Loss of humoral tolerance to red blood cell (RBC)-specific antigens may lead to the development of pathogenic autoantibodies and result in autoimmune hemolytic anemia, a severe and sometimes fatal disease. Recently, it has been demonstrated that although RBC-specific autoreactive B cells escape central tolerance (and can secrete autoantibodies), stringent T cell tolerance is a stopgap to autoimmunity; however, T cell tolerance mechanisms utilized against RBC antigens is largely unknown.
Thymic central tolerance is a process by which newly-formed autoreactive T cells undergo negative selection by deletion, anergy, or diversion into a regulatory T cell (Treg). This process ensures that newly-minted T cells are self-tolerant and functional. Elimination of >95% of thymocytes occurs during central tolerance. However, while rigorous, central tolerance sometimes fails and autoreactive T cells escape to the periphery. The youngest peripheral T cells, recent thymic emigrants (RTEs), are functionally distinct from mature T cells and are more susceptible to peripheral tolerance mechanisms in response to autoantigens. Herein, we assess central and peripheral T tolerance to RBC-specific autoantigens.
HOD transgenic mice express an RBC-specific fusion protein consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and human blood group, Duffy (D). To assess tolerance, HOD mice were bred with OTII mice, CD4+ T cell receptor (TCR) transgenic mice with specificity for OVA (contained within HOD). HODxOTII F1s were immunized with 100ug of OVA/CFA. To assess central tolerance, thymi from 8-12 week old HODxOTII F1 mice were harvested and stained with antibodies to identify OTIIs and Tregs. For RTE analysis, HOD mice were bred with OTII.Rag2p-GFP (referred to as HODxRTE mice), OTII T cells that express GFP during TCR rearrangement. GFP+ OTII RTEs were stained with antibodies against CD73, FR4, and PD1. To assess function, GFP+ OTII RTEs were sorted and 3x105 RTEs were infused into naïve B6 mice. Recipient mice received a 100uL packed HOD RBC transfusion and RTE proliferation was assessed at 3 days by Ki67 staining.
HOD+OTII+ mice had profound tolerance to the HOD RBC autoantigen; immunization with OVA/CFA elicited strong antibody responses in HOD-OTII+ littermates, but not autoreactive HOD+OTII+ mice. To determine whether OTIIs underwent central tolerance, thymi were analyzed. No significant differences in total numbers of thymocytes or CD4+ OTIIs was observed in autoreactive HOD+OTII+ mice, compared to control HOD-OTII+. Additional phenotyping for co-expression of CD25 and FoxP3 (a phenotype consistent with Tregs) revealed no difference, suggesting autoreactive OTII T cells were not diverted into a regulatory program. Thus, negative selection of RBC autoreactive CD4+ T cells does not occur in the thymus.
Given that RTEs are subject to tolerization in response to peripherally expressed autoantigens, HODxRTE F1s were used to assess whether RBC-specific autoreactive OTIIs are tolerized as RTEs. RTEs were divided into GFPhi (< 1 week in the periphery), GFPint (2-3 weeks in the periphery), and GFPneg (mature T cells). In HOD+RTE+ mice, GFPhi RTEs had significantly higher levels of PD1, a marker of exhaustion, compared to controls. As RTEs matured, PD1 expression slightly decreased, but remained high compared to controls. Additionally, as autoreactive OTII RTEs matured, a significant number OTIIs expressed CD73hiFR4hi, a phenotype consistent with anergy. On average, 5% of GFPneg OTIIs from HOD-RTE+ mice expressed CD73hiFR4hi, compared to over 60% in autoreactive HOD+RTE+ mice. To assess function, sorted RTEs from HODxRTE mice were adoptively transferred into naïve mice and assessed for proliferation post HOD RBC transfusion. All RTEs sorted from HOD-RTE+ mice proliferated (>95% of OTIIs were Ki67+). However, RTEs derived from HOD+RTE+ mice had diminished proliferative capacity upon maturation (Ki67+ RTEs: GFPhi 65%, GFPint 35%, GFPneg 15%).
These data demonstrate that RBC-specific autoreactive CD4+ T cells are neither deleted nor diverted into a Tregs during thymic central tolerance, rather peripheral tolerance mechanisms occurring during RTE maturation is required. Together, these data provide insight into RBC-specific T cell tolerance mechanisms, pinpoint when they occur, and demonstrate that if RTE maturation does not occur, RBC autoimmunity may ensue.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.