ABSTRACT
BACKGROUND: Polyclonal antithymocyte globulins (ATGs) are used clinically to prevent and treat acute allograft rejection and are believed to modulate the immune response primarily by depleting T cells. However, nondepleting mechanisms may also be important mediators of graft survival. In the present study, 14 lots of thymoglobulin (rabbit ATG) were analyzed and compared for nondepletive immunomodulatory activities in vitro. METHODS: Coincubation of human peripheral blood mononuclear cells with thymoglobulin induces CD4+CD25(high)Foxp3+ regulatory T cells, which were evaluated for consistent ability to suppress T-cell activation in mixed lymphocyte reactions. The consistency of CD2, CD3, CD11a, and CD45 antigen specificities in thymoglobulin was determined using flow cytometry to measure inhibition of fluorescent monoclonal antibody binding to Jurkat T cells. A transwell chemotaxis assay was established and used to evaluate ATG-mediated inhibition of stromal cell-derived factor (SDF)-1alpha-driven Jurkat T-cell migration. RESULTS: Physiologic levels of thymoglobulin produced nondepletive immunomodulatory activities, which were consistent from batch to batch. All lots of thymoglobulin induced functionally immunosuppressive regulatory T cells and inhibited monoclonal antibody binding to key T-cell surface antigens. In addition, these studies provide the first demonstration that thymoglobulin effectively inhibits CXCR4/SDF-1alpha-driven T-cell chemotaxis. CONCLUSIONS: This novel, systematic in vitro analysis of 14 different manufactured lots of thymoglobulin demonstrates the overall consistency of this product and provides further insights into nondepletive mechanisms by which thymoglobulin may generate durable immunoregulation and allograft survival.
Subject(s)
Antilymphocyte Serum/pharmacology , Immunosuppressive Agents/pharmacology , Animals , Antibodies, Monoclonal/analysis , Antigens/immunology , Antilymphocyte Serum/therapeutic use , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Flow Cytometry , Graft Rejection/immunology , Graft Rejection/prevention & control , Humans , Immunophenotyping , Immunosuppression Therapy/methods , Immunosuppressive Agents/therapeutic use , Jurkat Cells/immunology , Lymphocyte Depletion , Rabbits , T-Lymphocytes/immunology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunologyABSTRACT
Self tolerance to MHC class I-restricted nonmutated self Ags is a significant hurdle to effective cancer immunotherapy. Compelling evidence is emerging that altered peptide ligands can be far more immunogenic than their corresponding native epitopes; however, there is no way to reliably predict which modifications will lead to enhanced native epitope-specific immune responses. We reasoned that this limitation could be overcome by devising an empirical screen in which the nearly complete combinatorial spectrum of peptides of optimal length can be rapidly assayed for reactivity with a MHC class I-restricted cytotoxic T cell clone. This method, solid-phase epitope recovery, quantitatively ranks all reactive peptides in the library and allows selection of altered peptide ligands having desirable immunogenic properties of interest. In contrast to rationally designed MHC anchor-modified peptides, peptides identified by the present method are highly substituted in predicted TCR contact residues and can reliably activate and expand effector cell populations in vitro which lyse target cells presenting the wild-type epitope. We demonstrate that solid-phase epitope recovery peptides corresponding to a poorly immunogenic epitope of the melanoma Ag, gp100, can reliably induce wild-type peptide-specific CTL using normal donor T cells in vitro. Furthermore, these peptides can complement one another to induce these responses in an overwhelming majority of normal individuals in vitro. These data provide a rationale for the design of superior vaccines comprising a mixture of structurally diverse yet functionally convergent peptides.
