Programmed cell death-1, PD-1, is dysregulated in T cells from children with new onset type 1 diabetes

Abstract

Background: Programmed death cell 1 (PD-1) is an inhibitor of T cell activation and is also functionally linked to glycolysis. We hypothesized that PD-1 expression is defective in activated T cells from children with type 1 diabetes (T1D), resulting in abnormal T cell glucose metabolism.

Methods: In this pilot study, we enrolled children with new onset T1D within 2 weeks of diagnosis (T1D), unaffected siblings of T1D (SIBS), unaffected, unrelated children (CTRL), children with new onset, and untreated Crohn disease (CD). We repeated the assays 4-6 months post-diagnosis in T1D (T1D follow up). We analyzed anti-CD3/-CD28-stimulated peripheral blood mononuclear cells (PBMC) subsets for PD-1 expression by flow cytometry at baseline and after 24 h in culture. We measured cytokines in the culture medium by multiplex ELISA and glycolytic capacity with a flux analyzer.

Results: We enrolled 37 children. T cells derived from subjects with T1D had decreased PD-1 expression compared to the other study groups. However, in T1D follow-up T cells expressed PD-1 similarly to controls, but had no differences in PBMC cytokine production. Nonetheless, T1D follow up PBMCs had enhanced glycolytic capacity compared to T1D.

Conclusions: Activated T cells from T1D fail to upregulate PD-1 upon T-cell receptor stimulation, which may contribute to the pathogenesis of T1D. T1D follow up PBMC expression of PD-1 normalizes, together with a significant increase in glycolysis compared to T1D. Thus, insulin therapy in T1D children is associated with normal PD1 expression and heightened glycolytic capacity in PBMC.

Fig 1. T Cells from T1D fail to upregulate PD-1 upon stimulation.

Freshly isolated PBMCs were stained with antibodies against CD3, CD4, CD8, and PD-1 and analyzed by flow cytometry. Lymphocytes were identified by forward and side scatter and interrogated for CD4 and CD8 expression (bottom of right panel, lymphocyte gate). Another gate with higher side scatter identified larger cells that include monocytes and activated T cells (top of right panel, large gate). Mean percentage expression of PD-1 was compared by one-way ANOVA on each of the PBMC subpopulations (A-F). Percentage PD-1 surface expression in PBMC subsets is shown, at time zero (0) and after 24 h in culture with (S) and without (NS) stimulation (* indicate p < 0.05).

Fig 2. PD-1 expression recovers in T1D follow up.

A. Subpopulations of T cells, one that down regulates PD-1 and the other that upregulates PD-1 B. Expression of PD-1 in T cells from T1D after 4–6 months post diagnosis (T1D Follow up) (* indicate p < 0.05). Top left CD3⁺ CD4⁺ lymphocyte gate, top right CD3⁺ CD8⁺ lymphocyte gate, bottom left CD3⁺ CD4⁺ large gate, and bottom right CD3⁺ CD8⁺ large gate.

Fig 3. Cytokine production is not increased in T1D compared to T1D follow up.

Cells were cultured for 24 h with and without stimulation with anti CD3/CD28, the supernatant collected and assayed by multiplex cytokine ELISA. Data were log-transformed to obtain normal distributions. A. T1D NS (grey circles) vs S (black circles) Cytokines that showed statistically significant different in T1D between unstimulated and stimulated PBMCs. B. T1D F/U NS (grey circles) vs S (black circles) Cytokine concentration in PBMCs from T1D Follow up. C. T1D S (black circles) vs T1D F/U S (black squares) Comparison of cytokine expression on stimulated PBMCs from subjects with T1D and T1D Follow up. (* indicate p < 0.05).

Fig 4. PBMCs from T1D follow up had enhanced glycolytic capacity compared to T1D.

A. ECAR (mpH/min) in PBMCs from T1D (black circles) and T1D Follow up (open circles) after adding glucose, oligomycin and 2-DG. B. Integration of area under the curve showing comparisons in ECAR between T1D and T1D Follow up with additives, as shown.