Lab 19: Cell-Mediated Lysis (Natural cell-mediated cytotoxic effectors)
In the innate system there are several of mechanism that are involved with immune surveillance, where tumor cells are controlled and killed. Two of these mechanisms are Natural Killer (NK) and Natural Cytotoxic (NC). NK and NC activities belong to a family of Natural Cell Mediated Cytotoxic (NCMC) effector cells. Being part of the innate immune system allows them to kill certain cells without prior exposure, immunization, or activation. It is known that NK cells are involved in killing virus and intracellular pathogen infected cells, tumor-killing, and activation of naïve CD4 T cells to differentiate into inflammatory T cell (TH1). NC cells are involved in killing tumor cells and help fight chemical carcinogenesis.
Burnet’s Immune Surveillance Theory describes how the immune system responds to tumor cells. The theory states that the immune system recognizes tumor antigens and causes the tumor cells to die. In immunodeficient animal’s tumor cells are able to proliferate and tumor cells which have developed mechanisms to avoid immune surveillance are able to proliferate and become established tumors meaning that natural immunity plays a major role in immune surveillance. It also appears that T cells mediate surveillance against tumors expressing viral antigen but not against spontaneous or chemically induced tumors which are killed by NC or NK cells.
NK cells are large granular non-T, non-B lymphoid cells. It kills target cells by lytic granules (perforin and granzymes), and kill target cells which have Fas (CD95) since NK cells express Fas Ligand on surface. NK cells also produce granulysin to directly kill pathogens. NK cells make up a small fraction of peripheral blood lymphoid cells and are able to recognize and kill abnormal cells. Although NK cells can kill sensitive targets can be isolated from uninfected individuals activity is increased by 20 to 100-fold when exposed to IFN-alpha and Beta, or to the NK cell-activation factor, IL-12.
NK cells have two types of surface receptors:
NC kill target cells via membrane-bound (surface-bound) TNF (tumor necrosis factor).
Characteristics of both NK and NC and NCMC (cell mediated immunity):
Sensitivity of NK is Yac-1 (H-2a/a), mouse T cell lymphoma
Sensitivity of NC is 10 ME (H-2d/d), mouse fibrosarcoma, L929 (H-2k/k), mouse fibrosarcoma.
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 (with 10ME, Yac-1, and L88)) were cultivated and NC and NK cells were added. A Chromium assay was set up to see when the foreign antigens will be killed (either 6 or 18 hours) and which type of Ag will be killed at each interval.
The Chromium assay works by exposing the target cells to 51Cr, allowing the target cells to absorb the chromium. When the NK and NC cells kill the target cells, the Cr will then be released. Based on the amount of Cr in the solution, one can quantitatively determine the amount of cells killed.
Procedure:
Follow Lab Manual
Effector cells: spleen cells which contain NCMC/NK cells
Target cells: Yac-1 (NK target), 10 ME (NC target), and L88 (NC and NK resistant).
Changes in procedure:
Part A and Part B done by TA
Count the live spleen cells in Part C
Part D will not be done by TA
Part E, the TA will add radiolabeled target cells
Formula for calculating % spontaneous release and % specific 51Cr release for NK and NC assays:
Discussion:
For this lab, only one part (in vitro) was done because no mice were available.
In this lab we examined the mechanisms and differences of NC and NK killing by using a chromium assay. Yac-1 is more sensitive to NK cells, while 10 ME and L88 is sensitive to NC cells. It was also seen that each NCMC effector cell killed at different times. The NC kills at 18 hours, while the NK cells killed at 6 hours.
From the results obtained, the predicted trend was seen. For the NK killing, Yak-1 had the most killing since it had the highest number of Chromium present when compared to the 10ME and the L88. For the NC the 10ME had the most killing, followed by Yac-1 (for 20:1 and 80:1) and the L88 had the least killing, since it was NC and NK resistant.
For the SR (spontaneous release) % most of the numbers stayed under 30% meaning that there was no additional factor causing the target cells to be killed other than the NC and NK.