Immunology

γδ T cells play a critical role in homeostasis and diseases such as infectious diseases and tumors in both mice and humans. They can be categorized into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells. While CD27 expression segregates these two subsets in mice, little is known about human γδT17 cell differentiation and expansion. Previous studies have identified γδT17 cells in human skin and mucosal tissues, including the oral cavity and colon. However, human γδ T cells from peripheral blood mononuclear cells (PBMCs) primarily produce IFN-γ. In this protocol, we describe a method for in vitro expansion and polarization of human γδT17 cells from PBMCs.

Key Features

• Expansion of γδ T cells from peripheral blood mononuclear cells.

• Human IL-17A-producing γδ T-cell differentiation and expansion using IL-7 and anti-γδTCR.

• Analysis of IL-17A production post γδ T-cell expansion.

The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However, when ART is suspended, the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently, the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes, thus, restricting them from a broader level application. The novel TILDA, labelled as U-TILDA ('U' for universal), can detect all the major genetic subtypes of HIV-1 unbiasedly, and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies.

Graphical abstract:

Regulatory T cells (Tregs) suppress immune responses via a variety of mechanisms and can be used as a cellular therapy to induce tolerance. The function of Tregs is commonly assessed in vitro using assays that measure suppression of effector T cell proliferation and/or cytokine production. However, Tregs can also suppress the function of antigen presenting cells, creating a need for methodology to routinely measure this aspect of their function. This protocol describes a method to measure human Treg-mediated suppression of CD80 and CD86 expression on mature, monocyte-derived dendritic cells. Representative data show suppression mediated by polyclonal Tregs as well as antigen-specific Tregs generated using chimeric antigen receptor (CAR) technology. This method can be used in parallel to T cell suppression assays to measure the functional activity of human Tregs.

Thymic T_reg cell differentiation occurs via a two-step process. Step one generates T_reg cell progenitors (T_regP) and is driven by strong TCR interactions with antigens presented in the thymus. Step two is initiated by activation of STAT5 via IL-2, or IL-15, leading to mature T_reg cells capable of emigrating from the thymus and mediating immune tolerance and homeostasis in peripheral tissues. Herein we describe an in vitro T_regP cell differentiation assay that models the second, cytokine dependent, step of thymic T_reg cell development. It can be utilized with relative ease to determine if a population of thymocytes represents a potential progenitor population for T_reg cells as well as test how different cytokines or chemical inhibitors modulate the differentiation of known T_regP cell populations into mature T_reg cells.

To track recent thymic emigrants (RTEs) or study T cell development in the thymus, intra-thymic injection of a cellular tag or precursor cells for various T cell lineages is often desired. However, the traditional surgical approach to expose the thymus for intra-thymic injection is time-consuming and can cause a high level of pain and stress to animals, which might disrupt immune homeostasis, potentially confounding the results. Here, we introduce an ultrasound guided intra-thymic injection procedure, which is non-surgical and minimally invasive to animals. This technique is relatively easy to learn and offers an efficient and accurate tool to track RTEs or perform intra-thymic transfer of various cell types to investigate their differentiation.

This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), with T-cell depleted donor bone marrow bearing a different congenic marker (CD45.2) to that of the host mouse (CD45.1). New cell production post-BMT can thus be tracked by measuring the fraction of CD45.2⁺ cells over time within a population of interest (Hogan et al., 2015; Gossel et al., 2017).

Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing maintenance of effector functions in long-lived memory T cells during antigen-independent proliferation, human naïve and memory CD8 T cells can be sorted from peripheral blood mononuclear cells (PBMCs), labeled with the proliferation-tracking dye carboxyfluorescein succinimidyl ester (CFSE), and then purified based on their levels of cell division. This allows investigators to assess differences in the desired molecular target in cells that have undergone cytokine-driven proliferation. We provide here a protocol for assessing epigenetic programs in divided and undivided human naïve and memory CD8 T cells following 7 days in culture with IL-7 and IL-15 to illustrate how this approach can shed light on the mechanism(s) that governs the preservation of effector functions during homeostasis of long-lived memory CD8 T cells.

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24⁺ invariant natural killer T (Vα24⁺iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24⁺iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8⁺ cytotoxic T lymphocytes to eliminate MHC^- and MHC⁺ tumor cells, respectively. Here we describe a robust protocol to reprogram human Vα24⁺iNKT cells into iPSC, and then to re-differentiate them into Vα24⁺iNKT cells (iPS-Vα24⁺iNKT). We further provide a protocol to measure the activity of iPS-Vα24⁺iNKT cells.