Dear all,
I am wondering if you have ever used (HLA-transfected) HEK293T cells in co-culture with HLA mismatched PBMC to study alloimmune responses? Do you think co-stimulation in addition to IL-2 (like CD28 activation) might be necessary to induce alloreactive T cell activation?
Thank you so much in advance!
Kind regards,
Kevin
HEK293T cells have been used successfully to present alloantigens and activate mismatched T cells when transfected with appropriate HLA alleles. They lack normal costimulatory capacity though.
Adding costimulatory ligands like CD80, CD86, CD40 etc via transfection or bead-immobilization can enhance allostimulation significantly. CD28 activation alone may help. Beyond CD28, consider 4-1BB, OX40, and ICOS pathway activation for optimal T cell stimulation and survival.
Cytokine support with IL-2 is good, but IL-7 and IL-15 could also promote better T cell expansion.
Make sure to match the optimal HLA allele combinations between stimulators and responders.
Also, Ratio of aAPCs to T cells matters - start with 1:10 to 1:100 then optimize. May need to sort/enrich for memory T cells to see best alloreactivity.
Thank you for your input, Salah A. Alshehade - very helpful! Could you please tell me which of your proposed approaches worked best in your hands using HEK293T and PBMC for investigating alloimmune responses? Thanks, Kevin
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