COP9 signalosome component JAB1/CSN5 is necessary for T cell signaling through LFA-1 and HIV-1 replication.

To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication

Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front.

Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)-a collection of components characteristic of the CRC iTME. Enrichment of PD-1+CD4+ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains

Snapin, positive regulator of stimulation- induced Ca²⁺ release through RyR, is necessary for HIV-1 replication in T cells.

To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca²⁺ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca²⁺/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4⁺ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca²⁺ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology

JAB1 overcomes the inhibition of HIV-1 replication in cognate peptide expressing SupT1 cells.

<p>(A) pBMN-control IRES-Lyt2α’ or pBMN-JAB1 IRES-Lyt2α’ retrovirus vectors were transduced into SupT1 cells that expressed either C-Pep1 or Pep24. (B) pBMN-control IRES-Lyt2α’ or pBMN-JAB1 IRES-Lyt2α’ retrovirus vectors were transduced into SupT1 cells. These cells were challenged with HIV-1 (NL4-3) at a dose 400 TCID₅₀ per 5×10⁴ cells. p24^gag levels in culture supernatants were assayed from four wells on the indicated days after infection. p24^gag levels were normalized to cell number determined using an XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. * indicates <i>p</i><0.05, Control C-Pep-1 SupT1 versus Control Pep24 SupT1, and # indicates <i>p</i><0.05, Control Pep24 SupT1 versus JAB1 Pep24 SupT1 by <i>t</i> test. (C) JNK inhibitor (SP600125) inhibits HIV-1 replication. SupT1 cells were treated JNK inhibitor (SP600125) for 30 min before HIV-1 challenge. These cells were challenged with HIV-1 (NL4-3) at a dose 400 TCID₅₀ per 5×10⁴ cells. p24^gag levels in culture supernatants were assayed from five wells on the indicated days after infection. p24^gag levels were normalized for cell number using XTT assay. Data are presented as the average ± SE per 10⁶ cells. Similar results were observed in three independent experiments. *indicates <i>p</i><0.05, No treatment versus 10 µM SP600125, and # indicates <i>p</i><0.05, No treatment versus 30 µM SP600125 by <i>t</i> test.</p

JNK is one of kinases on which HIV-1 depends.

<p>Jurkat cells expressing C-Pep1 or Pep24 were treated with indicated stimuli. Cells were stained with pERK-Alexa 647 or pJNK-Alexa 647 phosphospecific antibodies and analyzed by flow cytometry. (A) Histograms are colored according to the different stimuli. (B) Fold change was approximated by calculating the log2 ratio of mean fluorescence intensity of stimulated versus unstimulated cells.</p

Proposed model for the inhibition of signalling pathway by Pep24.

<p>(A) After LFA-1 activation, JAB1 relocalizes into cytoplasm and nucleus, activates AP-1 and induces gene activation, and finally activates HIV-1 replication. (B) When Pep24 binds to JAB-1, JAB1 relocalization is blocked inhibiting downstream signals and HIV-1 replication.</p

Pep24 blocks LFA-1-induced JAB1 relocalization.

<p>Jurkat cells expressing indicated peptides were adhered to either anti-LFA-1 mAb or control IgG coated plates for 30 min. After cross-linking stimulation, cells were stained for JAB1 (red). GFP (green) is an indicator of peptide expression. (A–C) C-Pep1-expressing cells without stimulation. (D–F) C-pep1-expressing cells with LFA-1 cross-linking stimulation. (G–I) Pep24-expressing cells without stimulation. (J–L) Pep24-expressing cells with LFA-1 cross-linking stimulation.</p

Selected peptide, Pep24, preferentially inhibits the NFAT signaling pathway.

<p>(A) The sequences of control peptides and selected peptide. (B–D) Reporter plasmids (B) p55-IgκLuc, (C) NFAT Luc, or (D) AP-1 Luc were transfected into Jurkat cells expressing indicated peptides with pBMN LacZ as the internal control plasmid. Cells treated for 3 hr (8 hr for AP-1) with or without indicated agents (2 µg/ml PHA, 10 ng/ml PMA, and 10 ng/ml TNF-α) prior to measurement of luciferase activity. The experiments were repeated three times and the average is plotted ± SE. Jurkat cells transfected with reporter without treatment were assigned a value of 1 and were used to calculate the fold activation. Transfection efficiencies were normalized to a co-transfected lacZ plasmid.</p

Pep24 specifically binds to JAB1.

<p>The indicated biotin-conjugated peptides were incubated with GST or with the GST-fused JAB1. The complex of peptide-GST fusion protein immobilized on streptavidin-agarose was detected by western blotting with anti-GST antibody.</p

Pep80 inhibits the interaction between Snapin and RyR in T cells.

<p>After crosslinking the ER fraction from Jurkat cells and from C-Pep-1- or Pep80-expressing Jurkat cells, samples were immunopreciptated with anti-RyR3 antibody and immunoblotted with anti-Snapin antibody.</p