Weekly Digests
‹ Back to February

ICAM-1 helps tumor exosomes suppress immunity

February 2, 2022

Tumors employ diverse tactics to suppress CD8+ T cell immunity, one of which is secreting tumor-derived extracellular vesicles (TEVs) that express PD-L1. However, exactly how TEV PD-L1 interacts with CD8+ T cells remains unknown. Recently reported in Developmental Cell, Zhang and Zhong et al. uncovered a novel role of the adhesion molecule ICAM-1 on TEVs for dampening CD8+ T cell activity.

To begin, Zhang, Zhong, and the team characterized ICAM-1 expression on TEVs from human melanoma cell lines. They found that ICAM-1 was specifically enriched in melanoma-derived exosomes (as opposed to microvesicles) and upregulated along with PD-L1 in response to IFNγ exposure. Initially observed through Western blotting, this result was recapitulated using an ELISA the authors developed in house to characterize TEV-expressed proteins. Further confirming that ICAM-1 is expressed in TEVs, the authors found that ICAM-1 co-localized and co-immunoprecipitated with Hrs, a critical component of the EV loading and release pathway, within melanoma cells. shRNA knockdown of Hrs or Rab27a, another regulator of exosome release, reduced ICAM-1 levels in secreted exosomes. Altogether, these results demonstrate the presence of ICAM-1 on TEVs.

Next, the researchers questioned whether ICAM-1 colocalizes with PD-L1 on TEVs. Using immuno-electron microscopy, they visualized expression of these molecules on the same exosomes. Consistent with their prior findings, the fraction of ICAM-1+PD-L1+ exosomes increased with IFNγ treatment. Furthermore, depletion of ICAM-1+ exosomes using anti-ICAM-1 Dynabeads reduced PD-L1 levels compared to non-depleted exosomes and the bead-enriched exosomes. Interestingly, ELISA analysis found higher ICAM-1 expression on PD-L1+ exosomes isolated from the plasma of metastatic melanoma patients compared to those isolated from healthy patients. Thus, ICAM-1 and PD-L1 can be co-expressed on exosomes and may play supporting roles in tumor progression.

The team next characterized TEV interactions with CD8+ T cells. CFSE-stained exosomes could bind to CD8+ T cells, and significantly better when the cells were pre-stimulated. Blocking ICAM-1, either through an anti-ICAM-1 mAb or shRNA-mediated ICAM-1 knockdown, diminished this binding. To determine which receptor could be binding ICAM-1, the researchers targeted LFA-1 or Mac-1 – known ICAM-1 binding partners. Blocking LFA-1, but not Mac-1 reduced TEV binding of CD8+ T cells, and accordingly, LFA-1 was upregulated on CD8+ T cells after stimulation. These results indicated that ICAM-1 on TEVs may bind CD8+ T cells, specifically through LFA-1, which is upregulated by T cells upon their activation.

The authors then questioned whether ICAM-1 on TEVs plays a role in CD8+ T cell suppression. First, they confirmed that exposure to melanoma TEVs reduced CD8+ T cell functionality. Indeed, NFAT activation by a Jurkat reporter cell line; T cell proliferation; Ki67 and GzmB expression; and IL-2, TNFα, and IFNγ secretion were all diminished upon incubation of T cells with TEVs. Importantly, blockade of ICAM-1 (either through an anti-ICAM-1 mAb or shRNA knockdown) on the TEVs restored these functional parameters. These results were not unique to melanoma; colon and lung cancer TEVs had similar suppressive effects that were reversed by ICAM-1 blockade. PD-L1 was necessary for this suppression, but not involved in T cell binding, as, compared to wild-type melanoma TEVs, PD-L1-/- TEVs bound T cells similarly, but had reduced suppressive capacity.

Next, Zhang and Zhong et al. explored their findings in vivo using YUMM1.7 melanoma tumors. Tumor-bearing mice injected intravenously with TEVs showed accelerated tumor growth, decreased CD8+ T cell infiltration, and reduced Ki67 and GzmB expression among intratumoral PD-1+ CD8+ T cells, compared to control mice that were not given TEVs. However, these effects were lost when the infused exosomes were derived from ICAM-1-/- YUMM1.7 cells or wild-type exosomes pre-treated with anti-ICAM-1 mAb. These findings were mirrored in the MC38 colon cancer model. These in vivo results complemented the team’s prior studies, further indicating a role of ICAM-1 in abetting TEV-mediated immunosuppression.

Finally, the researchers developed an exosome–cell receptor binding assay to confirm the role of ICAM-1 in T cell suppression through PD-L1. They leveraged SrtA, a bacterial protein used in labeling studies that can transfer a biotin-labeled peptide motif to a target bearing a GlyGlyGlyGlyGly (G5) tag. The authors engineered melanoma cells to express SrtA fused to PD-L1, and Jurkat T cells to express the G5 tag fused to PD-1. In theory, the melanoma cells would produce TEVs expressing the PD-L1–SrtA fusion, which would be capable of labeling G5–PD-1 on the T cells when brought in proximity. The labeling performed as expected when the engineered T cells and TEVs were co-incubated, confirming that PD-L1 on the TEVs and PD-1 on the T cells do come in contact. Importantly, pre-treatment of TEVs with anti-ICAM-1 mAb or ICAM-1 knockdown reduced this labeling, suggesting that ICAM-1 may be required for optimal PD-L1–PD-1 binding between TEVs and T cells.

In summary, Zhang and Zhong et al. found a critical role of the adhesion molecule ICAM-1 on tumor cell-derived exosomes in enabling PD-L1-mediated immunosuppression of CD8+ T cells. Further understanding mechanisms of tumor resistance to endogenous immunity or immunotherapy could support advances in immune checkpoint blockade and other cancer therapies.

Write-up by Alex Najibi, image by Lauren Hitchings


Zhang W., Zhong W., Wang B., Yang J., Yang J., Yu Z., Qin Z., Shi A., Xu W., Zheng C., Schuchter L.M., Karakousis G.C., Mitchell T.C., Amaravadi R., Herlyn M., Dong H., Gimotty P.A., Daaboul G., Xu X., Guo W. ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression. Dev Cell. 2022 Jan 25.

In the Spotlight...

Short-course neoadjuvant in situ vaccination for murine melanoma

Aiken et al. demonstrated that neoadjuvant vaccination with an intratumorally delivered anti-GD2-targeted IL-2 (IT-IC; an immunocytokine) did not inhibit primary tumor growth, but led to increased CD8+ T cell infiltration, a higher CD8:Treg ratio, and development of immunologic memory against contralateral flank rechallenge post-resection in a B78 melanoma model. Immunological memory development was independent of challenge timing after surgery. Neoadjuvant IT-IC in GD2+ B16 melanoma led to less toxic wound healing (compared to radiation therapy plus IT-IC), and reduced lung metastasis in a CD4+ T cell-dependent manner after rechallenge with GD2- B16 cells.

Contributed by Shishir Pant

SLAMF3 and SLAMF4 are immune checkpoints that constrain macrophage phagocytosis of hematopoietic tumors

Li et al. showed that grafts of nucleated hematopoietic cells lacking expression of homotypic SLAM family receptors (SFR), notably SLAMF3 and SLAMF4, were cleared by phagocytic WT macrophages triggered in part by their LRP1 “eat-me receptors” to induce mTORC1 and Syk through SHP-1/2. SFR deficiency of hosts reduced hematopoietic tumor burden in murine models, and led to more severe TLR agonist-induced murine hemocytopenia. CAR-modified macrophages from SFR-negative hosts, or antibody blockade of SLAMF2 (a ligand for SLAMF3) and/or SLAMF3 increased phagocytosis of CD19+  targets in vitro.

Contributed by Paula Hochman

REVIEW: B Cell Function in the Tumor Microenvironment

B cells are often left out of conversations about the TME, and while older studies suggested that they may support tolerance in tumors, particularly through IL-10, more recent evidence has illuminated the heterogeneity of B cells and their potential antitumor functions. This review by Downs-Canner et al. discussed the complex range of B cell phenotypes, which may be more dependent on their environment (such as in TLSs) than on lineage-defined subsets. There is a need to better understand the roles of B cells and to identify markers for different B cell states, as B cells may contribute to antitumor immunity, responses to immunotherapy, and adverse events.

Contributed by Lauren Hitchings

Magnesium sensing via LFA-1 regulates CD8+ T cell effector function

Lötscher et al. demonstrated the magnesium dependence of co-stimulatory cell-surface molecule LFA-1-mediated activation and effector function of CD8+ T cells. Upon interaction with extracellular magnesium, LFA-1 adopts its active conformation and initiates outside-in signaling on CD8+ T cells, leading to increases in calcium flux, metabolic reprogramming, immune synapse formation, and cytotoxicity. LFA-1-sensed magnesium sufficiency improved infection/tumor-specific T cell function and enhanced the effectiveness of immunotherapeutic modalities. In patients treated with CAR T cells and ICI, low serum magnesium levels correlated with poor prognosis.

Contributed by Shishir Pant

Effector memory CD4+ T cells induce damaging innate inflammation and autoimmune pathology by engaging CD40 and TNFR on myeloid cells

McDaniel, Chawla, and Jain et al. investigated mechanisms of inflammatory cytokine pathology common to T cell-mediated autoimmune diseases and cancer immunotherapies. Effector memory CD4+ T cells (TEM) were shown to interact with myeloid cells, such as DCs and macrophages, and to stimulate CD40- and TNFR-dependent inflammation, which was independent of pattern recognition receptor activation. Blocking TNF and CD40 signaling in several models of T cell-driven inflammation rescued animals from cytokine storm and toxicity, suggesting TEM-driven innate myeloid cell activation is a key trigger of immunopathology. 

Contributed by Katherine Turner

Lung tumor MHCII immunity depends on in situ antigen presentation by fibroblasts

MHC-II+ fibroblasts (apCAFs), detected in abundance in TME of human NSCLC, triggered activation of autologous, freshly sorted CD4+ NSCLC TILs upon coculture. Activation was MHC-II-dependent. Human and mouse apCAFs were phenotypically similar, and a requirement for IFNγ and in vivo acquisition of tumor antigen was shown for apCAFs in mice. C1q overexpression was detected on human apCAFs, and its ligand, C1qbp, was detected by FACS on primary human TILs. Both human and mouse CD4+ T cells were rescued from apoptosis by C1q – implicating C1q as an extrinsic pro-survival signal.

Contributed by Margot O’Toole

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.