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PREreview of Acquisition of an immunosuppressive microenvironment after CAR-T therapy drives T-cell dysfunction and resistance
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Brief summary of the study - a sentence summarizing the study and general comments that apply across the full paper
By investigating B-ALL patients before and after CAR T cell treatment using flow cytometry, hematology analyses and scRNA-seq, the authors give new insights into the tumor microenvironment changes induced by CAR T treatment, notably the expansion of the myeloid and myeloid-derived suppressor cell compartment.
Major comments - Comments on the validity or strength of the methodology, experiments and analyses, strength of the conclusions
The patient analysis reporting is very confusing throughout the manuscript. For instance, in the first analysis, peripheral blood from 15 patients who received CAR T treatment is used for flow cytometry and/or hematologic assays. In STable1, 49 patients are annotated. 6 of these were annotated for flow cytometry AND hematology. Which are the other 9 patients? (see also comments in manuscript below) Please clarify why patients were used only for some, not all of the analyses (e.g. in the case for flow cytometry).
scRNA-seq data from 3 patients is not sufficient to draw meaningful conclusions. Although some hypotheses from this data is validated using other assays, the study would greatly benefit from adding more patients to this cohort, or alternatively phrasing conclusions more carefully.
Minor comments - Clarifications to statements in the text, interpretation of the results, presentation of the data/figures
Please add an abbreviation list
In The statistical analysis section in the supplementary file, ANOVA statistics, the post hoc test was not mentioned, the significant p-value was not identified
Conflicts of interest of reviewers
No conflicts of interest
Inline commenting section
Please add comments on the preprint below via comments. You can add comments on the full paper, sections or only individual fragments. Any comments added here will be reviewed for inclusion in the public review section if relevant, but will not be posted publicly in any way that can identify the commenter for individual comments.
Introduction section: “However, the interaction between the BM microenvironment and CAR T cells in B-ALL has not been investigated”
Comments:
There have been studies on the TME and CAR Ts in B-ALL, e.g. https://pubmed.ncbi.nlm.nih.gov/37718626/
The manuscript would benefit from discussing these and similar previous findings.
Methods section “Detailed information is provided in the Supplementary Materials file (available on the Blood Web site).”
Comments:
the methods section including the statistical analysis section is one of the chief sections of research articles so its details should be included in the main text not in the supplementary files. please revise and correct
Results section “To assess how CAR T cells influence the kinetics of the immune reconstitution following lymphodepletion, we analyzed CAR T cells and total CD3+ T cells by flow cytometry in parallel to common hematology laboratory parameters in the peripheral blood (PB) of 15 patients with B-ALL. Patient characteristics are reported in STable 1. CAR T-cell engraftment was detected with a median time to maximal expansion of 14 days (range 10-28 days) and a median peak expansion of 56 cells/mmc (range, 2-887), followed by a decrease in the third and fourth weeks post-infusion. A similar dynamic of expansion and contraction was observed in endogenous CD3+ T cells, with a median peak expansion 836 cells/mmc (range, 68-4395) at day 21 (range, 7-28, Figure 1A). ”
Comments:
1. Statistical Methodology Issues:
- No justification is provided for the sample size of 15 patients
- No statistical test is reported for comparing CAR T-cell and CD3+ T cell expansion kinetics
- The use of median and range is appropriate for non-normally distributed data, but no test for normality is mentioned
2. Missing Information:
- No mention of how missing data points were handled
- Sampling frequency and timing not clearly specified
- No discussion of potential confounding factors
Results section “To assess how CAR T cells influence the kinetics of the immune reconstitution following lymphodepletion, we analyzed CAR T cells and total CD3+ T cells by flow cytometry in parallel to common hematology laboratory parameters in the peripheral blood (PB) of 15 patients with B-ALL. Patient characteristics are reported in STable 1”
Comments:
In supplementary table 1, there are only 6 cases that underwent Hematology and flow cytometry analysis (not 14)
Please specify which "common hematology laboratory parameters" were acquired and include them in the methods section in the supplementals.
there are 49 patients in the table please specify the patient group (STable 1).
Results section “was confirmed in 21 patients treated with CARCIK-CD19 (Figure 1E and SFigure 1A-B). At day 28, a significant decrease in ALC and AMC was observed compared to the peak of expansion (Figure 1F, n=32 patients)”
Comments:
please explain the basis of patient selection (highlighted numbers) for each investigation
Figure 2.
Comments:
With regards to panel B - Why was the gating strategy not kept consistent between BM pre- and post-infusion samples? I.e. why was a CD45-low SSClow population included in the pre-infusion, but not the post-infusion samples? Also, was a live/dead stain (f.e. DAPI) used as done for the CAR T-cell product?
With regards to panels E and F - is it surprising that the CAR CD8 cluster (dark blue?) is only partially enriched in the post-infusion samples? Shouldn't all of it light up as red and not be present at all in the pre-infusion samples?
Do the authors have any sense of where those different immune subsets reside in the bone marrow? Do they co-localize with different BM niche cell types (vascular, stromal etc.).
Also with regards to the actual B-ALL cancer cells?
Result section “The identified 11 subpopulations were visualized using UMAP (Figure 2E).”
Comments:
Please identify the abbreviation as Uniform Manifold Approximation and Projection (UMAP)
Figure 3.
Comments:
Related to panel G - which cell types make CCL2, the ligand to recruit CCR2+ monocytes in this context?
Figure 4.
Comments:
Why not add the column of CAR T cells in panel D to the rest, so everything is normalized the same way and directly comparable?
the volcano plot in panel E for CAR T cells looks a bit unusual with its leftmost tail - which genes come up in said tail? These are data from several patients i.e. replicates, correct? And if pre-filtering and the like has been done according to standards, might an MA plot be a better representation of the data in this case?
Result section “The generalized increase in HIF1α, TGFB1, and TGFBR2 expression (Figure 5C) was validated by digital droplet (dd)-PCR in purified CD3+ and CD3- BM cell subsets (SFigure11A)The generalized increase in HIF1α, TGFB1, and TGFBR2 expression (Figure 5C) was validated by digital droplet (dd)-PCR in purified CD3+ and CD3- BM cell subsets (SFigure11A)”
Comments:
In SFigure11A, there is no significant increase for HIF1a or VEGFA, only TGFB1. TGFBR2 was not investigated in SFigure11A
Figure 5
Comments
I wonder if smth like a circos plot might a better way to visualize receptor:ligand analysis results instead or in addition to the presented heatmaps?
Line “… are available into the ArrayExpress collection in BioStudies with the accession number “E-MTAB-14549” (https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14549).”
Comments
No access
Competing interests
The authors declare that they have no competing interests.