cytokine production. The surface expression of 2B4 and other activating natural killer cell receptors is down-modulated after receptor engagement, which results in a weaker response to consecutive stimulation. We tested whether this regulatory mechanism applies to all SLAM-related receptors expressed by primary human natural killer cells. After co-culture with target cells expressing the respective ligands different effects on receptor surface expression were observed. While 2B4 ex-pression was strongly reduced, NTB-A showed less prominent down-modulation and the expression level of CRACC remained unchanged. The expression levels of the receptor-proximal signaling molecules SAP, EAT-2 and FynT did not change after receptor engagement. Co-culture with target cells expressing the ligands for NTB-A or CRACC had no impact on subsequent NTB-A or CRACC-mediated NK cell activation.

Upregulation of Fas and downregulation of CD94/NKG2A inhibitory receptors on circulating natural killer cells in patients with new-onset psoriasis

Psoriasis has been considered as a T-helper 1 cell-mediated autoimmune disease driven by collaboration with multiple components of innate and acquired immune cells. Natural killer (NK) cells have been shown to bridge innate and acquired immunity, and thus could potentially contribute to the pathophysiology of psoriasis. To investigate the phenotypic changes of circulating NK cells in patients with new-onset psoriasis. Fifteen patients with plaque psoriasis (eight women and seven men) who visited our clinic after their first episode of psoriasis and did not have a history of previous systemic therapy or phototherapy participated in this study. Peripheral blood mononuclear cells were isolated and stained with a panel of antibodies against cell surface receptors expressed on T and/or NK cells and analysed by flow cytometry. As compared with normal healthy volunteers, patients with new-onset psoriasis showed no significant changes in numbers of peripheral NK, NK-T or T cells. NK activating receptors 2B4, CD48, NKG2D, CD16 and CD56 were found to be unchanged in new-onset psoriasis. However, the expression of Fas (activation-induced death receptor) was upregulated, whereas the expression of the NK inhibitory receptors CD94 and NKG2A was dramatically reduced on NK cells of new-onset psoriasis. These changes occurred at the level of mean fluorescent intensity, but minimally affected percentages of cells expressing Fas, CD94 and NKG2A. Our findings demonstrate that changes in the expression of Fas and CD94/NKG2A receptors on NK cells may occur during new-onset psoriasis, and are likely to contribute to the pathogenesis of psoriasis.