Weekly Digests
‹ Back to July

Turning the tide: reprogrammed tumor cells become APCs

July 26, 2023

One immune evasion mechanism that limits antitumor immunity and response to immunotherapy is the downregulation of antigen processing and presentation in tumors. Zimmermannova and Ferreira et al. aimed to reverse this mechanism by forced expression of dendritic cell transcription factors in tumor cells to induce an antigen-presenting cell (APC) phenotype. Their results were recently published in Science Immunology.

To induce direct cell reprogramming, the researchers used a lentiviral polycistronic vector encoding mouse or human transcription factors PU.1, IRF8, and BATF3 (termed PIB). Having previously shown that PIB can reprogram fibroblasts into type 1 conventional dendritic cells (cDC1), the researchers tested PIB in he poorly immunogenic Lewis lung carcinoma (LLC) and B16F10 melanoma (B16) murine tumor lines. Transduced cells expressed CD45 and MHC-II, as well as the cDC1-specific marker CLEC9A, suggestive of a cDC1-like phenotype. Moving to human cells, the researchers used a panel of 28 solid tumor cell lines and 5 hematological cancer lines. Reprogramming with PIB induced a population of CD45+HLA-DR+ cells, which also expressed CLEC9A, CD226, and CD11c, and had a DC-like morphology, suggestive of induction of the cDC1 phenotype. These cells were therefore named tumor-APCs.

To assess gene expression changes after reprogramming, bulk RNAseq was performed on sorted reprogrammed cells. The reprogrammed cells showed a shift in transcriptome mapping that was similar to natural cDC1s, which allowed the creation of a human tumor-APC signature based on the cDC1 genes that were commonly upregulated in these lines during reprogramming. The upregulated genes in this signature were related to antigen processing and presentation, and immune interactions.

The reprogramming occurred fast, with some tumor-APCs arising as early as day 3 after transduction. To map the kinetics of the reprogramming, cells from the human glioblastoma T98G cell line were transduced with PIB-encoding lentivirus. When reprogrammed and partially reprogrammed tumor-APCs from various time points were subjected to RNAseq and ATAC sequencing, all time points clustered separately from parenteral cells, and the cells obtained at days 7 and 9 clustered closest to peripheral cDC1s. Epigenetic remodeling occurred mostly in the first three days, and chromatin remodeling was induced, as genes associated with cDC1 function had closed chromatin in parental cells, and open chromatin regions after reprogramming in transduced cells.

Mouse tumor-APCs had increased expression of β2-microglobulin and MHC-I, and human tumor-APCS had increased levels of HLA-ABC and HLA-DR, suggesting increased tumor immunogenicity. To determine if tumor-APCs could present tumor-associated antigens, the researchers applied mass spectrometry-based immunopeptidomics on B16-derived tumor-APCs. Tumor-APCs presented more peptides than controls, which were predicted to bind MHC-I, and included peptides that originated from known melanoma-associated antigens.

PIB transduction also induced costimulatory molecules on both mouse and human tumor cells. Coculture experiments of tumor-APCs (derived from B16 and LLC lines) and OT-1 cells showed that the tumor-APCs primed naive OT-1 CD8+ T cells. When B16-OVA tumor-APCs were cocultured with untreated B16-OVA cells with increasing ratios of activated OT-I cells, the tumor-APCs were more susceptible to T cell-mediated killing. At high T cell to target cell ratios, there was also killing of the non-target population, potentially caused by a bystander killing effect.

Further assessing the cDC1 function of tumor-APCs, the researchers found that the cells secreted IL-12p70, IL-29, CXCL10, and TNFα in response to Poly(I:C) and LPS. Tumor-APCs were also able to take up protein (OVA) and dead cells and process internalized antigens. Pulsing of naive OT-1 CD8+ T cells with the OVA peptide SIINFEKL while in coculture with tumor-APCs induced proliferation of the T cells. Tumor-APCs were also shown to cross-present antigens to CD8+ T cells in a coculture in which OVA protein was pulsed.

Zimmermannova and Ferreira and team then determined whether primary tumor cells could also be reprogrammed to tumor-APCs. To test this, samples were collected from patients with various solid cancer types. In all primary samples, CD45 and HLA-DR expression was induced in subsets of cells. Reprogrammed cells were purified and subjected to single-cell RNAseq. Most reprogrammed cells clustered closely to peripheral blood cDC1 and separate from parenteral cells. Reprogrammed cells had increased expression of cDC1 canonical markers, reprogramming markers, costimulatory molecules, endogenous IRF8 and BATF3, and the tumor-APC signature. Melanoma tumor-APCs were then stimulated with HLA-A2-restricted CMV pp65 and MART-1 peptides and cocultured with CD8+ T cells from HLA-A2+CMV+/MART-1+ donors. This led to an induction of memory CMV+CD8+ T cells and naive CD8+ T cells, similar to results obtained using monocyte-derived DCs.

Next, the tumor-APCs from primary melanoma were cocultured with tumor-infiltrating lymphocytes (TIL) from the same patient. After 8 hours, the TIL had increased expression of CD107a and CD137, and the cytokines IFNγ and TNFα, which correlated with increased cytotoxicity against the reprogrammed tumor cells. These TIL also had higher expression of BTLA, TIM3, LAG3, PD-1, CD28, and CD69. When anti-PD-1 or anti-CTLA-4 was added during the cultures, there was a small increase in cytolysis of the reprogrammed cells.

RNAseq of tumor-APCs showed that genes related to cell cycle progression and proliferation downregulated over time after reprogramming, and tumor suppressor genes were activated. In vitro experiments confirmed that reprogrammed cells had slower cell division than parental cells, and their colony formation was dramatically reduced. These data suggest that the reprogrammed cells lost some of their malignant characteristics.

Finally, in vivo experiments were conducted in syngeneic mice with B16-OVA tumors. The researchers generated B16-derived tumor-APCs, which were pulsed with OVA protein and stimulated with Poly(I:C), after which they were injected intratumorally. This treatment delayed tumor growth and prolonged survival. Increased numbers of tumor-specific T cells were detected in the blood and in tumor-draining lymph nodes (TDLN) after treatment. In the tumors, there was an increase in T and NK cells, including CD44+PD-1+ and CD44+PD-1- CD8+ and CD4+ T cells. Adding anti-PD-1 and anti-CTLA-4 treatment further reduced tumor growth and improved survival, suggesting a synergistic effect.

These data show that tumor cells can be reprogrammed into cDC1-like cells to activate tumor antigen presentation and overcome immune evasion mechanisms. The synergistic results seen in this study with immune checkpoint blockade suggest that this strategy might be a promising treatment in tumors with a “cold” immune environment.

Write-up by Maartje Wouters, image by Lauren Hitchings

Meet the Researcher

This week, co-first authors Olga Zimmermannova and Gabriela Ferreira answered our questions.

The Pereira lab behind the study (from left to right): Olga Zimmermannova, Gabriela Ferreira, Filipe Pereira, Ervin Ascic, and Ilia Kurochkin.

What was the most surprising finding of this study for you?
We were initially surprised that the reprogramming process could be initiated in cancer cells from multiple tumor types, and that this process was extremely fast. However, we got most excited by the level of synergism that reprogrammed tumor antigen-presenting cells (tumor-APCs) reached in combination with immune checkpoint inhibitors in vivo. The game-changing moment was to see our first tumor-bearing animal being completely cleared of cancer with the tumor-APC reprogramming strategy. On the other hand, the most relieving moment was when we confirmed that reprogramming abolished tumorigenic properties in cancer cells.

What is the outlook?
In this study, we provided proof of principle that mouse and human cancer cells could be reprogrammed into antigen-presenting cells, and that reprogrammed tumor-APCs can initiate robust antitumor immune responses in vivo. The next step is to elicit the reprogramming of cancer cells in situ within tumors. We will place our efforts in translating our findings into an innovative cancer immunotherapy based on in vivo reprogramming of cancer cells. This spans from the development of systems allowing efficient delivery of the reprogramming factors into the tumors, preclinical safety studies, and the development of spheroid-based platforms to predict efficacy of tumor-APC based immunotherapy in patients.

What was the coolest thing you’ve learned (about) recently outside of work?
OZ: In general: We can smell petrichor (odor of soil after rain) better than sharks can smell blood. Concerning my summer plans: that you no longer need a visa to travel to Uzbekistan.
GF: There is such a thing as too much water for aloe vera. Learned it the hard way. Learned this week: Saturn's rings might be the result of a collapsed moon, and we are just lucky to live at the same time we can see them, because they might disappear over time.


Zimmermannova O, Ferreira AG, Ascic E, Velasco Santiago M, Kurochkin I, Hansen M, Met Ö, Caiado I, Shapiro IE, Michaux J, Humbert M, Soto-Cabrera D, Benonisson H, Silvério-Alves R, Gomez-Jimenez D, Bernardo C, Bauden M, Andersson R, Höglund M, Miharada K, Nakamura Y, Hugues S, Greiff L, Lindstedt M, Rosa FF, Pires CF, Bassani-Sternberg M, Svane IM, Pereira CF. Restoring tumor immunogenicity with dendritic cell reprogramming. Sci Immunol. 2023 Jul 14.

In the Spotlight...

Chimeric TIM-4 receptor-modified T cells targeting phosphatidylserine mediates both cytotoxic anti-tumor responses and phagocytic uptake of tumor-associated antigen for T cell cross-presentation

Cieniewicz et al. transduced T cells to express the chimeric receptor CER-1236, consisting of the extracellular domain of TIM-4, which recognizes phosphatidylserine (PS) as a phagocytic target, fused with the intracellular signaling domains TLR2/TIR, CD28, and CD3ζ. CER-1236 T cells showed in vitro phagocytic and cytotoxic activity against MCL and EGFR-mutated NSCLC expressing PS, expression of which could be enhanced by treatment with BTK or EGFR inhibitors, respectively. CER-1236 T cells could cross-present antigens in in vitro assays, and infusion into NSG mice bearing MCL or NSCLC tumors induced tumor regression and improved survival.

Contributed by Maartje Wouters

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity

Koyama et al. showed that steady-state expression of MHC-II by ileal intestinal epithelial cells (IEC) differed in genetically identical mice, depending on the vendor of origin. Analysis of fecal and ileal samples identified vancomycin-sensitive bacterial taxa, transferrable by cohousing, that positively associated with IFNγ-induced IEC MHC-II expression. Taxa negatively regulating IEC MHC-II expression were also identified, but were not transferred by cohousing. Pre-graft oral vancomycin treatment of recipient mice reduced IEC MHC-II expression and CD4+ T cell-mediated GVHD. The pre-transplant fecal microbiota profile correlated with outcomes for a clinical cohort receiving allogeneic stem cell transplants.

Contributed by Paula Hochman

Clec4A4 Acts As A Negative Immune Checkpoint Regulator to Suppress Antitumor Immunity

Uto et al. showed that when the homotypic C-type lectin receptor Clec4A4, found on certain cDC subsets, was knocked out in mice, the mice exhibited reduced tumor progression and enhanced antigen-specific priming of adoptively-transferred T cells in TdLNs. The TMEs of Clec4A4 KO mice had more activated cDCs and TILs, fewer MDSCs, and lower expression of suppressive soluble factors than the TMEs of WT mice. Treating human CLEC4A-Tg mice with a blocking anti-human CLEC4A mAb inhibited progression of established tumors and Treg cell accumulation in tumor tissues, boosted antitumor responses without inducing adverse events, and had additive effects when combined with anti-PD-1.

Contributed by Paula Hochman

Immunosurveillance shapes the emergence of neo-epitope landscapes of sarcomas, revealing prime targets for immunotherapy

Using the relative increase in MHC binding of mutated versus native neoepitopes (DAI), Osei-Hwedieh et al. demonstrated that patients with osteosarcomas presented significantly more high-DAI neoepitopes than leiomyosarcomas or liposarcomas. Evidence for effective immunosurveillance was dependent on antigenicity, as patients with osteosarcomas mounted poor antitumor immune responses (based on TCR analysis), allowing the retention of higher-DAI antigens, whereas patients with leiomyosarcomas mounted strong T cell responses. Vaccination with tumor-derived gp96 (a heat shock protein), alone or in combination with checkpoint blockade, prolonged survival in a mouse osteosarcoma model.

Contributed by Shishir Pant

S-531011, a novel anti-human CCR8 antibody, induces potent anti-tumor responses through depletion of tumor-infiltrating CCR8-expressing regulatory T cells

Nagira et al. investigated the impact of selective reduction of CCR8+ tumor-infiltrating Tregs on antitumor immunity, and identified a high-affinity, humanized, anti-human CCR8 mAb, S-531011 that selectively bound CCR8+ cells, neutralized CCR8 signaling, and exhibited potent ADCC (but not CDC) towards tumor-infiltrating CCR8+ Tregs, but not Tregs from PBMC. In a human CCR8 knock-in mouse tumor model, S-531011 combined with anti-PD-1 selectively depleted tumor-infiltrating Tregs, but not effector T cells, and suppressed tumor growth compared to anti-PD-1 alone, with no observable adverse effects, suggesting translational potential.

Contributed by Katherine Turner

ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

Mota et al. demonstrated that the spontaneous ALK-specific T cell responses generated in mice with ALK+ lung tumors were insufficient to achieve an effective antitumor response and were not enhanced by ICIs. Vaccination with a single ALK peptide increased intratumoral ALK-specific CD8+ T cells, delayed tumor progression, eradicated lung tumors in combination with ALK TKIs, and prevented the metastatic spread to the brain. In vaccinated mice, tumors escaped due to reversible MHC-I downregulation. ALK immunogenic peptides were identified in the context of two frequent HLA molecules (HLA-A*02:01 and HLA-B*07:02) from human tumor samples.

Contributed by Shishir Pant

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.