Activation of autoreactive lymphocytes in the lung by radioresistant cells expressing a STING gain-of-function mutation

Gain-of-function mutations in the dsDNA sensing adaptor STING lead to a severe autoinflammatory syndrome known as STING-associated vasculopathy with onset in infancy (SAVI). Patients with SAVI develop interstitial lung disease (ILD) and produce autoantibodies that are commonly associated with systemic autoimmune diseases. Mice expressing the most common SAVI mutation, STING V154M (VM), similarly develop ILD but exhibit severe T and B cell lymphopenia and low serum Ig titers, and they lack autoantibodies. Importantly, lethally irradiated VM hosts reconstituted with WT stem cells (WT→VM) still develop ILD. In this study, we find that WT→VM chimeras had restored B cell function, produced autoantibodies, and thereby recapitulated the loss of tolerance seen in patients with SAVI. Lymphocytes derived from both WT and BCR or TCR transgenic (Tg) donors accumulated in the extravascular lung tissue of WT+Tg→VM mixed chimeras, but lymphocyte activation and germinal center formation required WT cells with a diverse repertoire. Furthermore, when T cells isolated from the WT→VM chimeras were adoptively transferred to naive Rag1-deficient secondary hosts, they trafficked to the lung and recruited neutrophils. Overall, these findings indicated that VM expression by radioresistant cells promoted the activation of autoreactive B cells and T cells that then differentiated into potentially pathogenic effector subsets.


Supplemental Figure 1. Gating strategy for lung resident B cells in WT+MD4 mixed chimeras
Example flow cytometry from a VM CD45 .1/.2 mouse irradiated and reconstituted with 80% MD4 Rag1 - /-CD45 .2/.2 and 20% WT CD45 .1/.1 bone marrow.Text above each bivariate plot describes the gate highlighted in red.Arrows indicate that this population was then sequentially gated on by the indicated markers in the adjacent right plot.The second and third row then stratify the analysis by either WT or MD4 donor status.Gates of populations shown in Figure 3 are highlighted in purple.Extravascular (EV); Plasma Cell (PC), Germinal Center (GC).

Supplemental Figure 2. Unsupervised clustering of B cell markers identifies repertoire and VM host dependent phenotypes.
1000 Live CD45 + CD45IV neg CD19 + lung extravascular (EV) B cell flow cytometry events per mouse from WT+MD4→WT (n=4) and WT+MD4→VM (n=6) mice were concatenated after excluding CD45.1 single positive radioresistant events.(A) A UMAP was generated using MHCII, IgD, Fas, CD23, CD69, GL7, CD138, CD21, CD86, B220, CD19, IgM, AA4.1, and CD11c.(B) Unsupervised clustering using Phenograph identified 10 clusters in this dataset, which were manually annotated based on their expression profile (Supplemental Table 1) and projected onto the UMAP alongside their annotated name.(C) shows heatmap projections for each parameter used to generate the UMAP and perform unsupervised clustering.(D) UMAPs depicting events from WT donor-derived cells in either WT (WT→WT) or VM (WT→VM) hosts, as well as events from MD4 donor-derived cells in either WT (MD4→WT) or VM (MD4→WT) hosts.Regions of the UMAP enriched in WT→VM events are highlighted with rings corresponding to the Fas + cluster (red), the germinal center (GC) cluster (green), and the plasma cell (PC) cluster (black).

Supplemental Figure 3. Gating strategy for lung resident T cells in WT+OTI/II mixed chimeras
Example flow cytometry from a VM CD45 .1/.2 mouse irradiated and reconstituted with 40% OT-I Rag2 - /-CD45 .2/.2 , 40% OT-II Rag2 -/-CD45 .2/.2 ,and 20% WT CD45 .1/.1 bone marrow.Text above each bivariate plot describes the gate highlighted in red.Arrows indicate that this population was then sequentially gated on by the indicated markers in the adjacent right plot.The second and third row then stratify the analysis by either WT or OT-I/II donor status.Gates of populations shown in Figure 4 are highlighted in purple.Extravascular (EV); Effector (Eff); Effector Memory (EM).
Cohorts described in Figure 7 were assessed for (A) the total number of CD3 + splenic T cells and CD19 + splenic B cells.(B) %CD69 + in T cells from the spleen and resident within the lung.(C) %IgD + in B cells from the spleen and resident within the lung.Nonparametric Mann-Whitney U-tests were used for pair-wise comparisons to determine statistical significance, (ns p>0.05).
Supplemental Table 3