Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1, a cell surface glycoprotein expressed on MM cells. In preclinical models, elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein, we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1–CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary, human MM cells. Taken together, these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone.     

Regulation of Ly49D/DAP12 signal transduction by Src-family kinases and CD45

Activating, DAP12-coupled members of the Ly-49 family of NK cell receptors help control viral infections in mice. However, the kinases and/or phosphatases mediating tyrosine phosphorylation of Ly-49D-associated DAP12 have not been elucidated. In this study, we show for the first time that Src family tyrosine kinases are physically and functionally associated with Ly-49D/DAP12 signaling in murine NK cells. Specifically, we demonstrate the following: 1) inhibition of Src family kinases suppresses DAP12 phosphorylation and downstream DAP12 signals; 2) both Fyn and Lck are capable of phosphorylating DAP12; and 3) both kinases coimmunoprecipitate with the Ly-49D/DAP12 complex in NK cells. Although we detect enhanced phosphorylation of Fyn upon Ly-49D cross-linking in NK cells, Ly-49D-mediated events in both Fyn-/- and Fyn/Lck-/- mice appear normal, reinforcing the theme of redundancy in the ability of Src family kinases to initiate activation events. In contrast to disruption of specific Src family enzymes, Ly-49D/DAP12-mediated calcium mobilization and cytokine production by CD45 null NK cells are defective. Although others have ascribed the effects of CD45 mutation solely on the suppression of Src family activity, we demonstrate in this study that DAP12 is hyperphosphorylated in CD45 null NK cells, resulting in uncoordinated tyrosine-mediated signaling upon Ly-49D ligation. Therefore, although our data are consistent with a Src kinase activity proximally within DAP12 signaling, DAP12 also appears to be a substrate of CD45, suggesting a more complex role for this phosphatase than has been reported previously.     

Inappropriate recruitment and activity by the Src homology region 2 domain-containing phosphatase 1 (SHP1) is responsible for receptor dominance in the SHIP-deficient NK cell

We have previously demonstrated that the NKR repertoire is profoundly disrupted by SHIP deficiency. This repertoire disruption is characterized by receptor dominance where inhibitory signals from 2B4 repress killing of complex targets expressing MHC class I and activating ligands. In this study, we examine the molecular basis of receptor dominance in SHIP-/- NK cells. In this study, we show that in SHIP-/- NK cells there is a pronounced bias toward the 2B4 long isoform. We have also characterized signaling molecules recruited to 2B4 in SHIP-/- NK cells. Interestingly, we find that approximately 10- to 16-fold more Src homology region 2 domain-containing phosphatase 1 (SHP1) is recruited to 2B4 in SHIP-/- NK cells when compared with wild type. Consistent with SHP1 overrecruitment, treatment with sodium orthovanadate or a novel inhibitor with micromolar activity against SHP1 restores the ability of SHIP-/- NK cells to kill Rae1+ RMA and M157+ targets. These findings define the molecular basis for hyporesponsiveness by SHIP-deficient NK cells.     

Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8⁺ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users. S. Calpe and E. Erdős contributed equally to this work     

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.     

Coligation of the B cell receptor with complement receptor type 2 (CR2/CD21) using its natural ligand C3dg: activation without engagement of an inhibitory signaling pathway

C3dg is a cleavage product of the C3 component of complement that can facilitate the coligation of the complement receptor 2 (CR2/CD21) with the BCR via C3dg/Ag complexes. This interaction can greatly amplify BCR-mediated signaling events and acts to lower the threshold for B cell activation. Although previous studies have used anti-CR2 Abs or used chimeric Ags in the context of BCR transgenic mice as surrogate C3d-containing ligands, we have used a physiological form of C3d to study signaling in B cells from wild-type C57BL/6 mice. We find that while CR2-enhanced BCR signaling causes intracellular Ca2+ mobilization and total pTyr phosphorylation of an intensity comparable to optimal BCR ligation using anti-IgM Abs, it does so with limited activation of inhibitory effectors (such as CD22, Src homology region 2 domain containing phosphatase 1, and SHIP-1) and without substantial receptor cross-linking. In summary, we demonstrate that CR2-enhanced BCR signaling may proceed not only through the previously described amplification of positive signaling pathways, but is potentially augmented by a lack of normal inhibitory/feedback signaling.     

An intracellular multi-effector complex mediates somatostatin receptor 1 activation of phospho-tyrosine phosphatase eta

The receptor-like phosphotyrosine phosphatase eta (PTPeta) is an important intracellular effector of the cytostatic action of SST. Here we characterize, in Chinese hamster ovary-k1 cells, the intracellular pathway that from somatostatin receptor 1 (SSTR1), leads to the activation of PTPeta and that involves, in a multimeric complex and sequential activation, the tyrosine kinases Janus kinase (JAK) 2 and Src, and the cytosolic phosphotyrosine phosphatase SHP-2. We show that inhibitors of JAK2 and Src and dominant-negative mutants of SHP-2 and Src abolished the SSTR1-mediated PTPeta activation, suggesting that all these effectors participate in the activation of PTPeta. In basal conditions, JAK2 forms a multimeric complex with SHP-2, Src and PTPeta. In response to SST, JAK2 is activated in a G protein-dependent manner, dissociates from and phosphorylates SHP-2, increasing its activity. Subsequently, SHP-2 dissociates from Src, dephosphorylates the Src inhibitory tyrosine-529, and causes an autocatalytical increase of the phosphorylation of Src tyrosine 418, located inside its kinase activation loop. Active Src, in turn, controls the activity of PTPeta, via a direct interaction and phosphorylation of the phosphatase. These data for the first time depict an intracellular pathway involving a precise sequence of interactions and cross-activation among tyrosine phosphatases and kinases acting upstream of PTPeta. In particular the sequential activation of JAK2, SHP-2, and Src conveys the molecular signaling from SSTR1 to the activation of this phosphatase that is responsible for the final biological effects of SST.     

Inhibition of the Jun N-terminal protein kinase pathway by SHIP-1, a lipid phosphatase that interacts with the adaptor molecule Dok-3

Dok-3 is a Dok-related adaptor expressed in B cells and macrophages. Previously, we reported that Dok-3 is an inhibitor of B-cell activation in A20 B cells and that it associates with SHIP-1, a 5' inositol-specific lipid phosphatase, as well as Csk, a negative regulator of Src kinases. Here, we demonstrate that Dok-3 suppresses B-cell activation by way of its interaction with SHIP-1, rather than Csk. Our biochemical analyses showed that the Dok-3-SHIP-1 complex acts by selectively inhibiting the B-cell receptor (BCR)-evoked activation of the Jun N-terminal protein kinase (JNK) cascade without affecting overall protein tyrosine phosphorylation or activation of previously described SHIP-1 targets like Btk and Akt/PKB. Studies of B cells derived from SHIP-1-deficient mice showed that BCR-triggered activation of JNK is enhanced in the absence of SHIP-1, implying that the Dok-3-SHIP-1 complex (or a related mechanism) is a physiological negative regulator of the JNK cascade in normal B cells. Together, these data elucidate the mechanism by which Dok-3 inhibits B-cell activation. Furthermore, they provide evidence that SHIP-1 can be a negative regulator of JNK signaling in B cells.     

Requirement for CD45 in fine-tuning mast cell responses mediated by different ligand–receptor systems

The receptor-like protein tyrosine phosphatase CD45, the most abundant cell surface phosphatase on all nucleated hemopoietic cells, is a critical regulator of the activation status of Src family kinases (SFKs). To study the impact of CD45 on mast cell function, we compare bone marrow-derived mast cells (BMMCs) from CD45-deficient mice and from mice expressing an activating point mutation (E613R) in the juxtamembrane wedge of CD45. In response to Ag-triggered FcεR1-mediated activation, CD45-deficient BMMCs exhibit increased inhibitory Lyn phosphorylation and drastically reduced effector functions (degranulation and cytokine secretion). In contrast, CD45 E613R BMMCs show stronger effector functions after Ag-triggering than wild-type (WT) BMMCs. Despite these dichotomous phenotypes, phosphorylation of the inhibitory tyrosine in the SFK Lyn of CD45 E613R BMMCs is comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fyn-regulated phosphatidylinositol-3-kinase pathway. Interestingly, depending on the receptor system addressed, CD45-deficient and CD45 E613R BMMCs show uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or SF is enhanced compared to WT BMMCs. Together, the data indicate that CD45 plays a complex and essential role in fine-tuning mast cell responses mediated by different ligand–receptor systems.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

The chicken SLAM family

The signaling lymphocytic activation molecule (SLAM) family of receptors is critically involved in the immune regulation of lymphocytes but has only been detected in mammals, with one member being present in Xenopus. Here, we describe the identification, cloning, and analysis of the chicken homologues to the mammalian SLAMF1 (CD150), SLAMF2 (CD48), and SLAMF4 (CD244, 2B4). Two additional chicken SLAM genes were identified and designated SLAMF3like and SLAM5like in order to stress that those two receptors have no clear mammalian counterpart but share some features with mammalian SLAMF3 and SLAMF5, respectively. Three of the chicken SLAM genes are located on chromosome 25, whereas two are currently not yet assigned. The mammalian and chicken receptors share a common structure with a V-like domain that lacks conserved cysteine residues and a C2-type Ig domain with four cysteines forming two disulfide bonds. Chicken SLAMF2, like its mammalian counterpart, lacks a transmembrane and cytoplasmic domain and thus represents a glycosyl-phosphatidyl-inositol-anchored protein. The cytoplasmic tails of SLAMF1 and SLAMF4 display two and four conserved immunoreceptor tyrosine-based switch motifs (ITSMs), respectively, whereas both chicken SLAMF3like and SLAMF5like have only a single ITSM. We have also identified the chicken homologues of the SLAM-associated protein family of adaptors (SAP), SAP and EAT-2. Chicken SAP shares about 70 % identity with mammalian SAP, and chicken EAT-2 is homologous to mouse EAT-2, whereas human EAT-2 is much shorter. The characterization of the chicken SLAM family of receptors and the SAP adaptors demonstrates the phylogenetic conservation of this family, in particular, its signaling capacities.     

Molecular analysis of NTB-A signaling: a role for EAT-2 in NTB-A-mediated activation of human NK cells

Engagement of NTB-A on human NK cells by homophilic interaction with NTB-A-expressing target cells can trigger NK cell cytotoxicity, cytokine production, and proliferation. To better understand how NTB-A can activate NK cells, we analyzed the molecular mechanisms of NTB-A signaling. We show that NTB-A is tyrosine phosphorylated in unstimulated human NK cells and associates with SLAM-associated protein (SAP) and EAT-2. This phosphorylation of NTB-A is mediated by Src family kinases and is most likely a result of the homophilic interaction of NTB-A among neighboring NK cells. Stimulation of NK cells by NTB-A-positive targets results in increased NTB-A phosphorylation. The cytoplasmic tail of NTB-A contains three tyrosines, two of which are embedded within an immunoreceptor tyrosine-based switch motif. We generated a NTB-A-negative NK cell line, in which we expressed different mutants of NTB-A. Functional studies showed that the second tyrosine is sufficient and essential for NTB-A-mediated cytotoxicity. EAT-2, but not SAP, is recruited to this second tyrosine, indicating that SAP may be dispensable for this NTB-A function. To further investigate this, we silenced SAP expression in NK cell lines. Functional analysis of these cells showed that NTB-A can mediate NK cell cytotoxicity in the absence of SAP, probably via EAT-2. In contrast, NTB-A-mediated IFN-gamma production was greatly reduced in the absence of SAP, demonstrating that cytokine production and cytotoxicity are differentially dependent on SAP and possibly EAT-2.     

Activation by SLAM Family Receptors Contributes to NK Cell Mediated “Missing-Self” Recognition

Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells’ ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated “missing-self” reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.     

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.     

Subdomain X of the kinase domain of Lck binds CD45 and facilitates dephosphorylation

CD45 is a transmembrane, two-domain protein-tyrosine phosphatase expressed exclusively in nucleated hematopoietic cells. The Src family kinase, Lck, is a major CD45 substrate in T cells and CD45 dephosphorylation of Lck is important for both T cell development and activation. However, how the substrate specificity of phosphatases such as CD45 is achieved is not well understood. Analysis of the interaction between the cytoplasmic domain of CD45 and its substrate, Lck, revealed that the active, membrane-proximal phosphatase domain of CD45 (CD45-D1) bound to the phosphorylated Lck kinase domain, the SH2 domain, and the unique N-terminal region of Lck. The second, inactive phosphatase domain (CD45-D2) bound only to the kinase domain of Lck. CD45-D2 was unable to bind phosphotyrosine, and its interaction with the kinase domain of Lck was independent of tyrosine phosphorylation. The binding of CD45-D2 was localized to subdomain X (SD10) of Lck. CD45-D2 bound similarly to Src family kinases but bound Csk to a lesser extent and did not bind significantly to the less related kinase, Erk1. CD45 dephosphorylated Lck and Src at similar rates but dephosphorylated Csk and Erk1 at lower rates. Replacement of Erk1 SD10 with that of Lck resulted in the binding of CD45-D2 and the conversion of Erk1 to a more efficient CD45 substrate. This demonstrates a role for CD45-D2 in binding substrate and identifies the SD10 region in Lck as a novel site involved in substrate recognition.     

CD300F blocks both MyD88 and TRIF-mediated TLR signaling through activation of Src homology region 2 domain-containing phosphatase 1

CD300F is known to exhibit inhibitory activity in myeloid cells through its intracellular ITIM. To investigate the effect of CD300F stimulation on TLR signaling, the human acute monocytic leukemia cell line THP-1 was treated with CD300F-specific mAbs or two synthetic peptides that represented the ITIM-like domains of CD300F. Treatment with these agents blocked TLR2-, 3-, 4-, and 9-mediated expression of proinflammatory mediators such as IL-8 and matrix metalloproteinase-9. The luciferase reporter assay in 293T cells and Western blot analysis of THP-1 cells revealed that these inhibitory actions were effective in pathways involving MyD88 and/or TRIF of TLR signaling and associated with marked suppression of IκB kinase activation, phosphorylation/degradation of IκB, and subsequent activation of NF-κB. Use of specific inhibitors and immunoprecipitation analysis further indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatase-1, a protein tyrosine phosphatase with inhibitory activity in hematopoietic cells. These data indicate that CD300F is an active regulator of TLR-mediated macrophage activation through its association with Src homology 2 domain-containing phosphatase-1 and that the synthetic peptides can be applied for the regulation of immune responses that are induced by TLRs.