Michael A. Norcross

Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity.

Background:Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B*57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B*57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B*57:01 from abacavir-treated cells. Design and methods:An HLA-B*57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B*57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results:Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B*57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B*57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B*57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B*57:01 with high affinity that was not altered by abacavir addition. Conclusion:Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B*57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.

Dimerization of CXCR4 in living malignant cells: control of cell migration by a synthetic peptide that reduces homologous CXCR4 interactions

Chemokine receptor CXCR4 (CD184) may play a role in cancer metastasis and is known to form homodimers. However, it is not clear how transmembrane regions (TM) of CXCR4 and receptor homotypic interactions affect the function of CXCR4 in living cells. Using confocal microscopy and flow cytometric analysis, we showed that high levels of CXCR4 are present in the cytoplasm, accompanied by lower expression on the cell surface in CXCR4 transfectants, tumor cells, and normal peripheral blood lymphocytes. CXCR4 homodimers were detected in tumor cells, both on the cell surface membrane and in the cytoplasm using fluorescence resonance energy transfer and photobleaching fluorescence resonance energy transfer to measure energy transfer between CXCR4-CFP and CXCR4-YFP constructs. Disruption of lipid rafts by depletion of cholesterol with methyl-β-cyclodextrin reduced the interaction between CXCR4 molecules and inhibited malignant cell migration to CXCL12/SDF-1α. A synthetic peptide of TM4 of CXCR4 reduced energy transfer between molecules of CXCR4, inhibited CXCL12-induced actin polymerization, and blocked chemotaxis of malignant cells. TM4 also inhibited migration of normal monocytes toward CXCL12. Reduction of CXCR4 energy transfer by the TM4 peptide and methyl-β-cyclodextrin indicates that interactions between CXCR4s may play important roles in cell migration and suggests that cell surface and intracellular receptor dimers are appropriate targets for control of tumor cell spread. Targeting chemokine receptor oligomerization and signal transduction for the treatment of cancer, HIV-1 infections, and other CXCR4 mediated inflammatory conditions warrants further investigation. [Mol Cancer Ther 2006;5(10):2474–83]

Identification of Human Macrophage Inflammatory Proteins 1α and 1β as a Native Secreted Heterodimer

Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1α and 1β (MIP-1α and MIP-1β) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1α or MIP-1β precipitated a protein complex containing both MIP-1α and MIP-1β under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH2-terminal truncated MIP-1α (residues 5–70) together with either intact MIP-1β or NH2-terminal truncated MIP-1β (residues 3–69), respectively. The secreted MIP-1α/β heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH2-terminal truncated MIP-1α and full-length MIP-1β in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1α and MIP-1β is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1α/β may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.

Natural truncation of the chemokine MIP-1β/CCL4 affects receptor specificity but not anti-HIV-1 activity

Activated lymphocytes synthesize and secrete substantial amounts of the β-chemokines macrophage inflammatory protein (MIP)-1α/CCL3 and MIP-1β/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1β secreted by activated human peripheral blood lymphocytes (MIP-1β(3–69)) lacks the two NH2-terminal amino acids of the full-length protein. This truncated form of MIP-1β has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1β(3–69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1β(3–69) showed that the truncated protein not only shares the ability of intact MIP-1β to induce Ca2+ signaling through CCR5, but unlike the full-length protein, it also triggers a Ca2+ response via CCR1 and CCR2b. These results demonstrate that NH2-terminally truncated MIP-1β functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.

CD26 expression correlates with entry, replication and cytopathicity of monocytotropic HIV–1 strains in a T–cell line

Experiments to identify cell determinants involved in HIV-1 tropism revealed a specific decrease in the expression of the T–cell activation antigen CD26 after monocytotropic (M–tropic) but not T–cell line–tropic (T–tropic) virus infection of the PM1 T–cell line. The level of CD26 expression in single–cell clones of PM1 correlated with the entry rate and cytopathicity of M–tropic HIV–1 variants, resulting in preferential survival of cells with low CD26 levels after infection. Experiments with recombinant viruses showed that the third hypervariable region of the envelope gp120 plays an important role in this selection process. This study identifies CD26 as a key marker for M–tropic human immunodeficiency virus type 1 (HIV–1) infection and suggests a mechanism for the early loss of CD26–expressing cells in HIV–1–infected individuals.

Control of Adaptive Immune Responses by Staphylococcus aureus through IL-10, PD-L1, and TLR2

Microbes induce innate immune responses in hosts. It is critical to know how different microbes control adaptive responses through innate pathways. The impact of gram-positive bacteria on the innate and adaptive responses is unclear. Herein we report that Staphylococcus aureus induces IL-10, Th17-inducing cytokines IL-6 and IL-23, chemokines, and regulates dendritic cell markers. S. aureus inhibits T-cell IL-2 responses through modulation of HLA-DR, CD86 and PD-L1. IFN-gamma, Src kinase inhibitors, or TLR2 antibodies prevented the down-modulation of HLA-DR by S. aureus. Our data demonstrate that innate TLR signaling induces multi-dimensional inhibition of adaptive immune responses, which may contribute to the lack of protective immunity to bacteria or microbe tolerance. IL-10 and PD-L1 antagonists may boost immunity to vaccines for S. aureus and other microbes.

A transgenic mouse model for HLA-B*57:01–linked abacavir drug tolerance and reactivity

&NA; Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA‐B*57:01. However, not all HLA‐B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA‐linked ADRs in vivo, we generated HLA‐B*57:01‐Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA‐B*57:01‐dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy‐like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC‐reactive CD8+ T cells with an effector‐like and skin‐homing phenotype along with CD8+ infiltration and inflammation in drug‐sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA‐B*57:01‐restricted, ABC‐reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.