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Pesce et al. · Frontiers in Immunology · 2023

TMEM123 a key player in immune surveillance of colorectal cancer

Pesce Elisa, Cordiglieri Chiara, Bombaci Mauro, Eppenberger-Castori Serenella, Oliveto Stefania, Manara Cristina, Crosti Mariacristina, Ercan Caner, Coto Mairene, Gobbini Andrea, Campagnoli Susanna, Donnarumma Tiziano, Martinelli Manuele, Bevilacqua Valeria, De Camilli Elisa, Gruarin Paola, Sarnicola Maria L., Cassinotti Elisa, Baldari Ludovica, Viale Giuseppe, Biffo Stefano, Abrignani Sergio, Terracciano Luigi M., Grifantini Renata

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The study

What was asked, and what was found

Pesce et al., Frontiers in Immunology, 2023 followed a protein they had found on tumour infiltrating lymphocytes, TMEM123, and asked what it does there. Staining of colorectal cancer tissue microarrays showed the protein on infiltrating CD4 and CD8 T cells, and the presence of TMEM123 positive CD8 T cells tracked with better overall and metastasis free survival. Receptor stimulation induced it, peaking at days 2 and 3 with 60-70% of cells positive, and it collected in the protrusions the cell uses to move and to grip.

Testing that meant knocking the protein down in primary human T cells. The team used AUMsilence sdASO. Four oligonucleotides were designed against the target and two were carried forward, both sequences printed in the Methods, with a matched scramble control from the same supplier. They were added straight to the growth medium at 10 and 15 μM. The paper states plainly that the oligonucleotides are taken up by primary cells on their own, with no transfection reagent and no other stress, and that silencing reduced the protein without disturbing the activation state of the cells.

With the protein down, the cells stopped moving properly. Migration of Jurkat cells and of primary CD8 T cells toward colorectal cancer cells was impaired in every condition tested, and passage through a confluent endothelial monolayer fell significantly. Adhesion failed within the first 15 to 30 minutes of imaging, actin protrusions were lost, paxillin dropped, and phosphorylation of focal adhesion kinase and ERK1/2 dropped with it. The actin nucleation machinery followed: less phosphorylated Rac1 and Cdc42, less WAVE2, less Arp3, more N-WASp, and more inactive phosphorylated cofilin. Silenced cells also released less Th1 effector cytokine, mainly tumour necrosis factor alpha but also interferon gamma and interleukin 2. The endpoint was a co-culture of patient derived colorectal cancer organoids with the same patients' CD8 T cells, followed for 7 days: silenced T cells migrated toward the organoids far less, and the organoid cell counts were almost unaltered, where control T cells still carrying TMEM123 had reduced them.

Key findings

  • Silencing TMEM123 with AUMsilence sdASO cut the migration of Jurkat cells and of primary human CD8 T cells toward colorectal cancer cells in every condition tested.(Results, TMEM123 is involved in T lymphocytes motility, chemotaxis and trans-endothelial migration)
  • Silenced T cells also crossed an endothelial monolayer far less readily, so the effect held in the step that gets a lymphocyte out of the bloodstream and into tissue.(Results, TMEM123 is involved in T lymphocytes motility, chemotaxis and trans-endothelial migration)
  • The oligonucleotides were taken up by primary human T cells on their own, with no transfection reagent and no other stress applied, and knocked the protein down without changing the cells' activation state.(Results, TMEM123 expression is associated with activated/effector phenotypes in T cells)
  • Silencing reached the cytoskeletal machinery: paxillin fell, and phosphorylation of focal adhesion kinase and of ERK1/2 fell with it.(Results, Reduction of TMEM123 expression alters cytoskeleton organization and downstream signaling pathways)
  • In silenced CD8 T cells the actin nucleation pathway was down across the board, with less phosphorylated Rac1 and Cdc42, less WAVE2 and less Arp3, and more N-WASp.(Results, Reduction of TMEM123 expression alters cytoskeleton organization and downstream signaling pathways)
  • Silenced T cells released less effector cytokine, with the largest fall in tumour necrosis factor alpha.(Results, TMEM123 expression is associated with activated/effector phenotypes in T cells)
  • The functional endpoint came from patient derived colorectal cancer organoids co-cultured with the same patients' CD8 T cells: silenced T cells migrated toward the organoids less, and the organoid cell counts were almost unaltered where control T cells still carrying TMEM123 had reduced them.(Results, TMEM123 drives migration and clustering of CD8+T lymphocytes in tumor organoids and promotes killing of cancer cells)
  • Silencing also left cofilin phosphorylated at serine 3, the inactive state in which it can no longer act on actin filaments, which completes the picture of a cell that has lost its actin machinery.(Results, Reduction of TMEM123 expression alters cytoskeleton organization and downstream signaling pathways)
  • The authors read the cytokine result as a loss of cytotoxic function, naming tumour necrosis factor alpha first and interferon gamma and interleukin 2 after it.(Discussion)

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