Multinucleation resets human macrophages for specialized functions at the expense of their identity

Abstract Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign‐body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome‐mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7‐H3 (CD276) and can form granuloma‐like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell–cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC‐specific functionality.

For osteoclasts, various markers exist for their biological characterization, for instance the ability to resorb bone. What, apart from the arrangement and number of nuclei, were the biological parameters that confirmed that the cells made by addition of IFN or IL-4 were LCG and FBGC? In fig 2c: did the authors perform stainings with isotype control antibodies? In my experience, quite often, antibodies stain mononuclear cells much intenser, since the cytoplasm is much more condense, less spread over a large area. Resorption assay in 6 is not clear. It is weird that osteoclasts apparently display so limited resorption? Also the traces are not typical for osteoclasts. Please explain. Provide a better image Supplementary 2A, even at 250% the lettering is vague. What do the colours in 2A mean? *CROSS-CONSULTATION COMMENTS* I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology! 4. In Figure4, the authors showed that transcripts in LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.
5. Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway? **Minor comments:** 1. Supplementary tables 1-6 were not provided.

Significance (Required)
Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.

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Evidence, reproducibility and clarity:
Evidence, reproducibility and clarity (Required) **Summary:** The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variantsosteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below. **Minor comments:** • The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts.
• The overall decrease in phagocytic identity of all the MGCs, and the specific upregulation of phagocytic pathways in the FBGCs are conflicting. Are there subsets of phagocytic pathways that were down and upregulated during the formation of FBGCs?
• What are the identities of the mononuclear cells in each of the MGC experiment? They appeared to be quite heterogeneous based on the DEGs identified, beyond the common phagocyte signature. Can the authors comment on the difference between the mononuclear cells and whether this will affect the DEG analysis?
• The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study **Major comments:** • As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues.

Significance:
Significance (Required) **Significance:** • The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored.
• My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg LGCs and FBGCs is limited.
3. How much time do you estimate the authors will need to complete the suggested revisions:

Estimated time to Complete Revisions (Required) (Decision Recommendation)
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General Statements [optional]
[Authors' reply]. We thank all the reviewers for their very constructive comments. We note that our findings were unanimously received as novel and unique. The reviewers highlighted the rigor in data analysis (Reviewer 1) and the improvement of our understanding of the fusion process and the underlying biology (Reviewer 1, 2 and 3) -a characteristic of macrophages among all other immune cells. We have addressed all the points raised by the reviewers and provide a fully revised version of our manuscript.

Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)): Summary: The authors compared the various multinucleated cells, osteoclasts, LCG and FBGC. Overall, the manuscript shows rigor in the analyses, and also very interesting approaches for retrieving mononuclear cells, for instance using DC-STAMP siRNA. This work adds very much to understanding the biological differences, as summarized in figure 6h. A lot of work in osteoclast field with for instance qPCR is hampered because, inevitably, a mix of mononuclear and multinucleated cells is always measured. Here, a solid attempt to separate those mixes with cell sorting and subsequent analysis on the mononuclear and multinucleated isolates, really adds. Choice of figures is good, also the extra info of the supplementary figures is relevant and makes it easy to read.
Major and minor concerns:  [1] and CD86 [2], respectively. As per the biological parameters, we indeed confirm that FBGCs show enhanced phagocytosis properties ( Figure 5C) while LGCs can form granuloma-like clusters in vitro ( Figure 4D and E) We did not observe a potential artefact of staining in multinucleated cells when compared to mononuclear cells. In fact, some markers of multinucleation such as B7-H3 is augmented in LGCs ( Figure 4E).  Figure 2A with better resolution. In STRING protein-protein interaction analysis, there is no particular meaning of the node color itself.

CROSS-CONSULTATION COMMENTS
I have read the comments of the other two reviewers, and together. I absolutely agree with their additions, Indeed, supplementary tables are lacking, as well as there could be a bit more emphasis on the fact that it is all in vitro work. Together, I think the three of us are complementary in our comments, with good overlap as well. Any effort to stain for instance pathology material with the markers that have been found, would be great, especially for the LGC and the FBGC, that are much less studied in the field of MNGs. Having said that ,I can also live without this addition, but then it could be highlighted in the discussion that these are the future avenues that should be considered. Collaborate with Pathology!

[Authors' reply].
We appreciate that the reviewer provides cross-consultation comments which we address in our revised manuscript. As such, we discuss future avenues regarding the translatability of these results to human pathology involving MGCs.
Reviewer #1 (Significance (Required)): This manuscript is particularly interesting to those who are interested in the BIOLOGY of MNCs. In essence, three types of MNCs were cultured and compared, with each of them a specific function.
I am an osteoclast expert (76 publications), and have two publications on FBGCs

[Authors' reply].
We sincerely thank the reviewer for his/her pertinent comments, enthusiasm for our findings and for providing us an overall summary of our findings in view of all other reviewer comments.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: In this manuscript, the authors performed a comparative transcriptome analysis of mononuclear and multinuclear human osteoclasts, LGCs and FBGCs. They found that multinucleation triggers a significant downregulation of macrophage identity in all three types of MGCs. Furthermore, RNA-seq data and in-vitro functional analysis of multinucleated cells showed that macrophage cell-cell fusion and multinucleation enhance phagocytosis and contribute to lysosome-dependent intracellular iron homeostasis. Furthermore, multinucleation of osteoclasts promoted mitochondrial activity and oxidative phosphorylation, resulting in maximal respiration. This unique Full Revision 4 and interesting study addresses the fundamental question of how cell-cell fusion and multinucleation contribute to cellular activity and biological homeostasis.
Major comments #1 The authors generated mature multinucleated cells by stimulating human PBMC-derived macrophages with either IFN-g, IL-4, or RANKL. However, no quantitative data have been presented to determine how effectively IL-4, IFN-g, and RANKL can induce multinucleated giant cells from mononuclear macrophages. Quantitative data showing induction efficiency would provide a more detailed picture of the overall experiment.

#2
The authors mentioned, "The distinct morphological appearance of these three types of MGCs ( Figure 1B) suggested cell type-specific functional properties and shared mechanisms underlying macrophage multinucleation". However, there is no discussion or data showing how the nuclear arrangement and intracellular location affect the biological function of multinucleated cells.

[Authors' reply]. This is good point and is now discussed in the revised manuscript (see highlighted text in revised manuscript and below).
Whether MGC-specific nuclear arrangements and/or numbers are indicative of specialized function is currently unclear. Intracellular nuclei arrangement is likely to be important for the sealing zone formation in a polarized bone-resorbing osteoclast. Furthermore, whether distinct transcriptional activities are assigned to different nuclei of the MGC also remain to be tested. Recent elegant work performed in multinucleated skeletal myofibers suggest transcriptional heterogeneity among the different nuclei of the polykaryon [3].
#3 Based on the results of DC-stamp knockdown experiments, the authors concluded that cellcell fusion and multinucleation suppress the mononuclear phagocytic gene signature. However, to strengthen this hypothesis, it would be necessary to provide at least data showing that DCstamp knockdown reduces the number of multinucleated cells. Figure 2F). For human osteoclasts, the data was included in our previously published paper ( [4] and below).

A B
LGCs

#4 In Figure4, the authors showed that transcripts in
LGCs were enriched for antigen presentation and adaptive immune system pathways. In addition, multinucleation of LGCs increased the surface expression of B7-H3 (CD276) and colocalized with CD3+ cells, suggesting that LGC multinucleation potentiates T cell activation. However, the authors did not present enough data to demonstrate the antigen-presenting ability of LGCs or their specific T cell activating capacity.
[Authors' reply]. We agree with the reviewer that our data on a potential role of LGCs' on T cell activation is based on increased surface expression of B7-H3 and the unique CD3+ cluster forming ability of LGCs. In order to check for further markers of antigen presentation, we have performed MHC-1 and MHC-2 quantification by ImageStream in 3 types of MGCs (see below).

Although there was no difference in MHC-I/MHC-2 between the mononucleated and multinucleated macrophages, the mean fluorescent intensity (MFI) range was the highest in IFN-
γ-stimulated macrophages, suggesting that LGCs may be better equipped for antigen presentation than the other 2 types of MGCs. A more comprehensive analysis of antigen presentation requires enzymatic digestion and isolation and phenotyping of LGCs from clusters in vitro and human tissues in vivo. This is a program of research that we have initiated as part of a separate study, which will focus on the in vivo relevance of the current findings such as the unique Ag presentation ability of LGCs in a non-sterile tissue environment.
# 5 Figure 6 clearly shows that mature multinucleated osteoclasts exhibit increased ATP production and maximal respiration. However, the glycolytic pathway did not differ between mononuclear and multinuclear osteoclasts. No explanation for this observation has been provided. It is easy to understand that osteoclasts acquire ATP through aerobic respiration during multinucleation. But how NADPH, which is essential for its redox reaction, is produced? Is it by acquiring αKG from the glutamine pathway?
[Authors' reply]. This is a point worth expending (see also discussion; highlighted text). Osteoclast multinucleation is characterized by increased mitochondrial gene expression which also translates into increased spare respiratory capacity (SRC or maximal respiration). This metabolic rewiring does not modify glycolysis and basal respiration rate. As the reviewer correctly states, increased SRC may be a way to supply more ATP to the energy-demanding polykaryon.
As per the production of NAD(P)H as an electron source for ETC, it could indeed be through glutamine rather than glucose usage in multinucleated osteoclasts. Furthermore, as iron is an essential cofactor for ETC activity through activity of iron-sulfur clusters, the mitochondrial concentration of iron is likely to be critical for the mitochondrial activity of multinucleated osteoclasts (see also discussion).
[Authors' reply]. We apologize for this. The revised versions of supplementary tables are provided as part of the revised manuscript.
[Authors' reply]. Thanks for pointing this out. We have now replaced Figure 2D with a more pronounced DAPI+ nuclei.
Reviewer #2 (Significance (Required)): Although it is well known that multinucleation of cells constantly occurs, especially in osteoclasts, skeletal muscle, and trophoblasts of the placenta, the biological significance of multinucleation and the intracellular functions of multinucleation are not well understood. In this unique study, three types of multinucleated cells were generated from human peripheral blood to elucidate the genetic and functional differences between mononucleated and multinucleated cells. Furthermore, by demonstrating the possibility that the morphological peculiarity of multinucleation can regulate cell function, this paper provides clues to understanding the underlying biology of multinucleated cells and how they maintain cell function in homeostatic and pathological settings.
[Authors' reply]. We thank the reviewer for finding our study unique and biologically meaningful. We also thank the reviewer for all the suggestions that improved significantly the overall message of the manuscript.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary: The manuscript of Ahmadzadeh and Pereira et al is an interesting study of the fusion process key to the formation of multinucleated giant cells (MGCs). Our current ability to discriminate between different types of MGCs is limited, and there are gaps in our understanding of the molecular determinants of cell fusion. In this study, the authors isolated different MGC variants -osteoclasts, Langhans giant cells (LGCs) and foreign body giant cells (FBGCs) and identified common, as well as MGC-specific genes and pathways involved in the process of cell fusion. The approach of isolating and comparing different types of MGCs is novel, and the manuscript is well presented and written. However, due to the in vitro nature of the study, the physiological significance of the findings is unclear. I have further minor and major points for the authors to address, as detailed below. 7 Minor comments: 1. The approach to isolate the different MGCs using FACS and imaging technique is highly novel. However the difference between MGC subtypes isolated isn't immediately apparent beyond the morphological comparisons. In my opinion some of the results of MGC-specific assays from Figures 4, 5 and 6 can be included in Figure 1, e.g. TRAP staining and hydroxyapatite resorption for osteoclasts, to provide evidence of purity and specificity of each MGC subtype early on in the manuscript. Classical or canonical genes associated with each MGC subtype can also be highlighted in the volcano plots in Figure 1C, e.g ACP5, CTSK, TNFRSF11A for osteoclasts.  Figure 1B and C). For instance, TRAF3-dependent IRF activation pathway is specific to mononucleated IFN-γdifferentiated macrophages (Supplementary Figure 1B). 4. The authors should also frame/discuss the findings in the context of diagnostic and therapeutic potentials to highlight the clinical significance of this study. Major comments: • As mentioned before, the physiological significance of the findings is unclear. Some form of in vivo data is needed to support some of the key conclusions of the study, e.g validating some of the markers of the pathways identified (common and MGC subtype-specific), and the role of lysosome-mediated iron homeostasis in multinucleation. The authors can make use of the FACs and imaging approaches they developed to look at MGCs in relevant tissues.

[Authors' reply]. This is an important point that we would like to explore in a comprehensive way. We have initiated a 2-year program to undertake a Multiplexed Immunohistochemistry (mIHC)
Full Revision 9 using MILAN (Multiple Iterative Labeling by Antibody Neodeposition) https://www.lpcm.be/multiplex-ihc-milan approach in human biopsies using >100 antibodies. The current study is pivotal in selecting the gene targets (i.e. common and MGC-specific markers) for prioritization. We foresee to gain critical pathophysiological information about the tissue characteristics of MGCs. The reviewer would acknowledge that these high-throughput and biopsy-based initiatives are lengthy and not the primary scope of our current findings which set the foundation of major cellular events governing multinucleation in macrophages.
Reviewer #3 (Significance (Required)): Significance: • The approach of isolating and comparing different types of MGCs is novel, and the findings certainly improved our understanding of the fusion processes of MGCs. However the physiological role of these processes in health and disease that involve MGCs is still unclear due to the lack of in vivo data. The findings were discussed in quite a bit of detail in the context of current literature, though clinical impact was not explored.

[Authors' reply]. We are grateful to Reviewer 3 for raising relevant and constructive points regarding the main findings. His/her review significantly improved the clarity of the overall manuscript.
We recognize our study lacks human clinical association but we highlight the prospective translatability of our findings and the usage of donor-based human macrophages throughout the manuscript. As also recommended by Reviewer 1 in his/her cross-consultation, we discuss the potential clinical impact of our findings in the Discussion of our revised manuscript.

• My background is bone biology with a very keen interest in osteoclast biology so arguably my knowledge on other MGCs eg
LGCs and FBGCs is limited.

7th Nov 2022 1st Editorial Decision
Dear Prof. Behmoaras, Thank you for transferring your revised manuscript from Review Commons to EMBO reports. I have now received the reports from the three referees that had assessed your study at RC that were asked to re-evaluate the manuscript. Please find their comments below. As you will see, the referees now fully support the publication of your study.
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Please use this link to submit your revision: https://embor.msubmit.net/cgi-bin/main.plex Thank you for the submission of your revised manuscript to our editorial offices. We are nearly done. Before I will proceed with formal acceptance, I have these further editorial requests I also ask you to address in a final revised manuscript: -We updated our journal's competing interests policy in January 2022 and request authors to consider both actual and perceived competing interests. Please review the policy https://www.embopress.org/competing-interests and update your competing interests if necessary. Please name this section 'Disclosure and Competing Interests Statement' and put it after the Acknowledgements section. -In Figure 6C the lower panels in the first two rows (IFN-g and IL-4) are completely empty, there is no image noise. For microscopy images, there should be some image noise. The source data is exactly the same and has no image noise. Could you please comment on this?
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-In Figure 6C the lower panels in the first two rows (IFN-g and IL-4) are completely empty, there is no image noise. For microscopy images, there should be some image noise. The source data is exactly the same and has no image noise. Could you please comment on this?
These are high-contrast images and show the resorbed areas by osteoclasts in black against a white background. The output are binary images of each individual well that is subjected to automated analysis. A total white background represents no resorptive activity of the cell which is the case for IFN-g (LGCs) and IL-4 (FBGCs).
-Please provide a completed source data checklist (attached again).

DONE
-No source data for Fig 6D was provided. Please do that or provide an explanation (in the checklist).
Apologies for the mistake. Uploaded now.
-Please note that all corresponding authors are required to supply an ORCID ID for their name upon submission of a revised manuscript. Please do that for cocorresponding author Carine Wouters. Please find instructions on how to link your ORCID ID to your account in our manuscript tracking system in our Author guidelines: http://www.embopress.org/page/journal/14693178/authorguide#authorshipguidelines<ht tps://ddec1-0-enctp.trendmicro.com:443/wis/clicktime/v1/query?url=http%3a%2f%2fwww.embopress.org %2fpage%2fjournal%2f14693178%2fauthorguide%23authorshipguidelines&umid=af20d 579-8b43-4d42-b71b-4a1d96d2f94b&auth=3eee6d57317a38631073652579c10dc620ca2b41-0acc92e0ce3472ecde99d4ca5b66b0c9be3158b7> OK. Carine Wouters ORCID ID is 0000-0002-6426-8845 https://orcid.org/0000-0002-6426-8845?lang=en I have told the author to do this on the manuscript tracking system -if you can link the above ID to her name/email, that would be great too -Please find attached a word file of the manuscript text (provided by our publisher) with changes we ask you to include in your final manuscript text. Please use the attached file as basis for the revision and provide your final manuscript file with track changes, in order that I can see any modifications done.
OK. This is now uploaded. I also responded to specific queries regarding biological/technical replicates -all changes indicated as track changes.
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Reporting Checklist for Life Science Articles (updated January 2022)
ideally, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay. plots include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates.
the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
a statement of how many times the experiment shown was independently replicated in the laboratory.
-common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; Please complete ALL of the questions below. Select "Not Applicable" only when the requested information is not relevant for your study.
if n<5, the individual data points from each experiment should be plotted. Any statistical test employed should be justified. Source Data should be included to report the data underlying figures according to the guidelines set out in the authorship guidelines on Data Presentation.
Each figure caption should contain the following information, for each panel where they are relevant: a specification of the experimental system investigated (eg cell line, species name). the assay(s) and method(s) used to carry out the reported observations and measurements. an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.

Study protocol
Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Include a statement about sample size estimate even if no statistical methods were used. Yes Figure Legends (p17-19) Were any steps taken to minimize the effects of subjective bias when allocating animals/samples to treatment (e.g. randomization procedure)? If yes, have they been described?

Yes
Material and Methods (p11) Include a statement about blinding even if no blinding was done.

Yes
Material and Methods (p14) Describe inclusion/exclusion criteria if samples or animals were excluded from the analysis. Were the criteria pre-established?
If sample or data points were omitted from analysis, report if this was due to attrition or intentional exclusion and provide justification.

Not Applicable
For every figure, are statistical tests justified as appropriate? Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it. Is there an estimate of variation within each group of data? Is the variance similar between the groups that are being statistically compared?
Yes Figure Legends and Material and Methods (p16)

Sample definition and in-laboratory replication Information included in the manuscript?
In which section is the information available?
(Reagents and Tools Table, Materials