Dick, Christopher J.

Background: Beside fibril-forming proteins, amyloid deposits contain other proteins which can influence organ toxicity, disease initiation and clearance. We investigated the association between the amyloid plaque proteomic signature of diagnostic renal biopsies from patients achieving deep and sustained hematologic remissions and renal organ response in AL amyloidosis. Methods: Retrospective study of newly diagnosed AL amyloidosis patients between 2010-2020. Patients were eligible if; (1) they had a baseline renal biopsy which had undergone laser micro-dissection and mass spectrometry, (2) were assessable for renal response [RenR, non-selective proteinuria ≥1g /24 hours, predominantly albumin], (3) were not on renal replacement therapy and did not experience renal progression within 3-months of diagnosis and (4) had RenR measurements, at least twice annually in the first year from treatment and annually thereafter. Patients who did not obtain a renal complete response (RenCR) required a hematologic very good partial response (VGPR) or better within 6 months of treatment initiation, sustained for a minimum of 6 months. Hematological and renal responses were graded according to consensus criteria. Overrepresentation analyses of differentially expressed proteins were performed using the WebGestalt 2019 and the reactome pathway database. A false detection rate corrected p value <0.05 was used to identify significantly overrepresented proteins and pathways. Results: We defined two organ response categories, to capture the extreme ends of the spectrum: renal suboptimal responders (n=24), patients for whom the best renal response was ≤partial response (RenPR), and renal complete responders (n=29), patients who obtained a RenCR at best response. Overall, the median follow-up was 6.7 years (IQR: 5.4-9.2), 6.6 years (IQR: 5.4-8.1) for complete responders, and 8.4 years (IQR: 6.1-11.3) for suboptimal responders. Between renal suboptimal responders and complete responders there was no significant difference in median age (63 vs 63 years, p=0.7), lambda light chain isotype (66% vs 79%, p=0.3), first-line therapy received and renal stage (Stage III: 14% vs 8%, p=0.9). The median time to RenCR was 52-months (IQR: 31-63). Protein pathways over-represented in renal complete responders included 14-3-3 proteins (YWHAG, YWHAH, YWHAQ, YWHAZ), early components of the complement cascade and complement regulatory proteins (C3, C4A, C4B, C8A, complement factor H), keratins, cytoskeletal proteins (tubulin, actin, filamen) and collagen IV. Protein pathways over-represented in renal suboptimal responders included terminal/effector components of the complement cascade (C5, C6, C8B, C9), amyloid signature proteins (ApoA1 and E, vitronectin, clusterin), proteoglycans (heparan sulfate proteoglycan), transgelin and metallopeptidase inhibitor 3. Proteins with the highest-fold increase in renal complete responders included serine protease inhibitor (SERPINA3) and F-box and leucine rich repeat protein (FBXL6), 9.0 x 106 & 7.7-fold, respectively. Proteins with the highest-fold increase in renal suboptimal responders included complement 6 and 8B, 3.5 & 3.6-fold, respectively. Conclusion: This is the first study to identify proteomic determinants of organ response in AL amyloidosis. Molecular chaperones involved in protein aggregate homeostasis, such as the 14-3-3 proteins are upregulated in renal complete responders, similar to what we have shown in our prior work. This suggests that organ responders may have a more favorable microenvironment which “primes” them for organ response after the amyloidogenic light chain has been cleared from the circulation. Renal suboptimal responders (≤RenPR) have elevated levels of terminal complement proteins including membrane attack complex proteins and reduced levels of early components or complement regulatory proteins, which may be responsible for reno-toxic and reno-protective effects, respectively.

Abstract NKG2D is expressed on cytotoxic lymphocytes and is an important immunosurveillance receptor that recognizes stress ligands such as MICA on tumor cells or virally infected cells. Unlike traditional immune activating receptors, NKG2D couples to the non-ITAM-containing transmembrane adaptor DAP10, which recruits PI3K and Grb2 to initiate target cell lysis. Current understanding predicts an ITAM-independent mechanism of NKG2D-mediated cytotoxicity. However, we show that in primary human NK cells, the ITAM-containing adaptors DAP12, FcϵR1γ, and CD3ζ selectively regulate the Grb2-Vav1 branch of NKG2D-mediated signal transduction and cytotoxicity. Furthermore, this ITAM-adaptor regulation of NKG2D is independent of ITAM activation. Thus our data reveal a more complex interplay between NKG2D and other activating receptors than was previously imagined, and we have uncovered a novel mechanism of ITAM-adaptor regulation of cell-mediated cytotoxicity. A more thorough understanding of immunoreceptor signaling networks will lead to more precise therapies when fighting diseases such as cancer, autoimmunity, and infection.

Abstract The NK cell activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell - target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D coated beads is impaired in the absence of CrkL. Antibody-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras-family GTPase Rap1 is required for NK cell killing and is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization and granule release.

Abstract Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcγRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-γ. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and “natural cytotoxicity,” thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.

Abstract Selective cell death plays a critical role in the development of the immune repertoire and in the elimination of target cells expressing foreign Ags. The apoptosis induced by ligation of the Fas Ag, a member of the TNFR/nerve growth factor receptor superfamily, contributes to both of these modes of cell loss. However, in spite of the molecular cloning of the Fas Ag and the identification of a specific cytoplasmic domain required for its function, it remains unclear as to which Fas-induced second messengers mediate the development of programmed cell death. We, therefore, evaluated Fas-initiated signal transduction in susceptible cell types. We determined that Fas ligation induces the rapid tyrosine phosphorylation of multiple cellular proteins. These phosphorylation events occur within 1 min and decline toward baseline by 30 min. In addition, Fas ligation increases the in vitro protein kinase activity of the tyrosine phosphorylated proteins. Pharmacologic inhibitors of protein tyrosine kinases block, in a concentration-dependent manner, Fas-induced DNA fragmentation and prolong cell survival. These results suggest that protein tyrosine kinase activation is an early and obligatory signal in Fas-induced apoptosis.

Abstract Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.

Abstract Protein tyrosine kinases play fundamental roles in the transduction of signals that regulate cell growth, differentiation, and functional responses to a diversity of external stimuli. It is therefore likely that understanding protein tyrosine kinase activity in NK cells will be crucial in further defining the intracellular regulation of their unique and specialized functions. We investigated the role of protein tyrosine phosphorylation in receptor-mediated signal transduction using stimuli known to play major roles in regulating NK cell activation. Immunoblot analyses with antiphosphotyrosine antibodies demonstrated that IL-2, a potent stimulus for NK cell proliferation and an agent that enhances NK cytotoxic function, induced the tyrosine phosphorylation of at least eight proteins in clonal CD16+/CD3-human NK cells. In contrast, IL-4, which modulates NK cell function without inducing proliferation, had no apparent effect on protein tyrosine phosphorylation. Because protein kinase C (PKC) activation plays a prominent, yet distinct role in NK cell-mediated cytolytic reactions, we next investigated whether PKC activation affects NK cell protein tyrosine phosphorylation. Surprisingly, PKC-activating agents, including the phorbol esters 12-O-tetradecanoylphorbol-13-acetate and 4 beta-phorbol 12, 13-didecanoate, as well as the synthetic diacylglycerol,1-oleoyl-2-acetylglycerol, also induced the tyrosine phosphorylation of a distinct set of proteins. The 4 beta-phorbol 12,13-didecanoate homolog, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, also failed to induce protein tyrosine phosphorylation. Further, the PKC inhibitor, 1-O-hexadecyl-2-O-methylglycerol blocked tyrosine phosphorylation induced by 1-oleoyl-2-acetylglycerol. In subsequent studies, both CD8+ and CD8- NK clones were found to express the src-family tyrosine kinase, p56lck, which was detected by immunoblot analysis with anti-p56lck antiserum. In both types of clonal NK cell lines, IL-2 and 12-O-tetradecanoyl-phorbol appeared to stimulate the differential phosphorylation of p56lck as evidenced by the appearance of higher molecular mass isoforms on SDS-polyacrylamide gels. Thus, our results identify and characterize a potential role for tyrosine phosphorylation and for the lymphocyte-specific tyrosine kinase p56lck in the signaling events that regulate NK cell activation.

Abstract Urinary extracellular vesicles (uEVs) have been used as a biomarker tool for detection of low abundance immunoglobulin-based targets in uEVs with heterogeneous high protein mixtures. This protocol outlines a pre-incubation method to minimize non-specific secondary antibody binding. Additionally, we include our approach for standardization of uEV amounts used for sample comparison. We propose that these and knowledge of the detection limit for target proteins are strategies applicable to other laboratories working with complex heterogeneous samples.