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:

Macrophages are key cells in the innate immune system and play a role in a variety of diseases. However, macrophages are terminally differentiated and difficult to manipulate genetically via transfection or through CRISPR-Cas9 gene editing. To overcome this limitation, we provide a simplified protocol for the generation of mouse embryonic stem cells-derived macrophages (ESDM). Thus, genetic manipulation can be performed using embryonic stem cells, selecting for the desired changes, and finally producing macrophages to study the effects of the previous genetic manipulation. These studies can contribute to many areas of research, including atherosclerosis and inflammation. Production of ESDM has been previously achieved using embryoid body (EB) intermediates. Here, we optimized the EB method using a simplified medium, reducing the number of recombinant proteins and medium recipes required. Our EB-based differentiation protocol consists of three stages: 1) floating EB formation; 2) adherence of EBs and release of floating macrophage progenitors; and, 3) terminal differentiation of harvested macrophage progenitors. The advantages of this protocol include achieving independent floating EBs in stage 1 by using a rocker within the tissue culture incubator, as well as the exclusion of small EBs and cell clusters when harvesting macrophage progenitors via cell filtration.

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.

The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo has been limited and no in vitro system has been available that resembles B cell reaction in GC. Here we describe the protocol for a culture system named “induced GC B (iGB) culture system” which can induce massive expansion of B cells that exhibit GC B cell-like phenotype, and thus it mimics the GC reaction. This protocol can be useful to elucidate the molecular mechanisms of GC B cell differentiation.

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.

The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), there has not been a method to evaluate corresponding high-dimensional functionomic data (relating to cellular functions) from single cells. Here, we present a protocol to quantitatively measure the differentiation potentials of single human hematopoietic stem and progenitor cells, and then cluster the cells according to these measurements. High dimensional functionomic analysis of cell potential allows cell function to be linked to molecular mechanisms within the same progenitor population.

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.