IntroductionTumor progression in patients and mice is associated with increasing levels of a population of suppressor cells known as myeloidderived suppressor cells (MDSCs). MDSCs suppress antitumor immunity by blocking the activation of CD4 ϩ and CD8 ϩ T cells, 1-3 skewing cytokine production toward a type 2 phenotype, 4 inhibiting natural killer-cell cytotoxicity, 5,6 promoting the accumulation of immune suppressive regulatory T cells, 7,8 and perturbing lymphocyte trafficking. 9 As a result, MDSCs are a significant obstacle to cancer immunotherapies that require activation of the host's immune system.A hallmark of tumor-driven MDSCs is their elevated presence in the BM, spleen, blood, lymph nodes, and primary and metastatic tumor sites. 1,10 Their accumulation is attributed to multiple proinflammatory factors, including IL-1␤, 11,12 IL-6, 13 prostaglandin E 2 (PGE 2 ), 14,15 S100A8/A9 proteins, 10, 17 and VEGF,18,19 driving their differentiation from hemopoietic progenitor cells. These inflammatory mediators are produced by tumor cells 13,[20][21][22] or host cells 23,24 or both. In persons with cancer, tumor cells are the predominant inducers because removal of tumor causes a rapid decrease in MDSCs. 25,26 In contrast to induction of MDSCs, the factors that regulate MDSC maintenance and turnover are not well understood.Accumulation For personal use only. on May 11, 2018. by guest www.bloodjournal.org From Mass spectrometryMDSCs were obtained from the peripheral blood of tumor-bearing mice 14 (Ͼ 90% Gr1 ϩ CD11b ϩ cells; 5 ϫ 10 6 -10 7 cells/mouse) and lysed at a final concentration of 0.1% Rapigest acid-cleavable detergent (Waters) in 100mM NH 4 HCO 3 , pH 8.4. Cell lysates were digested with sequencing grade-modified trypsin (1:50 trypsin-to-protein ratio; Promega) for 1 hour at 37°C, after which trifluoroacetic acid was added to a final pH of ϳ 3. Lysates were incubated for 1 hour at 37°C, freeze-thawed at Ϫ80°C, and microfuged at 13 200 rpm for 5 minutes (Eppendorf 5415 D), and the trifluoroacetic acid-precipitated material was discarded. 27 Supernatant fluid containing tryptic peptides was collected and brought to pH 7, and peptide concentration was measured by OD 280. Peptides (30 g) were desalted (C18 spin cartridges; Pierce) and analyzed with a LTQ-FT Ultra mass spectrometer (ThermoFischer) interfaced with an Agilent 1100 nanoLC system. Tandem mass spectra were searched against the National Center for Biotechnology Information (NCBI) mouse protein database 28 with the use of MASCOT data analysis software (v2.1; Matrix Science) with the following conditions: peptide mass tolerance at 10 ppm; fragment mass tolerance at 1.5 Da; a maximum of 2 allowed missed cleavages; and methionine oxidation and disulfide as the variable modifications. Proteins identified by Ն 2 peptides with a MASCOT MOWSE (molecular weight search) score Ͼ 30 were considered reliable identifications. 29 Antibodies, flow cytometry, confocal microscopyFluorescently coupled mAbs to Gr1, CD11b, Fas, FasL, CD69, CD3, CD4, DO11.10 TCR (clon...

Myeloid-derived suppressor cells (MDSC) accumulate in patients and animals with cancer where they mediate systemic immune suppression and obstruct immunebased cancer therapies. We have previously demonstrated that inflammation, which frequently accompanies tumor onset and progression, increases the rate of accumulation and the suppressive potency of MDSC. To determine how inflammation enhances MDSC levels and activity we used mass spectrometry to identify proteins produced by MDSC induced in highly inflammatory settings. Proteomic pathway analysis identified the Fas pathway and caspase network proteins, leading us to hypothesize that inflammation enhances MDSC accumulation by increasing MDSC resistance to Fas-mediated apoptosis. Immunotherapies aimed at activating the host's immune system are promising strategies for the treatment of cancer because of their potential for minimal toxicity to healthy cells and their ability to induce immune memory that may protect against metastatic disease (1). Disappointingly, clinical trials of most cancer vaccines or other active T-cell mediated immunotherapies have not yielded significant patient responses (2). Because most cancer patients are immune suppressed, these failures are most likely because of the inability of cancer patients to immunologically respond to the immunotherapy agents. Although multiple mechanisms contribute to immune suppression in individuals with cancer (3), myeloid-derived suppressor cells (MDSC) 1 accumulate in virtually all cancer patients and are a major cause of tumor-induced immune suppression because of their inhibition of both adaptive and innate immunity (4). Because of their widespread presence and potent immune suppressive effects, identifying the cellular and molecular mechanisms responsible for MDSC accumulation and suppressive activity may facilitate the development of effective immunotherapy strategies.Chronic inflammation frequently precedes tumor onset (5) and many cancer cells produce pro-inflammatory mediators, suggesting that chronic inflammation contributes to tumorigenesis and tumor progression (6). We and others have previously demonstrated that inflammation via the proinflammatory molecules interleukin (IL)-1␤ (7, 8), toll-like receptor 4 (TLR4) (9), IL-6 (10), prostaglandin E2 (11, 12), and S100A8/A9 proteins (13, 14) increases either the number or the suppressive potency of MDSC, or both. This causative relationship between inflammation and MDSC induction led us to hypothesize that MDSC not only are an obstacle to immunotherapy, but also contribute to the onset and progression of tumors by inhibiting immune surveillance of newly transformed cells and by blocking natural immunity to established tumors (15).We are studying the effects of inflammation on tumor progression and MDSC development using the mouse BALB/cderived, spontaneously metastatic 4T1 mammary carcinoma (16) transfected with the IL-1␤ gene (4T1/IL-1␤) (7). When 1 The abbreviations used are: MDSC, myeloid-derived suppressor cells; DAPI, 4Ј,6-diamidino-2-phenylin...

Immune suppressive myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit innate anti-tumor immunity and are a significant obstacle to cancer immunotherapy. Inflammation is a known inducer of Gr1(+)CD11b(+) MDSC; however, the factors/conditions that regulate MDSC survival and half-life have not been identified. We have used mass spectrometry (MS) and proteomic analysis to identify proteins and pathways that regulate MDSC survival. This analysis revealed high expression of caspase family proteins and the Fas-FasL, p38 MAPK, and TGFβ pathways, suggesting that Fas-FasL apoptosis regulates MDSC survival. Flow cytometry, confocal microscopy, and western blot analyses confirmed the MS findings and demonstrated that Fas(+) MDSC are susceptible to Fas-mediated killing in vitro. In vivo studies with FasL-deficient and Fas-deficient mice demonstrated that Fas-FasL interactions are essential for MDSC apoptosis and for rejection of established metastatic disease and survival and that FasL(+) T cells are the effector population mediating MDSC apoptosis. MS findings validated by biological experiments demonstrated that inflammation increases MDSC levels by protecting MDSC from Fas-mediated apoptosis, possibly by activating p38 MAPK. These results demonstrate that MDSC half-life in vivo is regulated by FasL(+) T cells and that inflammation increases MDSC levels by conferring resistance to Fas-mediated apoptosis and identifies T cells as the relevant effector cells causing MDSC apoptosis in vivo. This newly recognized mechanism for regulating MDSC levels identifies potential new targets for decreasing MDSC in cancer patients.

CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFNγ under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis – multiple reaction monitoring mass spectrometry (MRM-MS). We used MRM-MS to detect and quantify predicted phospho-peptides derived from T-bet. By analyzing activated murine WT and Rheb deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify 6 T-bet phosphorylation sites. Five of these are novel, and 4 sites are consistently dephosphorylated in both Rheb deficient CD4+ T-cells and T-cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the 6 phosphorylation sites was tested for the ability to impair IFNγ expression. Single phosphorylation site mutants still support induction of IFNγ expression, however simultaneous mutation of 3 of the mTORC1-dependent sites results in significantly reduced IFNγ expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation.

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti–TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti–TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti–TIM-3 Ab. In these conditions, anti–TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti–TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti–TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen

Due to their optical properties and morphology, thin films formed of nanoparticles are potentially new platforms for soft laser desorption/ionization (SLDI) mass spectrometry. Thin films of gold nanoparticles (with 12±1 nm particle size) were prepared by evaporation-driven vertical colloidal deposition and used to analyze a series of directly deposited polypeptide samples. In this new SLDI method, the required laser fluence for ion detection was equal or less than what was needed for matrix-assisted laser desorption/ionization (MALDI) but the resulting spectra were free of matrix interferences. A silicon microcolumn array-based substrate (a.k.a. black silicon) was developed as a new matrix-free laser desorption ionization surface. When low-resistivity silicon wafers were processed with a 22 ps pulse length 3× Nd:YAG laser in air, SF 6 or water environment, regularly arranged conical spikes emerged. The radii of the spike tips varied with the processing environment, ranging from approximately 500 nm in water, to ~2 μm in SF 6 gas and to ~5 μm in air. Peptide mass spectra directly induced by a nitrogen laser showed the formation of protonated ions of angiotensin I and II, substance P, bradykinin fragment 1-7, synthetic peptide, pro14-arg, and insulin from the processed silicon surfaces but not from the unprocessed areas. Threshold fluences for desorption/ionization were similar to those used in MALDI. Although compared to silicon nanowires the threshold laser pulse energy for ionization is significantly (~10) higher, the ease of production and robustness of microcolumn arrays offer complementary benefits.

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