Experiment 17: Cell Mediated Lysis Assays: Cytotoxic T Lymphocytes

CTLs serve as one of the mechanisms in the innate system. They target cells that are virally infected and cause apoptosis. CTLs also play an important role in the rejection of allografts. CTLs kills target cells when the antigens are displayed on MHC I molecules.

Naïve mature T cells can be activated by:

Specific antigents + APC’s
Alloantigen
Mitogen

Activation of naïve mature T cells require 2 independent signals

Binding of TCR and co-receptor molecules to generate a signal to the T cell that the antigen has been recognize. If there is no second signal then anergy occurs
Co-stimulatory signal delivered by APC to stimulate T cell growth.

Only T cells that are self MHC restricted (ie. already learned the self/non-self discrimination) will be able to kill. Once activated, T cells synthesize and secrete the T-cell growth factor IL-2 to drive clonal expansion. After activation, the T cells proliferate/differentiate into armed effectors cells.

Generation of effectors (CTL’s) inolve the cooperation of at least three types of cells:

Ag-specific T effector percursrs
Ag-specific T amplifiers
nonspecific accessory cells.

In this lab CTLs are to be generated from mature naïve CD8+ T cells (spleen cells from C57 B1/6 mouse) by using allogeneic cells (spleen cells from F1 mouse = Balb/c X C57 B1/6). In the mixed lymphocyte culture (MLC) suspensions of responder T cells are cultured with allogeneic stimulator lymphocytes. IF the stimulator cell population contains T cells, their differentiation must be prevented by irradiation or treatment with mitomycin C which blocks mitosis and ensures only the desired effectors (responder cells) may multiply.

MLR (mixed lymphocyte reaction) may be unidirectional meaning that one population of allogeneic cells serves as Ag (stimulator cells) and is inhibited from proliferating therefore only the proliferation of the second population (responder cells) will be measured.

In this experiment, spleen cells (in vitro) and mice (in vivo) will immunize with cells of a different MHC (H-2) haplotype.

Stimulator cells are the F1 spleen cells (H-2)

Responder cells are C57 B1/6

Changes in Procedure:

Part A: In vivo part was cancelled.

Part B: In vitro is unidirectional MLR. Students to do in vitro immunization of C57 by obtaining spleen cells from C57 mice.

Change in procedure: instead of counting C57 spleen cells and determining cell concentration in step 4, count 1*10⁸ spleen cells per spleen and make appropriate dilution for step 5.

The mixing of 2 population of allogeneic lymphoid cells results in T cell proliferation (MLR).

Activating stimulus is the foreign histocompatibility Ag expressed on the allogeneic stimulator cells.

Results:

After pouring off the media:

Data for the plates (only had two due to lack of cells [we dropped our sample])

	Plate One	Plate Two
Control	0 Foci	0 Foci
10 uL	3 Foci	0 Foci
100 uL	16	0 Foci

Class data:

	Plate One	Plate Two	Plate Three
100 uL	79	67	16
100 uL	81		0
10 uL	89	76	0
CTL		77	3
Control	8	63	0
L88.3 (only)	77	70	0

Discussion

In this lab we wanted to examine the effects of CTLs on a cell’s population by studying in an in vivo condition and in an in vitro condition. The in vivo portion was not performed however the in vitro.

There are two parts of this experiment. The first was in vivo in which spleen cells (b/b) were harvested and then injected into another mouse (a/a), causing an immune response since the spleen cells injected did not match the host cells. From there spleen cells are again harvested and place back into b/b. The second part was done in vitro where cells in a similar fashion, so that the only difference was that for in vivo the mice served as the culture dish while for in vivo a Petri dish served as a culture dish.

If the in vivo part was done, it would be expected that for the trials where the CTL concentrations are the highest, there would be the most killing, while the control (with no CTLs) will have no killing.

In our experiment group, we experienced unexpected results. For our control, we obtained no foci which should not be possible since there were no CTLs, therefore there should be cell growth, and hence foci. Other groups, however seemed to experience a similar problem although the magnitude was not as great. As you increase the concentration of CTLs, you would expect that there would fewer foci, since there are more CTLs (“cell killers”) out there. This was seen when comparing the class data between the 10 and 100 uL (and not control). There seems to be a slight decrease in the number of foci in the cell cultures. For our group’s data, however, this was not the case and the 100uL actually had the most CTLs.

Possible reasons as to why this occurred is due to possible contamination in the plates, so there will be more foci than expected, cell death via apoptosis (due to no co-stimulation), and/or washing off the cells after the incubation period.