SerpinB1 regulates homeostatic expansion of IL-17+ γδ and CD4+ Th17 cells

Abstract

SerpinB1 is an endogenous inhibitor of serine proteases recognized for its anti-inflammatory and host-protective properties. Although loss of serpinB1 in mice does not result in gross immune deregulation, serpinb1a(-/-) mice display increased mortality and inflammation-associated morbidity upon challenge with influenza virus. Here, we show that IL-17A(+) γδ and CD4(+) Th17 cells are already expanded in the lungs of serpinb1a(-/-) mice at steady-state. Both γδ and αβ(+) CD4(+) CCR6(+) T cells isolated from the lungs of naive serpinb1a(-/-) mice displayed a skewed transcriptional profile relative to WT cells, including increased Th17 signature transcripts [Il17a, l17f, and Rorc (RORγt)] and decreased Th1 signature transcripts [Ifng, Cxcr3, and Tbx21 (T-bet)] in γδ T cells. In addition to the lung, IL-17A(+) γδ and CD4(+) Th17 cells were increased in the spleen of naive serpinb1a(-/-) mice, despite normal αβ and γδ T cell development in the thymus. Within the γδ T cell compartment, loss of serpinb1a prompted selective expansion of Vγ4(+) and Vγ6/Vδ1(+) cells, which also displayed elevated expression of the proliferating cell nuclear antigen, Ki-67, and IL-17A. Given that serpinb1a is preferentially expressed in WT IL-17A(+) γδ and CD4(+) Th17 cell subsets vis-à-vis other T cell lineages, our findings reveal a novel function of serpinB1 in limiting untoward expansion of lymphocytes with a Th17 phenotype.

Keywords: cell proliferation; cytokine regulation; inflammation; lymphocytes; population size.

Figure 1.. Increase of IL-17⁺ γδ and CD4 T cells in lungs of naive serpinb1a^−/− mice.

Matched groups of WT and serpinb1a^−/− mice were killed without infection or as a control on Day 2 of sublethal infection with influenza virus. Suspensions of lung cells were cultured with PMA and ionomyin (and brefeldin A) and stained with surface antibodies and intracellularly for IL-17 and IFN-γ. Cells were gated on lymphocytes (low side-scatter, CD45⁺CD11b^neg) and then on γδ TCR or CD4. (A) γδ T cells. Shown left to right are representative contour plots, followed by quantitation of total, IL-17⁺, and IFN-γ⁺ γδ T cells. (B) CD4 T cells. Shown are contour plots followed by quantitation of total, IL-17⁺, and IFN-γ⁺ CD4 cells and Tregs (CD4⁺FoxP3⁺). Numbers in quadrants indicate percentage in each. Means ± sem for eight to 12 mice/group from two to three experiments. *P < 0.05; ***P < 0.001. sb1^−/−, serpinb1a^−/−. Counts of monocytes, macrophages, and neutrophils in naive lungs are shown in Supplemental Fig. 1.

Figure 2.. Transcriptome analysis of T cell lineages in lungs of naive WT and serpinb1a^−/− mice.

(A) Transcription levels of serpinb1a in three populations of WT T cells expressed as arbitrary units (A.U.). (B) Principal components (PC) analysis of the six analyzed populations. PC1 accounts for 85.4% of the genotype variation, PC2 for 9.9%, and PC4 for <1%. PC3, which is not displayed, accounts for 3.3%, but no genotype-dependent differences were seen. (C) Heat map of all 2131 genes. The data were analyzed using hierarchical clustering. Mean normalized values from two independent analyses were used for cluster analysis. (D) Transcriptional levels of signature genes differentially expressed between serpinb1a^−/− [knockout (KO)] and WT γδ T cells (upper) and CCR6⁺ CD4 cells (lower). (E) Increase of CCR6⁺ γδ and CD4 T cells and (F) CD27^neg CCR6⁺ γδ T cells in lungs of WT and serpinb1a^−/− mice. (A–D; mean of duplicates) Data represent evaluations of RNA from two isolates of lung cells, each from five or more mice/cell type. (E and F) Means ± sem or representative data for eight mice/group from two experiments. *P < 0.05; ***P < 0.001.

Figure 3.. Increase of IL-17⁺ and CCR6⁺ γδ T cells in spleens of serpinb1a^−/− mice.

Splenocytes of 4- and 10-week-old, naive WT and serpinb1a^−/− mice were evaluated for surface antigens or IL-17 expression as in Fig. 1. Cells were gated on lymphocytes (CD45⁺CD11b^neg) and then on γδ TCR or CD4. (A) γδ Cells. Shown are total, IL-17⁺, and CCR6⁺ γδ T cells. (B) CD4 cells. Shown are total, IL-17⁺, and CCR6⁺ CD4 cells. Means ± sem for eight mice/group at 4 weeks and six mice/group at 10 weeks, each from two experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Related findings are in Supplemental Fig. 2.

Figure 4.. Selective increase of Vγ4⁺ and Vγ6/Vδ1⁺ γδ T cells in lung and spleen of serpinb1a^−/− mice.

Lung (left) and spleen (right) cells of WT and serpinb1a^−/− mice were stained with GL-3, followed by antibodies for Vγ1, Vγ4, Vγ5, and 17D1, which detect Vγ5/Vδ1 and Vγ6/Vδ1. Shown are absolute counts of total, Vγ1⁺, Vγ4⁺, and Vγ6/Vδ1⁺ γδ T cells. No Vγ5⁺ cells were detected (data not shown), and thus, 17D1 antibody staining identified Vγ6/Vδ1⁺ cells. The data are means ± sem for eight mice/group in three experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Similar results were obtained for Vγ1⁺, Vγ4⁺, and Vγ6/Vδ1⁺ spleen cells of 4-week-old mice (data not shown).

Figure 5.. Increased percentage of IL-17⁺-producing cells within the serpinb1a^−/− Vγ4⁺ and Vγ6/Vδ1⁺ subsets.

Lung and spleen cells of WT and serpinb1a^−/− mice were cultured with PMA and ionomycin. The cells were surface-stained as in Fig. 4 and intracellularly for IL-17. Events were gated on lymphocytes and then on specific γδ subsets. (A) IL-17⁺ cells quantified as percentage of the Vγ1⁺, Vγ4⁺, and Vγ6/Vδ1⁺ subsets. (B) Representative contour plots of IL-17-stained Vγ4⁺ and Vγ6/Vδ1⁺ cells; there were no IL-17⁺ Vγ1 cells. (C) Enumeration of IL-17⁺ Vγ1⁺, Vγ4⁺, and Vγ6/Vδ1⁺ cells. Means ± sem for four mice/genotype. **P < 0.01; ***P < 0.001. Related findings are in Supplemental Figs. 4 and 5.

Figure 6.. Selectively increased proliferation of serpinb1a^−/− Vγ4⁺ and Vγ6/Vδ1⁺ γδ T cells.

Freshly isolated lung (A and B) and spleen (A–D) cells of naive WT and serpinb1a^−/− mice were surface-stained with γδ subset antibodies, as in Fig. 4, and intracellularly for (A, B, and D) the proliferation marker Ki-67 and (C and D) RORγt. (A) Dot plots showing Ki-67 staining of Vγ1⁺, Vγ4⁺, and Vγ6/Vδ1⁺ subsets. (B) Ki-67⁺ cells quantified as percentage within each subset. (C) RORγt staining of Vγ1⁺ and Vγ4⁺ cells. (Left) Flow cytometry plots. (Right) RORγ⁺ cells quantified within the Vγ4⁺ subset. (D) Vγ4 cells costained for Ki-67 and RORγt. (Left) Contour plots. (Right) Quantitation of Ki-67⁺ RORγ^neg and Ki-67⁺ RORγ⁺ cells within the Vγ4 subsets. Means ± sem or representative data for four mice/group. **P < 0.01; ***P < 0.001.