Myosin IIA is required for cytolytic granule exocytosis in human NK cells.

Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector-target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.

Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination.

Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.

Mechanisms and Emerging Therapies for Treatment of Seizures in Pediatric Autoimmune Encephalitis and Autoinflammatory/Autoimmune-Associated Epilepsy.

There has been increasing understanding of the role of inflammation in seizures and epilepsy, as well as targeted immunomodulatory treatments. In children, immune-mediated seizures often present acutely in the setting of autoimmune encephalitis and are very responsive to immunotherapy with low rates of subsequent epilepsy. Conversely, seizures in autoimmune-associated epilepsies, such as Rasmussen syndrome, can remain refractory to multimodal therapy, including immunomodulation. In this review, the authors discuss the presentations of immune-mediated seizures in children, underlying mechanisms, and emerging therapies.

Expansion of the clinical and neuroimaging spectrum associated with NDUFS8-related disorder.

Biallelic pathogenic variants in NDUFS8, a nuclear gene encoding a subunit of mitochondrial complex I, result in a mitochondrial disorder characterized by varying clinical presentations and severity. Here, we expand the neuroimaging and clinical spectrum of NDUFS8-related disorder. We present three cases from two unrelated families (a girl and two brothers) homozygous for a recurrent pathogenic NDUFS8 variant [c.460G>A, p.(Gly154Ser)], located in the [4Fe-4S] domain of the protein. One of the patients developed auto-antibody positive diabetic ketoacidosis. Brain MRIs performed in two of the three patients demonstrated diffuse cerebral and cerebellar white matter involvement including corticospinal tracts, but notably had sparing of deep gray matter structures. Our report expands the neuroimaging phenotype of NDUFS8-related disorder to include progressive leukodystrophy with increasing brainstem and cerebellar involvement, with relative sparing of the basal ganglia. In addition, we describe autoimmune diabetes in association with NDUFS8-related disorder, though the exact mechanism of this association is unclear. This paper provides a comprehensive review of case presentation and progressive neuroimaging findings of three patients from two unrelated families that have an identical pathogenic NDUFS8 variant, which expands the clinical spectrum of NDUFS8-associated neurological disease.

A Late Phase of Long-Term Synaptic Depression in Cerebellar Purkinje Cells Requires Activation of MEF2.

The MEF2 family of transcription factors restricts excitatory synapse number in an activity-dependent fashion during development, yet MEF2 has not been implicated in long-term synaptic depression (LTD), which is thought to initiate synapse elimination. Mutations in MEF2 pathways are implicated in autism spectrum disorders, which include cerebellar dysfunction. Here, we test the hypothesis that cerebellar LTD requires postsynaptic activation of MEF2. Knockdown of MEF2D produces suppression of the transcription-dependent late phase of LTD in cultured Purkinje cells. The late phase of LTD is also completely blocked in Purkinje cells derived from MEF2A+MEF2D null mice and rescued with plasmids that drive expression of MEF2D but not phosphatase-resistant mutant MEF2D S444D. Wild-type Purkinje cells transfected with a constitutively active form of MEF2 show no alterations of synaptic strength. Thus, postsynaptic activation of MEF2 by S444 dephosphorylation is necessary, but not sufficient, for the late phase of cerebellar LTD.

Chromatin Environment and Cellular Context Specify Compensatory Activity of Paralogous MEF2 Transcription Factors.

Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes, but how TFs dynamically respond to paralog depletion on a genome-wide scale in vivo remains incompletely understood. Using single and double conditional knockout of myocyte enhancer factor 2 (MEF2) family TFs in granule neurons of the mouse cerebellum, we find that MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. Although both TFs are highly expressed in granule neurons, transcriptomic analyses show MEF2D is the predominant genomic regulator of gene expression in vivo. Strikingly, genome-wide occupancy analyses reveal upon depletion of MEF2D, MEF2A occupancy robustly increases at a subset of sites normally bound to MEF2D. Importantly, sites experiencing compensatory MEF2A occupancy are concentrated within open chromatin and undergo functional compensation for genomic activation and gene expression. Finally, motor activity induces a switch from non-compensatory to compensatory MEF2-dependent gene regulation. These studies uncover genome-wide functional interdependency between paralogous TFs in the brain.

MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers.

Organismal development requires the precise coordination of genetic programs to regulate cell fate and function. MEF2 transcription factors (TFs) play essential roles in this process but how these broadly expressed factors contribute to the generation of specific cell types during development is poorly understood. Here we show that despite being expressed in virtually all mammalian tissues, in the retina MEF2D binds to retina-specific enhancers and controls photoreceptor cell development. MEF2D achieves specificity by cooperating with a retina-specific factor CRX, which recruits MEF2D away from canonical MEF2 binding sites and redirects it to retina-specific enhancers that lack the consensus MEF2-binding sequence. Once bound to retina-specific enhancers, MEF2D and CRX co-activate the expression of photoreceptor-specific genes that are critical for retinal function. These findings demonstrate that broadly expressed TFs acquire specific functions through competitive recruitment to enhancers by tissue-specific TFs and through selective activation of these enhancers to regulate tissue-specific genes.

CD151 accelerates breast cancer by regulating alpha 6 integrin function, signaling, and molecular organization.

CD151, a master regulator of laminin-binding integrins (alpha(6)beta(4), alpha(6)beta(1), and alpha(3)beta(1)), assembles these integrins into complexes called tetraspanin-enriched microdomains. CD151 protein expression is elevated in 31% of human breast cancers and is even more elevated in high-grade (40%) and estrogen receptor-negative (45%) subtypes. The latter includes triple-negative (estrogen receptor, progesterone receptor, and HER2 negative) basal-like tumors. CD151 ablation markedly reduced basal-like mammary cell migration, invasion, spreading, and signaling (through FAK, Rac1, and lck) while disrupting epidermal growth factor receptor (EGFR)-alpha(6) integrin collaboration. Underlying these defects, CD151 ablation redistributed alpha(6)beta(4) integrins subcellularly and severed molecular links between integrins and tetraspanin-enriched microdomains. In a prototypical basal-like mammary tumor line, CD151 ablation notably delayed tumor progression in ectopic and orthotopic xenograft models. These results (a) establish that CD151-alpha(6) integrin complexes play a functional role in basal-like mammary tumor progression; (b) emphasize that alpha(6) integrins function via CD151 linkage in the context of tetraspanin-enriched microdomains; and (c) point to potential relevance of CD151 as a high-priority therapeutic target, with relative selectivity (compared with laminin-binding integrins) for pathologic rather than normal physiology.

TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16- NK cells with similarities to decidual NK cells.

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and cultured in vitro, were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition, Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.

Contrasting effects of EWI proteins, integrins, and protein palmitoylation on cell surface CD9 organization.

CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of alpha3beta1 and alpha6beta4 integrins and by palmitoylation of the CD9 and beta4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression.

Human decidual NK cells form immature activating synapses and are not cytotoxic.

In early pregnancy invading fetal trophoblasts encounter abundant maternal decidual natural killer cells (dNK). dNK express perforin, granzymes A and B and the activating receptors NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as LFA-1. Even though they are granular and express the essential molecules required for lysis, fresh dNK displayed very reduced lytic activity on classical MHC I negative targets K562 and 721.221, approximately 15% of that of peripheral NK cells. dNK formed conjugates and activating immune synapses with 721.221 and K562 cells in which CD2, LFA-1 and actin were polarized toward the contact site. However, in contrast to peripheral NK cells, they failed to polarize their microtubule organizing centers and perforin-containing granules to the synapse, accounting for their lack of cytotoxicity.

The mature activating natural killer cell immunologic synapse is formed in distinct stages.

Natural killer (NK) cells form a structure at their interface with a susceptible target cell called the activating NK cell immunologic synapse (NKIS). The mature activating NKIS contains a central and peripheral supramolecular activation cluster (SMAC), and includes polarized surface receptors, filamentous actin (F-actin) and perforin. Evaluation of the NKIS in human NK cells revealed CD2, CD11a, CD11b and F-actin in the peripheral SMAC (pSMAC) with perforin in the central SMAC. The accumulation of F-actin and surface receptors was rapid and depended on Wiskott-Aldrich syndrome protein-driven actin polymerization. The accumulation at and arrangement of these molecules in the pSMAC was not affected by microtubule depolymerization. The polarization of perforin, however was slower and required intact actin, Wiskott-Aldrich syndrome protein, and microtubule function. Thus the process of CD2, CD11a, CD11b, and F-actin accumulation in the pSMAC and perforin accumulation in the central SMAC of the NKIS are sequential processes with distinct cytoskeletal requirements.