24 – Diagnostics Tests II

Question 1

What are some measurable characteristics of T lymphocyte response?

Answer 1

-activation marker expression (flow cytometry)
-proliferation responses
-cytokine secretion
-cytotoxicity of target cells (CD8 cells)

Question 2

Flow cytometry:

Answer 2

-used to characterize individual cells
-expression of cell surface proteins or intracellular cytokines can be measure

Question 3

Basic principle of flow cytometry:

Answer 3

-passage of cells in front of a laser so they can be detected and counted
-cell proteins of interest are fluorescently labeled and excited by the laser
>measure the fluorescence to determine amount and type of cells present in sample
*labeled cells can be sorted into specific tubes

Question 4

Clonal expansion:

Answer 4

-antigen present to T cells via MHC need 3 signals
-start to proliferate
-maturing T and B cells=immunoblasts
*can measure the proliferating cells

Question 5

Measurement of proliferation responses:

Answer 5

-lymphocytes blastogenesis in response to specific ANTIGEN
>add antigen to interested culture of lymphocytes and APCs
>measure uptake of radioactive thymidine to determine proliferation response
*amount of radioactivity is proportional to proliferation

Question 6

Measurement of proliferation responses: mitogens

Answer 6

-lymphocytes also proliferate in response to MITOGENS

Question 7

Mitogens:

Answer 7

-generally lectins
>plant derived proteins that bind to CHO residues on cell surface glycoproteins and cause lymphocytes to divide

Question 8

Examples of mitogens:

Answer 8

-concanavalin A (Con A): T cells
-Phytohemagglutinin (PHA): T cells
-Pokeweed Mitogen (PWM): T and B cells

Question 9

Advances in proliferation measurements:

Answer 9

-radio-labeled thymidine can be replaced by CFSE (fluorescent compound)
*CFSE brightness can be measured by flow cytometry
>non proliferating cells=single CFSE peak=0
>proliferating cells=multiple peaks=1-5 generations

Question 10

Measurement of cytokine production in response to mitogens or antigens:

Answer 10

1. Bioassays
2. ELISA-based assays for cytokines
   *culture lymphocytes with mitogen or antigen and measure amount of cytokine of interest released into cell culture
   *mainly measures activity of Th population that secrete the cytokines

Question 11

ELISpot assay:

Answer 11

-show IFN-gamma release response of peripheral blood mononuclear cells to a target antigen
*bound cytokines are detected using enzyme-linked antiglobulin
*spots where cytokines are=number in sample

Question 12

Cytotoxicity assays:

Answer 12

measure the T cell-mediated killing of target cell population
*measures activity of CD8
*BOTH antigen and MHC 1 restricted

Question 13

Measurement of T cell cytotoxicity:

Answer 13

1. Combined radio-labeled target cells and ‘effector’ lymphocyte population
2. Culture the populations together (if MHC I presentation of antigen =some target cells will be killed)
3. Dead target cells release radioactivity (or fluorescence)
4. Measure amount of radioactivity (fluorescence) released to determine T cell cytotoxicity

Question 14

In vivo measurement of cell-mediated immunity: DTH testing

Answer 14

1. Inject antigen into subcutaneous tissue and it is processed by APCs
2. Th1 effector cell recognizes antigen and releases cytokines
3. Recruitment of phagocytes and plasma to site=visible lesion