Rosario Billetta

Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25bright cells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.

Clustering of T Cell Ligands on Artificial APC Membranes Influences T Cell Activation and Protein Kinase C θ Translocation to the T Cell Plasma Membrane

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase θ, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

Modulation of T cell function by combination of epitope specific and low dose anticytokine therapy controls autoimmune arthritis.

Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by generating and maintaining inflammation, which leads to tissue damage. Current biological therapies target innate immunity, eminently by interfering with single pro-inflammatory cytokine pathways. This approach has shown excellent efficacy in a good proportion of patients with Rheumatoid Arthritis (RA), but is limited by cost and side effects. Adaptive immunity, particularly T cells with a regulatory function, plays a fundamental role in controlling inflammation in physiologic conditions. A growing body of evidence suggests that modulation of T cell function is impaired in autoimmunity. Restoration of such function could be of significant therapeutic value. We have recently demonstrated that epitope-specific therapy can restore modulation of T cell function in RA patients. Here, we tested the hypothesis that a combination of anti-cytokine and epitope-specific immunotherapy may facilitate the control of autoimmune inflammation by generating active T cell regulation. This novel combination of mucosal tolerization to a pathogenic T cell epitope and single low dose anti-TNFα was as therapeutically effective as full dose anti-TNFα treatment. Analysis of the underlying immunological mechanisms showed induction of T cell immune deviation.

Theoretical and practical aspects of antigenized antibodies

Antigenization of antibody is a new immunological concept (Zanetti 1992) that exemplifies MecthnikofTs words (Metchnikoff, reprinted in 1989) in a paradigmatic way. It was conceived with a two-fold purpose. One was to investigate directly the molecular basis of antibody antigenicity and immunogenicity. The other was to test from a molecular perspective one of the most controversial postulates ofthe idiotype network theory (Lindenmann 1973, Jeme 1974): antibodies as internal images of antigens. It soon became apparent, however, that antigenization of antibody was also a new, powerful way to provide a stable conformation to oligopeptides of general biological interest. Understanding the molecular and cellular requirements for antigenicity and immunogenicity of proteins is still a central issue in immunology. Antigenization of antibody may represent, therefore, a new alternative way to solve some of the puzzling aspects of immunology that still elude our understanding (e.g., the relationship between amino acid sequence, three-dimensional structure and function of soluble proteins and cell-surface receptors).

Heat shock protein-derived T-cell epitopes contribute to autoimmune inflammation in pediatric Crohn's disease.

Pediatric Crohns disease is a chronic auto inflammatory bowel disorder affecting children under the age of 17 years. A putative etiopathogenesis of Crohns disease (CD) is associated with disregulation of immune response to antigens commonly present in the gut microenvironment. Heat shock proteins (HSP) have been identified as ubiquitous antigens with the ability to modulate inflammatory responses associated with several autoimmune diseases. The present study tested the contribution of immune responses to HSP in the amplification of autoimmune inflammation in chronically inflamed mucosa of pediatric CD patients. Colonic biopsies obtained from normal and CD mucosa were stimulated with pairs of Pan HLA-DR binder HSP60-derived peptides (human/bacterial homologues). The modulation of RNA and protein levels of induced proinflammatory cytokines were measured. We identified two epitopes capable of sustaining proinflammatory responses, specifically TNF〈 and IFN© induction, in the inflamed intestinal mucosa in CD patients. The responses correlated positively with clinical and histological measurements of disease activity, thus suggesting a contribution of immune responses to HSP in pediatric CD site-specific mucosal inflammation.

Epitope-specific immune tolerization ameliorates experimental autoimmune encephalomyelitis

The availability of glatiramer acetate (GA) for inducing immune tolerance is a significant advancement in the treatment of multiple sclerosis (MS). However, a sizable proportion of patients maintain active disease, regardless of treatment. Another approach to induce T-cell tolerance is therefore still an unmet medical need. We hypothesized that induction of mucosal tolerance toward a pro-inflammatory T-cell epitope derived from a heat shock protein (HSP) (RatP2) could translate into clinical benefit. We found that treatment of experimental autoimmune encephalomyelitis (EAE, a model of MS) with the peptide RatP2 determined a significant clinical improvement, which was comparable to the standard tolerization treatment (an MBP-derived peptide pool) and superior to GA. Histological analysis demonstrated a reduction of brain and spinal cord inflammatory lesions in treated animals. Moreover, with immunological analysis we identified biomarkers associated with clinical response. This work provides proof-of-concept to support the further testing of this approach as a possible complement to currently available therapies for MS.

Ligand expression using antigenization of antibody: Principle and methods

Here we describe a new method for the expression of oligopeptides of immunological and biological interest, antigenization of antibody. This approach consists of grafting by protein engineering techniques discrete oligopeptide sequences of proteins in the hypervariable loops of a host antibody molecule. The main advantage of this approach is the possibility of expressing oligopeptides within the physicochemical constraints of the fold of an immunoglobulin molecule. Detailed here are the methods that we have developed for the engineering, expression, and testing of antigenized antibodies and an example of how this new methodology may help to create biologically active ligands. In the future antigenized antibodies may become valuable tools for a better and faster understanding of ligand:receptor interactions, a step that may speed up the rational design of new drugs.

Engineered idiotypes. Immunochemical analysis of antigenized antibodies expressing a conformationally constrained Arg-Gly-Asp motif

We report on the immunochemical characterization of two antibodies engineered to express RGD, a peptide from adhesive proteins of the extracellular matrix. One or three RGD motifs were introduced in the third complementarity-determining region (CDR) of a murine heavy (H) chain variable (V) region gene yielding two antibodies, gamma 1RGD and gamma 1(RGD)3. A murine monoclonal antibody (mAb) raised against an RGD-containing synthetic peptide bound in Western blot the H chain of both gamma 1RGD and gamma 1(RGD)3. Pronectin F, a genetically-engineered polymer containing RGD, abrogated this binding. Anti-idiotypic antibodies against the (RGD)3 loop were generated in a rabbit by immunization with gamma 1(RGD)3. Anti-idiotype antibodies purified by affinity-chromatography on the synthetic peptide GRGDSPC reacted in ELISA with gamma 1(RGD)3 and human fibronectin. Adhesive proteins, unlike RGD-containing synthetic peptides, were able to interfere with the interaction between gamma 1(RGD)3 and the anti-idiotypic antibodies. These results suggest that it is possible to genetically engineer the hypervariable loops of immunoglobulins and confer them new idiotypic characteristics. These results support the concept of antibody mimicry.

Idiotype Vaccines by Antibody Engineering: Structural and Functional Considerations

The idea of utilizing antibodies as noninfectious vaccines originates from the concept of idiotypes as imperfect chemical copies of antigens (1, 2) and is based on the property that antibodies can be immunogenic (3). This has been demonstrated in heterologous (4), homologous (3), syngeneic (5), and autologous (6) systems. Historically, the existence of immunoglobulin molecules related to antigenic determinants, not by fitting them but rather by resembling them, was first hypothesized by Lindemann (1). Borrowing his words there exist two different varitypes* related to one particular antigenic determinant: a varitype leading to a combining site matching that determinant, and which is conventionally called antibody; another varitype leading to an idiotypic site cross-reacting with that determinant, and for which we propose the name of homobody. Accordingly, antigenic determinants are projected into the world of immunoglobulin molecules in two manners: as negative images, these define antibodies; as positive images, these define homobodies.