NLRP6 regulates ZAP-70-erk signaling in both murine and human T cells and influences acute graft-versus-host disease(GVHD)

Abstract The Nlrp6 (NOD-like receptor family pyrin domain-containing 6) inflammasome is essential for innate immune responses and maintaining homeostasis in intestinal epithelial cells. We were thus surprised to find that Nlrp6 deficiency in host non-hematopoietic cells, including intestinal epithelial cells, ameliorated acute graft-versus-host disease (aGVHD) in an inflammasome and gut microbiome independent manner (Toubai, et al. Nat Microbiol 2019). Nlrp6 is also expressed in T cells, and donor T cells are critical for aGVHD pathogenesis. We previously reported that Nlrp6 deficient (Nlrp6-/-) T cells exacerbate GVHD in multiple murine models in an inflammasome independent manner by increasing the differentiation of pathogenic Th1 cells (Blood 138: 2766, Transplant Cell Ther 28(3): S276, 2022, Transplant Cell Ther 29: S60, 2023). Here we determined the mechanisms of Nlrp6-dependent regulation of donor T cell-driven aGVHD, investigated whether these mechanisms influenced allogeneic T cell-driven graft-versus-tumor (GVT) responses, and assessed the association of Nlrp6 expression in T cells of patients with aGVHD. We first tested whether Nlrp6 influenced T cell receptor (TCR) signal transduction pathways, focusing on downstream activation of Zap-70 and Erk because they enhance Th1 differentiation. After non-specific TCR stimulation, unphosphorylated Zap-70 decreased to a greater extent in Nlrp6-/- than WT CD4+ T cells, suggesting that phosphorylated Zap-70 (pZap-70) may have increased to a greater extent in Nlrp6-/- CD4+ T cells. Consistent with this, pZap-70 was significantly higher in Nlrp6-/- than WT CD4+ T cells. Hence, we measured phosphorylated Erk and found increased p Erk1/2 in resting and nonspecific TCR-stimulated Nlrp6-/- CD4+but not CD8+ T cells. There was no difference in the phosphorylation of Lck and NFAT in Nlrp6-/- T cells, indicating that Nlrp6 deficiency did not globally alter TCR signaling. To further test whether the Zap-70-Erk pathway was necessary for enhanced activation of Nlrp6-/-T cells, we treated Nlrp6-/- and WT T cells with an Erk inhibitor, which significantly reduced Erk signaling following nonspecific stimulation of Nlrp6-/- but not WT T cells in vitro. GVHD and graft-versus tumor (GVT) responses are tightly linked, making it difficult to target aGVHD without increasing the risk for relapse. CD8+ cytolytic activity is critical for GVT responses. With this in mind, we were intrigued when CD8+ donor T-cell expansion, activation, and cytokine production 7 days post murine allo-BMT as well as proliferation after non-specific TCR stimulation were increased in Nlrp6-/- T cells, yet Nlrp6 deficiency did not affect CD8+ T cell-mediated aGVHD-related mortality. Thus, we hypothesized that Nlrp6 would not influence GVT responses. To test this, we determined whether deficiency of Nlrp6 in allogeneic T cells altered their anti-leukemia activity. We used a C57BL/6 into BALB/c bone marrow transplant (BMT) model and added Baf/3-ITD leukemia cells at the time of BMT. Consistent with our hypothesis, GVT responses were equivalent in recipients of allogeneic WT or Nlrp6-/- T cells. In addition, there was no difference in the anti-leukemia effect of Nlrp6-/- compared to WT T cells using an in vitro cytotoxic T lymphocyte (CTL) killing assay. Furthermore, degranulation of in vitro activated Nlrp6-/- and WT CD8+ T cells was similar as was the production of the cytotoxic molecules granzyme B and perforin. These data indicated that Nlrp6 in donor T cells was not required for the GVT effect. To test whether decrease Nlrp6 expression in donor T cells was associated with aGVHD in human allogeneic hematopoietic cell transplant recipients, we measured its expression in donor T cells from patients with aGVHD relative to healthy controls. Consistent with our murine data, Nlrp6 expression was significantly decreased in peripheral blood CD4+ and CD8+ T cells from patients with aGVHD. These data suggested that decreased donor T cell Nlrp6 expression is associated with aGVHD in both humans and mice. Collectively, these data indicate Nlrp6 negatively regulates allogeneic donor T cell Zap-70-Erk signaling, resulting in reduced aGVHD and maintenance of GVT responses. As such, these studies provide mechanistic insights into the cell type-specific and context-dependent antagonistic biological functions of Nlrp6 on controlling aGVHD inflammatory responses.