In new paper in @SciImmunology, Steven Rosenberg's group from @NIH asks why are #Tregs accumulate in tumor tissues and if it has something to do with #cancer #antigen recognition. But how to study #Foxp3+ Tregs in humans? No reliable surface marker exist yet.
— David Usharauli (@3DiMMUNE) January 14, 2019
1. First, they sorted fix/perm T cells from #tumor samples based on Foxp3+ expression to separate #Tregs from non-Tregs and sent the sorted samples to @AdaptiveBiotech (this is what all do now days looks like) for deep #TCR #sequencing and alpha/beta TCR #pairing.
— David Usharauli (@3DiMMUNE) January 14, 2019
2. They found differences between Treg-TCRs usage and non-Treg TCR usage. OK. Then what? #Sequencing or even TCR pairing by itself have no value unless one knows what these #TCR recognize. Many labs don't even bother to do anything about it, but #Rosenberg's people did.
— David Usharauli (@3DiMMUNE) January 14, 2019
3. To do it, they took top #Foxp3+ #Treg TCRs pairs and #transfected them individually into #autologous T cells and tested their #reactivity to autologous tumor samples. Some #TCR specifically recognized something in autologous tumor samples. They thought it must #antigen. pic.twitter.com/fbj8esBJSq
— David Usharauli (@3DiMMUNE) January 14, 2019
4. To identify antigen recognized by Treg TCRs, they used whole-#exome and RNA sequencing (RNA-seq) to detect somatic mutations and make mutant peptide pool. Indeed one Treg TCR from one patient and another Treg TCR from another patient recognized mutant #annexin A1 and #CCL5. pic.twitter.com/FRiibvwYTX
— David Usharauli (@3DiMMUNE) January 14, 2019
5. But here is the twist. None of these #Treg #TCRs recognized autologous tumor samples. Interestingly, for #annexin A1 the authors suggested its expression was too low in tumor samples and for #CCL5 they found that tumor cells actually do not express CCL5 at #RNA level.
— David Usharauli (@3DiMMUNE) January 14, 2019
Basically, Treg TCR could recognize lab #synthesized #mutant peptides from patient samples but not natural #tumor cells. So, the authors ended up back in square one. #Tregs go to tumor tissues but the methods we have right now does not allow us to know what Ag they see it there.— David Usharauli (@3DiMMUNE) January 14, 2019
