Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P3 production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P3 production in RBL-2H3 cells.

The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the β2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the β2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal α-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal α-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with different in vivo half-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+ T cells.

Ocular cicatricial pemphigoid antigen: Partial sequence and biochemical characterization

Ocular cicatricial pemphigoid (OCP) is an autoimmune disease that affects mainly conjunctiva and other squamous epithelia. OCP is histologically characterized by a separation of the epithelium from underlying tissues within the basement membrane zone. Immunopathological studies demonstrate the deposition of anti-basement membrane zone autoantibodies in vivo. Purified IgG from sera of patients with active OCP identified a cDNA clone from a human keratinocyte cDNA library that had complete homology with the cytoplasmic domain of β4-integrin. The sera recognized a 205-kDa protein in human epidermal, human conjunctiva, and tumor cell lysates that was identified as β4-integrin by its reaction with polyclonal and monoclonal antibodies to human β4-integrin. Sera from patients with bullous pemphigoid, pemphigus vulgaris, and cicatricial pemphigoid-like diseases did not recognize the 205-kDa protein, indicating the specificity of the binding. These data strongly implicate a role for human β4-integrin in the pathogenesis of OCP. It should be emphasized that multiple antigens in the basement membrane zone of squamous epithelia may serve as targets for a wide spectrum of autoantibodies observed in vesiculobullous diseases. Molecular definition of these autoantigens will facilitate the classification and characterization of subsets of cicatricial pemphigoid and help distinguishing them from bullous pemphigoid. This study highlights the function and importance of β4-integrin in maintaining the attachment of epithelial cells to the basement membrane.

Immune response to a differentiation antigen induced by altered antigen: A study of tumor rejection and autoimmunity

Recognition of self is emerging as a theme for the immune recognition of human cancer. One question is whether the immune system can actively respond to normal tissue autoantigens expressed by cancer cells. A second but related question is whether immune recognition of tissue autoantigens can actually induce tumor rejection. To address these issues, a mouse model was developed to investigate immune responses to a melanocyte differentiation antigen, tyrosinase-related protein 1 (or gp75), which is the product of the brown locus. In mice, immunization with purified syngeneic gp75 or syngeneic cells expressing gp75 failed to elicit antibody or cytotoxic T-cell responses to gp75, even when different immune adjuvants and cytokines were included. However, immunization with altered sources of gp75 antigen, in the form of either syngeneic gp75 expressed in insect cells or human gp75, elicited autoantibodies to gp75. Immunized mice rejected metastatic melanomas and developed patchy depigmentation in their coats. These studies support a model of tolerance maintained to a melanocyte differentiation antigen where tolerance can be broken by presenting sources of altered antigen (e.g., homologous xenogeneic protein or protein expressed in insect cells). Immune responses induced with these sources of altered antigen reacted with various processed forms of native, syngeneic protein and could induce both tumor rejection and autoimmunity.

CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins

Neisseria gonorrhoeae (GC) or Escherichia coli expressing phase-variable opacity (Opa) protein (Opa+ GC or Opa+ E. coli) adhere to human neutrophils and stimulate phagocytosis, whereas their counterparts not expressing Opa protein (Opa− GC or Opa− E. coli) do not. Opa+ GC or E. coli do not adhere to human lymphocytes and promyelocytic cell lines such as HL-60 cells. The adherence of Opa+ GC to the neutrophils can be enhanced dramatically if the neutrophils are preactivated. These data suggest that the components binding the Opa+ bacteria might exist in the granules. CGM1a antigen, a transmembrane protein of the carcinoembryonic antigen family, is exclusively expressed in the granulocytic lineage. The predicted molecular weight of CGM1a is ≈30 kDa. We observed specific binding of OpaI+ E. coli to a 30-kDa band of polymorphonuclear leukocytes lysates. To prove the hypothesis that the 30-kDa CGM1a antigen from neutrophils was the receptor of Opa+ bacteria, we showed that a HeLa cell line expressing human CGM1a antigen (HeLa-CGM1a) bound Opa+ E. coli and subsequently engulfed the bacteria. Monoclonal antibodies (COL-1) against CGM1 blocked the interaction between Opa+ E. coli and HeLa-CGM1a. These results demonstrate that HeLa cells when expressing the CGM1a antigens bind and internalize OpaI+ bacteria.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

Use of soluble peptide–DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis

Considerable evidence indicates that CD4+ T cells are important in the pathogenesis of rheumatoid arthritis (RA), but the antigens recognized by these T cells in the joints of patients remain unclear. Previous studies have suggested that type II collagen (CII) and human cartilage gp39 (HCgp39) are among the most likely synovial antigens to be involved in T cell stimulation in RA. Furthermore, experiments have defined dominant peptide determinants of these antigens when presented by HLA-DR4, the most important RA-associated HLA type. We used fluorescent, soluble peptide–DR4 complexes (tetramers) to detect synovial CD4+ T cells reactive with CII and HCgp39 in DR4+ patients. The CII-DR4 complex bound in a specific manner to CII peptide-reactive T cell hybridomas, but did not stain a detectable fraction of synovial CD4+ cells. A background percentage of positive cells (<0.2%) was not greater in DR4 (DRB1*0401) patients compared with those without this disease-associated allele. Similar results were obtained with the gp39-DR4 complex for nearly all RA patients. In a small subset of DR4+ patients, however, the percentage of synovial CD4+ cells binding this complex was above background and could not be attributed to nonspecific binding. These studies demonstrate the potential for peptide–MHC class II tetramers to be used to track antigen-specific T cells in human autoimmune diseases. Together, the results also suggest that the major oligoclonal CD4+ T cell expansions present in RA joints are not specific for the dominant CII and HCgp39 determinants.

Melan-A/MART-151–73 represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+ T cells

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+ cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+ T cells. The Melan-A/MART-151–73 peptide was able to induce the in vitro expansion of specific CD4+ T cells derived from normal DR4+ donors or from DR4+ patients with melanoma when pulsed onto autologous dendritic cells. CD4+ responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73 peptide and DR4+ melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+ T cell immunoreactivity against the Melan-A/MART-151–73 peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35 reactive CD8+ T cells in freshly isolated blood harvested from HLA-A2+/DR4+ patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+ T cell responses against melanoma in vivo.

Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is a processivity factor required for DNA polymerase δ (or ɛ)-catalyzed DNA synthesis. When loaded onto primed DNA templates by replication factor C (RFC), PCNA acts to tether the polymerase to DNA, resulting in processive DNA chain elongation. In this report, we describe the identification of two separate peptide regions of human PCNA spanning amino acids 36–55 and 196–215 that bind RFC by using the surface plasmon resonance technique. Site-directed mutagenesis of residues within these regions in human PCNA identified two specific sites that affected the biological activity of PCNA. Replacement of the aspartate 41 residue by an alanine, serine, or asparagine significantly impaired the ability of PCNA to (i) support the RFC/PCNA-dependent polymerase δ-catalyzed elongation of a singly primed DNA template; (ii) stimulate RFC-catalyzed DNA-dependent hydrolysis of ATP; (iii) be loaded onto DNA by RFC; and (iv) activate RFC-independent polymerase δ-catalyzed synthesis of poly dT. Introduction of an alanine at position 210 in place of an arginine also reduced the efficiency of PCNA in supporting RFC-dependent polymerase δ-catalyzed elongation of a singly primed DNA template. However, this mutation did not significantly alter the ability of PCNA to stimulate DNA polymerase δ in the absence of RFC but substantially lowered the efficiency of RFC-catalyzed reactions. These results are in keeping with a model in which surface exposed regions of PCNA interact with RFC and the subsequent loading of PCNA onto DNA orients the elongation complex in a manner essential for processive DNA synthesis.

Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.

Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: Implications for molecular mimicry in autoimmune disease

The immunodominant, CD8+ cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein–Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide—RSKFRQIV—located in a serine/threonine kinase and a bacterial peptide—RRKYKQII—located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted αβ TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8+ CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such as Mycobacterium tuberculosis and Leishmania major is the requirements to sustain memory/effector Th1 cells in vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity to L. major after vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generated in vivo to mediate long-term protection to an intracellular pathogen.