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.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

The latent HIV-1 viral reservoir in resting CD4⁺ (rCD4⁺) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant’s blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4⁺ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Group 2 Innate Lymphoid Cells (ILC2) play an important role in immune responses at barrier surfaces, notably in the lung during airway allergic inflammation or asthma. Several studies have described methods to isolate ILC2s from wild-type naive mice, most of them using cell sorting to obtain a pure population. Here, we describe in detail, a simple, efficient method for isolation and culture of lung mouse ILC2s. Lungs from Rag2^-/- mice pretreated with IL-33 are collected and processed into single cell suspensions. Lymphoid cells are then recovered by density gradient separation. Lin^-CD45⁺ cells are selected by depletion of lineage positive cells followed by positive selection of CD45⁺ cells. Culture of the isolated cells for several days results in a highly purified ILC2 population expressing typical cell surface markers (CD90.2, Sca1, CD25, CD127, and IL-33R). These cells can be expanded in culture for up to 10 days and used for diverse ex vivo assays or in vivo adoptive transfer experiments.

Regulatory T cells (Tregs), a subset of CD4⁺CD25⁺ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25⁺ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.

Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4⁺CD25⁺ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4⁺ T cells are enriched by negative selection, followed by positive selection of CD25⁺ T cells. Tumor-induced purified Tregs (CD3⁺CD4⁺CD25⁺FoxP3⁺) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.

Several studies have shown that the detrimental influence of abdominal obesity on metabolic processes is mediated by the intra-abdominal fat depot. Visceral adipose tissue has been shown to be an independent risk factor for coronary heart disease, hypertension, impaired glucose tolerance and Diabetes Mellitus Type 2 (DM2). Diet-induced obesity in mice, primarily of the C57BL/6J strain, is a commonly used method to study the development of insulin resistance as a model for DM2. The white or visceral adipose tissue (here referred to as VAT), especially the fat around the gonads, is a commonly used organ of study in this model, as it accumulates large numbers of lymphocytes in response to diet-induced obesity. The protocol below describes the isolation of lymphocytes from the stromal vascular fraction (SVF) from VAT.

The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells will emerge from the bone marrow. Thus all B cells that were exposed to the antigen and still alive after at least 4 weeks (8 weeks or more is ideal), are memory B cells.

Splenic B cells from B1-8 mice were prepared with an EasySep Mouse B Cell Enrichment Kit according to the manufacturer’s protocol. Single-cell suspensions were transferred intravenously into tail veins of recipient mice. Approximately 1 million NP+ B cells were transferred per mouse. Approximately 12-24 h after transfer, mice were immunized intra-peritoneally with 50 µg of NP-CGG precipitated in alum.

The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to separate the intracellular vesicles such as endosomes or lysosomes, which could be useful to identify the subcellular localization of the signaling complexes.

Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3^neg, CD14^neg, CD19^neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with target cells or cytokines. This method can be used to assess the general functional capacity of NK cells in peripheral blood mononuclear cells from a wide range of study participants.

Autoreactive T cells restricted to CD1 molecules and specific for endogenous lipids are abundant in human blood (de Jong et al., 2010; de Lalla et al., 2011). A few self-lipid molecules recognized by diverse individual T cell clones and accumulated within APCs following stress signals or cell transformation have been identified so far (de Jong et al., 2010; Chang et al., 2008; Lepore et al., 2014). These findings suggested that auto-reactive CD1-restricted T cells display broad lipid specificities and may play critical roles in different types of immune responses including cancer immune surveillance, autoimmunity and antimicrobial immunity. Therefore, the identification of the repertoire of self-lipid molecules recognized by T cells is important to study the physiologic functions of this T cell population and to assess their therapeutic potential (Lepore et al., 2014). Here we describe the protocol we established to isolate and identify endogenous lipids derived from leukemia cells, which stimulate specific autoreactive CD1c-restricted T lymphocytes (Lepore et al., 2014). This protocol can be applied to isolate lipid antigens from any type of target cells and to investigate the self-lipid antigen specificity of autoreactive T cells restricted to all CD1 isoforms (Facciotti et al., 2012).