Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

Reviewer #1 (Public Review):

Farhat-Younis and colleagues demonstrate tumor-specific IgM's capacity to induce tumor cell death in monocyte-derived dendritic cell cultures. They subsequently designed a chimeric receptor based on high-affinity FcRI. However, the authors found that the transfection process was more efficient when either the variable light or heavy chain was transfected individually rather than the entire scFv. This scFv construct led to an endoplasmic reticulum (ER) stress response and scFv degradation. A considerable portion of the manuscript is dedicated to the negative scFv expression results. The authors pivoted to a modified FcgRI capable of transmitting IgM signals. This represents a tremendous amount of work in the development of this chimeric receptor, the critical experiment showing efficacy in vivo was not presented, and instead various in vitro assays are shown. Thus, this manuscript will markedly benefit from showing improved responses to tumors in vivo when macrophages express FcgRI-IgM.

1. In a mouse tumor model, the authors demonstrated that monocyte-derived dendritic cells (MoDCs) treated with IgG immune complexes (ICs) were more effective at preventing tumor growth compared to those treated with IgM ICs (as shown in Figure 1B). In Figure 1C, their in vitro experiments revealed that IgM resulted in tumor cell death, as well as increased production of nitric oxide (NO) and granzyme B.
How do the authors reconcile IgG IC-treated MoDCs performing better in preventing tumors in vivo than IgM IC-treated MoDCs, despite the in vitro results with IgM-ICs. The authors speculate that IgG IC-treated MoDCs might trigger T cell immunity but do not show T cell involvement.

2. The authors report distinct functional consequences of MoDCs incubated with tumor-IgG complexes and tumor IgM complexes. Tumor growth was inhibited and T cell immunity induced with the former. The latter, however, elicited robust anti-tumor killing. What happens if MoDCs are incubated with both IgG and IgM complexes? If this combined treatment induces effective killing and T cell memory, would this impact the design of the chimeric receptor to include IgG responsiveness as well?

3. In Figure 5H, the authors demonstrate the ability of the chimeric receptor construct to deplete tumor cells in vitro. The ms would improve if the authors could show the chimeric receptor construct results in tumor cell death and/or prevention in an in vivo model. Similarly, if combined stimulation with IgG and IgM complexes enhances tumor response, this should be incorporated into the therapeutic strategy.

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.

In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:
In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Weaknesses:

Nevertheless, there are several minor issues that should be addressed:

1- TCR fragments are commonly used to induce ER stress in non-immune cells. Therefore, it would be interesting to investigate whether TCR fragments can be expressed in myeloid cells and if they induce ER stress. Addressing this issue would support the notion that these cells lack the ER chaperones required for folding immunoglobulin variable chains.
2- It would be valuable to determine whether, after the degradation of scFv fragments by myeloid cells, they are presented on MHC-I and MHC-II.
3- Some methodological details, such as the vaccination protocol and high-resolution microscopy procedures, are missing from the text.