Lee-Chang C, Bodogai M, Moritoh K, et al. Aging Converts Innate B1a Cells into Potent CD8⁺ T Cell Inducers. J Immunol. 2016;196(8):3385-3397. doi:10.4049/jimmunol.1502034

This is a follow up paper to an earlier observation by the group that a subset of B cells that accumulate in elderly mammals (4-1BBL⁺). These cells are shown to induces anti-tumor CD8⁺ T-cells in mice. In this report, they investigate a mechanistic hypothesis as to how these cells develop during aging. Through their experiments they demonstrate that certain cytokines produced by aging myeloid cells induce a subset of B cells which can activate CD8⁺ T-cells in an antigen dependent manner to express GrB. They also show limited data which support that a similar subset of B cells in humans also respond in the same way to aging myloid cells and induce a similar subset of T-cells.

This is method which is heavily relied on in immunology. It relies on a microfluidic device known as a flow cytometer and a variety of fluorescent antibodies for various intracellular and extracellular molecules. Immune cells can be identified by the various molecules they express. With flow cytometry cells prepared from various sources into a single cell suspension can be counted and even sorted for collection.

Antibodies recognize a specific molecular pattern which is referred to as its ligand (similar to the terminology to receptors). In order to differentiate cells, a fluorescent pigment molecule (fluorophore) is attached to an antibody specific for a identifying molecule. This molecule could be a inside the cell (intracellular) or outside the cell (extracellular). Often intracellular ligands are secreted signalling molecules, but could also include enzymes, transcription factors, the intracellular portions of receptors etc. etc. Extracellular ligands are cell surface features, most often receptors. Before cells are stained they must have all non-specific binding patterns blocked. This is especially important in immunology where many cells are able to bind the F_c (constant) region of antibodies. To access intracellular ligands, cells are fixed and permeabilized.

It may be helpful to know what the various ligands are so here's a table:

If one thing can be gleaned from this section on labelling it may be that there are many cells involved in the immune system. A typical basic research programme defines the cells they are studying using antibodies to label cells. As more studies are performed on different subsets more functional data becomes linked to various molecular identities and more mechanistic theories about the larger system can be proposed.

Once cells have been labelled they are passed though a microfuidics device called a flow cytometer. This creates a stream of liquid where each cell is more or less lined up in the stream. Lasers excite the labelled cells and this can be detected as counts of specific fluorophore signal. Multiple labels can be detected on a single cell (with multiple lasers!). Some machines have the ability to direct the flow of the cells so that they can be collected for later experiments.

To test the response of certain cell to specific stimulation the can be purified from the samples on the basis of the same labels used in flow cytometry. These labels are immobilized to a substrate and the cells are passed over. Cells can be collected on the basis of not sticking to the labels (negative selection) or later dislodged from the substrate and thus collected on the basis of adhering (positive selection).

Stimulation could come from a specific antibody which engages a receptor cytokines, other cells (a mix of different receptor ligands and cytokines), antigens and/or some combination of these factors. These stimulations are carried out in plastic dishes in the tissue culture incubator. Factors interact with the cells in question and elicit a response. Changes in the cells are quantified by flow cytometry.

A common quantifiable response is proliferation. This can be measured by dyeing the subset of cells of interest. If the cells divide upon stimulation they dilute the dye. This can be detected on a flow cytometer.

In a population of antigen responding cells a very small proportion can actually detect the antigen of interest. The immune system relies on various amplification methods to ensure that the small number of cells that recognize a particular antigen can proliferate and engage the necessary responses. For the purposes of research the number of cells which are triggered by antigen are too small to measure accurately.

To test the role of antigen specific stimulation in a living body, transgenic mice are used. There are two lines used in this study: pme1 and OT-1. The both are similar in that their T-cells recognize a specific antigen rather than a random selection. This means when that antigen is injected into the mouse it elicits a large population of cells to be come active, thus allowing better study.

— Daniel Chan 2020/01/13 19:23