Tolerization of CD4+ Recent Thymic Emigrants Is Required to Prevent RBC-Specific Autoimmunity

Background

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.

Methods

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.

Results

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 GFP^hi (< 1 week in the periphery), GFP^int (2-3 weeks in the periphery), and GFP^neg (mature T cells). In HOD+RTE+ mice, GFP^hi 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 CD73^hiFR4^hi, a phenotype consistent with anergy. On average, 5% of GFP^neg OTIIs from HOD-RTE+ mice expressed CD73^hiFR4^hi, 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: GFP^hi 65%, GFP^int 35%, GFP^neg 15%).

Conclusions

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.