Tumor-derived extracellular vesicles regulate tumor-infiltrating regulatory T cells via the inhibitory immunoreceptor CD300a

  1. Yuta Nakazawa
  2. Nanako Nishiyama
  3. Hitoshi Koizumi
  4. Kazumasa Kanemaru
  5. Chigusa Nakahashi-Oda
  6. Akira Shibuya

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Abstract

Although tumor-infiltrating regulatory T (Treg) cells play a pivotal role in tumor immunity, how Treg cell activation are regulated in tumor microenvironments remains unclear. Here, we found that mice deficient in the inhibitory immunoreceptor CD300a on their dendritic cells (DCs) have increased numbers of Treg cells in tumors and greater tumor growth compared with wild-type mice after transplantation of B16 melanoma. Pharmacological impairment of extracellular vesicle (EV) release decreased Treg cell numbers in CD300a-deficient mice. Coculture of DCs with tumor-derived EV (TEV) induced the internalization of CD300a and the incorporation of EVs into endosomes, in which CD300a inhibited TEV-mediated TLR3–TRIF signaling for activation of the IFN-β-Treg cells axis. We also show that higher expression of CD300A was associated with decreased tumor-infiltrating Treg cells and longer survival time in patients with melanoma. Our findings reveal the role of TEV and CD300a on DCs in Treg cell activation in the tumor microenvironment.

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  1. Version published to 10.7554/elife.61999 on eLife
  2. eLife

    This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on October 30 2020, follows.

    Summary

    Using a mouse model of melanoma, this report demonstrates the relevance of the CD300a immunoreceptor, specifically in dendritic cells (DCs), in tumor growth. It shows that the absence of CD300a is correlated with a higher number of regulatory T cells (Tregs) within the tumor microenvironment and therefore the tumor grows faster and survival decreases. Based on additional experiments, the authors propose a mechanism by which tumor-derived extracellular vesicles (TEVs) interact with CD300a in DCs, decreasing IFNbeta production which subsequently reduces the number of Tregs. In addition, data from melanoma patients show a correlation between overall survival and higher levels of CD300a expression in the tumor.

    Essential Revisions

    1. It is highly recommended to clearly demonstrate the role of IFNbeta in the proposed mechanism. In addition to using an anti-IFNbeta mAb in an in vitro culture (Figure 3D), other experiments must be performed, such as in vivo experiments with the anti-IFNbeta mAb. The authors have used this mAb in their previously published article (Nakahashi-Oda et al., Nature Immunology, 2016). Alternatively, in vivo experiments could also be performed with IFNAR1-like (IFN alpha and beta receptor 1 subunit) KO animals.

    In addition, is the observed increase in Tregs within the tumor in CD300a-/- animals due only to an increase in IFNbeta production by DCs? Are there other cytokines and/or cell-cell contact that may play a role? At least this should be discussed.

    1. Why are not all the experiments performed on CD300afl/fl Itgax-Cre mice instead of CD300a-/- mice? The experiments in Figures 2a, S2C, 3 and 4 should have been performed on CD300afl/fl Itgax-Cre mice. This is very important to state unequivocally that only CD300a in DCs is involved in the induction of an immune response capable of inhibiting tumor development.

    2. The authors found expansion of tumor-infiltrating Tregs in mice deficient in CD300a. However, no increase in Tregs was observed in tumor-draining lymph nodes. Did authors assess the expression of Treg activation and proliferation molecular markers, such as CD25, CTLA4, GITR, CD39, CD73 or Ki67? If indeed, Treg expansion as a result of CD300a-deficiency is the cause of enhanced tumor growth, authors should provide more evidence of Treg suppressive response. For example, authors can consider measuring the levels of co-stimulatory molecules (e.g. CD40, CD80 and CD86) on dendritic cells, which generally correlate with Treg activitiy and/or tumoral IL-2 concentration.

    3. PD-1 is the only marker analyzed to assess the exhausted status of CD8+ T cells infiltrating tumor lesion of CD300a-/- mice. Additional evidence of this functional status could be provided, such as for instance expression of CTLA4, TIM3 or other immune checkpoints, or low Ki67 levels. Indeed, particularly in reference of the human setting, PD-1 is also a sign of T cell activation, usually expressed in T cells infiltrating highly immunogenic and hot tumors. Hence, it would be useful having a broader characterization of immune effectors associated with progressing tumor microenvironment when CD300a is lost.

    4. Since authors have Foxp3-reporter mice, they should confirm their data in Fig. 3D with natural / freshly isolated Tregs, unless they are suggesting that CD300a mainly prevents in situ conversion of intra-tumoral CD4+Foxp3- Tconv cells into Tregs.

    5. Given that the interaction between CD300a and phosphatidylserine (PS) is critical to CD300a activation, PS co-localization with CD300a ought to be included in confocal microscopy. In addition, the binding of CD300a to PS and PE, which are both upregulated in dead cells, implies that apoptotic bodies could also be shuttling comparable signaling, Can the authors exclude that these particles are present in the EV preparations? Furthermore, does tumor supernatant lose any effect when depleted of EVs? The latter evidence could significantly strengthenthe exclusive involvement of exosomes in the process.

    6. Did authors validate the importance of PS in the context that they propose with an anti-PS blocking antibody? There are not many anti-PS blocking antibodies available and they might not block engagement with CD300a (see Nat Commun. 2016 Mar 14;7:10871). Nonetheless, this would be a good assay to demonstrate PS as the ligand that triggers CD300a to inhibit TLR3 and subsequent IFN-β production.

  3. Version published to 10.1101/2020.11.10.376715v1 on bioRxiv