Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

MS4A4A: a novel cell surface marker for M2 macrophages and plasma cells.

MS4A4A is a member of the membrane-spanning, four domain family, subfamily A (MS4A) that includes CD20 (MS4A1), FcRß (MS4A2) and Htm4 (MS4A3). Like the first three members of this family, transcription of MS4A4A appears to be limited to hematopoietic cells. To evaluate expression of the MS4A4A protein in hematopoietic cell lineages and subsets we generated monoclonal antibodies against extracellular epitopes for use in flow cytometry. In human peripheral blood we found that MS4A4A is expressed at the plasma membrane in monocytes but not in granulocytes or lymphocytes. In vitro differentiation of monocytes demonstrated that MS4A4A is expressed in immature but not activated dendritic cells, and in macrophages generated in the presence of interleukin-4 ('alternatively activated' or M2 macrophages) but not by interferon-γ and lipopolysaccharide ('classically' activated or M1 macrophages). MS4A4A was expressed in the U937 monocytic cell line only after differentiation. In normal bone marrow, MS4A4A was expressed in mature monocytes but was undetected, or detected at only a low level, in myeloid/monocytic precursors, as well as their malignant counterparts in patients with various subtypes of myeloid leukemia. Although MS4A4A was not expressed in healthy B lymphocytes, it was highly expressed in normal plasma cells, CD138+ cells from multiple myeloma patients, and bone marrow B cells from a patient with mantle cell lymphoma. These findings suggest immunotherapeutic potential for MS4A4A antibodies in targeting alternatively activated macrophages such as tumor-associated macrophages, and in the treatment of multiple myeloma and mantle cell lymphoma.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

CD40 translocation to lipid rafts: signaling requirements and downstream biological events.

CD40, a member of the TNF receptor family, is expressed on a variety of immune and non-immune cells. Its interaction with its ligand, CD154, plays a pivotal role in humoral and cell-mediated immunity. A low level of CD40 is constitutively associated within membrane lipid rafts and, upon engagement, this level is significantly enhanced. In this study, our objective is to evaluate the process of CD40/lipid raft association in terms of the signals required for its initiation and the resulting biological outcomes. Here, we show the CD40/lipid raft association to be independent of PI-3-kinase, Src family kinases and p38 MAPK pathways. Moreover, CD40 lacking its intracellular domain, which is usually required for CD40-mediated signaling, still localizes to lipid rafts upon engagement, confirming that the CD40/lipid raft association is independent of signaling events. As to the biological outcomes of the CD40/lipid raft association, we show that disrupting lipid raft integrity selectively abolishes CD40-mediated Akt phosphorylation. In addition, replacing the transmembrane domain of CD40 with that of CD45 (a protein excluded from lipid rafts) dramatically reduced CD40-mediated Akt phosphorylation and B7.1 upregulation, while not influencing p38, ERK and JNK activation. Together, these findings clarify the requirements for CD40/lipid raft association and the signals triggered upon CD40 engagement by CD154.

CD20 homo-oligomers physically associate with the B cell antigen receptor. Dissociation upon receptor engagement and recruitment of phosphoproteins and calmodulin-binding proteins.

B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation, and activation of B lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using coprecipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers. Additionally, the function of CD20 was explored by examining its association with surface-labeled and intracellular proteins before and after BCR signaling. Two major surface-labeled proteins that coprecipitated with CD20 were identified as the heavy and light chains of cell surface IgM, the antigen-binding components of the BCR. After activation, BCR-CD20 complexes dissociated, and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. These data provide new evidence of the involvement of CD20 in signaling downstream of the BCR and, together with the previously described involvement of CD20 in calcium influx, the first evidence of physical coupling of the BCR to a calcium entry pathway.

The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism.

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.

Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure.

In vivo ablation of malignant B cells can be achieved using antibodies directed against the CD20 antigen. Fine specificity differences among CD20 monoclonal antibodies (mAbs) are assumed not to be a factor in determining their efficacy because evidence from antibody-blocking studies indicates limited epitope diversity with only 2 overlapping extracellular CD20 epitopes. However, in this report a high degree of heterogeneity among antihuman CD20 mAbs is demonstrated. Mutation of alanine and proline at positions 170 and 172 (AxP) (single-letter amino acid codes; x indicates the identical amino acid at the same position in the murine and human CD20 sequences) in human CD20 abrogated the binding of all CD20 mAbs tested. Introduction of AxP into the equivalent positions in the murine sequence, which is not otherwise recognized by antihuman CD20 mAbs, fully reconstituted the epitope recognized by B1, the prototypic anti-CD20 mAb. 2H7, a mAb previously thought to recognize the same epitope as B1, did not recognize the murine AxP mutant. Reconstitution of the 2H7 epitope was achieved with additional mutations replacing VDxxD in the murine sequence for INxxN (positions 162-166 in the human sequence). The integrity of the 2H7 epitope, unlike that of B1, further depends on the maintenance of CD20 in an oligomeric complex. The majority of 16 antihuman CD20 mAbs tested, including rituximab, bound to murine CD20 containing the AxP mutations. Heterogeneity in the fine specificity of these antibodies was indicated by marked differences in their ability to induce homotypic cellular aggregation and translocation of CD20 to a detergent-insoluble membrane compartment previously identified as lipid rafts.