CTL Activity Can Be Demonstrated by CML

Development of the cell-mediated lympholysis (CML) assay was a major experimental advance that contributed to understanding of the mechanism of target-cell killing by CTLs. In this assay, suitable target cells are labeled intracellularly with chromium-51 (51Cr) by incubating the target cells with Na251 CrO4. After the 51Cr diffuses into a cell, it binds to cytoplasmic proteins, reducing passive diffusion of the label out of the cell. When specifically activated CTLs are incubated for 1-4 h with such labeled target cells, the cells lyse and the 51Cr is released. The amount of 51Cr released correlates directly with the number of target cells lysed by the CTLs. By means of this assay, the specificity of CTLs for allogeneic cells, tumor cells, virus-infected cells, and chemically modified cells has been demonstrated (Figure 14-17).

The T cells responsible for CML were identified by selectively depleting different T-cell subpopulations by means of antibody-plus-complement lysis. In general, the activity of CTLs exhibits class I MHC restriction. That is, they can kill only target cells that present antigen associated with syn-geneic class I MHC molecules. Occasionally, however, class II-restricted CD4+ T cells have been shown to function as CTLs.

Strain X

Spleen

Strain X

Spleen

Lymphocytes (responder cells)

Strain Y

plee

Strain Y

plee

Spleen Treat with x-rays or

Lymphocytes ¿ (stimulator cells)

mitomycin C

A

I J

18-24 h

o o o o o

[3H] thymidine

Microwell cultures

[3H] thymidine

Time, days

FIGURE 14-16

Time, days

FIGURE 14-16

Count 3H incorporated into DNA

One-way mixed-lymphocyte reaction (MLR). (a) This assay measures the proliferation of lymphocytes from one strain (responder cells) in response to allogeneic cells that have been x-irradiated or treated with mitomycin C to prevent proliferation (stimulator cells). The amount of [3H] thymidine incorporated into the DNA is directly proportional to the extent of responder-cell proliferation. (b) The amount of [3H]-thymidine uptake in a one-way MLR depends on the degree of differences in class II MHC molecules between the stimulator and responder cells. Curve 0 = no class II MHC differences; curve 1 = one class II MHC difference; curve 2 = two class II MHC differences. These results demonstrate that the greater the class II MHC differences, the greater the proliferation of responder cells.

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