Kim Schroder

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet β cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR α- and β-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528–539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC50 > 100 μM), a larger 16-mer fragment, 526–541, shows activity in the low micromolar range (EC50 = 2.3 μM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2.

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (109) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2+ MUC1+ MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were “consensus” agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-α, IFN-γ, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.

Acquired thymic tolerance and experimental allergic encephalomyelitis in the rat. I. Parameters and analysis of possible mechanisms.

Intrathymic injection of guinea pig myelin basic protein (MBP) or the immunodominant, ence phalitogenic fragment of MPB, 68 – 86, without otherwise compromising the peripheral lymphocyte pool in adult LEW rats, dramatically inhibits onset of experimental allergic encephalomyelitis (EAE) caused by the usual peripheral inoculation with MBP in complete Freunds adjuvant. This surprising finding demonstrates that interaction of antigen and one or more components of an intact thymus can down‐regulate systemic responses by mature T cells already existing in the peripheral lymphocyte pool. How this happens is not known. In studies designed to explore possible mechanisms: (a) adult thymectomized animals remain susceptible to active EAE, thus EAE cannot be attributed solely to recent thymic emigrants that might be inactivated by antigen deposited in the thymus; (b) heterotopic isografts of injected thymic lobes transfer thymic tolerance to secondary recipients, thus the tolerance effect is dominant over an intact, non‐treated thymus; (c) T cells from made thymic tolerant but not immunized donors are less effective in causing EAE following adoptive transfer into, and active immunization of, secondary, irradiated recipients; and (d) animals resistant to active EAE as a consequence of thymic tolerance are fully vulnerable to adoptive EAE caused by already activated MBP‐specific T cell subpopulations. These results rule out a possible mechanism previously proposed for acquired thymic tolerance, i.  e., that potentially pathogenic T cells traffic to the antigen‐injected thymus where they are inactivated or eliminated.

Analysis of TCR β chains in Lewis rats with experimental allergic encephalomyelitis. II. Vβ8.2+ T cells with limited CDR3 N region additions derive from the adult thymus

Immunization of Lewis (LEW) rats with guinea pig myelin basic protein (MBP) induces a population of encephalitogenic CD4 T cells having specificity for the dominant immunogenic peptide of MBP, 68 – 86. The TCR β chains of these disease‐causing T cells show three distinct features: they are almost exclusively Vβ8.2, they use AspSer as the first two amino acid residues of the third complementarity‐determining region (CDR3) and these junctional region sequences show few if any non‐germline N‐region nucleotide additions. This last feature raises the possibility that these autoimmune T cell precursors derive from TCR gene rearrangements occurring during early, perinatal ontogeny, a period when the enzyme terminal deoxynucleotidyl transferase (TdT), responsible for N region additions, is not expressed. An alternative possibility is that these features of the TCR of MBP 68 – 86‐reactive T cells are dictated by considerations of antigen selection throughout ontogeny both in the thymus and in the periphery – i.e., that such β chains are conformationally the most appropriate for triggering by an epitope of 68 – 86 complexed to class II RT1.Bl MHC molecules. We show here that active experimental allergic encephalomyelitis, while delayed in onset, occurs in heavily irradiated animals, but not in the absence of a thymus, a finding indicating that this autoimmune disease is caused by a T cell subpopulation derived from the post‐irradiation adult thymus. These disease‐causing T cells are heavily Vβ8.2+ , CDR3 AspSer+ and use few N region additions. We conclude that T cells with these TCR β chain features can be generated in the adult thymus and most likely reflect requirements imposed by antigen selection.

Natural mutants of HIV CTL epitopes with enhanced immunogenicity as potential vaccine candidates

Introduction remains a serious, ultimately lethal pandemic disease in much of the developing world. Significant treatment advances have been made with antiretroviral agents, but major problems remain. These treatments are expensive and difficult to apply especially for developing world nations that lack adequate finances and infrastructure, do not eliminate all virus and therefore require life long treatment regimens, carry significant toxic side effects and in some quarters becoming less effective due to viral mutation. There remains a major need to identify effective measures to prevent new HIV infections, especially in areas of the world where it is a pandemic. Based on the history of vaccine development, it remains a strong consensus in the biomedical research community that immunization will provide the most effective and affordable long term-approach to controlling the spread of HIV/AIDS. Strong cytotoxic T-lymphocyte (CTL) responses are thought to be important for the development of an effective human immunodeficiency virus (HIV) vaccine; their benefit has been established in simian immunodeficiency virus (SIV) vaccination studies and in natural infections [1,2]. One of our major goals is to identify optimized, highly immunogenic peptides for the design of prophylactic or therapeutic vaccines for HIV infection. Our strategy derives from the hypothesis that the rarer mutant CTL epitopes of HIV may be more immunogenic than the common ones and that these rarer mutant sequences of HIV epitopes might be more effective in generating cross reactive anti-HIV CTL responses against both the rare and common mutant sequences.