0992: FoxP3^Lo CD4⁺ T cells are functionally suppressive and expanded in the immune-mediated fibrotic diseases IgG4-related disease and systemic sclerosis

Background/Purpose: The absence of regulatory T cells (Tregs) results in multiorgan autoimmunity in the context of monogenic “Tregopathies,” but their role in mediating polygenic autoimmune diseases remains less clearly defined. Antigen-experienced human T cells expressing FoxP3, a transcription factor driving regulatory function, can be divided into FoxP3^Lo and FoxP3^Hi subsets. FoxP3^Lo T cells are expanded in the context of autoimmunity, but there is a paucity of data defining the function of FoxP3^Lo T cells or their relevance to immune-mediated fibrosis. Previous studies suggest they are non-suppressive and primarily composed of activated effector T cells. We examined FoxP3⁺ T cells across three different immune-mediated fibrotic diseases, systemic sclerosis (SSc), IgG4-related disease (IgG4-RD), and sarcoidosis.

Methods: We used spectral flow cytometry to examine circulating Treg subsets in PBMCs from patients with SSc (n=18, median age 55; 10 diffuse, 7 limited, 1 sine), IgG4-RD (n=27, median age 65, 15 head and neck, 10 pancreatobiliary, 2 retroperitoneal fibrosis, 6 kidney), sarcoidosis (n=17, median age 66, 7 only pulmonary, 10 extra-pulmonary involvement) and healthy donors (HD, n=17, median age 64). Bulk RNA sequencing of flow purified T cell populations was used to define the transcriptional differences between Treg subsets. Purified Treg subsets were used for in vitro assays to measure the capacity to suppress T cell proliferation.

Results: FoxP3^Lo T cells were preferentially expanded in SSc and IgG4-RD (Fig 1C). FoxP3^Lo T cell expansion correlated with disease severity, including early disease, skin score and interstitial lung disease in SSc and internal organ involvement in IgG4-RD (Fig 1 D-H). Expression of the transcription factor Helios reliably excluded IL2 and IFNg-producing effector T cell contaminants from the FoxP3^Lo gate, as has been previously suggested (Fig 3A). Helios⁺ FoxP3^Lo T cells are also expanded in SSc and IgG4-RD, indicating that the FoxP3^Lo expansion is not driven by activated effector T cells (Fig 3C). FoxP3^Lo T cells expressed decreased levels of the suppressive protein, CTLA4, and activation marker, ICOS, relative to FoxP3^Hi cells, as previously reported. Transcriptionally, CD127^LoCD25^Lo (analogous to FoxP3^Lo) T cells expressed decreased levels of the activation markers TNFRSF9 (CD137), IL2R (CD25), TNFRSF8 (CD30), CD70, and HLA-DR, as well as PTPN22, relative to CD25^Hi cells, suggesting a difference in TCR binding affinity or activation state between these subsets (Fig 2B). The inhibitory receptor, PDCD1 (PD1), was uniquely upregulated in CD25^Lo T cells. Despite differences in activation status and expression of CTLA4, CD127^LoCD25^Lo T cells showed an equal ability to suppress T cell proliferation in vitro when compared to their CD25^Hi counterparts (Fig 3D).

Conclusion: We show that FoxP3⁺ T cells expressing low levels of FoxP3 are legitimate regulatory T cells but have reduced expression of certain markers of activation and Treg function. FoxP3^Lo T cells are expanded in the immune-mediated fibrotic diseases SSc and IgG4-RD and correlate with disease severity, suggesting either a pathogenic role in the context of fibrosis or an induced regulatory response.