Identifying Regulators of the Immune Response to Dying Cells
The work of Greten and colleagues supports a mechanism of immune cell activation in which dying cells release proteasome-processed antigenic peptides. In the presence of active peptidases, such as DPP-3 and TOP-1, no cytotoxic T cell activation occurs. If the peptidases are inactivated, however, a robust dendritic cell-mediated priming of T cells takes place.
Cytotoxic T cells are responsible for carrying out antigen-mediated immune responses against virally-infected and malignant cells. In both cases, cytotoxic T cells are stimulated by interacting with antigen presenting cells, such as dendritic cells (DCs). Infected cells produce virus-specific antigens and pathogen associated molecular patterns, which are recognized by DCs and lead to robust T cell activation. Dead or dying uninfected cells, on the other hand, release damage associated molecular patterns, but their release does not always appear to be sufficient to induce cytotoxic T cell activity. Tim Greten, M.D., of CCR’s Medical Oncology Branch, and a group of international collaborators set out to understand how immune responses against dying cancer cells are regulated. These processes are likely to be important for improving the efficacy of cancer treatment vaccines, which induce an immune reaction against a patient’s cancer cells.
The researchers began their studies by investigating the ability of necrotic melanoma cells to initiate an immune response. They inoculated mice with DCs, which were incubated with melanoma cells that had been frozen and thawed three times (FT) or frozen and thawed three times then heated (FTheat) to induce cell death. These mice were then treated with live melanoma cells. All the mice vaccinated with the FT-loaded DCs developed tumors. In contrast, 80 percent of the mice given FTheat-stimulated DCs did not develop tumors over more than five months, suggesting they had established an active immune response. Indeed, the mice that received FTheat-treated DCs had higher levels of antigen-specific cytotoxic T cells, and those cells showed higher antigen-specific lysis. Similar results were observed with inoculation of FT or FTheat cells alone.
To begin to understand the mechanism of this induced immune response, the investigators took soluble fractions from the FT and FTheat melanoma cells and incubated them with splenocytes. They saw increased cytokine secretion and cell proliferation from the FTheat fractions but not the FT fractions. Similar results were seen with FT and FTheat colon cancer cells, suggesting this mechanism is not restricted to melanoma cells. The scientists also tried directly incubating T cells with the necrotic melanoma cells but saw no increased proliferation, indicating that DCs are essential for necrosis-induced cytotoxic T cell activation.
Based on these results, it was not clear whether FT cells fail to activate an immune response or actively block it. To address this issue, the researchers inoculated mice with a combination of FT and FTheat melanoma cells and found that the T cells showed reduced antigen-specific lysis. Likewise, culturing splenocytes with FT and FTheat cells prevented T cell proliferation, suggesting that some factor from the FT cells blocks cytotoxic T cell activation. Interestingly, this activation was restored by digesting the soluble fraction from FT cells with trypsin, pointing to a trypsin-sensitive protein as the regulatory factor. This was true in a variety of cell types, including mouse colon cancer cells, human tumor cells, and mouse embryonic fibroblasts.
To identify the factors involved in regulating the immune response to necrotic cells, the investigators separated the FT cell soluble fraction using gel filtration. They then tested the ability of fractions to induce cytotoxic T cell activity. Fractions 9-23 strongly activated T cells when incubated with an antigen and splenocytes. Fractions 9-23 alone did not activate T cells but had to be combined with antigen and DCs to stimulate T cells. Because the proteins in these fractions are of high molecular weight and affect the antigen, the scientists hypothesized that the proteasome, a large protein complex that degrades proteins, could be responsible for the activity in these fractions.
Pretreating fractions 9-23 with lactacystin, an irreversible proteasome inhibitor, reduced T cell proliferation when mixed with antigen and splenocytes. These results suggest that proteasome peptide generation may play a role in cytotoxic T cell activation by dying cells when the negative regulator is absent. In support of this idea, the researchers found that pretreating melanoma cells with lactacystin then inducing necrosis with FTheat reduced T cell proliferation when incubated with splenocytes. Likewise, mice inoculated with lactacystin-treated FTheat cells had reduced antigen-specific immune responses compared to mice treated with FTheat cells.
The investigators then characterized the remaining gel filtration fractions to identify key inhibitory proteins. They found that fractions 37-45 prevented cytotoxic T cell activation in the presence of antigen and fractions 9-23. Treating fractions 37-45with low pH or high temperature eliminated this activity. Preincubating DCs with fractions 37-45 or adding them after fractions 9-23 and antigen were mixed with DCs did not affect T cell activation, suggesting that fractions 37-45 affect the antigen and/or proteins in fractions 9-23.
To determine the specific regulators, the scientists performed additional separation steps on fractions 37-45. Three fractions from the final step blocked cytotoxic T cell activation and were analyzed by liquid chromatography and tandem mass spectrometry. The researchers found a number of peptidases, which degrade peptides, in these fractions. The fraction with the most activity correlated with high intensities of two potential candidate regulators, dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1).
To study the activities of DPP-3 and TOP-1, the scientists generated recombinant proteins and found that both could block T cell activation in splenocytes treated with FTheat melanoma cells. Heating either peptidase blocked its inhibitory activity, suggesting that heat allows for cytotoxic T cell activation by protecting peptides from digestion. The investigators also found that inoculating mice with recombinant TOP-1 or DPP-3 and FTheat melanoma cells reduced antigen-specific lysis. Knocking down TOP-1 in melanoma cells partially reduced the T cell inhibitory effect with FT. Knocking down DPP-3, however, did not have much effect, suggesting that the proteins have redundant activities.
Finally, the researchers asked whether these peptidases control cytotoxic T cell activation for mechanisms of cell death besides FT. They found that TOP-1 knock down enhanced T cell activation in gamma-irradiated colon cancer cells in vitro. Likewise, mice bearing lymphoma cells with reduced TOP-1 had enhanced antigen-specific immune responses following X-ray irradiation compared to control tumors with normal TOP-1 expression.
Taken together, these results support a model where DPP-3 and TOP-1 in necrotic cells control cytotoxic T cell activation, which also relies on proteasome-dependent antigenic peptide formation. Blocking the activity of these peptidases may enhance the efficacy of cancer treatment vaccines.Summary Posted: 10/2013
Gamrekelashvili J, Kapanadze T, Han M, Wissing J, Ma C, Jaensch L, Manns MP, Armstrong T, Jaffee E, White AO, Citrin DE, Korangy F, Greten TF. Peptidases released by necrotic cells control CD8+ T cell cross-priming. Journal of Clinical Investigation. October 8, 2013 PubMed Link