Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
The authors were attempting to describe whether trained innate immunity would modulate antibody-dependent cellular phagocytosis (ADCP) and/or efferocytosis.
Strengths:
The use of primary murine macrophages, and not a cell line, is considered a strength. The trained immunity-mediated changes to phagocytosis affected both melanoma and breast cancer cells. The broad effect is consistent with trained immunity.
Weaknesses:
The most significant weakness, also noted by the authors in the discussion, is the lack of in vivo data. Without these data, it is not possible to put the in vitro data in context. It is unknown if the described effects on efferocytosis will be relevant to the in vivo progression of cancer.
We thank the reviewer for these comments. To examine the role of trained immunity on the modulation of macrophage efferocytosis in vivo, we performed immunostaining analysis in sections from B16F10 tumour samples.
Importantly, we found that macrophage efferocytosis of apoptotic tumour cells was significantly decreased in the tumour tissue that was excised from mice treated with β-glucan 7 days prior to tumour inoculation (supplementary Figure 3). These data are consistent with our findings using co-culture assays further strengthening the impact of our key findings in this report.
Reviewer #2 (Public review):
Summary:
The authors follow up their preclinical work on beta-glucan-induced trained immunity in murine tumor models that they published in Cell in 2020. In particular, they focus on the role of trained immunity and efferocytosis of cancer cells
Strengths:
While properly conducted, the work is underwhelming and fully depends on in vitro observations performed with co-cultures of bone marrow derived macrophages from beta-glucantreated mice and tumor cell lines. From these in vitro studies, the authors conclude that trained immunity induction has no effect on antibody-dependent cellular phagocytosis, while it decreases efferocytosis.
Weaknesses:
It would be important to study these phenomena in tumor mouse models in vivo. The authors clearly have the expertise as they have shown in previous studies. Especially because the in vitro observation appears to conflict with the in vivo anti-tumor found in mice prophylactically treated with beta-glucan. Clearly, trained immunity is associated with diverse cellular responses and mechanisms, some of which may promote tumor growth, as the current manuscript suggests, but in the absence of in vivo studies, it is merely a mechanistic exercise of which the relevance is difficult to determine.
We thank the reviewer for raising this important comment. We have followed reviewer’s suggestion and examined the role of trained immunity on the modulation of macrophage efferocytosis in vivo. As mentioned in our response to Reviewer 1, we demonstrate that efferocytosis of apoptotic melanoma cells in situ was attenuated in tumour samples from ‘trained’ mice as compared to those from controltreated mice.
Efferocytosis displays a pro-tumour and immunosuppressive role, therefore both our in vitro co-culture (Figure 1) and in vivo (supplementary Figure 3) findings are consistent with our previously published in vivo data supporting the tumour-suppressive role of prophylactic treatment with β-glucan (Kalafati, Kourtzelis et al, PMID: 33125892).
Reviewer #3 (Public review):
Summary:
Chatzis et al showed that β-glucan trained macrophages have decreased phagocytic activity of apoptotic tumor cells and that is accompanied by lower levels of secreted IL-1β using a mouse model. Strengths: This finding has a potential impact on designing new cancer immunotherapeutic approaches by targeting macrophage efferocytosis.
Weaknesses:
Whether this finding could be applied to other scenarios is underdetermined.
(1) Does the decrease of efferocytosis also occur in human monocytes/macrophages after training?
(2) Both β-glucan and BCG are well-trained innate immunity agents, the authors showed that β-glucan decreased efferocytosis via IL-1 β, so it is interesting to know whether BCG has a similar effect.
We thank the reviewer for these comments. Our data suggest that induction of trained immunity with β-glucan contributes to decreased macrophage efferocytosis of tumour cells based on co-culture and in vivo approaches in a mouse setting.
We agree with the reviewer that utilisation of a human setting would be important to provide additional validation of our findings.
Induction of trained immunity entails epigenetic and metabolic reprogramming of hematopoietic stem and progenitor cells (HSPCs). As such, the elucidation of mechanisms that modulate trained immunity in human cells would require the establishment of a macrophage differentiation model based on the use of HSPCs rather than the stimulation of monocytes or macrophages with β-glucan.
Additionally, the investigation of the impact of BCG in trained immunity-dependent phagocytosis would require the assessment of all different types of phagocytic cargos (apoptotic melanoma and breast cancer cells, apoptotic neutrophils, microbial bioparticles) as we did in the case of the β-glucan. The capacity of different molecules to induce trained immunity in the efferocytosis setting requires further investigation that would be beyond the scope of this study. Therefore, we plan to address these very interesting points in a future study.
Additional text was added in the Discussion section to clarify the reviewer's points. In addition, we provide a more specific title that reflects better the specificity of our findings.
