Global profiling of human blood ILC subtypes reveals that NK cells produce homeostatic cytokine amphiregulin and sheds light on HIV-1 pathogenesis
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
The interrelatedness of human blood innate lymphoid cell (ILC) subsets, and how they are perturbed by HIV-1, remains unclear. Transcriptional and chromatin profiling separated blood ILCs into ILC2s, ILCPs, one cluster that included CD56 dim and CD56 − NK cells, and CD56 hi NK cells that have features of both CD56 dim/– NK cells and ILCs. In contrast to mice, human NK cells expressed tissue repair protein amphiregulin (AREG), with greater production by CD56 hi NK cells than by ILCs. AREG was induced by TCF7/WNT signaling, IL-2, or IL-15, but not by inflammatory cytokines, and was inhibited by TGFB1, a cytokine elevated in people living with HIV-1. NK cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. In people living with HIV-1, AREG + NK cell percentage correlated with numbers of ILCs and CD4 + T cells, and correlated inversely with inflammatory cytokine IL-6. RNA-Seq showed increased antiviral gene expression in all ILC subsets from people who were HIV-1 viremic, and increased expression of anti-inflammatory gene MYDGF in CD56 hi NK cells from elite controllers. Functionally-defective CD56 − NK cells were increased in people living with HIV-1 in inverse correlation with CD56 dim NK cells, ILCs, and CD4 + T cells. Experiments with human PBMCs ex vivo and in humanized mice revealed that CD4 + T cells and their production of IL-2 prevented CD56 dim transition to CD56 − NK cells by activating mTOR, and, in people living with HIV-1, plasma IL-2 correlated with CD4 + T cell number but not with CD8 + T cells. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including homeostatic functions of NK cells discovered here.
Graphical Abstract
Article activity feed
-
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
To the Editor,
We thank the reviewers for their generous efforts on behalf of our manuscript. We were very pleased to see that reviewer #1 considered our manuscript a “very detailed, high quality paper” that “serves as an important resource for the field.” And that Reviewer #2 concurred, writing that our manuscript is “valuable because it generates large datasets on the NK/ILC family from human blood that can be deposited in repositories”, and that it is of “special relevance to the HIV field because it examines viral infection effects on these subsets.”
We believe the revisions made in response to the reviewers suggestions have improved the presentation of …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
To the Editor,
We thank the reviewers for their generous efforts on behalf of our manuscript. We were very pleased to see that reviewer #1 considered our manuscript a “very detailed, high quality paper” that “serves as an important resource for the field.” And that Reviewer #2 concurred, writing that our manuscript is “valuable because it generates large datasets on the NK/ILC family from human blood that can be deposited in repositories”, and that it is of “special relevance to the HIV field because it examines viral infection effects on these subsets.”
We believe the revisions made in response to the reviewers suggestions have improved the presentation of our data. Our responses to each reviewer comment are listed below in bold font. References mentioned here are listed in a bibliography at the end of this document.Changes to the text are also highlighted in the manuscript.
1. Point-by-point description of the revisions
This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Wang and colleagues present a very detailed, high quality paper describing phenotyping, transcriptional, epigenetic and functional differences between NK cells and ILCs in human peripheral blood. They overlay these studies which descriptions of differences in these populations in healthy and HIV infected (viremia, ART-treated, controllers), extending a their previous 2020 study.
Overall, this paper serves as an important resource for the field. There are areas in which the manuscript could be modified to improve clarity and detail regarding rigor.
My comments below are all addressable and therefore I class them all as 'major'.
- The authors describe ILCs as being 'permanently deleted' in HIV infection. As written, this can be misinterpreted to suggest complete ablation of this cell subset. This is clearly not the case. Moreover, there looks to be some restoration of ILCs in ART-treated participants. I suggest revising text to quantify the level of cell loss or replace the word deleted with reduced. The reviewer is correct that ILCs are not completely ablated. In response to this important point we have clarified the text, as follows:
- As indicated on page 5, we have changed the text to, “ILCs are decreased in the blood and intestinal lamina propria in people living with HIV-1, even after viremia has been suppressed by antiviral therapy”.
- In the Results on page 13, we have changed the text to “ILCs were decreased in all subgroups of people living with HIV-1”.
__- The text on page 18 was changed to, “The percentage of ILCs in the Lin–CD56– population decreased from 37.5% in HIV-1– controls to 7.34% in people living with HIV-1 who are viremic; the reduction in ILCs was not fully restored by ART (24.38%) or in spontaneous controllers (18.16%)” __
- As indicated on page 26 in the Discussion, we have changed the text to “HIV-1 infection permanently reduces ILCs but not NK cells”.
- The HIV EC are not clearly discussed in the paper and are not distinguished from viremic controllers. If ILCs are permanently reduced in this group, what does this suggest for the role of this cell subset in HIV control? Plasticity between ILC and NK cells is described. Is this plasticity relevant at all for HIV control, elite or otherwise? We thank the reviewer for asking us to clarify these important points in the manuscript:
- __Though elite controllers suppress HIV-1 viremia to undetectable levels, ILC numbers are still decreased in these individuals. Additionally, we do not detect an inverse correlation between ILC numbers and viremia. Further, our previous publication showed that ILC reductions in HIV-1 infection correlate inversely with markers of systemic inflammation, for example sCD14 ____(Wang ____et al____, 2020b)____. Like other people living with HIV-1 infection, elite controllers have elevated microbial translocation, suggestive of disturbed gut homeostasis ____(Brenchley ____et al____, 2006)____. Some studies have even reported higher rates of cardiovascular disease in____ elite controllers, presumably as a result of higher levels of systemic inflammation____ (Crowell et al. 2015; Caetano et al. 2022)____.____ Taken together, these observations suggest that ILCs play no direct role in control of HIV-1 replication. These points are discussed on page 26-27. __
- __The fascinating question of plasticity between ILCs and NK cells is one we have explored extensively, both in our previous work and in the current manuscript. Plasticity has been reported between intraepithelial ILC1s and NK cells from tumor tissue, and between ILC3s and NK cells in tonsil ____(Moreno-Nieves ____et al____, 2021; Raykova ____et al____, 2017; Cortez ____et al____, 2017)____. When ILC2s are cultured in vitro with IL-12, IFN-γ and TBX21 expression are upregulated to the levels of CD56hiNK cell ____(Lim ____et al____, 2016)____, indicating possible plasticity between ILC2 and CD56hiNK cells under certain inflammatory conditions. In HIV-1 infection, we do not detect correlations between the decrease in ILCs and increase in NK cells. In fact, the total NK cells did not change in HIV-1+ people who are viremic, on ART, or viremic controllers [(Figure 2A and 2B in ____(Wang ____et al____, 2020b)____]. Our transcriptome analysis indicates that, during HIV-1 infection, ILCs are likely depleted by inflammation-induced apoptosis (Figure 4I and Supplemental Table 6). However, whether blood ILCs (ILCPs or ILC2s) can upregulate TBX21 and EOMES to give rise to NK cells during HIV-1 infection is an interesting question worth further investigation. We now discuss the reviewer’s question on pages 27. __ III. The authors write that ILCs have a primary role in cytokine secretion and then distinguish the ILCs from NKs....but NKs also secrete cytokines. Can the authors please clarify their text?
__The reviewer is correct that, in this context, our statement is confusing. We have therefore deleted the sentence “Typical of cells with a primary role in secretion of cytokines…” on page 10. This change helps to focus the text on the specific genes that distinguish these subsets. __
- Discussion (an also Results) - The authors use pseudotime analysis to show that CD56+NK cells sit between the ILCs and CD56-NKs. When initially described in the Discussion they don't really interpret this in terms of their (and others' observations) that CD56-NKs are increased in disease. Other groups have suggested CD56-NKs are dysfunctional. Here you show different granzyme profiles between CD56+ and CD56- NKs. Elsewhere you distinguish CD56dim and CD56- NKs in terms of functionality in HIV+ donors, though these cells cluster on the pseudotime analysis. The IL-2 and CD4+ T cell data add another layer of complexity.
- Altogether, I am unclear on the authors' interpretation of their collective data.
- Can you please clarify whether you are suggesting that CD56dimNKs are a distinct from CD56+ NKs but functional as opposed to CD56-NKs that are dysfunctional? __It would be best to respond to these two questions together. It is important to remember that our manuscript describes the characterization of these cells using several techniques. __
- By flow cytometry, NK cells can be separated into CD56hi, CD56dim, and CD56neg populations (Figure 1F). Pseudotime analysis of our single cell RNA-Seq data showed that, at the transcriptional level, CD56dim and CD56neg NK cells are very similar to each other and cluster together (Figures 3A and 3F). Nonetheless, as compared with CD56dim NK cells, CD56neg NK cells express lower levels of GZMA and PRF1 (Supplemental Figure 4A), and produce less IFN-γ upon cytokine stimulation (Figures 8H and 8J). And when CD56neg NK cells are sorted and assessed by Bulk RNA-Seq, which gives deeper coverage than single cell RNA-Seq, metabolic gene expression is altered with respect to CD56dim NK cells (Figure 9).
__- Our pseudotime analysis based on single cell RNA-Seq showed that CD56hiNK cells form a distinct cluster, but that they share some transcriptional features with both the ILC cluster and the CD56dim/CD56–NK cell cluster (Figures 3A-3C and 3F). __
- In people living with HIV-1, as assessed by flow cytometry, CD56neg NK cells increase in number at the expense of CD56dim NK cells (Figure 7C). And yes, we show here that CD56neg NK cells expand in tissue culture if CD4+ T cells are depleted from the PBMCs, and addition of exogenous IL-2 prevents the switch from CD56dimNK cells to CD56–NK cells (Figures 8A-8C). These points are covered in the Results and Discussion on pages 11 and page 29-30.
Are CD56-NKs end-stage or can they be rescued? IL-2 treatment of CD56–NK cells from people living with HIV-1 can be converted back to CD56dimNK cells, though function is not fully restored to the level of CD56dimNK cells ____(Mavilio ____et al____, 2005)____. ____In our manuscript, we showed that IL-2 or IL-15 treatment can prevent CD56dimNK cells from becoming dysfunctional CD56–NK cells, and that these cytokines maintain mTOR activity (Figure 8A-D, 8G, 8I and Figure 10). Taking together our data with the published data, CD56–NK cells appear to be an end-stage dysfunctional cell. Whether conditions can be found for full restorage of function is an important question that is worth pursuing. This point is now stated on page 30.
CD8 T cell number remain high post ART and very much drive the ongoing inverted CD4:CD8 ratio. Have the authors consider how and if the need of these cells for IL-2 impact recovery of NK cell populations? __Two orthogonal experiments indicate that IL-2 secreted by CD4+ T cells, but not from CD8+ T cells, is responsible for the recovery of CD56dimNK cells after treatment with ART. In our ex vivo culture of PBMCs, depletion of CD4+ T cells , but not of CD8+ T cells, was associated with increased numbers of CD56–NK cells (Supplemental Figure 6A). Additionally, the IL-2 concentration in plasma from people living with HIV-1 correlated with CD4+ T cell numbers, but not with CD8+ T cell numbers (Figures 8N and 8O). __
Discussion: It would be very helpful if the authors can pull this large volume of data together in a summary paragraph and possibly also a graphic. __We thank the reviewer for the suggestion. We have now summarized our data in the discussion (page 31). Additionally, we have added bullet points with a 2 sentence summary to accompany our graphic abstract (page 3). __
"Thus, GZMK+CD8+T cells appear to be the adaptive counterpart of CD56hiNK cells, representing an intermediate state between cytokine producing CD4+T cells and cytotoxic CD8+T cells" For me, the 'cytokine-producing CD4+ T cells' comes from in from left field here. Can the authors please clarify or was the intent to write cytokine-producing CD8+ T cells? To clarify what we meant we need to discuss two points:
__First, NK cells can be considered as the innate immune counterparts of CD8+T cells, in that both cell types are cytotoxic killer cells. In contrast, ILCs may be considered as the innate counterparts of non-cytotoxic CD4+ T helper cells. For example, like Th1 cells, ILC1 cells are TBX21+ producers of IFN-γ. And like Th2 cells, ILC2s are GATA3+ producers of IL-13. The relationship between ILCs and NK cells is similar to the relationship between CD4+T help cells and CD8+ cytotoxic T cells ____(Vivier ____et al____, 2018)____. __
Second, previous studies showed that GZMK+CD8+T cells are distinguished from other CD8+T cells by higher expression of IL7R, TCF7, IFN-γ and TNF-α, and by decreased cytotoxic activity ____(Jonsson ____et al____, 2022)____. Thus, the relationship between GZMK+CD8+T cells and GZMK–CD8+T cells may be similar to the relationship between CD56hiNK cells (GZMK+, IL7R+, TCF7+, higher IFN-γ production and lower cytotoxicity) and cells from the CD56dim and CD56–NK cell cluster (GZMK–, IL7R–, TCF7–, lower IFN-γ production and higher cytotoxicity). In response to this comment we have modified the text on page 25-26 to make these points more clear.
VII. Methods: Were Pearson correlations applied because of the large 'n' or were data first tested for normality?
After checking the normality, nonparametric spearman correlation was performed for panels that failed the normality test. For panels with smaller n, the normality test may be not applicable, and Pearson correlation was performed.
VIII. Methods: Please clearly justify the use of t-tests throughout the manuscript rather than non-parametric based tests.
__We tested the normality before performing parametric or nonparametric test. The t-test, Wilcoxon test or Mann-Whitney test used in our analysis was now specified for each panel in the legend. For panels that calculate cell percentage or numbers with smaller n, normality test may be not applicable, t-test was performed as done previously in these papers, Figure 7C and 7D in ____(Wang ____et al____, 2020a)____, and in Figure 3 and 4 in ____(Xue ____et al____, 2022)____. We confirmed these analyses with two statisticians in our institute. __
Methods: How many cells were acquired in flow cytometry? How many ILCs were acquired in healthy/HIV+ donors for these studies? Did this create limitations on interpretation of phenotypic data? ILCs constitute roughly 1,000 cells per million PBMCs. To assess ILCs, we acquired data from 500,000 PBMCs, or roughly 500 ILCs per sample. NK cells constitute roughly 100,000 cells per million PBMCs, we acquired data from 200,000 PBMCs, or roughly 20,000 NK cells per sample. For each patient group, whether HIV-1-negative, HIV-1 viremic, etc, we analyzed PBMCs from at least 19 blood donors. This information is now stated in the “Flow cytometry” section in “Methods”, on page 35.
Methods: How was the cytokine concentrations used for in vitro assays determined? The cytokine concentrations were determined according to previous publication and our previous studies ____(Romee ____et al____, 2016, 2012; Wang ____et al____, 2020b; Silverstein ____et al____, 2022)____. We have added the related references to the “Stimulation conditions” section in “Method”, on page 36.
Figure: In Figure 4A, should it be '+ILC- ' or '+ILC' (no negative symbol)? __Yes, the reviewer is correct. We have deleted the typo “-”. __
XII. It's unclear from you single cell analysis how many cells were acquired for each of the 4 subsets. I assume less ILCs were analyzed. If so, can you please clarify for the non-bioinformatician how your bioinformatic analysis took these differences into account?
Indeed, we were concerned that we might have too few ILCs and therefore set up conditions so that similar numbers of each cell type were assessed. To accumulate enough ILCs for single cell analysis, ILCs were sorted from 3 donors. ILCs, CD56hi, CD56dim and CD56–NK cells were sorted in parallel from the same 3 donors. The sorting strategy is shown in Supplemental Figure 1D. Equal numbers of ILCs, CD56hi, CD56dim and CD56–NK cells were sorted and then mixed together before library preparation. In total, libraries were generated from 5,210 single cells using 10 x genomics. 1,478 ILC2s, 897 ILCPs, 1,116 CD56hiNK cells, and 1,486 CD56dim and CD56–NK cells were shown in UMAP. We have added this information in Figure 3 legend.
Reviewer #1 (Significance (Required)):
Overall, this paper serves as an important resource for the field.
There are areas in which the manuscript could be modified to improve clarity and detail regarding rigor (see above). The overall message of this work for understanding HIV pathogenesis is unclear but can be addressed.
__We appreciate the reviewer’s efforts on behalf of our manuscript. By carefully addressing the reviewer’s questions and comments, we believe that the rigor of our manuscript is improved and that the message regarding HIV-1-induced abnormalities of ILCs and NK cells are clarified. __
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: This manuscript makes use of RNA sequencing, ATAC-Seq and single cell sequencing to compare transcriptomes and identify relationships between various ILC/NK cell subsets in blood from uninfected controls. The authors describe differences in genes and pathways between subsets and determine that CD56high NK cells are an intermediate-connecting cell type between ILC progenitors and CD56dim NK cells. The authors show how HIV infection (viremic, ART+ or controllers) effect ILC/NK cell gene expression and pathways. One of the genes enhanced in NK cells is AREG which the authors demonstrate to be upregulated in IL/NK subsets in response to PMAI/I stimulation or stimulation with IL-2 or IL-15. The authors demonstrate that TGFB1 or knockout of RUNX3 modulates the frequency of AREG+ NK cells, implicating the Wnt signaling pathway in AREG regulation. Authors show that in PLWH, the frequency of AREG+ NK cells is altered. Authors also report that with HIV infection there is an increase in CD56neg NK cells with corresponding loss of CD56dim cells. Using healthy control PBMCs, the authors suggest differentiation of CD56dim into CD56neg is prevented by CD4 T cell production of IL-2 but promoted by TGFB1. Also in healthy controls, the authors run metabolomics to describe how CD56dim are more metabolically functional than CD56neg and link this to MTOR activity.
Major:
- Which population(s) of NK cells (high, dim or negative for CD56) are responding to stimulation with IL-2 and IL-15 by upregulating AREG in Figure 5? Does this NK population have higher expression of receptors for IL-2 and IL-15 (in single cell seq data or by flow) and if so how does receptor expression change in PLWH? We thank the reviewer for these excellent questions. In response, we have added the new figure below to Supplemental Figure 4E. In general, CD56hi NK cells have higher AREG RNA and protein than do the other NK cell subsets. After IL-2 or IL-15 stimulation, all three NK cell populations upregulate AREG, though CD56hiNK cells produce significantly higher levels of AREG than do either CD56dim or CD56negNK cells.
__Consistent with the above results, the expression of IL2RB, IL2RG and IL15RA was higher in CD56hiNK cells than in CD56dimNK cells (new Supplemental Figure 4F). __
We examined IL2RA, IL2RB, IL2RG, and IL15RA expression in the different NK cell subsets from the three groups of people living with HIV-1, ART, viremic, and controllers. As compared with cells from HIV-1-negative people, the only clearly significant difference for these four genes was a reduction in IL2RB expression in CD56– and CD56dim NK cells from viremic people. These points are now mentioned on page 15.
- Does signaling through IL-2 or IL-15 converge on the TCF7/Wnt signaling pathway? For example, is it possible to knockdown TCF7 then stimulate with IL-2 or IL-15 and measured AREG+ cells with the idea that loss of TCF7 would result in reduced cytokine-induced activation of AREG if mediated through the Wnt pathway? Indeed, this question was very important to us and we attempted to answer it with lentiviral vector-mediated knockdown with shRNA and with electroporation of Cas9 ribonucleoprotein complexes (RNPs). Though we were successful in decreasing TCF7 protein, to render NK cells competent for lentiviral transduction or for electroporation with the Cas9 RNPs, NK cells must first be cultured for at least 7 days in high concentration IL-2. This treatment with IL-2 activates AREG production before TCF7 protein is decreased. These points are now discussed in the manuscript on page 29.
In Figure 8 the authors demonstrate that CD4 T cells promote the maintenance of CD56dim NK cells likely through production of IL-2. Was AREG expression examined in these studies and if so could you conclude that CD4 T cells also promote AREG expression in this population? Yes, indeed, CD4+T cells, or IL-2, promote AREG production by CD56dimNK cells. Depletion of CD4+T cells from PBMCs decreased AREG production and addition of exogenous IL-2 was sufficient to restore AREG production by NK cells when CD4+ T cells were depleted (see figure below, left panel). Additionally, IL-2 neutralizing antibodies decreased AREG production by NK cells in total PBMC cultures (right panel). These results are now included as Supplemental Figure 6G and 6H in the revised manuscript.
In Figure 8D, although significant, the increase in percentage from 2-4% of the CD56neg is a minimal change. Could a higher dose of IL-2 be tested to see more substantial changes? Similar comment for Figure 10D, although significant, the increase in percentage from 2-4% of the NK population to CD56neg is not a large change with rapamycin. Could a higher dose be tested (that does not kill the cells) or a different inhibitor be used to see more substantial changes? More robust changes would strengthen the conclusions. First to clarify, as concerns figure 8D, we believe the reviewer is referring to IL-2 neutralizing antibody, not IL-2. Regarding Figure 8D, which used IL-2 neutralizing antibody at 4 ug/ml, we tried a higher dose (10 ug/ml) but this caused cell death. We also tried longer culture time with 4 ug/ml IL-2 neutralizing antibody, and found the cell viability decreases after 7 days culture. Similarly, the limited in vitro culture time in the presence of anti-IL-2 antibody restricted experimental conditions in Figure 10D.
In Figure 10, was the increase in pmTOR with IL-2/IL-15 stimulation specifically observed in CD56dim cells (rather than total NK cells or CD56neg cells)? This would strength the conclusion that CD56dim is more metabolically functional than CD56neg. Our data indicate that the mTOR response to IL2 in CD56–NK cells was similar to that in CD56+NK cells (including CD56dim and CD56hi) (see figure below). This result is consistent with a previous report showing that, when stimulated with exogenous IL-2, sorted CD56– NK cells become CD56dimNK cells ____(Mavilio ____et al____, 2005)____. Our data extend this observation by showing that upregulation of CD56 on CD56neg NK cells by IL-2 correlates with mTOR upregulation.
Minor
- Approximately how many ILC from PLWH were submitted for single cell sequencing: Since this cell population was depleted in HIV+, was there a sufficient number of cells to interpret/publish DEG results in Figure 4? In Figure 4, ILCs were sorted from HIV-1 negative donors and different groups of PLWH, and then these cells were subjected to bulk-RNA seq using CEL-Seq2. In Figure 4, for most blood donors, whether HIV-1 negative or PLWH, well over 1,000 ILCs were sorted and used to construct high quality libraries (see figure below, was included as new Supplemental Figure 3C). PCA plots also showed that all ILC libraries clustered together as a distinct population (Figure 4A), indicating the quality of all ILC libraries was comparable.
On page 13 of text, it is a bit disconcerting to jump to Figure 7 then back to Figure 4. Would recommend reorganizing text or figure panels to flow better. __We have deleted the premature mention of Figure 7A. __
Supplemental Figure 1 and 2: Dots for ILC population are purple in color but legend is mislabeled as pink color.
Supplemental Figure 4: Dots for HIV+ controller are purple in color, but legend is mislabeled as pink in color. Thank you, these mistakes in the labeling of the colors have been corrected.
Would recommend adding quantification of Figure 5A for all donors tested. In fact, the AREG production by CD56hi, CD56dim, CD56–NK cells and ILCs from HIV-1 negative donors were quantified in Figure 6A.
Figure 5B for IFNg it appears that part of the positive population is not gated on and thus percentages are likely higher if the gate is adjusted. We thank the reviewer for the suggestion. The gating for IFN-γ has been adjusted in the revised manuscript, as suggested. The original Figure 5B and associated histogram Figure 5C were replaced accordingly. Please see the newly gated figure below:
For Figure 5F, need to add to text and panel that PMA/I was used to stimulate as described in figure legend. Current figure and text read that the Wnt agonist alone was responsible for levels depicted in NK subsets. We now state that PMA+ionomycin was used to stimulate the cells in Figure 5F and 5G, and in the text (page 16 and 28).
On the x-axis of Figure 6F-J does "Lin-TBX21+" refer to Total NK cells? If so then would recommend sticking with nomenclature "Total NK" as in Figure 6A-B. The x-axis of Figure 6F-J was changed to “total NK (Lin–TBX21+)”.
Would recommend labeling gates for populations of interest in Supplemental Figure 5B-C, Supplement Figure 6A, Figure 8A. The populations of interest (CD56dimNK, CD56–NK, and ILCs) were labeled as suggested.
Other groups have shown that CD56neg are increased in HIV, functionally impaired and correlate with loss of CD4 T cells. Would recommend citing their studies. The original manuscript had mentioned only Mavilio et al. The revised manuscript now has a more complete list of references ____(Cao ____et al____, 2021; Mavilio ____et al____, 2005; Cocker ____et al____, 2022; Alter ____et al____, 2005; Barker ____et al____, 2007)____. These references are cited in the Introduction (page 5) and in the Results (page 20).
Would recommend adding quantification of Figure 8A for all donors tested. Also, for the 3rd flow plot does "NK+CD4" mean purified NK cells + autologous CD4 T cells? If so, then would clarify in figure legend. It may strengthen conclusion for IL-2/IL-15 to show differentiation of NK cells is not contact dependent with T cell via transwell assay. __Quantification for all donors in Figure 8A has been added to the revised manuscript (Figure 8A, right panel). The figure legend has been modified accordingly. As far as the comment about contact-dependence, the fact that CD56dimNK cells were maintained by IL-2 in the absence of CD4+ T cells demonstrates that direct contact with CD4+ T cells is not required. __
Reviewer #2 (Significance (Required)):
Significance: This study is valuable because it generates large datasets on the NK/ILC family from human blood that can be deposited in repositories and of special relevance to the HIV field because it examine show viral infection (controlled or not) effects these subsets.
The strengths of the study are the cohort of PBMC samples utilized (HIV-, HIV+ viremic, HIV+ ART+ and HIV+ controlled), the multi-omics approach for transcriptome and epigenetics and the in vitro mechanistic studies identifying key regulators of NK cell differentiation/function.
The study advances the field "mechanistically" by providing key targets that may be subject to therapeutic modulation in PLWH such as AREG, IL-2 signaling, IL-15 signaling, Wnt signaling, and mTOR activity (although more work will need to be done to examine these pathways using PBMCs from HIV+). This study advances the field "conceptually" by providing large datasets for others to mine if deposited.
The audience for this study "basic research" such as immunologists in the HIV field or immunologist interested in ILC/NK biology.
My field of expertise is infectious disease (HIV, SARS-CoV-2), basic immunology (ILC, NK, B cell) and autoimmune disease. Would recommend additional reviewers for assessment of metabolic genes/pathways in Figure 9-10.
__We were very pleased to see that the reviewer thought our manuscript makes a valuable contribution to HIV-1 immunology and ILC/NK biology, that it advances mechanistic understanding of pathogenesis in people living with HIV-1, and that it provides a valuable data resource. __
- In Figure 9, the metabolism related genes were defined as canonical targets of, or regulated by, MTOR signaling, in previous publications ____(Saxton & Sabatini, 2017; Bayeva ____et al____, 2012)____.
- In Figure 10, we used pMTOR, pAKT, p4EBP1, pS6 and CD71 to monitor MTOR pathway activation as reported previously by others ____(Marçais ____et al____, 2014)____. We have cited this paper on pages 23 and 24.
References for Response to Reviewers
Alter G, Teigen N, Davis BT, Addo MM, Suscovich TJ, Waring MT, Streeck H, Johnston MN, Staller KD, Zaman MT, et al (2005) Sequential deregulation of NK cell subset distribution and function starting in acute HIV-1 infection. Blood 106: 3366–3369
Barker E, Martinson J, Brooks C, Landay A & Deeks S (2007) Dysfunctional natural killer cells, in vivo, are governed by HIV viremia regardless of whether the infected individual is on antiretroviral therapy. AIDS 21: 2363–2365
Bayeva M, Khechaduri A, Puig S, Chang H-C, Patial S, Blackshear PJ & Ardehali H (2012) mTOR regulates cellular iron homeostasis through tristetraprolin. Cell Metab 16: 645–657
Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z, Bornstein E, Lambotte O, Altmann D, et al (2006) Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12: 1365–1371
Caetano DG, Ribeiro-Alves M, Hottz ED, Vilela LM, Cardoso SW, Hoagland B, Grinsztejn B, Veloso VG, Morgado MG, Bozza PT, et al (2022) Increased biomarkers of cardiovascular risk in HIV-1 viremic controllers and low persistent inflammation in elite controllers and art-suppressed individuals. Sci Rep 12: 6569
Cao W-J, Zhang X-C, Wan L-Y, Li Q-Y, Mu X-Y, Guo A-L, Zhou M-J, Shen L-L, Zhang C, Fan X, et al (2021) Immune Dysfunctions of CD56neg NK Cells Are Associated With HIV-1 Disease Progression. Front Immunol 12: 811091
Cocker ATH, Liu F, Djaoud Z, Guethlein LA & Parham P (2022) CD56-negative NK cells: Frequency in peripheral blood, expansion during HIV-1 infection, functional capacity, and KIR expression. Front Immunol 13: 992723
Cortez VS, Ulland TK, Cervantes-Barragan L, Bando JK, Robinette ML, Wang Q, White AJ, Gilfillan S, Cella M & Colonna M (2017) SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling. Nat Immunol 18: 995–1003
Crowell TA, Gebo KA, Blankson JN, Korthuis PT, Yehia BR, Rutstein RM, Moore RD, Sharp V, Nijhawan AE, Mathews WC, et al (2015) Hospitalization Rates and Reasons Among HIV Elite Controllers and Persons With Medically Controlled HIV Infection. J Infect Dis 211: 1692–1702
Jonsson AH, Zhang F, Dunlap G, Gomez-Rivas E, Watts GFM, Faust HJ, Rupani KV, Mears JR, Meednu N, Wang R, et al (2022) Granzyme K+ CD8 T cells form a core population in inflamed human tissue. Sci Transl Med 14: eabo0686
Lim AI, Menegatti S, Bustamante J, Le Bourhis L, Allez M, Rogge L, Casanova J-L, Yssel H & Di Santo JP (2016) IL-12 drives functional plasticity of human group 2 innate lymphoid cells. J Exp Med 213: 569–583
Marçais A, Cherfils-Vicini J, Viant C, Degouve S, Viel S, Fenis A, Rabilloud J, Mayol K, Tavares A, Bienvenu J, et al (2014) The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells. Nat Immunol 15: 749–757
Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, Marcenaro E, Angeline O’Shea M, Kinter A, Kovacs C, Moretta A, et al (2005) Characterization of CD56–/CD16+ natural killer (NK) cells: A highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci U S A 102: 2886–2891
Moreno-Nieves UY, Tay JK, Saumyaa S, Horowitz NB, Shin JH, Mohammad IA, Luca B, Mundy DC, Gulati GS, Bedi N, et al (2021) Landscape of innate lymphoid cells in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment. Proc Natl Acad Sci U S A 118
Raykova A, Carrega P, Lehmann FM, Ivanek R, Landtwing V, Quast I, Lünemann JD, Finke D, Ferlazzo G, Chijioke O, et al (2017) Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells. Blood Adv 1: 2679–2691
Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, et al (2016) Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci Transl Med 8: 357ra123
Romee R, Schneider SE, Leong JW, Chase JM, Keppel CR, Sullivan RP, Cooper MA & Fehniger TA (2012) Cytokine activation induces human memory-like NK cells. Blood 120: 4751–4760
Saxton RA & Sabatini DM (2017) mTOR Signaling in Growth, Metabolism, and Disease. Cell 169: 361–371
Silverstein NJ, Wang Y, Manickas-Hill Z, Carbone C, Dauphin A, Boribong BP, Loiselle M, Davis J, Leonard MM, Kuri-Cervantes L, et al (2022) Innate lymphoid cells and COVID-19 severity in SARS-CoV-2 infection. Elife 11
Vivier E, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, Koyasu S, Locksley RM, McKenzie ANJ, Mebius RE, et al (2018) Innate Lymphoid Cells: 10 Years On. Cell 174: 1054–1066
Wang J, Xu Y, Chen Z, Liang J, Lin Z, Liang H, Xu Y, Wu Q, Guo X, Nie J, et al (2020a) Liver Immune Profiling Reveals Pathogenesis and Therapeutics for Biliary Atresia. Cell 183: 1867–1883.e26
Wang Y, Lifshitz L, Gellatly K, Vinton CL, Busman-Sahay K, McCauley S, Vangala P, Kim K, Derr A, Jaiswal S, et al (2020b) HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells. Nat Immunol 21: 274–286
Xue R, Zhang Q, Cao Q, Kong R, Xiang X, Liu H, Feng M, Wang F, Cheng J, Li Z, et al (2022) Liver tumour immune microenvironment subtypes and neutrophil heterogeneity. Nature 612: 141–147
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
This manuscript makes use of RNA sequencing, ATAC-Seq and single cell sequencing to compare transcriptomes and identify relationships between various ILC/NK cell subsets in blood from uninfected controls. The authors describe differences in genes and pathways between subsets and determine that CD56high NK cells are an intermediate-connecting cell type between ILC progenitors and CD56dim NK cells. The authors show how HIV infection (viremic, ART+ or controllers) effect ILC/NK cell gene expression and pathways. One of the genes enhanced in NK cells is AREG which the authors demonstrate to be upregulated in IL/NK subsets in …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
This manuscript makes use of RNA sequencing, ATAC-Seq and single cell sequencing to compare transcriptomes and identify relationships between various ILC/NK cell subsets in blood from uninfected controls. The authors describe differences in genes and pathways between subsets and determine that CD56high NK cells are an intermediate-connecting cell type between ILC progenitors and CD56dim NK cells. The authors show how HIV infection (viremic, ART+ or controllers) effect ILC/NK cell gene expression and pathways. One of the genes enhanced in NK cells is AREG which the authors demonstrate to be upregulated in IL/NK subsets in response to PMAI/I stimulation or stimulation with IL-2 or IL-15. The authors demonstrate that TGFB1 or knockout of RUNX3 modulates the frequency of AREG+ NK cells, implicating the Wnt signaling pathway in AREG regulation. Authors show that in PLWH, the frequency of AREG+ NK cells is altered. Authors also report that with HIV infection there is an increase in CD56neg NK cells with corresponding loss of CD56dim cells. Using healthy control PBMCs, the authors suggest differentiation of CD56dim into CD56neg is prevented by CD4 T cell production of IL-2 but promoted by TGFB1. Also in healthy controls, the authors run metabolomics to describe how CD56dim are more metabolically functional than CD56neg and link this to MTOR activity.
Major:
- Which population(s) of NK cells (high, dim or negative for CD56) are responding to stimulation with IL-2 and IL-15 by upregulating AREG in Figure 5? Does this NK population have higher expression of receptors for IL-2 and IL-15 (in single cell seq data or by flow) and if so how does receptor expression change in PLWH?
- Does signaling through IL-2 or IL-15 converge on the TCF7/Wnt signaling pathway? For example, is it possible to knockdown TCF7 then stimulate with IL-2 or IL-15 and measured AREG+ cells with the idea that loss of TCF7 would result in reduced cytokine-induced activation of AREG if mediated through the Wnt pathway?
- In Figure 8 the authors demonstrate that CD4 T cells promote the maintenance of CD56dim NK cells likely through production of IL-2. Was AREG expression examined in these studies and if so could you conclude that CD4 T cells also promote AREG expression in this population?
- In Figure 8D, although significant, the increase in percentage from 2-4% of the CD56neg is a minimal change. Could a higher dose of IL-2 be tested to see more substantial changes? Similar comment for Figure 10D, although significant, the increase in percentage from 2-4% of the NK population to CD56neg is not a large change with rapamycin. Could a higher dose be tested (that does not kill the cells) or a different inhibitor be used to see more substantial changes? More robust changes would strengthen the conclusions.
- In Figure 10, was the increase in pmTOR with IL-2/IL-15 stimulation specifically observed in CD56dim cells (rather than total NK cells or CD56neg cells)? This would strength the conclusion that CD56dim is more metabolically functional than CD56neg.
Minor
- Approximately how many ILC from PLWH were submitted for single cell sequencing: Since this cell population was depleted in HIV+, was there a sufficient number of cells to interpret/publish DEG results in Figure 4?
- On page 13 of text, it is a bit disconcerting to jump to Figure 7 then back to Figure 4. Would recommend reorganizing text or figure panels to flow better.
- Supplemental Figure 1 and 2: Dots for ILC population are purple in color but legend is mislabeled as pink color.
- Supplemental Figure 4: Dots for HIV+ controller are purple in color, but legend is mislabeled as pink in color.
- Would recommend adding quantification of Figure 5A for all donors tested.
- Figure 5B for IFNg it appears that part of the positive population is not gated on and thus percentages are likely higher if the gate is adjusted.
- For Figure 5F, need to add to text and panel that PMA/I was used to stimulate as described in figure legend. Current figure and text read that the Wnt agonist alone was responsible for levels depicted in NK subsets.
- On the x-axis of Figure 6F-J does "Lin-TBX21+" refer to Total NK cells? If so then would recommend sticking with nomenclature "Total NK" as in Figure 6A-B.
- Would recommend labeling gates for populations of interest in Supplemental Figure 5B-C, Supplement Figure 6A, Figure 8A.
- Other groups have shown that CD56neg are increased in HIV, functionally impaired and correlate with loss of CD4 T cells. Would recommend citing their studies.
- Would recommend adding quantification of Figure 8A for all donors tested. Also, for the 3rd flow plot does "NK+CD4" mean purified NK cells + autologous CD4 T cells? If so, then would clarify in figure legend. It may strengthen conclusion for IL-2/IL-15 to show differentiation of NK cells is not contact dependent with T cell via transwell assay.
Significance
This study is valuable because it generates large datasets on the NK/ILC family from human blood that can be deposited in repositories and of special relevance to the HIV field because it examine show viral infection (controlled or not) effects these subsets.
The strengths of the study are the cohort of PBMC samples utilized (HIV-, HIV+ viremic, HIV+ ART+ and HIV+ controlled), the multi-omics approach for transcriptome and epigenetics and the in vitro mechanistic studies identifying key regulators of NK cell differentiation/function.
The study advances the field "mechanistically" by providing key targets that may be subject to therapeutic modulation in PLWH such as AREG, IL-2 signaling, IL-15 signaling, Wnt signaling, and mTOR activity (although more work will need to be done to examine these pathways using PBMCs from HIV+). This study advances the field "conceptually" by providing large datasets for others to mine if deposited.
The audience for this study "basic research" such as immunologists in the HIV field or immunologist interested in ILC/NK biology.
My field of expertise is infectious disease (HIV, SARS-CoV-2), basic immunology (ILC, NK, B cell) and autoimmune disease. Would recommend additional reviewers for assessment of metabolic genes/pathways in Figure 9-10.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Wang and colleagues present a very detailed, high quality paper describing phenotyping, transcriptional, epigenetic and functional differences between NK cells and ILCs in human peripheral blood. They overlay these studies which descriptions of differences in these populations in healthy and HIV infected (viremia, ART-treated, controllers), extending a their previous 2020 study.
Overall, this paper serves as an important resource for the field. There are areas in which the manuscript could be modified to improve clarity and detail regarding rigor.
My comments below are all addressable and therefore I class them all as 'major'.
- The …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Wang and colleagues present a very detailed, high quality paper describing phenotyping, transcriptional, epigenetic and functional differences between NK cells and ILCs in human peripheral blood. They overlay these studies which descriptions of differences in these populations in healthy and HIV infected (viremia, ART-treated, controllers), extending a their previous 2020 study.
Overall, this paper serves as an important resource for the field. There are areas in which the manuscript could be modified to improve clarity and detail regarding rigor.
My comments below are all addressable and therefore I class them all as 'major'.
- The authors describe ILCs as being 'permanently deleted' in HIV infection. As written, this can be misinterpreted to suggest complete ablation of this cell subset. This is clearly not the case. Moreover, there looks to be some restoration of ILCs in ART-treated participants. I suggest revising text to quantify the level of cell loss or replace the word deleted with reduced.
- The HIV EC are not clearly discussed in the paper and are not distinguished from viremic controllers. If ILCs are permanently reduced in this group, what does this suggest for the role of this cell subset in HIV control? Plasticity between ILC and NK cells is described. Is this plasticity relevant at all for HIV control, elite or otherwise?
- The authors write that ILCs have a primary role in cytokine secretion and then distinguish the ILCs from NKs....but NKs also secrete cytokines. Can the authors please clarify their text?
- Discussion (an also Results) - The authors use pseudotime analysis to show that CD56+NK cells sit between the ILCs and CD56-NKs. When initially described in the Discussion they don't really interpret this in terms of their (and others' observations) that CD56-NKs are increased in disease. Other groups have suggested CD56-NKs are dysfunctional. Here you show different granzyme profiles between CD56+ and CD56- NKs. Elsewhere you distinguish CD56dim and CD56- NKs in terms of functionality in HIV+ donors, though these cells cluster on the pseudotime analysis. The IL-2 and CD4+ T cell data add another layer of complexity.
- a. Altogether, I am unclear on the authors' interpretation of their collective data.
- b. Can you please clarify whether you are suggesting that CD56dimNKs are a distinct from CD56+ NKs but functional as opposed to CD56-NKs that are dysfunctional?
- c. Are CD56-NKs end-stage or can they be rescued?
- d. CD8 T cell number remain high post ART and very much drive the ongoing inverted CD4:CD8 ratio. Have the authors consider how and if the need of these cells for IL-2 impact recovery of NK cell populations?
- Discussion: It would be very helpful if the authors can pull this large volume of data together in a summary paragraph and possibly also a graphic.
- "Thus, GZMK+CD8+T cells appear to be the adaptive counterpart of CD56hiNK cells, representing an intermediate state between cytokine producing CD4+T cells and cytotoxic CD8+T cells" For me, the 'cytokine-producing CD4+ T cells' comes from in from left field here. Can the authors please clarify or was the intent to write cytokine-producing CD8+ T cells?
- Methods: Were Pearson correlations applied because of the large 'n' or were data first tested for normality?
- Methods: Please clearly justify the use of t-tests throughout the manuscript rather than non-parametric based tests.
- Methods: How many cells were acquired in flow cytometry? How many ILCs were acquired in healthy/HIV+ donors for these studies? Did this create limitations on interpretation of phenotypic data?
- Methods: How was the cytokine concentrations used for in vitro assays determined?
- Figure: In Figure 4A, should it be '+ILC- ' or '+ILC' (no negative symbol)?
- It's unclear from you single cell analysis how many cells were acquired for each of the 4 subsets. I assume less ILCs were analyzed. If so, can you please clarify for the non-bioinformatician how your bioinformatic analysis took these differences into account?
Significance
Overall, this paper serves as an important resource for the field.
There are areas in which the manuscript could be modified to improve clarity and detail regarding rigor (see above). The overall message of this work for understanding HIV pathogenesis is unclear but can be addressed.
-
-
-