Peer review of: Reversal of pre-existing NGFR-driven tumor and immune therapy resistance (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author); expert on mouse models for melanoma and signaling: 

This paper by Boshuizen and colleagues demonstrates a convincing correlation of high 
CD271/NGFR expression in melanoma cells with (MART-1) T cell resistance and resistance to other 
melanoma drugs. High CD271 expression has been associated with resistance to BRAF/MEK 
inhibition before. Likewise, a correlation of high CD271 expression with reduced T cell infiltration 
has previously been shown. Of note, the point crucial for the present study – inhibition of the 
NGFRhigh state by blocking specifically CD271 (or e.g. by engineering CD271-specific T-cells) – 
has not been addressed and it remains open why CD271/NGFRhigh cells are resistant to T cells. 

Specific points: 

1) For the key functional experiments, the authors use HSP90i, which is not at all a specific NGFR 
inhibitor. Downregulation of NGFR upon HSP90i might be merely correlative. Specific NGFR 
inhibition, at (or during acquisition of) the TR state in vitro and in vivo, has to be performed to 
assess whether inhibition of the CD271high state indeed prevents relapse, i.e. whether resistance 
is indeed NFGR driven, as claimed in the title. 

2) In Figure 1, the authors present a nice method for how to generate TR melanoma cells. 
However, the TR state is ill defined and it is unclear why the authors picked CD271/NGFR for their 
further studies (as it stands, the authors selected NGFR mainly based on literature; for this, the 
work shown in Fig1a-e would not have been necessary). What is the molecular signature of the TR 
state obtained in the experiments of Figure 1 and how does it compare to the signatures shown in 
Figure 4? 

3) As shown by others before, resistance formation involves various cellular states rather than just 
one particular, homogeneous cell state. TR state analysis should optimally be performed at the 
single cell level. Rather than showing overall NGFR expression (Fig. 1 f), the numbers of 
CD271/NGFRhigh cells as compared to CD271/NGFRlow cells have to be determined upon T cell 
resistance. Likewise, how many cells within the clones shown in Figure 3c are CD271 positive? 

4) The choice of the cell lines used for a particular set of experiments is not consistent (e.g. Figure 
1c vs 1h; different cell lines used for the different panels of Figure 2). Each key experiment should 
be repeated with at least two cell lines. 

5) Why are CD271/NGFRhigh cells resistant to T cells? The mechanisms underlying resistance 
formation have to be addressed. 
Do CD271/NGFRlow cells play a role in this as well (e.g. by interaction with CD271/NGFRhigh 
cells)? 

6) In Figure 6b, does shNGFR already exhibit an effect in non-TR cells? This control experiment 
needs to be included. 

7) Figure 5: what about NGFR expression in biopsies from resistant patients? 

8) Minor point: in general, the labelling of figure axes could be improved; as an example, in Figure 
3b, it's not clear immediately that NGFR MFI is measured, etc. 

Reviewer #2 (Remarks to the Author); expert on PDX models, melanoma and immunotherapy: 

This is a nice manuscript with some interesting findings. I have only three major concerns: 

1. The immunotherapy experiments are not conducted with autologous tumor and TILs. Although 
HLA matched it is therefore risky that some of the effects are not general but only pertaining to 
allogeneic responses or regulation of antigen processing. The authors should consider repeating 
some of the data using paired autologous TILs and tumor cells. 
2. The extended data 5 suggest that there might be an off target problem with the shRNA 
targeting NGFR. Please repeat the experiment with CRISPR/Cas9 deletion of NGFR. 
3. Although the data are suggestive of the NGFR is necessary, which can be strengthened by 
CRISPR, is NGFR sufficient to drive resistance? If NGFR is transgenically overexpressed, will this 
result in resistance?