Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism

Abstract

Human autoimmune (AI) diseases are difficult to treat, because immunosuppressive drugs are nonspecific, produce high levels of adverse effects, and are not based on mechanistic understanding of disease. Destroying the rare autoreactive T lymphocytes causing AI diseases would improve treatment. In animal models, TNF selectively kills autoreactive T cells, thereby hampering disease onset or progression. Here, we seek to determine, in fresh human blood, whether TNF or agonists of TNF selectively kill autoreactive T cells, while sparing normal T cells. We isolated highly pure CD4 or CD8 T cells from patients with type 1 diabetes (n = 675), other AI diseases, and healthy controls (n = 512). Using two cell death assays, we found that a subpopulation of CD8, but not CD4, T cells in patients' blood was vulnerable to TNF or TNF agonist-induced death. One agonist for the TNFR2 receptor exhibited a dose-response pattern of killing. In type 1 diabetes, the subpopulation of T cells susceptible to TNF or TNFR2 agonist-induced death was traced specifically to autoreactive T cells to insulin, a known autoantigen. Other activated and memory T cell populations were resistant to TNF-triggered death. This study shows that autoreactive T cells, although rare, can be selectively destroyed in isolated human blood. TNF and a TNFR2 agonist may offer highly targeted therapies, with the latter likely to be less systemically toxic.

Fig. 1.

TNF treatment of purified human CD4 or CD8 T cells from type 1 diabetics (black bar) compared with controls (shaded bar) for viability vs. killing (A) TNF treatment of CD4 T cells (n = 9 pairs, Upper) or TNF treatment of CD8 T cells (n = 12 pairs, Lower), using the LDH assay. (B) TNF treatment of purified human CD8 T cells from larger samples (n = 23 pairs) of type 1 diabetics and controls, using the WST-1 assay.

Fig. 2.

Effect of TNFR1 vs. TNFR2 agonist antibodies on death of Type 1 diabetic (dark bar) compared with control (shaded bar) CD8 T cells (A) TNFR1 agonist antibody treatment of purified human CD8 T cells from 11 pairs of type 1 diabetics and controls. (B) TNFR2 agonist treatment of purified human CD8 T cells without TNF (left column) or with TNF (right column) in type 1 diabetics compared with controls. (i) iv TNFR2 agonist clone #1 (n = 8 paired samples, Left; n = 8 paired samples, Right), (ii, v) TNFR2 agonist clone #2 (n = 5 sets of paired samples, Left; n = 5 paired samples, Right), (iii, vi) TNFR2 agonist clone #3 (n = 5 paired samples on Left; n = 5 paired samples on Right). Except for TNFR2 clone #1 (and high doses in Fig 2vi, as explained in the text), all P values were >0.05 with or without TNF.

Fig. 3.

A subset of insulin autoreactive CD8 T cells can be identified in some long-term diabetics (A) Insulin B10–18 tetramer positive CD8 frequency in a highly pure preparation of CD8 T cells from a long-term diabetic (age of onset 13 years, duration of diabetes 8 years) and a paired control. Both patient and control were HLA-A2.1+. Representative two-color dot plots are presented. (B) Insulin B10–18 tetramer positive CD8 frequency in long-term type 1 diabetics (n = 11) both positive and negative patients and controls. (C) Clinical characteristics of HLA-A2 positive type 1 diabetics with and without insulin B10–18 tetramer staining compared with matched controls.

Fig. 4.

Treatment of insulin autoreactive CD8 T cells with TNFR2 agonist clone #1 kills the pathogenic cells. (A) Targeted elimination of insulin B10–18 tetramer+ T cells with a 6-h treatment with TNFR2 agonist in a long-term diabetic compared with matched control T cells (B) Insulin B10–18 specific CD8 T cells from type 1 diabetics consistently decrease in culture when treated with TNFR2 agonist in culture for 6 h. (C) Repeat analysis of the same tetramer insulin positive long-term diabetic over a 3-year period repeatedly reveals the insulin autoreactive T cells. Autoreactive CD8 T cells can be repeatedly eliminated with a brief TNFR2 agonist exposure to diabetic cells.