Superiority of thyroid peroxidase DNA over protein immunization in replicating human thyroid autoimmunity in HLA-DRB1*0301 (DR3) transgenic mice

Abstract

Murine experimental autoimmune thyroiditis (EAT), characterized by thyroid destruction after immunization with thyroglobulin (Tg), has long been a useful model of organ-specific autoimmune disease. More recently, porcine thyroid peroxidase (pTPO) has also been shown to induce thyroiditis, but these results have not been confirmed. When (C57BL/6 x CBA)F(1) mice, recently shown to be susceptible to mouse TPO-induced EAT, were immunized with plasmid DNA to human TPO (hTPO) and cytokines IL-12 or GM-CSF, significant antibody (Ab) titres were generated, but minimal thyroiditis was detected in one mouse only from the TPO + GM-CSF immunized group. However, after TPO DNA immunization of HLA-DR3 transgenic class II-deficient NOD mice, thyroiditis was present in 23% of mice injected with TPO + IL-12 or GM-CSF. We also used another marker for assessing the closeness of the model to human thyroid autoimmunity by examining the epitope profile of the anti-TPO Abs to immunodominant determinants on TPO. Remarkably, the majority of the anti-TPO Abs was directed to immunodominant regions A and B, demonstrating the close replication of the model to human autoimmunity. TPO protein immunizations of HLA-DR3 transgenic mice with recombinant hTPO did not result in thyroiditis, nor did immunization of other mice expressing HLA class II transgenes HLA-DR4 or HLA-DQ8, with differential susceptibility to Tg-induced EAT. Moreover, our efforts to duplicate exactly the experimental procedures used with pTPO also failed to induce thyroiditis. The success of hTPO plasmid DNA immunization of DR3(+) mice, similar to our reports on Tg-induced thyroiditis and thyrotropin receptor DNA-induced Graves' hyperthyroidism, underscores the importance of DR3 genes for all three major thyroid antigens, and provides another humanized model to study autoimmune thyroid disease.

Fig. 1

Proliferative response to TPO in B6 and DR3⁺ Ab⁰/NOD mice after TPO protein immunization. (a) B6 mice were immunized sc with 200 µg hTPO or rhTPO, or 20 µg hTg in CFA in the alternate thigh and hind footpad on days 0 and 7. Lymph node cells were obtained on day 35 and cultured (6 × 10⁵/well) for 5 days with 20 µg/ml hTPO or rhTPO, or 40 µg/ml hTg, in the presence of 1 × 10⁶/well irradiated spleen cells. [³H]-thymidine uptake is represented by cpm in culture with antigen. Background cpm without antigen ranged from 1500 ± 200–1900 ± 100. (b) DR3⁺ Ab⁰/NOD mice were immunized iv on days 0 and 7 with 100 or 200 µg rhTPO followed by 20 µg LPS. On day 28, spleen cells (6 × 10⁵/well) were cultured for 4 days with medium alone, mTg or hTg (40 µg/ml) or rhTPO (10 µg/ml).

Fig. 2

IgG isotype quantification of rhTPO antibodies. HLA or H2 transgenic mice, on the B10 background, were immunized iv with 100 µg rhTPO plus 20 µg LPS on days 0 and 7. Sera were obtained on days 28 or 35. Isotypes of IgG (⋄ IgG1; ▪ IgG2a, ▴ IgG2b; × IgG3) with specificity for rhTPO were quantified at various serum dilutions for (a) HLA-DR3⁺, (b) HLA-DR4⁺, (c) HLA-DQ8⁺ and (d) H2E⁺ mice.

Fig. 3

hTPO antibodies and thyroiditis in (C57BL/6 × CBA)F₁ mice after immunization with hTPO plasmid DNA with or without IL-12 or GM-CSF plasmid DNA. On weeks 0, 3 and 6, mice were injected with DNA, 5 days after cardiotoxin. Sera and thyroids were collected at the time of sacrifice (week 17). (a) IgG titres to rhTPO were determined from 1 : 4000 serum dilutions, except for control mice (1 : 1000 serum dilutions). Thyroid pathology was determined as in Methods. (b) Antibodies to immunodominant region (IDR) epitopes A and B of TPO. Since two mice from the TPO + IL-12 DNA immunized group showed no TPO Abs (A), no IDR-A versus -B determination could be made.

Fig. 4

hTPO antibodies and thyroiditis in DR3⁺ Ab⁰/NOD mice after immunization with hTPO plasmid DNA with IL-12 or GM-CSF plasmid DNA. On weeks 0, 3 and 6, mice were injected with DNA, 5 days after cardiotoxin. Sera and thyroids were collected at the time of sacrifice (week 14). (a) IgG titres to rhTPO were determined from 1 : 4000 serum dilutions, except for control and TPO + IL-12 immunized groups (1 : 100 serum dilutions). Thyroid pathology was determined as in Methods. Antibodies to immunodominant region (IDR) epitopes A and B of TPO, from TPO + GM-CSF DNA immunized mice, as determined by rabbit antisera (b) or hFabs (c).

Fig. 5

Mononuclear cell infiltration in the thyroid of hTPO plasmid DNA-immunized mice. Thyroid sections from (a) a vector control DR3⁺Ab⁰/NOD mouse and (b) a DR3⁺Ab⁰/NOD mouse immunized with TPO + GM-CSF plasmid DNA with infiltration involving >10–20% of the gland. (Magnification 200×).