High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.     

Inhibition of clathrin‐mediated endocytosis by knockdown of AP‐2 leads to alterations in the plasma membrane proteome

In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.     

A single common portal for clathrin-mediated endocytosis of distinct cargo governed by cargo-selective adaptors

Sorting of transmembrane cargo into clathrin-coated vesicles requires endocytic adaptors, yet RNA interference (RNAi)-mediated gene silencing of the AP-2 adaptor complex only disrupts internalization of a subset of clathrin-dependent cargo. This suggests alternate clathrin-associated sorting proteins participate in cargo capture at the cell surface, and a provocative recent proposal is that discrete endocytic cargo are sorted into compositionally and functionally distinct clathrin coats. We show here that the FXNPXY-type internalization signal within cytosolic domain of the LDL receptor is recognized redundantly by two phosphotyrosine-binding domain proteins, Dab2 and ARH; diminishing both proteins by RNAi leads to conspicuous LDL receptor accumulation at the cell surface. AP-2-dependent uptake of transferrin ensues relatively normally in the absence of Dab2 and ARH, clearly revealing delegation of sorting operations at the bud site. AP-2, Dab2, ARH, transferrin, and LDL receptors are all present within the vast majority of clathrin structures at the surface, challenging the general existence of specialized clathrin coats for segregated internalization of constitutively internalized cargo. However, Dab2 expression is exceptionally low in hepatocytes, likely accounting for the pathological hypercholesterolemia that accompanies ARH loss.     

Identification and characterization of fully human anti-CD22 monoclonal antibodies

CD22 is a member of the B cell receptor family and is implicated in B cell function and development. It is expressed on multiple forms of B cell lymphoma and is an attractive cancer therapeutic target. We report here the identification of two fully human anti-CD22 antibodies using phage display methodology. Both antibodies exhibit specific binding to cell surface-associated CD22 in multiple B cell lines. Through ELISA using mammalian cell-expressed sub-domains of CD22 as binding antigen, we mapped the binding epitopes of the newly identified CD22 antibodies to be within the Ig-like domains 5 to 7 of CD22. Their epitopes do not overlap with those of several therapeutic antibodies currently in preclinical or clinical development. These antibodies have potential as cancer therapeutic candidates and research reagents.     

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.     

Identification and characterization of fully human anti-CD22 monoclonal antibodies

CD22 is a member of the B cell receptor family and is implicated in B cell function and development. It is expressed on multiple forms of B cell lymphoma and is an attractive cancer therapeutic target. We report here the identification of two fully human anti-CD22 antibodies using phage display methodology. Both antibodies exhibit specific binding to cell surface-associated CD22 in multiple B cell lines. Through ELISA using mammalian cell-expressed sub-domains of CD22 as binding antigen, we mapped the binding epitopes of the newly identified CD22 antibodies to be within the Ig-like domains 5 to 7 of CD22. Their epitopes do not overlap with those of several therapeutic antibodies currently in preclinical or clinical development. These antibodies have potential as cancer therapeutic candidates and research reagents.Key words: human antibody, CD22, phage display, cancer, hematological     

The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and alpha 2-6 sialyltransferase, CD75, on B cells.

Functional maturation of B lymphocytes correlates with expression of the B lineage-specific cell surface glycoprotein CD22. Two CD22 polypeptides have been characterized and suggested to play a role in B cell-B cell interaction as well as in B cell adhesion to monocytes. In this work we provide evidence that CD22 is directly involved in the cognate interaction between B and T cells. One of the two CD22 polypeptides, CD22 beta, interacts with a specific ligand on a subpopulation of CD4+ T cells. Our results suggest that the T cell ligand of CD22 is CD45RO, an isoform of the leukocyte common antigen class of phosphotyrosine phosphatases associated with the helper T cell phenotype. We further demonstrate that CD22 recognizes a second ligand, CD75, expressed predominantly on activated B cells and shown to be a cell surface alpha 2-6 sialyltransferase.     

A Tandem Di-hydrophobic Motif Mediates Clathrin-dependent Endocytosis via Direct Binding to the AP-2 ασ2 Subunits

Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.     

The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein,via tyrosine-dependent interaction

The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.     

CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux

CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation.     

Regulation of Endocytic Sorting by ESCRT–DUB-Mediated Deubiquitination

Endocytosis of cell surface receptors mediates cellular homeostasis by coordinating receptor distribution with downstream signal transduction and attenuation. Post-translational modification with ubiquitin of these receptors, as well as the proteins that comprise the endocytic machinery, modulates cargo progression along the endocytic pathway. The interplay between ubiquitination states of cargo and sorting proteins drives trafficking outcomes by directing endocytosed material toward either lysosomal degradation or recycling. Deubiquitination by specific proteinases creates a reversible system that promotes spatial and temporal organization of endosomal sorting complexes required for transport (ESCRTs) and supports regulated cargo trafficking. Two dubiquitinating enzymes--ubiquitin-specific protease 8 (USP8/Ubpy) and associated molecule with the SH3 domain of STAM (AMSH)--interact with ESCRT components to modulate the ubiquitination status of receptors and relevant sorting proteins. In doing so, these ESCRT-DUBs control receptor fate and sorting complex function through a variety of mechanisms described herein.     

Cargo regulates clathrin-coated pit dynamics

Clathrin-coated pits (CCPs) are generally considered a uniform population of endocytic machines containing mixed constitutive and regulated membrane cargo. Contrary to this view, we show that regulated endocytosis of G protein-coupled receptors (GPCRs) occurs preferentially through a subset of CCPs. Significantly, GPCR-containing CCPs are also functionally distinct, as their surface residence time is regulated locally by GPCR cargo via PDZ-dependent linkage to the actin cytoskeleton. Such cargo-regulated CCPs show delayed recruitment of dynamin and can undergo an abortive event in which clathrin coats separate from the plasma membrane without concomitant receptor endocytosis. Segregation of cargo into CCP subsets, combined with cargo-dependent control of CCP dynamics, suggests a simple kinetic mechanism to generate functional specialization early in the endocytic pathway and reduce competition between diverse endocytic cargo.     

The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2)

CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.     

Lateral membrane protein associations of CD4 in lymphoid cells detected by cross-linking and mass spectrometry

Interactions of membrane proteins are important in various aspects of cell function. However, weak membrane protein-protein interactions are difficult to study using techniques such as co-immunoprecipitations. CD4 is a cell surface protein involved in T cell activation and the binding of the human immunodeficiency virus to HIV target cells. Here we report the use of cross-linking followed by affinity purification of CD4 in combination with mass spectrometry for identification of proteins that are in the proximity of CD4. Besides the components of the CD4 receptor complex, CD4 and lck, we have identified by tandem mass spectrometry 17 tryptic peptides from transferrin receptor CD71, three peptides from protein phosphatase CD45, and one peptide from 4F2 lymphocyte activation antigen CD98. The efficiency of the cross-linking did not correlate with the level of cell surface expression of the detected molecules, excluding a possible bias of the cross-linking toward the most abundant cell surface molecules. Whereas the association of CD4 with CD45 has been reported, the associations with CD71 and CD98 have not been previously described. We used small-scale immunoprecipitation after cross-linking in combination with fluorescence resonance energy transfer (FRET) measurements to investigate the association between CD4 and CD71. Our data show that CD71 self-associates on the cell surface, that a small fraction of CD4 can be detected by copurifying it with CD71 after cross-linking, and that the level of association between CD4 and CD71 significantly increases after phorbol 12-myristate 13-acetate-induced endocytosis of CD4. This suggests that a small fraction of CD4 associates with clusters of CD71. As both molecules undergo endocytic recycling, the association and cross-linking result from their clustering in the same pit and/or vesicle. The CD4-CD98 association probably results from nonspecific cross-linking.     

CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-gamma production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.     

Maintenance and loss of endocytic organelle integrity: mechanisms and implications for antigen cross-presentation

The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8⁺ T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.     

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.     

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.     

Imaging lysosomal enzyme activity in live cells using self-quenched substrates

Endocytosis, the internalization and transport of extracellular cargo, is an essential cellular process. The ultimate step in endocytosis is the intracellular degradation of extracellular cargo for use by the cell. While live cell imaging and single particle tracking have been well-utilized to study the internalization and transport of cargo, the final degradation step has required separate biochemical assays. We describe the use of self-quenched endocytic cargo to image the intracellular transport and degradation of endocytic cargo directly in live cells. We first outline the fluorescent labeling and quantification of two common endocytic cargos: a protein, bovine serum albumin, and a lipid nanoparticle, low-density lipoprotein. In vitro measurements confirm that self-quenching is a function of the number of fluorophores bound to the protein or particle and that recovery of the fluorescent signal occurs in response to enzymatic degradation. We then use confocal fluorescence microscopy and flow cytometry to demonstrate the use of self-quenched bovine serum albumin with standard fluorescence techniques. Using live cell imaging and single particle tracking, we find that the degradation of bovine serum albumin occurs in an endo-lysosomal vesicle that is positive for LAMP1.     

B cell antigen receptor and CD40 differentially regulate CD22 tyrosine phosphorylation

Cell surface molecules on lymphocytes positively or negatively modulate the Ag receptor signaling, and thus regulate the fate of the cell. CD22 is a B cell-specific cell surface protein that contains multiple ITIMs in the cytoplasmic tail, and critically regulates B cell activation and survival. CD22 regulation on B cell signaling is complex because CD22 can have both positive and negative roles in various contexts. We generated phosphospecific polyclonal Abs reacting four major CD22 tyrosine motifs (Y762, Y807, Y822, and Y842) and analyzed the pattern and intensity of phosphorylation of these tyrosine residues. The tyrosine motifs, Y762, Y822, and Y842, are considered as ITIM, whereas the other, Y807, is suggested to be important for Grb2 recruitment. Approximately 10% of the four tyrosine residues were constitutively phosphorylated. Upon anti-IgM ligation, CD22 Y762 underwent most rapid phosphorylation, whereas all four tyrosine residues were eventually phosphorylated equally at approximately 35% of all CD22 molecules in the cell. By contrast, anti-CD40 stimulation specifically up-regulated anti-IgM-induced phosphorylation of tyrosines within two ITIM motifs, Y762 and Y842, which was consistent with in vivo finding of the negative role of CD22 in CD40 signaling. Thus, CD22 phosphorylation is not only quantitatively but also qualitatively regulated by different stimulations, which may determine the outcome of B cell signaling.