Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund’s adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon γ levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Identification and characterization of an alternative cytotoxic T lymphocyte-associated protein 4 binding molecule on B cells.

To determine whether alternative cytotoxic T lymphocyte-associated protein 4 (CTLA4) binding proteins exist on B cells, we constructed (i) mCTLA4hIgG consisting of the extracellular region of a mouse CTLA4 molecule and the Fc portion of a human IgG1 molecule and (ii) PYAAhIgG, a mutant mCTLA4hIgG, having two amino acid substitutions on the conserved MYPPPY motif in the complementarity-determining region 3-like region and lacking detectable binding to both B7-1 and B7-2 molecules. Using these fusion proteins (mCTLA4hIgG and PYAAhIgG), we demonstrated that a mouse immature B-cell line, WEHI231 cells, expressed alternative CTLA4 binding molecules (ACBMs) that were distinct from both B7-1 and B7-2. ACBMs were 130-kDa disulfide-linked proteins. More importantly, ACBMs were able to provide costimulatory signal for T-cell proliferation in the presence of anti-CD3 monoclonal antibodies. In addition, we demonstrated that more than 20% of B220+ cells obtained from normal mouse spleen expressed ACBMs.

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes.

Using transgenic mice that replicate the hepatitis B virus (HBV) genome, we recently demonstrated that class I-restricted, hepatitis B surface antigen-specific cytotoxic T lymphocytes (CTLs) can noncytolytically eliminate HBV pregenomic and envelope RNA transcripts from the hepatocyte. We now demonstrate that the steady-state content of these viral transcripts is profoundly reduced in the nucleus and cytoplasm of CTL-activated hepatocytes, but their transcription rates are only slightly reduced. Additionally, we demonstrate that transcripts covering the HBV X coding region are resistant to downregulation by the CTL. These results imply the existence of CTL-inducible hepatocellular factors that interact with a discrete element(s) between nucleotides 3157 and 1239 within the viral pregenomic and envelope transcripts and mediate their degradation, thus converting the hepatocyte from a passive victim to an active participant in the host response to HBV infection.

Elimination of residual metastatic prostate cancer after surgery and adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy

Cancer relapse after surgery is a common occurrence, most frequently resulting from the outgrowth of minimal residual disease in the form of metastases. We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade as an adjunctive immunotherapy to reduce metastatic relapse after primary prostate tumor resection. For these studies, we developed a murine model in which overt metastatic outgrowth of TRAMP-C2 (C2) prostate cancer ensues after complete primary tumor resection. Metastatic relapse in this model occurs reliably and principally within the draining lymph nodes in close proximity to the primary tumor, arising from established metastases present at the time of surgery. Using this model, we demonstrate that adjunctive CTLA-4 blockade administered immediately after primary tumor resection reduces metastatic relapse from 97.4 to 44%. Consistent with this, lymph nodes obtained 2 weeks after treatment reveal marked destruction or complete elimination of C2 metastases in 60% of mice receiving adjunctive anti-CTLA-4 whereas 100% of control antibody-treated mice demonstrate progressive C2 lymph node replacement. Our study demonstrates the potential of adjunctive CTLA-4 blockade immunotherapy to reduce cancer relapse emanating from minimal residual metastatic disease and may have broader implications for improving the capability of immunotherapy by combining such forms of therapy with other cytoreductive measures including surgery.

Parasite-mediated nuclear factor κB regulation in lymphoproliferation caused by Theileria parva infection

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor κB (NFκB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFκB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IκB molecules which normally sequester NFκB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IκBα. However, IκBα reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFκB-mediated positive feedback loop which restores cytoplasmic IκBα. In contrast, T. parva mediated continuous degradation of IκBβ resulting in persistently low cytoplasmic IκBβ levels. Normal IκBβ levels were only restored following T. parva killing, indicating that viable parasites are required for IκBβ degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IκB degradation and consequent enhanced expression of NFκB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IκB levels or NFκB activation, indicating that the parasite subverts the normal IκB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

Specific Activation of Leukocyte β2 Integrins Lymphocyte Function–associated Antigen-1 and Mac-1 by Chemokines Mediated by Distinct Pathways via the α Subunit Cytoplasmic Domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (αMβ2) but not lymphocyte function–associated antigen-1 (LFA-1; αLβ2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1α in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1α were confirmed by expression of αM or αL in αL-deficient Jurkat cells. Moreover, expression of chimeras containing αL and αM cytoplasmic domain exchanges indicated that α cytoplasmic tails conferred the specific mode of regulation. Coexpressing αM or chimeras in mutant Jurkat cells with a “gain of function” phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the αL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of β2 integrins. Our data suggest that a specific regulation of β2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the α subunit cytoplasmic domains.

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-Å resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (VH) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human VH3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig VH regions and the T-cell receptor Vβ regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor Vβ backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (VH) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging.

Antioxidants may play an important role in preventing free radical damage associated with aging by interfering directly in the generation of radicals or by scavenging them. We investigated the effects of a high vitamin E and/or a high beta-carotene diet on aging of the anion transporter, band 3, in lymphocytes and brain. The band 3 proteins function as anion transporters, acid base regulators, C02 transporters, and structural proteins that provide a framework for membrane lipids and that link the plasma membrane to the cytoskeleton. Senescent cell antigen (SCA), which terminates the life of cells, is a degradation product of band 3. This study was conducted as a double-blind study in which eight groups of middle-aged or old mice received either high levels of beta-carotene and/or vitamin E or standard levels of these supplements in their diets. Anion transport kinetic assays were performed on isolated splenic lymphocytes. Immunoreactivity of an antibody that recognizes aging changes in old band 3 preceding generation of SCA was used to quantitate aged band 3 in brain tissue. Results indicate that vitamin E prevented the observed age-related decline in anion transport by lymphocytes and the generation of aged band 3 leading to SCA formation. beta-Carotene had no significant effect on the results of either assay. Since increased aged band 3 and decreased anion transport are initial steps in band 3 aging, which culminates in the generation of SCA and cellular removal, vitamin E prevents or delays aging of band 3-related proteins in lymphocytes and brain.

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.

Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires.

Immunological self-tolerance is ensured by eliminating or inhibiting self-reactive lymphocyte clones, creating physical or functional holes in the B- and T-lymphocyte antigen receptor repertoires. The nature and size of these gaps in our immune defenses must be balanced against the necessity of mounting rapid immune responses to an everchanging array of foreign pathogens. To achieve this balance, only a fraction of particularly hazardous self-reactive clones appears to be physically eliminated from the repertoire in a manner that fully prevents their recruitment into an antimicrobial immune response. Many self-reactive cells are retained with a variety of conditional and potentially flexible restraints: (i) their ability to be triggered by antigen is diminished by mechanisms that tune down signaling by their antigen receptors, (ii) their ability to carry out inflammatory effector functions can be inhibited, and (iii) their capacity to migrate and persist is constrained. This balance between tolerance and immunity can be shifted, altering susceptibility to autoimmune disease and to infection by genetic or environmental differences either in the way antigens are presented, in the tuning molecules that adjust triggering set points for lymphocyte responses to antigen, or in the effector molecules that eliminate, retain, or expand particular clones.