Background/Purpose: PD-1^hi CXCR5^– T peripheral helper (Tph) cells are highly expanded in RA and SLE patients. Like T follicular helper (Tfh) cells, human Tph cells express high levels of ICOS and MAF and secrete IL-21 to facilitate B cell function. While Tfh cells depend on CXCR5 to home to follicles, Tph cells frequently express chemokine receptors that enable migration to inflamed sites. Whether Tph cells expand in murine autoimmunity models, and what the role of Tph cells is in vivo remains unknown. Further, whether Bcl6, a master regulator for Tfh cells, is necessary for Tph cells has not been established. We investigated these questions in pristane-induced lupus.

Methods: We evaluated the cellular and molecular features of Tph cells in the tissues of pristane-treated mice. C57bl6 or CD4^creBcl6^fl/fl mice were injected with 0.4-0.5 ml pristane intraperitoneally once and harvested 2 weeks or >2 months later with age-matched controls. Tissues were processed for flow cytometry and histology.

Results: In pristane-treated mice, PD-1^hi CXCR5^– CD4⁺ T cells accumulated first in peritoneal fluid and spleen, and later in lung and peritoneal granulomas. These cells in the periphery expressed high levels of ICOS and Maf and produced IL-21, with an intermediate level of Bcl6. While lacking CXCR5, ~40% of PD-1^hi CXCR5^– T cells in pristane-treated mice expressed CXCR6 across multiple tissues. These results demonstrate that PD-1^hi CXCR5^– CD4⁺ T cells in this model closely resemble human Tph cells (Fig.1).

Accompanying Tph expansion, B cell responses were seen in pristane-treated mice. In spleen, frequencies of extrafollicular B cells, CD138⁺ plasmablasts, CD11c⁺CD21^– age-associated B cells (ABC), and CD73⁺IgD^– class-switched memory B cells increased, while the proportion of CD23^hiCD21⁺ follicular B cells decreased. The frequencies of plasmablasts, ABCs, and memory B cells also increased in lungs (Fig.2). Immunofluorescence microscopy revealed lymphoid aggregates within lungs of pristane-treated mice, which contained clusters of Tph cells and B cells.

To determine the role of Bcl6-dependent Tfh cells in this B cell response, we compared pristane-induced lupus in mice with conditional loss of Bcl6 in CD4⁺ T cells (CD4-Bcl6 KO) and littermate controls (WT). As expected, CD4-Bcl6 KO mice had reduced CXCR5⁺ Tfh cells, and also showed less expansion of extrafollicular, memory and CD138⁺ B cells in spleen, indicating a critical role for Tfh cells in B cell activation in spleen. In contrast, pristane-treated CD4-Bcl6 KO mice had increased frequencies of Tph cells, memory B cells and plasmablasts in lungs, suggesting that Tph accumulation and B cell responses within lungs can occur independently of Bcl6⁺ Tfh cells in pristane-induced lupus.

Conclusion: Tph cells are expanded in spleen, target tissues, and ectopic lymphoid structures in pristane-treated mice. Murine Tph cells express molecules involved in B cell activation and migratory receptors distinct from Tfh cells. In the absence of Bcl6 and Tfh cells, pristane-induced lupus mice developed increased Tph cells and B cell activation in lung. These results provide the first in vivo evidence of a role for Tph cells in peripheral B cell responses in autoimmunity.

Figure 1. Expanded PD-1hiCXCR5- CD4 Tph cells in murine lupus expressed factors associated with B cell help, including ICOS, Maf and IL-21. A). PD-1hiCXCR5-CD4 T cells expanded in peritoneal fluid and spleen at 2 weeks and were found in lung and granuloma after 2 months in pristane-treated lupus mice (Mann-Whitney test, n= 5 – 13); B). CXCR6 was more frequency expressed on Tph cells in peripheral tissues (PF, lung, and granuloma) than in spleen Tph/Tfh cells and peripheral PD-1- cells; Tph and Tfh cells expressed comparable levels of ICOS, both significantly higher than PD-1-CXCR5- DN cells; Bcl6 and c-Maf were highly expressed by spleen Tfh cells, intermediately expressed by Tph cells in spleen and peripheral tissue, greater than peripheral DN cells; PD-1-, PD1hiCXCR5- Tph, PD1hiCXCR5+ Tfh cells were sorted from pristane-treated mice and stimulated in vitro; Tph and Tfh expressed higher IL-21 than PD1- cells; ordinary ANOVA. Mean±SD shown, , *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. Pathologic B cells expanded in spleen, target organs, and ectopic lymphoid structures in pristane-treated mice. A) Increased extrafollicular B cells (mostly CD23loCD21hi marginal zone B cells) and decreased CD23hiCD21+ follicular B cells were found in the spleen of pristane-treated mice (n=9-13). B) CD11c+CD21- ABCs and CD73+IgD- class-switched memory B cells increased in PF and lung (n=6-12). CD138+ plasmablasts increased in spleen and lung (n=5-6). C) Immunofluorescence images of ectopic lymphoid aggregates in the lung of pristane-treated mice. Mean±SD shown, Mann-Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3. Pristane-treated CD4-Bcl6 KO mice had preferential expansion of Tph in the periphery with pathological B cell changes in the lung. A) Pristane-treated CD4-Bcl6 KO had decreased CXCR5+ CD4 cells compared to littermate controls; B) CD4-Bcl6 KO mice showed increased Tph expansion in lung (6% in CD4-Bcl6 KO and 3% in control) than littermate controls, with comparable Tph frequency in spleen. C) CD4-Bcl6 KO showed less expansion of extrafollicular B cells, class-switched memory B cells and CD138+ B cells in the spleen, while these populations were expanded in the lung compared to WT (n=2-6). Mean±SD shown, Mann-Whitney test, *p < 0.05, **p < 0.01.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expanded-t-peripheral-helper-cells-and-increased-pathologic-b-cell-lung-infiltration-in-pristane-induced-murine-lupus-in-the-absence-of-bcl6-tfh-cells/