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Sep 12, 2011

Free Radical Produced by Myeloid Cells Implicated in Blocking CTL Therapy

  • Studies in mice and evaluation of human tumor tissue have provided new insights into a mechanism responsible for the failure of CTL-based cancer immunotherapies to kill tumors. Researchers at the H. Lee Moffitt Cancer Center and collaborators found that myeloid cells infiltrating tumor sites modify the pMHC complexes on tumor cells, which reduces the capacity of the MHC molecules to bind antigenic peptides and subsequently be recognized by CTLs.

    The mechanism involves PNT production by myeloid-derived suppressor cells (MDSCs), which inhibits binding of processed peptides to tumor cell-associated MHC, causing resistance to antigen-specific CTLs, claim Dmitry Gabrilovich, M.D., and colleagues. Using a mouse model of tumor-associated inflammation in which the antitumor effects of antigen-specific CTLs are eradicated by tumor-cell expression of IL-1β, the researchers confirmed that therapeutic failure is not caused directly by IL-1β suppression of CTLs but rather by the presence of the free radical peroxynitrite  (PNT) produced by recruited MDSCs.

    The Moffitt team’s work is published in The Journal of Clinical Investigation in a paper titled “Tumor-infiltrating myeloid cells induce tumor cell resistance to cytotoxic T cells in mice.”

    Despite the fact that it is now possible to induce tumor-specific immune responses in most patients treated with various types of cancer immunotherapy, the proportion of patients who benefit clinically from these treatments remains small, Dr. Gabrolovich et al. report. The tumor environment is clearly one source of protection against even potent CTL responses, but results from mouse experiments and clinical trials in patients with adoptive transfer of antigen-specific T cells suggests that inhibition of CTLs at the tumor site may not be the actual cause.

    The researchers built on their previous work demonstrating that in mice, myeloid-derived suppressor cells (MDSCs) infiltrating the tumor are a source of the free radical peroxynitrite (PNT). Their finding suggested that MDSCs induceT-cell tolerance via production of PNT and nitration/nitrosylation of TCRs and CD8 molecules on the surface of T cells.

    In effect the TCRs lost the ability to recognize specific peptide/MHC (pMHC) complexes and perform their antitumor activity. Nitrosylation of tyrosine residues has been long recognized as a marker of PNT activity, the researchers add. Their previous findings, which combined studies demonstrasting high levels of nitrotyrosine (NT) in a range of cancers, led the team  to investigate whether increased levels of PNT at the tumor site causes post-translational modifications in tumor cells and render them resistant to CTLs.

    Their initial findings showed that OT-1 transgenic CD8+ T cells were rendered incapable of killing target peptide antigen-carrying murine EL-4 cancer cells when the cancer cells had been treated with either PNT or the PNT donor 3-morpholinosydnonimine hydrochloride (SIN-1).

    Importantly, this cancer-killing reduction was only evident when the tumor cells were treated with Sin-1 or PNT before addition of the peptide targeted by the CD8+ T cells. If target cells were first loaded with the peptide and then treated with SIN-1 or PNT, the CTL-mediated killing was not affected. The effects were similarly observed in different cancer cell lines.

    The initial results suggested that PNT might be affecting the binding of specific peptides to MHC class I on tumor cells. Indeed, staining experiments showed that pretreating EL-4 cells with PNT substantially reduced binding of peptide and formation of the pMHC cmplex. Conversely, PNT had no effect on the pMHC complexes when tumor cells were treated with PNT after peptide loading.

    They next evaluated whether direct NT modification of a peptide epitope would reduce its binding capacity to MHC. Indeed, the team found that  nitration of the tyrosine in a peptide epitope derived from telomerase reverse transcriptase (TERT) significantly reduced its binding to HLA-A2 in T2 cells, “demonstrating that nitration of either the MHC molecule or the peptide epitope can affect binding.”

    PNT thus seems to alter peptide binding MHC class I on the surface of tumor cells by impacting on the formation of pMHC complexes, diminishing effective recognition of the tumor cells by CTLs. However, they continue, in the natural cellular envinroment pMHC complexes are assembled intracellularly in the ER and are subsequently transported to the cell surface for presentation to CTLs.

    Yet the research suggested that while PNT blocked recognition by CTLS of naturally processed peptides, it had no effect on already made pMHC complexes. These observations suggested that PNT exerts its main effect on the recognition of naturally processed antigen during the assembly of the pMHC complex.

    They tested this using Lewis lung carcinoma (LLC) and B16-F10 melanoma cells transfected with a single-chain H-2Kb-SIINFEKL construct. In these cells the H-2Kb-SIINFEKL pMHC complex is synthesized as a fusion protein that does not require antigen processing and epitope assembly, the team notes. The cells were then treated with PNT and binding of the anti-pMHC antibody evaluated. PNT caused more than a 10-fold increase in the level of NT staining of tumor cells, but treatment did not affect the pMHC expression on the cell surface or killing of the LLC single-chain H-2Kb-SIINFEKL cells by targeted CTLs.

    “These data support the hypothesis that PNT affects the formation of pMHC complexes, preventing CTL killing of the tumor cells,” the researchers state.

    The next stage was to discover what could be releasing PNT into a tumor microenvironment to prevent CTL binding to target cells. The team looked at tumor tissues from patients with lung adencarcinoma, large cell lung cancer, breast ductal carcinoma, and pancreatic ductal carcinoma, and carried out NT staining as a marker of PNT production.

    In each tumor type NT staining was significantly higher in myeloid cells than in tumor cells or epithelial cells. The only exception was in breast cancer samples, in which normal ductal epithelial cells adjacent to breast cancer also showed moderate to strong NT positivity. “Thus, myeloid cells were the predominant source of NT in lung, pancreatic, and breast cancer patients,” the authors state.

    In LLC and EL-4 tumor tissues just about all of the PNT was associated with MDSCs, though to a lesser extent with F4/80+ macrophages.  “These results raised the question as to whether myeloid cells were able to cause tumor cell resistance to CTLs,” they add.

    To answer this they isolated MDSCs and macrophages from the spleens or tumor tissues of tumor-bearing mice and incubated the cells with EL-4 tumor cells. The myeloid cells were subsequently removed and binding of the fluorescently labeled peptide to the tumor cells was evaluated. MDSCs from spleen or tumor tissues had a significant inhibitory effect on the peptide binding to tumor cells, whereas immature myeloid cells (IMCs) from the spleen of naive mice didn’t affect peptide binding and the effects of macrophages were not significant. No effect of the myeloid cells on the expression of MHC class I on the surface of the tumor cells was detected.

    To assess the ability of MDSCs to cause tumor cell resistance to CTLs, the team inclubated EL-4 cells with either MDSCs or control IMCs, and after subsequent removal of myeloid cells the tumor cells were loaded with specific or control peptides and used as targets in a lysis CTL assay. The preincubation of tumor cells with MDSCs from spleens or tumors of EL-4 tumor-bearing mice significantly reduced the killing of specific peptide-loaded target cells whereas incubation with IMCs had no effect.

    Immunoprecipitation and confocal microscopy studies on PNT-cultured EL-4 cells confirmed that PNT resulted in nitrosylation and led to colocalization of MHC class 1 and NT. The expression of NT on tumor cells was also detected following incubation of tumor cells with MDSCs but not with the control IMCs. 

    The researchers used two models to establish a causal relationship between PNT production by MDSCs and their effect on tumor cells. In one MDSCs were generated from a tumor bearing mouse that lacked a component of the cellular machinery responsible for generating reactive oxygen species (ROS).

    In the other, normal MDSCs were treated with a compound, CDDO-Me, which inhibits ROS and PNT production. In both cases the MDSCs were unable to inhibit peptide binding to MHC molecules by EL-4 tumor cells, and the CDDO-Me-treated MDSCs were significantly less able to block the killing of tumor cells by CTLs.  

    Moreover, tumor cells isolated from cancer-bearing mice that had been treated by CDDO-Me demonstrated much higher peptide binding than those from control mice. To see whether these tumors were also more susceptible to CTL killing, tumors from two different strains of CDDO-Me-treated mice were removed, and their cells used as targets in CTL assays. In both cases CDDO-Me treatment had no effect on the expression of MHC class I on the tumor cells, but it did enhance CTL-mediated killing.

    In vitro and in vivo studies demonstrated that induction of MDSCs at tumor sites is triggered by IL-1β production by tumors. In experimental mice with tumors that produced high levels of IL-1β, the antitumor effects of transferred activated T cells was significantly decreased.

    The adoptive CTL transfer experiments in tumor-bearing mice were then repeated in animals treated with total body irradiation followed by bone marrow transplantation. During the first 12 days after adoptive transfer CTLs almost completely stopped the growth of the tumors in animals with tumors that didn’t produce IL-1β but were ineffective against tumors that did express IL-1β.

    Moreover, while levels of peripheral MDSCs in TBI-treated mice with IL-1β-nonexpressing tumors were barely detectable seven days after TBI, those in mice with IL-1β-expressing tumors were higher. In these animals the number of PNT-producing myeloid cells at the tumor site was only marginally reduced after TBI. And when T-cell transfer to tumor-bearing mice was delayed by a week to allow for partial reconstitution of the myeloid compartment after TBI, the IL-1β-expressing  tumors caused even stronger suppression of tumor killing capacity 

    The team hypothesized that if MDSC-derived PNT was indeed responsible for the tumor cell resistance to CTLs, then the blockade of PNT with CDDO-Me would improve the effect of adoptive T-cell therapy even in conditions of induced enhanced inflammation.

    Testing this in the experimental mice they found that adding CDDO-Me to the animals diet caused a significant decrease in the number of NT+ cells infiltrating both IL-1β-expressing tumors and IL-1β-nonexpressing tumors in relevant animals. When CDDO-Me-treated mice then underwent TBI and adoptive T cell therapy, those with IL-1β-nonxpressing displayed significant antitumor effects. In mice with IL-1β-expressing tumors, T-cell therapy completely stopped tumor progression for 4 weeks.

    “The results of this study suggest a novel concept regarding tumor escape in cancer associated with inflammation,” the authors conclude. "We propose that the myeloid cells infiltrating tumor sites induce tumor cell resistance to CTLs by modifying the pMHC complexes on tumor cells.”


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