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This article will be discussed on November 14th at Biotech without Borders as part of the Deep Dive series.

In the context of the review which this paper is paired with for Deep Dive, this is an example of “altered intercellular communication”. Specifically this is about the immune system behaving differently over time that is referred to as immunosenecence.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers

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.

Materials and methods

Flow cytometry

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 Fc (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:

After making it I realized it's pretty big, and maybe not entirely helpful so I hid it. But please feel free to familiarize yourself :P

After making it I realized it's pretty big, and maybe not entirely helpful so I hid it. But please feel free to familiarize yourself :P

Ligand short Full name Location Species specificity (used in this study) Short description
TNF-α Tumor necrosis factor alpha intra- human, mouse a pro-inflammatory cytokine
IFN-γ Interferon gamma intra- human, mouse a pro-inflammatory cytokine
CD19 B-lymphocyte antigen CD19 extra- human, mouse the antigen specific B-cell receptor, marker for B-cells
4-1BBL Induced by lymphocyte activation receptor ligand extra- human, mouse activator of CD137 (co-stimulatory receptor) on T-cells
CD86 Cluster of differentiation 86 extra- human, mouse activator of costimulatory receptors on T-cells which govern activation and survival
CD8 Cluster of differentiation 8 extra- human, mouse T-cell co-receptor for MHC, marker for cytoxic T-cells
GrB Granzyme B intra- human, mouse (cross-reactive) activator of apoptosis, marker of activated cytotoxic cells
CD11b Integrin alpha M extra- human, mouse (cross-reactive) half of the macrophage-1 antigen, marker for macrophages
CD120b Tumor necrosis factor receptor 2 extra- human, mouse receptor for TNF-α
CD27 Cluster of differentiation 27 extra- human co-stimulatory receptor on B and T cells
IL-10 Interlukin 10 intra- human anti-inflammatory cytokine
CD14 Cluster of differentiation 86 extra- human co-receptor for a common bacterial antigens on antigen presenting cells
CD3 Cluster of differentiation 3 extra- human T-cell co-receptor, marker for T-cells
CD138 Syndecan 1 extra- human cell matrix binding and signalling, marker for plasma cells in blood samples
CD43 Leukosialin extra- human found on a majority of leukocytes
CD69 Cluster of differentiation 69 extra- human
CD5 Cluster of differentiation 5 extra- mouse a marker for memory cells
CD119 IFN-γ receptor 1 extra- mouse stimulated by IFN-γ
CD23 Fc receptor CD23 extra- mouse low affinity receptor for the constant region of certain antibodies
CD21/35 Complement C3d/C3b/C4b receptor extra- mouse allows complement signals to pass to B-cells
CD1d Cluster of differentiation 1, group 2 extra- mouse primarily a marker for natural killer cells, quite mysterious

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.

In vitro stimulation

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 vivo antigen specific stimulation

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.


Aging milieu activated B1a cells

Aging converts B1a cells into 4BL cells

Myeloid cells induce the generate of 4BL cells that drive antigen specific cytotoxic effects

Putative mechanism of myeloid induction


Daniel Chan 2020/01/13 19:23

research-review/aging-converts-innate-b1a-cells-into-potent-cd8-t-cell-inducers.txt · Last modified: 2020/05/05 06:33 by marcos