The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion
, Frank Liang 2, Joakim Karlsson 3,4, Ulf Yrlid 2, Jonas A. Nilsson 3,4,*,†, Ola Nilsson 1,† and Lisa M. Nilsson 3,4
Round 1
Reviewer 1 Report
The manuscript is easy to comprehend and clearly written. The subject of the manuscript is pertinent and valuable. The experiments are well crafted and important controls are included. However, some more evidence is needed to support the conclusion drawn.
Major comments
1) Conclusions drawn from figure 6 need more evidence.
Authors state: We speculated that the poor take-rate and/or growth not only was due to the low 242 proliferation rate of SINETs, but that they may be immunologically suppressed by TILs residing within the tumours.
More evidence is needed to prove that the tumors are immunologically suppressed by TILs residing within the tumours.
2) Line 268: Authors state: This means that ACT could be a feasible alternative for the treatment of patients with metastatic SINETs.
Experimental evidence is needed to conclude the above statement.
Author Response
The manuscript is easy to comprehend and clearly written. The subject of the manuscript is pertinent and valuable. The experiments are well crafted and important controls are included. However, some more evidence is needed to support the conclusion drawn.
We thank the reviewer for critically assessing our manuscript. It has enabled us to revise conclusions of the study.
Major comments
1) Conclusions drawn from figure 6 need more evidence.
Authors state: We speculated that the poor take-rate and/or growth not only was due to the low 242 proliferation rate of SINETs, but that they may be immunologically suppressed by TILs residing within the tumours.
More evidence is needed to prove that the tumors are immunologically suppressed by TILs residing within the tumours.
We have moved Figure 6 to the supplementary section, narrowed the abstract and improved the statement on previous line 231 per reviewer #2’s suggestions. The statement that the tumors may be immunologically suppressed by TILs residing within the tumors has been removed. It served only to introduce a new section but we agree that we could use a more neutral statement so we changed it.
2) Line 268: Authors state: This means that ACT could be a feasible alternative for the treatment of patients with metastatic SINETs.
Experimental evidence is needed to conclude the above statement.
Since the PDXv2 did not work we were not able to provide more experimental evidence about the feasibility of ACT for patients. We have provided in vitro data though, which could support initiation of a clinical trial. We have now removed the sentence and thank the reviewer for helping us improving our study.
Reviewer 2 Report
The manuscript was aimed to examine the microenvironment of small neuroendocrine tumors (SNET) and to determine whether the various subsets of tumor-infiltrating lymphocytes (TILs) express PD-1 molecule and therefore might be considered as a potential target for the therapy of immune checkpoint inhibitors.
In general, the manuscript is very well written and represents a deep analysis of the mutational status of SNETs, and their immune microenvironment. Important, the authors immunophenotyped the TIL's subsets and demonstrated their potent functional activity after the expansion.
I have the following questions and suggestions regarding this manuscript:
Since the xenografts experiments failed, all of them should be included in the Supplementary section (Figure 6) and removed from the abstract (lines 29-31). The formulation "seemly viable cells" (line 231) is also not convincing.
Figure 1 shown in the text (lines 81, 82, and 89) fits with y Figure 2 and its legend. Similarly, Figure 2 in the text ( lines 91, 92, 95, and 97) fits with Figure 1 and its legend.
Did the authors have a chance to examine the expression of PDL-1 on the tumors cells?
How do the authors explain the absence of colocalization with T-lymphocyte markers and granzyme B (line 130)?
Figure? (line 184) should be corrected.
What are the cells that are shown as the double-positive for CD4 and 8? (line 190 and Figure 5C)?
Author Response
The manuscript was aimed to examine the microenvironment of small neuroendocrine tumors (SNET) and to determine whether the various subsets of tumor-infiltrating lymphocytes (TILs) express PD-1 molecule and therefore might be considered as a potential target for the therapy of immune checkpoint inhibitors.
In general, the manuscript is very well written and represents a deep analysis of the mutational status of SNETs, and their immune microenvironment. Important, the authors immunophenotyped the TIL's subsets and demonstrated their potent functional activity after the expansion.
We thank the reviewer for finding merit of our study and for the comments.
Since the xenografts experiments failed, all of them should be included in the Supplementary section (Figure 6) and removed from the abstract (lines 29-31). The formulation "seemly viable cells" (line 231) is also not convincing.
We thank the reviewer for this good suggestion. We have moved figure 6 to the supplementary section, removed lines 29-31 of the abstract and rephrased the statement on line 231.
Figure 1 shown in the text (lines 81, 82, and 89) fits with y Figure 2 and its legend. Similarly, Figure 2 in the text ( lines 91, 92, 95, and 97) fits with Figure 1 and its legend.
We apologize for this mistake and thank the reviewer for finding this error. We have now swapped the order of the figures to match the text.
Did the authors have a chance to examine the expression of PDL-1 on the tumors cells?
We have only studied the expression of PD-L1 on the tumor cells included in the manuscript (figure 1; 14 % positivity) using the FDA-approved methodology. Previous reports have studied PD-L1 in somewhat larger cohorts with proportions of PD-L1 positive tumors ranging from 0-39% (Lamarcka et al. 2018, da Silva et al. 2018, Cives et al. 2019).
How do the authors explain the absence of colocalization with T-lymphocyte markers and granzyme B (line 130)?
We meant to state that the T lymphocyte aggregations did not express granzyme to a larger extent compared to other T lymphocytes. We have now clarified this in the text.
Figure? (line 184) should be corrected.
We apologize for this mistake and thank the reviewer for finding this error. We have now deleted this word since it was redundant. The number of TILs are mentioned in the text.
What are the cells that are shown as the double-positive for CD4 and 8? (line 190 and Figure 5C)?
We have not investigated these in detail in SINET but they are known to be present in many different tumor-types such as melanoma (eg Desfrançois et al., 2010) and liver cancer (Zheng et al., 2020), or auto-immune disease (eg Wang et al., 2021). ScSeq suggest that they be more similar to CD8 cells but also have some CD4 features (Parnot et al., 2020). Interestingly, some studies indicate that they can also be tumor-reactive (Desfrançois et al., 2010).
Round 2
Reviewer 1 Report
The authors have made successful revisions addressing all the comments.
The article addresses vital questions of the field and is scientifically sound.
