Paul J. Conlon

Activation of macrophage tumoricidal activity by granulocyte-macrophage colony stimulating factor

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo- controlled, randomized phase II trial

In this ‘double-blind’, randomized, placebo-controlled phase II trial, we compared an altered peptide ligand of myelin basic protein with placebo, evaluating their safety and influence on magnetic resonance imaging in relapsing–remitting multiple sclerosis. A safety board suspended the trial because of hypersensitivity reactions in 9% of the patients. There were no increases in either clinical relapses or in new enhancing lesions in any patient, even those with hypersensitivity reactions. Secondary analysis of those patients completing the study showed that the volume and number of enhancing lesions were reduced at a dose of 5 mg. There was also a regulatory type 2 T helper-cell response to altered peptide ligand that cross-reacted with the native peptide.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin.

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia.

Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and 3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS.

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Human endometriosis is associated with plasma cells and overexpression of B lymphocyte stimulator

Endometriosis affects 10–20% of women of reproductive age and is associated with pelvic pain and infertility, and its pathogenesis is not well understood. We used genomewide transcriptional profiling to characterize endometriosis and found that it exhibits a gene expression signature consistent with an underlying autoimmune mechanism. Endometriosis lesions are characterized by the presence of abundant plasma cells, many of which produce IgM, and macrophages that produce BLyS/BAFF/TNFSF13B, a member of the TNF superfamily implicated in other autoimmune diseases. B lymphocyte stimulator (BLyS) protein was found elevated in the serum of endometriosis patients. These observations suggest a model for the pathology of endometriosis where BLyS-responsive plasma cells interact with retrograde menstrual tissues to give rise to endometriosis lesions.

Characterization of γ-Aminobutyric Acid Receptor GABAB(1e), a GABAB(1) Splice Variant Encoding a Truncated Receptor

We have identified a splice variant encoding only the extracellular ligand-binding domain of the γ-aminobutyric acid B (GABAB) receptor subunit GABAB(1a). This isoform, which we have named GABAB(1e), is detected in both rats and humans. While GABAB(1e) is a minor component of the total pool of GABAB(1) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABAB(1e) lacks the ability to bind the radiolabeled antagonist [3H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABAB(2). Interestingly, when co-expressed with GABAB(2), not only does the truncated receptor heterodimerize with GABAB(2), the association is of sufficient avidity to disrupt the normal GABAB(1a)/GABAB(2) association. Despite this strong interaction, GABAB(1e) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABAB(1a)/GABAB(2).

Control of experimental contact sensitivity.

Publisher Summary This chapter discusses controls operative in the expression of experimental contact sensitivity. The events leading to contact sensitivity, including the essential reactivity of the antigen with self molecules, the involvement of Langerhans cells of the skin, and the eventual development of effector T lymphocytes are presented. The chapter focuses on several mechanisms influencing the extent of a sensitivity response. Controlling factors include (1) route and amount of antigen presentation, which to a great degree determine whether sensitization or tolerance will develop, (2) production of soluble suppressor substances that may accompany tolerogenic regimes of antigen administration, (3) formation of inhibitory antiidiotypic antibodies, and (4) development of T suppressor cells. These multiple regulatory mechanisms operating in contact sensitivity are quite similar to those involved in other cellular immune responses as well as in humoral responses and are in keeping with the general elaborate regulation controlling immunity. The chapter describes the regulatory and suppressive mechanisms that control the induction, magnitude, expression, or duration of the contact allergic reaction. Experimental contact sensitivity is a highly regulated immune response. Sensitization depends upon application of reactive antigens that couple covalently to self tissues.

Molecular Determinants of Melanocortin 4 Receptor Ligand Binding and MC4/MC3 Receptor Selectivity

The molecular basis of ligand recognition by the melanocortin 4 receptor (MC4R) has not been fully defined. In this study, we investigated the molecular determinants of MC4R ligand binding, employing a large array of ligands, using three approaches. First, molecular modeling of the receptor was used to identify Phe284, in transmembrane (TM) 7, as a potential site of ligand interaction. Mutation of Phe284 to alanine reduced binding affinity and potency of peptides containing l-Phe by up to 71-fold but did not appreciably affect binding of linear peptides containingd-Phe, consistent with a hydrophobic interaction between the Phe7 of α-melanocyte-stimulating hormone and Phe284. Second, we examined the effect of a naturally occurring mutation in TM3 (I137T) that is linked to obesity. This mutation decreased affinity and potency of cyclic, rigid peptides but not more flexible peptides, consistent with an indirect effect of the mutation on the tertiary structure of the receptor. Third, we examined the residues that support ligand selectivity for the MC4R over the MC3R. Mutation of Ile125 (TM3) of the MC4R to the equivalent residue of the MC3R (phenylalanine) selectively decreased affinity and potency of MC4R-selective ligands. This effect was mirrored by the reciprocal MC3R mutation F157I. The magnitude of this effect indicates that this locus is not of major importance. However, it is considered that an isoleucine/phenylalanine mutation may affect the orientation of Asp122, which has been identified as a major determinant of ligand binding affinity. Thus, this study provides further characterization of the MC4R binding pocket.

Contribution of Individual Amino Acids Within MHC Molecule or Antigenic Peptide to TCR Ligand Potency

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.