IL-27 maintains cytotoxic Ly6C + γδ T cells that arise from immature precursors

  1. Robert Wiesheu
  2. Sarah C. Edwards
  3. Ann Hedley
  4. Marie Tosolini
  5. Marcelo Gregorio Filho Fares da Silva
  6. Nital Sumaria
  7. Yasmin Optaczy
  8. David G. Hill
  9. Alan J. Hayes
  10. Jodie Hay
  11. Anna Kilbey
  12. Alison M. Michie
  13. Gerard J. Graham
  14. Anand Manoharan
  15. Christina Halsey
  16. Gareth W. Jones
  17. Karen Blyth
  18. Jean-Jacques Fournie
  19. Daniel J. Pennington
  20. Vasileios Bekiaris
  21. Seth B. Coffelt

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Abstract

In mice, γδ T cells that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage in the thymus, and in the periphery, these cells play a critical role in host defence and anti-tumor immunity. Unlike αβ T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that immature CD27 + Ly6C — cells convert into mature CD27 + Ly6C + cells, and these mature cells control cancer progression while the immature cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27 + Ly6C + cells and human Vδ2 + cells, while IL-27 is dispensable for mouse CD27 + Ly6C — cells and human Vδ1 + cells. These data reveal increased complexity within IFNγ-producing γδ T cells, comprising of immature and terminally differentiated subsets, that offer new insights into unconventional T cell biology.

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    Referee #3

    Evidence, reproducibility and clarity

    Mouse models have contributed significantly to the description of the anti-tumor and the tumor-promoting activity of gd-T cells, which (in mouse) can be divided into an IFNg and an IL-17-producing subgroup based on their CD27 expression. The study describes Ly6C+ and Ly6C cells as "new" subsets of IFNg-producing murine CD27+ gd T cells and proposes Ly6C+ cells as mouse counterparts to human Vd2 T cells. The use of Ly6C expression as a potential marker for functionally distinct CD27+gd T cell subpopulations is based on single-cell transcriptomics of murine lung gd T cells and was validated by flow cytometry of surface antigens and assay of cells for proliferation/cell death after CD3/CD28 stimulation, killing of tumor cells in vitro and in vivo and analysis of the conversion of Ly6C- to Ly6C+ cells and vice versa. Ly6C cells were defined as "immature" and Ly6C cells as "mature". Ly6C+ cells show increased tumor cell kill in vitro and better IFNg production in vitro. In a tumor model, adoptively transferred ex vivo expanded Ly6C+ but not Ly6C- cells prolonged survival. Based on the data shown in Fig. 6, it is postulated that IL-27 is important for the maintenance of LyC6+ cells in vitro and in vivo and for the increased IFN-γ production of Ly6C+-derived cells. It also modulates killing of tumor cells in vitro. Finally, the effects of IL-27 on human Vd1 and Vd2 T cells are analyzed and Vg9Vd2 T cells are postulated as a human counterpart to Ly6C+ gd-T cells. At times, I found the work hard to read, and hope that as someone who doesn't work with mouse gd-T cells, I fully understood the work.

    I agree with the characterization of LyC6+ cells as a "more mature/differentiated" subset of IFNg-producing CD27 gd T cells. My doubts mainly concern the interpretation of the transcriptomic data (Fig. 1) and the data shown in Fig.6 and 7 (erroneously referred to as Fig. 8) which described the effects of IL-27 and the postulated similarity of LyC6+ cells and human Vg9Vd2 T cells.

    I find it confusing to talk about Ly6 expression of Ly6+ and Ly6- cells. Perhaps it would be better to talk about these expanded cells Ly6+ derived and Ly6- derived cells (or something similar).

    Fig. 1 While the identification of clusters 0 and 1 helps to identify LyC6 expression as a marker for CD27+ gd T cell differentiation, I do not understand how the gene signatures and the expression patterns in human lymphocyte subgroups "highlight the high similarity between gd T cells between species", since Fig. 1 E and 1 F also show high similarities between both clusters and non-gd T cells. In fact, the greatest similarities exist between cluster-0 cells and NK cells, followed by mature Vd1 and finally by total Vg9Vd2 T cells and CD8 cells. Naïve Vd1 and naïve (ab T) lymphocytes cells show mainly similarities to cluster 1 cells.

    Fig. 2 Please name the organ from which the cells were taken. Please also present in the extended data figures a representative staining with a complete gating strategy, starting with and live gating, to give a wider audience an idea of how the subpopulations of CD27+gd T cells were identified. I also wonder if the expression levels (not the frequency) of CD27 and Ly6C are the same for different organs.

    Fig. 3. It appears that Ly6C+ -derived cells show an effect against the E0771 tumor. In the Kaplan Meyer plot these were compared only with the PBS control. Please show the P values for Ly6C+ and Ly6C - derived cells.

    Fig. 4 Please indicate the absolute number of gd-T cells and CD27+gd-T cells in organs and tumors.

    Fig. 5 Please indicate the absolute number of gd-T cells and CD27+gd-T cells in organs and tumors.

    Fig. 6A-C What is meant by control? No cytokines or IL-2 and IL-15 as in the other experiments plus the indicated cytokines. Please specify.

    Fig. 6D-G Please indicate the absolute cell counts. Please give also cell counts and frequencies of CD27-Ly6C+ and Ly6C- cells directly taken ex vivo for WT and IL-27R-/-.

    Figs. 6E and G and extended data Fig. 4. Was the significance of the differences between WT and IL-27-/- also tested for Ly6C-derived cells, as it was done for L6C+-derived cells. At least in some cases, I would be surprised if such differences were not observed (e.g. Extended data Fig. 4A for NKG2A expression in LN and lung). In addition, in some cases, the lack of differences of for Ly6C- cells between WT and IL-27R-/- cells may be (in part) reflect to their low abundance, which makes it difficult to find statistically significant differences in Il-27 dependence.

    Fig. 6F is aimed to show that there is no statistically significant difference in the Ly6C expression of Ly6C- derived cells between IL-27R/- and WT animals, while there is such a difference for the Ly6C+ derived cells. To me, it looks like the lack of significance of differences between the Ly6C-derived WT and IL-27R-/- cells is due to a single outlier. Especially, since only three animals were analyzed compared to five or more in most other experiments I am not convinced of the biological (non-) significance of these differences.

    Fig. 6E is indeed "incongruent" since it contradicts the claim that IL-27 supports the anti-tumor response of CD27+ gdT cells, as stated in the ONE-SENTENCE-SUMMARY. To claim a positive effect of IL-27 on tumor control by CD27+ gdT cells, an experiment as shown in Fig. 3 with gd T cells from IL-27R/- and WT mice or with cells expanded with or without IL-27 is required. Another question concerns the purity of cells after CD3/CD28 stimulation. Could it be that contaminating cells (e.g. NK cells) contribute to killing and that IL-27 effects are due to such cells?

    Fig. 7 (incorrectly referred to as 8) and extended data Fig. 5. Does the MFI indicate the MFI of the positively gated cells or the alll cells? Please specify. Please also indicate the proportion of Vd1+ and Vd2+ cells before and after expansion for all donors. Fig. 5 A shows an approximately 30% inhibitory effect of IL-27 on Vd1 expansion and a 70-75% reduction in expansion of Vd2 cells. This contradicts the postulated (functional) Vg9Vd2 T cell homology with Ly6C+ cells, since in mice ( Fig. 6) IL-27 reduces the expansion of Ly6C cells and accelerates the expansion of Ly6C+ cells. Another problem could be the comparison of the mouse subgroups with the total Vd1 and Vg9Vd2 T cells. In view of the data in Fig. 1, it would be better to compare mature and naïve cells from both populations.

    Mat. and Meth. been mentions FTOC experiments but I did not find them?

    Mat. and Meth. also mentions that WT and Tcrb/- mice were used as a source of CD27+Ly6C- and CD27+Ly6C+ cells in the in vivo expansion experiment. I didn't find any mention of the use of Tcrb/- mice in the text of the illustrations. Please explain.

    Ly6C is encoded by the genes Ly6c1 and Ly6c2. Ly6c2 is the dominant expressed gene, but in some populations (CD4+ T cells), a significant proportion of Ly6C-positive cells express Ly6C1 https://doi.org/10.4049/immunohorizons.2100114. This should be mentioned in the text. An optional experiment would be to test the expression of mRNA for both genes in naïve and expanded gd-T cells.

    With respect to possible revisions. For the core of the paper description of the Ly6C (derived populations (Fig. 1-5) 1-2 months should be sufficient. More complicated is to address the issue of Il-27. Fig. 6. A good part of my concerns are on the evidence of the lack of an Il-27 response of Ly6C- derived cells. Putting less emphasis on this point and might be largely sufficient, since in my eyes it is not so central anyway. A problem is the statement on the importance of IL-27 in control of tumor by Ly6C derived cells. This would need in vivo experiments which may take much longer (-6 months?).

    Significance

    The work is certainly of interest to most immunologists working with mouse gd-T cells. The characterization of the Ly6C-defined subgroups of CD27+gd-T cells is plausible except for details and supported by the results. The data on IL-27 in Fig. 6 and extended Fig. 4A are less clear, in particular the lack of effect of IL-27 on Ly6C cells may have been overestimated sometimes. Also the importance of IL-27 for tumor control by gd-T cells in vivo has yet to proven. Both would broaden the audience, nd may increase translational/clinical relevance.

    The weakest part is the postulated similarity between human gd T cell groups and the two mouse gd T cell subgroups. The cell populations of the mouse could indeed have human counterparts, but this has yet to be shown. Given the strong species-specific differences between both species especially for unconventional T cells, it is also conceivable that non-gd T cells take over the functions of certain gd T cell populations.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript submitted by Wiesheu et al focuses on the intrinsic nature of murine gd T cells in tumor immune response. Authors used their previously published scRNA-seq data and using a refined strategy, they dissected the role of Ly6C subset-specific CD27+ gd T cells. The authors represented experimental evidence for the possible differentiation of CD27+Ly6C gd T cells and revealed the essential role of IL-27 for the maintenance of CD27+Ly6C+ gd T cells. Finally, the authors found parallels in cellular features of gd T-cell subsets between mice and humans. Overall, this study is well-designed and expands knowledge of gd T-cell biology. However, the authors are suggested to consider the following comments:

    Major:

    1. One of the major concerns is that the submitted manuscript is the extension of their previous work (Edwards et al 2022) published in J Exp Med. This previous work clearly delineated the differential role of PD-1 and TIM-3 using the scRNA-seq approach. Using the same scRNA-seq data, authors are now defining gd T-cell subsets within the CD27+ population. While providing the functional basis for Ly6C expression, the author shall also define the role of PD-1 or TIM3 in this context. Additionally, this suggestion is based on the literature reports Zhu et al, 2015 Nature Communications (https://doi.org/10.1038/ncomms7072) and Huang et al, 2019 J Exp Med (https://doi.org/10.1084/jem.20190173). It would be interesting to find out if IRF1 also plays a mechanistic role in gd T cells like CD8 T cells. So further experiments are needed in this context, which will complete the functional features of CD27+ gd T cells.
    2. As stated in the manuscript, Ly6C is a marker for the myeloid compartment in mice. Also, the differentiation (as authors described "conversion") phenomenon of monocytes based on the Ly6C marker is already reported by Mildner et al 2017 Immunity (https://doi.org/10.1016/j.immuni.2017.04.018). In the case of monocytes, Mildner et al showed that Ly6C+ monocytes differentiate into Ly6C- cells. But, in the current manuscripts, authors are proposing the opposite differentiation trajectory for gd T cells. Also, in the corresponding analysis with human scRNA-seq data (fig. 1E-F), are there no analogous expression of genes in the monocyte cluster. Could you please explain? Sorry, this is a bit unclear to me!
    3. I think, it is a bit strong statement that CD27+Ly6C- gd T-cell converts into CD27+Ly6C+ gd T cells. This statement needs to be supported and validated by more experiments:
      • (a) authors shall perform trajectory analysis in scRNA-seq data, which will, hopefully, support the conversion hypothesis;
      • (b) authors shall represent the purity and sorting strategy of flow cytometry experiments. For such experiments, it is very important to have the highest purity without any 'false negative' sorting!
      • (c) Usually, a conversion of one phenotype into another phenotype requires an intermediate stage. For example, Mildner et al 2017 Immunity reported three stages of monocytes (Ly6C+, Ly6Cint, Ly6C-). However, none of the flow cytometry plots in the current manuscript show any 'intermediate' population.
      • (d) check for protein expression of homing receptors like Ccr7, SELL, and S1pr1 to completely exclude that there is no 'homing'
      • (e) Can authors consider a third possibility that there is a loss/gain of Ly6C protein expression through molecule signal orchestrated by epigenetics mechanism? If yes, this possibility is worth testing!
      • (f) Last, but not least: One way to prove the conversion would be tagging Ly6C protein with a marker (e.g. GFP) to track its fate during the differentiation. Would it be possible?
    4. The statement in line 137 is not entirely convincing as Fig. 1C-D shows some overlap of Ly6C and CCR7 gene expression in opposing clusters. At least, there are no clear 'black-and-white' opposing expressing levels. Sorry, this is my observation. Is there any other way of representation to make it clearer?
    5. Authors have nicely shown the cytotoxic potential of CD27+Ly6C+ gd T cells in the in vivo setting. Would they observe the similar cytotoxic potential of expanded CD27+Ly6C+ gd T cells? Also, it would be great if memory CD8 T cells were added to the experimental set-up as a positive control, in addition to naïve CD8 as a negative control.

    Minor:

    1. Authors have used t-SNE representation. However, UMAP representation is preferred as UMAP preserves local and global structure in the data. Can authors show the similar segregation of clusters in UMAP representation?
    2. Authors are suggested to add a supplementary figure on Ly6C2 and CCR7 expression in CD27- gd T cells. This is just a supporting evidence.
    3. If I have understood the fig. 1E-F correctly, has the gene signatures mapped to the B-cell cluster as well, not only to naïve Vd1 and ab T cells?
    4. Line 281-283: I think, it is redundant and can be moved to supplement.
    5. Line 309: I am not sure if the authors are referring to Fig. 6F. It should be Fig. 6E, right?
    6. In all figures, labels need to be precise. For example, figure labels say "% Ly6C+ cells". I think, the authors want to represent "% Ly6C+ CD27+ gd cells", right? Similarly, for other protein markers.
    7. Fig. 3I needs to include a figure legend for color bars.

    Significance

    This study is a good example of single-cell genomic data can be used to find novel immune cell subsets and their functions. Such approaches can be combined with human immune cells. Thus, it brings direct significance and relevance to the basic understanding and translational approaches. However, such study needs in-depth examination of claims and observations.

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    Referee #1

    Evidence, reproducibility and clarity

    Wiesheu et al. studied the maturation and functional maintenance of IFNg-producing γδ T cells in a mouse tumor model and by using NGS methods. The study provides insights into underlying mechanisms in the differentiation from Ly6C- to Ly6C+ γδ cells and their anti-tumor function in tissues. The study identifies IL-27 as one key cytokine that maintains these cytotoxic Ly6C+ γδ T cells. They further compare transcriptomes of murine and human γδ T cells. The study is well performed, and provides new insights into the functional differentiation of gd T cells in the tumor response. I have few remarks as outlined below.

    Major comments:

    Comparison of murine and human γδ T cells: This is an important point and should be addressed in more detail. In Figure 1e-f, the transcriptomes of mouse cells are nicely mapped to human transcriptomes. I agree that there are similarities between naive Vδ1 T cells and αβ T cells, but in Figure 1f it appears that Vγ9Vδ2 T cells lie between the two clusters. Could the authors analyse this in more detail? It is well known that Vγ9Vδ2 T cells are more innate than other γδ T cell subsets (e.g. Vd1 cells). I suggest that this should also be taken into account in the comparison. How do these results relate to the in vitro assays on human γδ T cells in Figure 7? Second, do the authors see common pathways to IL-27 responsiveness of murine and human γδ T cells? Are the molecules described in Fig. 6H also upregulated in human γδ T cells? Similarities and differences could be better described/discussed.

    Survival of Ly6C+ γδ T cells and implications for functionality: In the adoptive transfer experiments, tumour-bearing mice receive repeated injections of Ly6C+ γδ T cells. Would the system work with one transfer to further test survival or survival supported by IL-27? Do the transferred cells remain functional or would they be exhausted?

    Minor comments:

    In Figure 2A the CCR7 staining looks very weak despite the use of FMO. In my opinion, an isotype control or a positive control for CCR7 (e.g. expression on ab T cells) should be included. The Figure 1f is poor in resolution.

    Significance

    The study provides new insights into the functional differentiation and maintenance of murine γδ T cells in the tumor response, and compares this to human γδ T cell subsets at steady-state. A special focus was set on Ly6C, which has been rarely addressed in the past (please see references within the manuscript). A more in depth comparison of murine and human γδ T cells would be beneficial to address a broad readership and an ongoing debate on the translation of findings in murine systems to human immunology.

  5. Version published to 10.1101/2023.10.26.564114v1 on bioRxiv