Diagnostic tests- looking for cell-mediated immunity

Question 1

What are some measurable characteristics of T cell responses?

Answer 1

• activation marker expression
• proliferation responses (clonal expansion)
• cytokine secretion
• cytotoxicity to target cells (CD8)

Question 2

Activation marker expression: Flow Cytometry

Answer 2

• characterizes individual cells
• pass cells through laser, detect and count them
• cells of interest will be fluorescently labelled and excited by laser
• sorted into specific tubes
• put into plots

Question 3

Proliferation responses: clonal expansion

Answer 3

• Antigen presented to T cells via MHC, needs 3 signals
• T cells proliferate and clonally expand
• Maturing T and B cells= immunoblasts
• culture with radioactive component (thymidine) and measure uptake = Count number of proliferating cells

Question 4

Mitogens

Answer 4

• plant derived lectins that bind CHO residues on cell surface glycoproteins and cause lymphocytes to divide

Question 5

Advances in proliferation measurements

Answer 5

radioactivity now replaced with CFSE fluorescent compound. CFSE brightness can be measured with flow cytometry

• huge peak= 0 proliferating cells
• smaller peaks= proliferating cells

Question 6

Cytokine secretion measurement

Answer 6

• Use ELISA based assays for cytokines. Culture with mitogens or cytokines
• ELISpot assay- shows IFN-gamma release response of peripheral blood mononuclear cells to a target antigen

Question 7

Cytotoxicity to target cells detection

Answer 7

Cytotoxicity assays: measure the T cell mediated killing of a target cell population
Antigen-specific and MHC I restricted

• combine radio labeled target cells and the effector T cell population (must have same MHCI so same patient)
• culture two cell populations together
• if antigen is presented on target cell, T cell will destroy and a release of radioactivity will occur
  ** measure amount of radioactivity released to find out T cell cytotoxicity**

Question 8

Measure Cell mediated immunity in living

Answer 8

Delayed type hypersensitivity Testing (DTH)

• antigen injected into tissues
• Th1 cell recognizes, releases cytokines
• recruitment of phagocytes occurs
• LESION occurs

“TB testing”- response indicates that exposure has occurred before because memory T cell response