Tu125 - Defective Positive and Negative Selection of Thymocytes Derived from Type 1 Diabetic Patient Hematopoietic Cells

Abstract Text: The possible role of defective thymic selection in driving Type 1 Diabetes (T1D) is unknown. Personalized Immune (PI) mice were established by transplanting thymectomized NSG mice with CD34+ hematopoietic stem cells (HSCs) from T1D or healthy control (HC) donors, along with human fetal thymic tissue sharing the T1D-associated class II HLA risk allele. This allows de novo generation of a human immune system and exploration of thymocyte development in T1D immune systems. Following immune reconstitution, thymocytes were harvested and analyzed using single-cell 5’ RNA sequencing (10X Genomics) and bulk TCR sequencing (Adaptive Biotechnologies) of various sorted maturation subsets.

T-cell receptor hydrophobicity, particularly in the mid-CDR3 (mCDR3) region, reflects autoreactivity. In HC thymocytes, average mCDR3 hydrophobicity increased during positive selection (CD4+ CD8+ CD69− to CD69+ cells) and remained stable in mature CD4 single-positive (SP) Tconv cells, indicating effective positive and negative selection. In contrast, T1D thymocytes showed a smaller hydrophobicity shift during positive selection and increased hydrophobicity in CD4 SP Tconv cells, suggesting impaired selection processes.

Single-cell RNA sequencing also revealed T1D-associated deficits in thymocyte development. Transcriptional profiling of the normal human thymus identified maturing thymocyte subsets, including clusters linked to positive (hs-DP-3) and negative (hs-Sig-4) selection. These clusters were statistically significantly reduced in T1D mice, consistent with defective selection. Pathway and gene set enrichment analyses revealed apoptotic features in these clusters, further supporting disrupted selection.

These findings provide independent evidence, both physico-chemical and transcriptional, of defective T cell selection in T1D mice. Ongoing studies aim to further investigate these mechanisms.