Virus-induced diabetes in a transgenic model: role of cross-reacting viruses and quantitation of effector T cells needed to cause disease

Abstract

Virus-specific cytotoxic T lymphocytes (CTL) at frequencies of >1/1, 000 are sufficient to cause insulin-dependent diabetes mellitus (IDDM) in transgenic mice whose pancreatic beta cells express as "self" antigen a protein from a virus later used to initiate infection. The inability to generate sufficient effector CTL for other cross-reacting viruses that fail to cause IDDM could be mapped to point mutations in the CTL epitope or its COO(-) flanking region. These data indicate that IDDM and likely other autoimmune diseases are caused by a quantifiable number of T cells, that neither standard epidemiologic markers nor molecular analysis with nucleic acid probes reliably distinguishes between viruses that do or do not cause diabetes, and that a single-amino-acid change flanking a CTL epitope can interfere with antigen presentation and development of autoimmune disease in vivo.

FIG. 1

Sequences of NP aa 1 to 201 of various LCMV strains. The sequences are presented in the single-letter code. See Materials and Methods for details.

FIG. 2

(A) Incidence of IDDM in transgenic mice expressing LCMV ARM NP in their β cells after inoculation (10⁵ PFU i.p.) with LCMV strains ARM 53b, E-350, Traub, or Pasteur; VV; or Pichinde virus. A minimum of 20 mice of both genders were inoculated with LCMV strains, and 7 to 10 mice were infected with VV or Pichinde virus, all when the mice were 8 weeks of age. IDDM is defined as a blood glucose level of >300 mg/dl, a pancreatic insulin level of <15 μg of insulin/mg of pancreatic tissue, and mononuclear cell infiltration into islets of Langerhans with destruction of β cells. (B) Incidence of IDDM in transgenic mice expressing LCMV ARM NP in their β cells after inoculation with LCMV ARM 53b or LCMV ARM clone 13 (C113) (2 × 10⁶ PFU i.v.), VV recombinant expressing LCMV ARM NP (10⁵ PFU i.p.), or LCMV ARM (10⁵ PFU i.p.) into CD8-deficient mice (CD8 knockout [ko]). Mice in groups of 7 to 10 were injected when 8 weeks old. A similar lack of IDDM was observed in mice whose CD8⁺ T-cell population was removed by antibodies (see Materials and Methods) given 10⁵ PFU of LCMV ARM i.p.

FIG. 3

Histopathologic analysis of islets of Langerhans from pancreatic tissue taken from representative transgenic mice at 6 months of age (Fig. 2 and Table 2). (A) Uninfected mouse. (B to G) Mice inoculated i.p. with 10⁵ PFU of ARM (B), E-350 (C), Traub (D), Pasteur (E), VV recombinant expressing LCMV ARM NP (VV/NP) (F), and ARM in a mouse deficient in CD8⁺ lymphocytes (G [compare panels B and G]). (H) Tissue from a mouse inoculated with 2 × 10⁶ PFU of LCMV ARM i.v. (I) Tissue from a mouse inoculated with 2 × 10⁶ PFU of ARM variant clone 13 i.v. Similar data were obtained from 7 to 20 individual mice from each experimental group analyzed. In addition, islets studied from 10-month-old uninfected mice; mice infected i.p. with 10⁵ PFU of Traub, Pasteur, or VV/NP; CD8-deficient mice given ARM, or mice of a similar age given 2 × 10⁶ PFU of clone 13 i.v. when 8 weeks old failed to show lymphocytic infiltrates.

FIG. 4

Relative affinities of CTL harvested from LCMV ARM (IDDM inducer) or LCMV Pasteur (non-IDDM inducer). LCMV NP peptide aa 118 to 127 was used to coat BALB clone (BALB/c) 7 targets over a range of 10⁻⁶ to 10⁻¹⁰ M. The data are recorded as means ± 1 standard error of triplicate determinations (see Materials and Methods and reference for experimental details).

FIG. 5

Defect in intracellular processing of Traub NP aa 116 to 131, but not this peptide's ability to coat target cells for lysis by anti-LCMV ARM CTL. The panel on the left shows that equivalent molar amounts of ARM or Traub peptides NP aa 116 to 131 coated H-2^d target cells for corresponding CTL lysis. Similar data were observed in two other experiments. The panel on the right shows that the transfected ubiquitinated ARM NP oligomer that encodes NP aa 116 to 140 (pCMV-U-MG-ARM) is processed inside BALB cells and traffics to and is expressed on the surface of BALB cells for recognition by day 7 primary (d7^po) MHC-restricted LCMV-specific CTL. In contrast, a similar transfection with ubiquitinated Traub NP (NP aa 116 to 140) (pCMV-U-MG-Traub) fails to present LCMV Traub NP to the cell's surface for CTL recognition. Percentages of ⁵¹Cr released at E/T ratios of 50:1, 25:1, and 12:1 are given.

FIG. 6

Intracellular cytokine staining (IFN-γ and TNF-α) of LCMV-specific CD8⁺ T cells indicates that transfection of H-2^d BALB cells with ubiquitinated Traub NP aa 116 to 140 leads to activation of significantly less CD8⁺ T cells than does activation by ubiquitinated ARM NP aa 116 to 140 (P value for IFN-γ expression, 0.0003; P value for TNF-α expression, 0.0027). However, when either peptide is externally added to coat target cells (Traub and ARM NP aa 116 to 131), equivalent numbers of cytokine-containing CD8⁺ T cells (i.e., 43 and 42% IFN-γ-expressing CD8⁺ T cells, respectively) are generated. The data are representative of three independent experiments.

FIG. 7

Defect in the intracellular processing of full-length NP Traub despite equivalent levels of cytosolic expression of Traub and ARM NP. The top panel shows that whereas transfected full-length ARM NP (pCMV-ARM [see Fig. 5 legend]) is processed correctly as judged by recognition of LCMV-specific CTL over several E/T ratios, there is no recognition of similarly transfected full-length Traub NP (pCMV-Traub). The numbers shown represent the mean value of triplicate samples. Variance was <10% in the 5-h ⁵¹Cr release assay. D7 p^o, day 7 primary immune response; splenic lymphocytes. The lower panels show that cytosolic extracts from the full-length ARM- or Traub-transfected constructs (above) express similar levels of NP by Western blotting and by densitometry analysis. The negative control (U) was processed with cytosolic extract from uninfected BALB cells, and the positive control (I) is from LCMV ARM-infected BALB cells. ARM, pCMV-ARM NP; Traub, pCMV-Traub NP (constructs used to transfect BALB cells). Different concentrations of protein from each sample were loaded, and detection was with monoclonal antibody to LCMV ARM NP (see reference and Materials and Methods).