Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1–4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection

Next-generation in situ hybridization approaches to define and quantify HIV and SIV reservoirs in tissue microenvironments

Abstract The development of increasingly safe and effective antiretroviral treatments for human immunodeficiency virus (HIV) over the past several decades has led to vastly improved patient survival when treatment is available and affordable, an outcome that relies on uninterrupted adherence to combination antiretroviral therapy for life. Looking to the future, the discovery of an elusive ‘cure’ for HIV will necessitate highly sensitive methods for detecting, understanding, and eliminating viral reservoirs. Next-generation, in situ hybridization (ISH) approaches offer unique and complementary insights into viral reservoirs within their native tissue environments with a high degree of specificity and sensitivity. In this review, we will discuss how modern ISH techniques can be used, either alone or in conjunction with phenotypic characterization, to probe viral reservoir establishment and maintenance. In addition to focusing on how these techniques have already furthered our understanding of HIV reservoirs, we discuss potential avenues for how high-throughput, next-generation ISH may be applied. Finally, we will review how ISH could allow deeper phenotypic and contextual insights into HIV reservoir biology that should prove instrumental in moving the field closer to viral reservoir elimination needed for an ‘HIV cure’ to be realized

Receptor Endocytosis of Isolated CD79a but not CD79b.

<p>Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed (<u>Panel A</u>) and quantitated (<u>Panel B</u>) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter proteins with both CD79 cytoplasmic domains and other reporter proteins.</p

The Ia.2 Epitope Defines a Subset of Lipid Raft-Resident MHC Class II Molecules Crucial to Effective Antigen Presentation

Previous work established that binding of the 11-5.2 anti–I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti–I-Ak mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a b-chain–tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.22 tethered peptide–class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell–T cell interactions

Ultrastructural Colocalization of Ligand-BCR Complexes and AP2 in Clathrin-Coated Pits.

<p>A20µWT B cells or primary murine splenocytes were pulsed with anti-IgM-btn followed by anti-biotin-15 nm gold (arrow heads), incubated 2 minutes at 37° and then plasma membrane rips were prepared as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone.0054938-Caballero1" target="_blank">[10]</a>. The exposed cytoplasmic face of the plasma membrane was stained with anti-AP2 and Protein A-5 nm gold (arrows). The percent of BCR-containing CCP that also stained for AP2 is indicated below each image. Inset: AP2 and BCR co-localization within electron dense membrane regions lacking discernable CCP architecture. Shown are representative images from 1 of 3 experiments, with 1,000+ BCR-bound gold particles or 100+ CCP photographed cumulatively.</p

CD79b Y195 Controls BCR Internalization.

<p><u>Panel A</u>, Amino acid sequences of the cytoplasmic domains of CD79a and CD79b. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, Confocal laser scanning microscopic analysis of the endocytosis of indicated MHC class II-CD79 chimeric proteins. Plasma membrane is yellow (anti-MHC class II-Alexa 488+ post-endocytic labeling of external MHC class II-CD79 with an Alexa 594-labeled antibody). Internalized MHC class II-CD79 is green (anti-MHC class II-Alexa 488 only). <u>Panel C</u>, Quantification of MHC class II-CD79 endocytosis. 100+ cells from across 3 independent experiments were scored for internalization. Reported is the percent of cells showing internalized MHC class II-CD79 for each construct. Statistical comparisons were made between the construct with both CD79 cytoplasmic domains and cells expressing other constructs.</p

AP2µ Directly Binds the YxxØ Motif Centered on the Membrane-Proximal Tyrosine of Isolated CD79a.

<p><u>Panel A</u>, The binding of AP2µ-btn to bead-captured GST bearing 21 amino acid peptides centered on the five CD79 YxxØ motifs was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of CD79a–AP2 interactions and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between CD79a and other samples. <u>Panel B</u>, For both CD79a and CD79b, a positive or negative regulatory motif lies within +/−10 amino acids of the tyrosine residue of the membrane-proximal YxxØ AP2 binding motif. In this example, the motif is arbitrarily depicted as being downstream of the YxxØ motif.</p

AP2µ Binds to Isolated CD79a but not CD79b.

<p><u>Panel A</u>, Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. <u>Panel C</u>, Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. <u>Panel D</u>, The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 <i>E. coli</i> cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to <i>in vitro</i> translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL)₃, and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL)₃, fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL)₃–AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.</p

C-terminal Deletions Localize the Motif that Drives AP2 Binding to the Membrane Proximal YxxØ Motif of CD79a.

<p>Binding of AP2µ-btn to bead-captured GST bearing the indicated C-terminal deletions of CD79a was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Data is the mean of 3 independent experiments ± S.E.M. and data were normalized to the binding of AP2 to full length CD79a.</p