Does anyone have the experiences on MLR experiment to measure the proliferation of naive CD4 T cells?
My experiment went like this: I isolated PBMCs from blood of donor A and separated naive CD4 T cells as responders (R) by MACS kit. The responders were then stained with CFSE (final conc. was 5 uM) to measure their proliferation. Another PBMCs isolated from blood of donor B were irradiated at the dosage 5000 cGy as stimulators (S). I cocultured these cells at different R-to-S ratio of 1 to 2, 1to 5, 1 to 10, and 1 to 20. The starting number of R was 5*10^4. The cells were cultured for 3 days and 7 days. If cultured for 7 days, half volume of the medium was removed and fresh cRPMI was supplemented carefully on day 4. The results were analyzed by FACS. However, I could not see the CFSE dilution after 3 and 7 days. There was a positive control that the R was stimulated with anti-CD3/anti-CD28 Abs-coated beads. I could see the strong proliferation from the positive control, but no CFSE dilution was detected in the MLR. Can anyone have suggestions for me? Thanks in advance!
Dear Chun-Hao Lu
I assume that as your T cells in positive control have proliferated, the problem must be something with the PBMC (simulator).
Therefore, at first, just make sure that the PBMCs are viable and active, since you might have put the cells to death while performing the irradiation or other preparative procedures.
Secondly, PBMCs mainly consist of monocytes and lymphocytes, while dendritic cells _which are majorly responsible for the stimulation of naïve T cells_ are rare, being only 1 – 2% of PBMCs. In spite of the fact that you are practicing an allogenic MLR evaluating T cells' MHC-independent unspecific response, unstimulated naïve T cells are not potent enough to respond and proliferate without a primary stimulation. I propose that you may gain a better result by providing a minimal level of an adjuvant stimulant (e.g. anti-CD3/anti-CD28 Abs-coated beads) for your MLR groups and check if you see the expected variation among the experimental groups (no variation could mean the results are merely an effect of the adjuvant stimulant activity).
On the other hand you can try a [at least] partial purification of PBMC, removing the CD3+ T lymphocytes (using MACS, nylon wool, …) that comprise 70-85% of the pool and may interfere in the leukocyte reaction. In this way, in each well, you will have a higher ratio of real stimulatory cells.
Wish you all the best in your research,
Atefeh
Atefeh Yaftiyan Dear Atefeh Yaftiyan, thank you for the awesome suggestions. I am also thinking about the low proportion of the cells which could provide activation signals to CD4 T lymphocytes. I can try the way you mentioned to add a minimal level of additional stimulants for the MLR groups, and also try to increase the proportion of stimulatory cells. Thank you!
Any time! I hope these strategies would help you!
By the way, as you know, activation of naive T cells by adding the adjuvant stimulator could be done after the T cell isolation (before the MLR assay) and/or while carrying out the co-culture (directly in the MLR well). I think the results might be different, and you'd better choose the best timing according to the aim of your study.
Best regards,
Atefeh
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