Steven Rosenberg and colleagues from @ NIH ask why # Tregs build up in tumor tissues and if it has anything to do with # cancer # antigen recognition in a recent paper published in @ SciImmunology. But how can # Foxp3 + Tregs in humans be studied? There isn’t a trustworthy surface marker yet.David Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
1. First, fix / perm T cells were separated from non-Tregs using Foxp3 + expression from # tumor samples, and the sorted samples were sent to @ AdaptiveBiotech( this is how everything looks these days ) for deep # TCR # sequencing and alpha / beta TCR # pairing.David Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
2. 2. They discovered variations between the use of Treg-TCRs and non-treg TCR usage. Okay, so what? Unless one is aware of what these # TCR recognize, # Sequencing or even TCR pairing by themselves are worthless. Many labs don’t even bother to take action, but # Rosenberg &# 39’s people did.David Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
3. 3. They did this by transfecting individual top # Foxp3 + # Treg TCR pairs into # autologous T cells and evaluating their # reactivity to autologously tumor samples. In autologous tumor samples, some # TCR specifically identified something. They reasoned that it had to # antigen. pic. fbj8esBJSq on twitter.comDavid Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
4.. 4. They used whole -# exome and RNA sequencing( RNA – seq ) to find somatic mutations and create mutant peptide pools in order to identify antigen recognized by Treg TCRs. Indeed, mutant # annexin A1 and # CCL5 were identified by a Treg TCR from one patient and another from another patient. pic. / FRiibvwYTX on TwitterDavid Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
5. 5. But now for the surprise. All of these # Treg # TCRs failed to identify samples of autologous tumors. It’s interesting to note that the authors found that tumor cells do not express CCL5 at the # RNA level for # annexin A1 and that # CCL5 expression was too low in tumor samples.David Usharauli(@ 3DiMMUNE ) January 14, 2019 & mdash
In essence, Treg TCR was unable to identify natural tumor cells but could identify lab # synthesized # mutant peptides from patient samples. The authors thus found themselves back at square one. # Tregs enter tumor tissues, but our current methods do not enable us to determine what Ag they detect there.— David Usharauli on January 14, 2019(@ 3DiMMUNE )