Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patients with Vasculitis.

NKT cells are defined as T cells that recognize hydrophobic antigens presented by class I MHC-like molecules, including CD1d. Among CD1d-restricted NKT cells, type I and type II subsets have been noted. CD1d-restricted type I NKT cells are regarded as pro-inflammatory cells in general. On the contrary, accumulated evidence has demonstrated an anti-inflammatory property of CD1d-restricted type II NKT cells. In our earlier study using a rat model with vasculitis, we demonstrated the pro-inflammatory function of CD1d-restricted type II NKT cells and identified that one such cell recognized P518-532 of rat sterol carrier protein 2 (rSCP2518-532 ), which appeared on vascular endothelial cells presented by CD1d. Based on this evidence, we attempted to detect human CD1d-restricted type II NKT cells in peripheral blood using hSCP2518-532 , the human counterpart of rSCP2518-532, together with a CD1d tetramer in flow cytometry. First, we determined the binding of hSCP2518-532 to CD1d. Next, we detected CD3-positive hSCP2518-532 -loaded CD1d (hSCP2518-532 /CD1d) tetramer-binding cells in peripheral blood of healthy donors. The abundance of TGF-ß-producing cells rather than TNF-α-producing cells in CD3-positive hSCP2518-532 /CD1d tetramer-binding cells suggests the anti-inflammatory property of SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between healthy individuals and patients with vasculitis in a pilot study. Interestingly, the percentage of TGF-ß-producing cells in SCP2/CD1d tetramer-binding type II NKT cells in vasculitic patients was significantly lower than that in healthy controls despite the greater number of these cells. Although further studies to clarify the mechanism and significance of this phenomenon are needed, SCP2/CD1d tetramer-binding type II NKT cells in peripheral blood should be examined in more detail to understand the pathophysiology of vasculitides in humans. © 2018 International Society for Advancement of Cytometry.

Identification of Non-classical Follicular T Cells.

Nonclassical T cells are a heterogeneous group of T lymphocytes that are activated during the early stages of infection and act as a bridge between the innate and adaptive immune system. Among them, Natural Killer T (NKT) cells have been extensively studied in the last two decades due to their unique ability to recognize foreign/self-lipid antigens in the context of CD1d, a nonclassical major histocompatibility complex molecule. In this chapter, we describe our protocols to track murine NKT cells in lymph nodes by flow cytometry and confocal microscopy.

CD24+ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition.

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24+ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24+ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.

CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.