ABSTRACT

T lymphocytes are established mediators of ischemia reperfusion (IR)-induced AKI, but traditional immune principles do not explain their mechanism of early action in the absence of alloantigen. Nrf2 is a transcription factor that is crucial for cytoprotective gene expression and is generally thought to have a key role in dampening IRinduced AKI through protective effects on epithelial cells. We proposed an alternative hypothesis that augmentation of Nrf2 in T cells is essential to mitigate oxidative stress during IR-induced AKI. We therefore generated mice with genetically amplified levels of Nrf2 specifically in T cells and examined the effect on antioxidant gene expression, T cell activation, cytokine production, and IR-induced AKI. T cell-specific augmentation of Nrf2 significantly increased baseline antioxidant gene expression. These mice had a high frequency of intrarenal CD25 increased T cell expression of Nrf2 were significantly protected from functional and histologic consequences of AKI. Furthermore, adoptive transfer of high-Nrf2 T cells protected wild-type mice from IR injury and significantly improved their survival. These data demonstrate that T cell-specific activation of Nrf2 protects from IR-induced AKI, revealing a novel mechanism of tissue protection during acute injury responses.

CD4−CD8− double negative (DN) αβ T cells are legitimate components of the normal immune system. However, they are poorly understood and largely ignored by immunologists because of their historical association with the lymphoproliferation that occurs in mice (lpr and gld) and humans (ALPS patients) with impaired Fas-mediated apoptosis where they are considered abnormal T cells. We believe that the traditional view that DN T cells that cause lymphoproliferation (hereafter referred to as lpr DN T cells) are CD4 and CD8 T cells that lost their coreceptor, conceived more than two decades ago, is flawed and that conflating lpr DN T cells with DN T cells found in normal immune system (hereafter referred to as nDN T cells) is unnecessarily dampening interest of this potentially important cell type. To begin rectifying these misperceptions, we will revisit the traditional view of lpr DN T cells and show that it does not hold true in light of recent immunological advances. In lieu of it, we offer a new model proposing that Fas-mediated apoptosis actively removes normally existing DN T cells from the periphery and that impaired Fas-mediated apoptosis leads to accumulation of these cells rather than de novo generation of DN T cells from activated CD4 or CD8 T cells. By doing so, we hope to provoke a new discussion that may lead to a consensus about the origin of lpr DN T cells and regulation of their homeostasis by the Fas pathway and reignite wider interest in nDN T cells.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants(1-6). DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function(7) due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney(8) in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney. Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d α-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.