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All Immunology questions

Question 17 of 20 Advanced Practical

How would you separate effects on T-cell proliferation from effects on effector function?

Short answer

Label cells with a division-tracking dye such as CFSE, so each division halves the fluorescence, and measure cytokines or killing in the same cells by division number. A treatment that blocks function without blocking division, or the reverse, then shows up directly instead of being mixed into one readout.

Why

A drop in total IFN-gamma could mean fewer cells, less cytokine per cell, or both. Quah and Parish describe how CFSE binds long-lived proteins inside the cell, and how each daughter receives half the dye, so peaks on a histogram mark successive divisions, up to about eight. Combining that with intracellular cytokine staining, degranulation markers such as CD107a, or a killing assay lets the analyst ask whether cells that have divided the same number of times still function.

Precursor frequency and division index summarize proliferation separately from the fraction of producers per division. Viable cell counts with counting beads keep death from masquerading as reduced division.

How to work it through

  1. Label purified T cells or PBMC with a titrated division dye and confirm a tight undivided peak at day zero.
  2. Stimulate with a defined signal and include unstimulated and positive controls, with and without the test agent.
  3. Harvest at several days, restimulate briefly with a secretion blocker for cytokine staining, and add a viability dye and counting beads.
  4. Gate live singlet T cells, then analyse cytokine and degranulation markers within each division peak.
  5. Report proliferation indices and per-division function separately.

An example

An inhibitor lowers IFN-gamma in culture supernatant by 60 percent at day 4. CFSE shows the same division profile as vehicle, with most cells in divisions 3 to 5. Within each division, the fraction of IFN-gamma positive CD8 T cells falls from about 40 percent to 15 percent.

The drug suppresses effector function rather than expansion. A second compound gives the opposite pattern, with fewer divided cells but normal cytokine per division.

Where the answer stops holding

CFSE is mildly toxic at high concentrations and needs titration. Its broad emission spills into neighboring channels. Very rapid early divisions or dye transfer can blur the peaks.

Brief restimulation changes the cells being measured, and in vitro proliferation may not predict expansion in a patient.

A strong answer covers

  • Division dye halves with each division
  • Function measured within each division peak
  • Viability dye and counting beads prevent confusion with death
  • Separate indices for proliferation and per-cell function
  • Controls and titration of the dye

Common mistakes

  • Inferring proliferation from total cytokine in supernatant
  • Using too much CFSE so the dye itself suppresses division
  • Ignoring cell death when interpreting fewer divided cells