Ag Specific T Lymphocyte Response Biology Essay

Published: Last Edited:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

T lymphocytes are an important part of the adaptive immune system. They derive from hematopoietic stem cells in the bone marrow and mature in the thymus. In the lymph nodes the T lymphocytes come in contact with Dendritic Cells (DCs). If the T lymphocyte receptor (TCR) on the surface of the T lymphocyte binds a specific antigen (Ag) presented by the DCs (Figure 2), the lymphocyte will be activated, proliferate and differentiate into an effector T lymphocyte. The total number of Ag-specific T lymphocytes expand profoundly until the peak of the immune response, when the number of Ag-specific T lymphocytes starts to decline.1 This decline is known as the contraction phase2-3. Only 5 to 10% of the

T lymphocytes observed at the peak of the immune response is left after this decline3. These T lymphocytes form the memory pool1,4. A schematic overview of the different stages of an Ag-specific response is given in figure 1.

Figure 1. The stages of an Ag-specific T lymphocyte response

In the priming/expansion phase, T lymphocytes proliferate and differentiate. In the contraction phase, Ag-specific T lymphocytes numbers decline. And the population that is left forms the T lymphocyte memory pool.

Figure used from Schepers et al.3

Because T lymphocytes play a pivotal role in the immune response towards a variety of infectious pathogens as well as malignancies5, a quantitative understanding of lymphocyte proliferation, differentiation and death in vitro and in vivo, is very important.

The magnitude of an antigen-specific T lymphocyte immune response is controlled by the following three basic parameters:

1.) The number of naive antigen-specific T lymphocytes that is activated to undergo proliferation.

2.) The average number of cell divisions that these activated T lymphocytes undergo.

3.) The fraction of lymphocytes that dies in between two cycles of cell division.

Thus, a T lymphocyte response may either be stronger because more naïve T lymphocytes participate, because these cells undergo a stronger proliferative burst, or because cells are less likely to die. Whereas current technologies, like cellular barcoding6 or labeling with fluorescent dyes7 allow us to delineate recruitment from expansion, dissection of the factors shaping expansion, i.e. proliferation and cell death, is not yet possible.

We aimed to develop a method allowing to dissect the relative contribution of proliferation and cell death, during the different stages of an Ag-specific response in T lymphocytes. In order to determine the extent of cell death we created a cell death sensor.

The cell death sensor contains two loxP sites. After Cre induced recombination, the excised fragment will form a minicircle, with a genetic tag. (Figure 3C) The minicircle lacks an origin of replication, so it will be passed on to only one of the daughter cells when it divides. The number cells containing a minicircle in a population will remain constant, as the minicircles do not replicate during division. Upon cell death the cells containing a minicircle will decline proportionally to the death rate. Quantification of the number of minicircles present in a population therefore allows the determination of the fraction of cells which have died over time.

The cell death sensors used for this project have two fluorochromes. One fluorochrome is placed next to the promoter and is flanked by loxP sites. (Figure 3A and figure 4) This fluorochrome will end up in the minicircle. After recombination the second fluorochrome is placed next to the promoter (Figure 3B). The second fluorochrome will be passed on to next generations and can be a useful addition to determine the fraction of cells that have recombined.


Figure 3. Cds2.1

A. Cds2.1in its original form. The loxP sites are underlined for clarity. For a bigger version of this figure, see appendix 2.

B. The recombined cds2.1, eGFP is cut out.

C. The eGFP minicircle that is left after recombination. This will be present in the original cells and be passed on to only one daughter cell when a cell divides



We used extension multiplex ligation-dependent probe amplification to try to detect the minicircle cut out of the cds in the presence of Cre. This is a technique that enables quantification of small differences in DNA that are a consequence of Cre mediated recombination. The reduction of original vectors and the increase of recombined vectors can be detected in one sample. The minicircle should be detectable up to 5 months after Cre activation.8

In figure 4 you can see a schematic representation of the two cell death sensors used in the experiments, cds2.0 and cds2.1. Before the start of the project cds2.0 was already developed.

Cds2.0 switches from blue (TagBFP) to red (Katushka). The fluorescent intensity of TagBFP in cds2.0 was observed to be very dim in FACS experiments done previously. For this reason we aimed to replace the TagBFP by the brighter fluorochrome eGFP. Cds2.1 (Figure 3A) switches from green (eGFP) to Red (Katushka)



Figure 4. Linear representation of cds2.0 and cds2.1

A. Cds2.0, with TagBFP flanked by loxP sites.

B. Cds2.1, with eGFP flanked by loxP sites.

The aim of this project was to replace the TagBFP in cds2.0 with an eGFP, to test if we could observe recombination in both cds in T lymphocytes and other cell types and to see if we can detect the minicircle. We will show that recombination occurs in both cds and discuss the detection of the minicircle by eMLPA.