Andrew P. Cope

Shedding kinetics of soluble tumor necrosis factor (TNF) receptors after systemic TNF leaking during isolated limb perfusion. Relevance to the pathophysiology of septic shock.

We examined the kinetics of shedding of the soluble TNF receptors (TNF-Rs) in response to TNF leakage during isolated limb perfusion procedures and correlated them to the resulting hemodynamic effects. Shedding of the TNF-Rs started 7 min after TNF leakage into the systemic circulation. Three waves of shedding were observed peaking at 1, 8-12, and 48-72 h both in vivo and in cell cultures. The soluble receptors prolonged the half-life of TNF in the systemic circulation to 2.5-6 h. Excess shedding of the p75 compared with p55 TNF-Rs was noted during the first wave. The amount and speed of shedding of the p75 TNF-Rs were proportional to the serum TNF levels (P < 0.001). A maximal shedding capacity was attained only during the first wave of shedding, at TNF concentrations of approximately 1.5 ng/ml. Above this level, the linearity between TNF and its soluble receptors was lost. TNF-induced hypotension coincided with the initial imbalance between the concentrations of TNF and its soluble receptors. Despite the spontaneous correction of this imbalance at 8-12 h, the hemodynamic and biochemical alterations persisted and were further aggravated at 18 h, suggesting that other factors induced earlier by TNF are responsible for the perpetuation of the hemodynamic instability. This study may provide the basis for a more physiological therapeutic approach to TNF neutralization in septic shock patients.

HLA class II transgenic mice: models of the human CD4+ T‐cell immune response

Summary: This review examines the field of current HLA class II transgenic mouse models and the individual approaches applied in production of these mice. The majority of these mice have been created with the objective of obtaining a disease model with clinical features mimicking human autoimmune disease. The development process of a different type of HLA class II transgenic mice, which are designed to function as a substitute for a normal human immune system in studies of human autoantigens, is described. Several HLA‐DR4 transgenic lines with normally expressed HLA‐DR4 molecules have been produced. To obtain adequate positive selection of the HLA‐DR4‐restricted CD4+ T‐cell repertoire in these mice it is essential both to introduce a human CD4 transgene. and to delete the murine major histocompatibility complex (MHC) class II molecules. These HLA‐DR4 transgenic mice have been used to determine the immunogenic CD4+ T‐cell epitopes of several human autoantigenic proteins.

Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes

Immunodominant T cell epitopes from the autoantigen human cartilage glycoprotein 39 have previously been mapped in the context of HLA‐DR*0401 and *0402, using mice expressing HLA‐DR4 transgenes. We measured the dissociation rates of these epitopes from soluble recombinant DR*0401 and DR*0402 to assess the relationship between peptide/HLA‐DR4 kinetic stability and immunogenicity. Experiments were performed at endosomal pH (5.5) and at cell surface pH (7), in the absence and presence of soluble recombinant HLA‐DM (sDM). All (4/4) immunodominant peptide/HLA‐DR complexes exhibit dissociation half‐times of 1 h to several days. In contrast, most (3/4) non‐immunodominant complexes dissociate with half‐times <30 min under at least one of these conditions. Interestingly, a complex which is stable except in the presence of HLA‐DM at pH 5.5 is immunogenic only following peptide immunization, while a complex which is stable at acidic but not at neutral pH, is non‐immunogenic following either whole protein or peptide immunization. These data indicate that kinetic stability of peptide/MHC complexes in vivo is a key determinant of immunogenicity.

Autoantigenic HCgp39 Epitopes Are Presented by the HLA-DM-Dependent Presentation Pathway in Human B Cells

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes “epitope editing” by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR*04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1*0401 but closely related allele DRB1*0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR*0401 or DR*0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4+ T cell hybridomas generated from DR*04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.

Humanized mice as a model for rheumatoid arthritis

Chapter Summary Genetic susceptibility to rheumatoid arthritis (RA), a common autoimmune disease, is associated with certain HLA-DR4 alleles. Treatments are rarely curative and are often tied to major side effects. We describe the development of a humanized mouse model wherein new, less toxic, vaccine-like treatments for RA might be pretested. This model includes four separate transgenes: HLA-DR*0401 and human CD4 molecules, a RA-related human autoantigenic protein (HCgp-39), and a T-cell receptor (TCRαβ) transgene specific for an important HCgp-39 epitope, eliciting strong Th1 responses in the context of HLA-DR*0401.