Immunosuppressive CD4 Research Articles

Clinical Remission of Sight-Threatening Non-Infectious Uveitis Is Characterized by an Upregulation of Peripheral T-Regulatory Cell Polarized Towards T-bet and TIGIT.

Non-infectious uveitis can cause chronic relapsing and remitting ocular inflammation, which may require high dose systemic immunosuppression to prevent severe sight loss. It has been classically described as an autoimmune disease, mediated by pro-inflammatory Th1 and Th17 T-cell subsets. Studies suggest that natural immunosuppressive CD4+CD25+FoxP3+ T-regulatory cells (Tregs) are involved in resolution of inflammation and may be involved in the maintenance of clinical remission. To investigate whether there is a peripheral blood immunoregulatory phenotype associated with clinical remission of sight-threatening non-infectious uveitis by comparing peripheral blood levels of Treg, Th1, and Th17, and associated DNA methylation and cytokine levels in patients with active uveitic disease, control subjects and patients (with previously active disease) in clinical remission induced by immunosuppressive drugs. Isolated peripheral blood mononuclear cells (PBMC) from peripheral blood samples from prospectively recruited subjects were analyzed by flow cytometry for CD3, CD4, FoxP3, TIGIT, T-bet, and related orphan receptor γt. Epigenetic DNA methylation levels of FOXP3 Treg-specific demethylated region (TSDR), FOXP3 promoter, TBX21, RORC2, and TIGIT loci were determined in cryopreserved PBMC using a next-generation sequencing approach. Related cytokines were measured in blood sera. Functional suppressive capacity of Treg was assessed using T-cell proliferation assays. Fifty patients with uveitis (intermediate, posterior, and panuveitis) and 10 control subjects were recruited. The frequency of CD4+CD25+FoxP3+ Treg, TIGIT+ Treg, and T-bet+ Treg and the ratio of Treg to Th1 were significantly higher in remission patients compared with patients with active uveitic disease; and TIGIT+ Tregs were a significant predictor of clinical remission. Treg from patients in clinical remission demonstrated a high level of in vitro suppressive function compared with Treg from control subjects and from patients with untreated active disease. PBMC from patients in clinical remission had significantly lower methylation levels at the FOXP3 TSDR, FOXP3 promoter, and TIGIT loci and higher levels at RORC loci than those with active disease. Clinical remission was also associated with significantly higher serum levels of transforming growth factor β and IL-10, which positively correlated with Treg levels, and lower serum levels of IFNγ, IL-17A, and IL-22 compared with patients with active disease. Clinical remission of sight-threatening non-infectious uveitis has an immunoregulatory phenotype characterized by upregulation of peripheral Treg, polarized toward T-bet and TIGIT. These findings may assist with individualized therapy of uveitis, by informing whether drug therapy has induced phenotypically stable Treg associated with long-term clinical remission.

Suboptimal stimulation with staphylococcal enterotoxin C1 induces immunosuppressive CD4+CD25+ regulatory T cells by differential expression of FOXP3 isoforms

Abstract Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis and preventing excessive tissue damage upon immune activation. However, pathogens could exploit Tregs for their successful pathogenesis. We demonstrated that immunosuppressive CD4+CD25+FOXP3+ Tregs were induced by staphylococcal enterotoxin (SE) produced by an important human pathogen, Staphylococcus aureus. CD4+CD25+FOXP3+ T cells were induced from stimulation with SEC1 at a high (1 μg/ml) and low (1 ng/ml) concentrations that induced optimal stimulation and suboptimal stimulation, respectively. However, CD4+CD25+FOXP3+ T cells induced from suboptimal stimulation were functionally immunosuppressive, not from optimal stimulation. Immunosuppressive CD4+CD25+FOXP3+ T cells induced from suboptimal stimulation showed typical Treg surface markers such as CTLA-4, GITR, TNFR2, and CD45RO and produced immunomodulatory cytokines such as TGF-β and IL-10. However, suppression was mainly mediated by galectin-1 in a contact-dependent manner. We found that CD4+CD25+ Tregs from suboptimal stimulation highly express FOXP3 isoform lacking exon 2 (ΔE2) and partially lacking exon3 (ΔpE3) preferably localized to the nucleus, compared to those from optimal stimulation. Lentiviral transduction of FOXP3 isoforms (full length, ΔE2, ΔpE3) to Jurkat T cells did not result in immunosuppressive function. By contrast, when cultured in the media generated from suboptimal T cell stimulation, transduced Jurkat T cells became more immunosuppressive in an order of ΔE2 ΔpE3, and full length FOXP3. These results suggest that soluble factors generated from suboptimal T cell proliferation play an important role in induction of immunosuppressive Tregs.

Immunosuppressive monocytes (CD14+/HLA-DRlow/−) increase in childhood precursor B-cell acute lymphoblastic leukemia after induction chemotherapy

In tumor microenvironment, immunosuppression is a common event and results from the inhibition of activated immune cells and generation of cells with immunosuppressive capacity, as some subtypes of monocytes. The aim of this study was to evaluate the presence of immunosuppressive CD14+/HLA-DRlow/- monocytes in pediatric patients with the diagnosis of B-cell acute lymphoblastic leukemia(B-ALL) and, moreover, verify whether the chemotherapeutic treatment has any effect on these cells. Peripheral blood(PB) and bone marrow(BM) samples were collected from 15 untreated pediatric patients. The presence of CD14+/HLA-DRlow/- monocytes was evaluated at diagnosis and in the end of induction chemotherapy by flow cytometry. CD14+/HLA-DRlow/- monocytes increase was observed in 60% (9/15) of the patients at the end of the induction therapy. We were able to detect an increase in CD14+/HLA-DRlow/- monocytes values in BM and PB samples of pediatric patients with B-ALL. This increase was observed in the end of induction chemotherapy, which leads us to believe that these changes probably could have been induced by the inflammatory process engendered by the cytotoxic treatment or by drugs used in the chemotherapy treatment. This finding may be useful to guide new therapeutic approaches contemplating immunomodulatory drugs that act in the depletion of immunosuppressive monocytes.

Induction of Immunosuppressive CD8+CD25+FOXP3+ Regulatory T Cells by Suboptimal Stimulation with Staphylococcal Enterotoxin C1.

Superantigens (SAgs) produced by Staphylococcus aureus at high concentrations induce proliferation of T cells bearing specific TCR Vβ sequences and massive cytokinemia that cause toxic shock syndrome. However, the biological relevance of SAgs produced at very low concentrations during asymptomatic colonization or chronic infections is not understood. In this study, we demonstrate that suboptimal stimulation of human PBMCs with a low concentration (1 ng/ml) of staphylococcal enterotoxin C1, at which half-maximal T cell proliferation was observed, induced CD8+CD25+ T cells expressing markers related to regulatory T cells (Tregs), such as IFN-γ, IL-10, TGF-β, FOXP3, CD28, CTLA4, TNFR2, CD45RO, and HLA-DR. Importantly, these CD8+CD25+ T cells suppressed responder cell proliferation mediated in contact-dependent and soluble factor-dependent manners, involving galectin-1 and granzymes, respectively. In contrast, optimal stimulation of human PBMCs with a high concentration (1 μg/ml) of staphylococcal enterotoxin C1, at which maximal T cell proliferation was observed, also induced similar expression of markers related to Tregs, including FOXP3 in CD8+CD25+ cells, but these T cells were not functionally immunosuppressive. We further demonstrated that SAg-induced TCR Vβ-restricted and MHC class II-restricted expansion of immunosuppressive CD8+CD25+ T cells is independent of CD4+ T cells. Our results suggest that the concentration of SAg strongly affects the functional characteristics of activated T cells, and low concentrations of SAg produced during asymptomatic colonization or chronic S. aureus infection induce immunosuppressive CD8+ Tregs, potentially promoting colonization, propagation, and invasion of S. aureus in the host.

Novel Role of p53 in Septic Immunosuppression: Involvement in Loss and Dysfunction of CD4+ T Lymphocytes

Background/Aims: Immunosuppression frequently occurs during the development of sepsis and is closely associated with poor outcome. Characteristics of immunosuppressive CD4⁺ T lymphocytes in sepsis have been reported to include dramatic cell loss and inactivation. p53 acts as a pivotal transcription factor in regulating cell proliferation and apoptosis, which control tumorigenesis. However, few studies have investigated the universal role of p53 in immune cells, especially in the development of sepsis. Methods: A mouse model of sepsis was produced by cecal ligation and puncture (CLP), and isolated splenic CD4⁺ T cells or Jurkat cells were exposed to lipopolysaccharide (LPS) stimulation in vitro. We used genetic knockout (p53^-/-) mice or the specific inhibitor pifithrin-α (PFT) to investigate the regulatory mechanisms of p53. Cell proliferation ability was assessed using a Cell Counting Kit-8 assay, and apoptotic cells were stained with annexin V/propidium iodide and then analyzed using a FACScan flow cytometer. Protein and mRNA expression levels were measured by western blotting and real-time PCR, and cytokine levels in culture supernatants were determined by enzyme-linked immunosorbent assay. Results: Splenic CD4⁺ T lymphocytes from CLP mice expressed gradually elevated p53 mRNA and protein levels, which resulted in extracellular regulated protein kinase 1/2 inactivation and expression of apoptotic molecules. Specific inhibition of p53 by PFT or genetic knockout (p53^-/-) maintained CD4⁺ T lymphocyte homeostasis, as indicated by protection from cell loss and restoration of immune function. A medium dose of PFT improved the survival rate of mice, while mortality rate showed only a slight improvement in p53^-/- mice compared with wild-type mice. The in vitro responses to LPS were consistent with these results, and upregulation of p53 clearly affected the proliferation, apoptosis, and immune dysfunction of CD4⁺ T lymphocytes. In addition, we confirmed the regulatory effect of p53 in Jurkat cells, and inhibition of p53 by either inhibition or short hairpin RNA transduction markedly protected cells from LPS stimulation. Conclusion: Elevation of p53 in T lymphocytes during sepsis or endotoxin challenge might be responsible for inhibiting cell proliferation and enhancing both apoptosis and immune dysfunction of T cells.

Targeting CD38 Suppresses Induction and Function of T Regulatory Cells to Mitigate Immunosuppression in Multiple Myeloma.

Purpose: We study CD38 levels in immunosuppressive CD4+CD25highFoxp3+ regulatory T cells (Treg) and further define immunomodulating effects of a therapeutic CD38 mAb isatuximab/SAR650984 in multiple myeloma.Experimental Design: We evaluated percentages of CD38-expressing subsets in Tregs from normal donors and multiple myeloma patients. Peripheral blood mononuclear cells (PBMC) were then treated with isatuximab with or without lenalidomide or pomalidomide to identify their impact on the percentage and immunosuppressive activity of Tregs on CD4+CD25- T cells (Tcons). We investigated the mechanism of increased Tregs in multiple myeloma patients in ex vivo cocultures of multiple myeloma cells with PBMCs or Tcons.Results: CD38 expression is higher on Tregs than Tcons from multiple myeloma patients versus normal donors. CD38 levels and the percentages of CD38high Tregs are increased by lenalidomide and pomalidomide. Isatuximab preferentially decreases Treg and increases Tcon frequencies, which is enhanced by pomalidomide/lenalidomide. Isatuximab reduces Foxp3 and IL10 in Tregs and restores proliferation and function of Tcons. It augments multiple myeloma cell lysis by CD8+ T and natural killer cells. Coculture of multiple myeloma cells with Tcons significantly induces Tregs (iTregs), which express even higher CD38, CD25, and FoxP3 than natural Tregs. This is associated with elevated circulating CD38+ Tregs in multiple myeloma patients versus normal donors. Conversely, isatuximab decreases multiple myeloma cell- and bone marrow stromal cell-induced iTreg by inhibiting both cell-cell contact and TGFβ/IL10. Finally, CD38 levels correlate with differential inhibition by isatuximab of Tregs from multiple myeloma versus normal donors.Conclusions: Targeting CD38 by isatuximab can preferentially block immunosuppressive Tregs and thereby restore immune effector function against multiple myeloma. Clin Cancer Res; 23(15); 4290-300. ©2017 AACR.

Abstract 4701: Blocking FSTL1 ameliorates immunity against osteosarcoma

Abstract Purposes: Osteosarcoma is a rare malignant tumor of bone in children and young adults, and it is very hard to successfully control the systemic metastases and life-threatening health problems more than a decade after the completion of the care due to the intensiveness of the conventional treatments including surgical resection of the primary tumor and chemotherapy. Therefore, non/low-toxic but advanced treatments have been expected for a long while. We recently found that a BMP family FSTL1 is highly and functionally expressed in murine and human osteosarcoma cell lines, and systemically expands pluripotent mesenchymal stem/stromal cells (MSCs), which play a central role in amplification of tumor heterogeneity that is consequence of reciprocal evolution together with the tumor microenvironment, including induction of immune suppression and dysfunction, desmoplasia, and angiogenesis. FSTL1 and its induced MSCs also directly confer higher invasive and metastatic properties on tumor cells leading to further cancer progression. In this study, we evaluated antitumor efficacy induced by anti-FSTL1 mAb on murine osteosarcoma models, and also compared the therapeutic efficacy with those induced by immune checkpoint inhibitory mAbs (ICIs). Results: We used two syngeneic tumor models that were subcutaneously implanted with murine osteosarcoma NHOS cells in BALB/c mice, or LM3 cells in C3H mice. These mice were intraperitoneally injected with anti-FSTL1 mAbs (10mg/kg) twice on day 5 and day 8 after tumor implantation, and were sacrificed for immunological analysis of tumor-infiltrating cells, spleen cells and bone marrow cells on day 15. In both osteosarcoma models, CD45- MSCs as well as other immunosuppressive CD4+Foxp3+ Tregs and CD11b+Gr1+ MDSCs systemically increased, indicating impairment of antitumor immunity in the mice. Treatment with the anti-FSTL1 mAb significantly induced potent tumor-specific CTLs through reduction of MSCs, resulting in tumor-free in some of the treated mice. Although the antitumor efficacy was similar to those induced by ICIs, the mode of action was totally distinct because MSCs and the MSC-inducible exhausted CD8+PD1+Tim3+ T cells were reduced only in the anti-FSTL1-treated mice, but not in the ICI-treated mice. Conclusions: These results suggest that blocking FSTL1 properly reprograms immunity toward elimination of osteosarcoma by reducing the tumor-supportive MSCs. Anti-FSTL1 mAb may be a promising drug for treating osteosarcoma more moderately but effectively in clinical settings. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Kazunori Aoki. Blocking FSTL1 ameliorates immunity against osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2017-4701

Abstract 1634: CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer

Abstract The cyclin D:cyclin-dependent kinases 4 and 6 (CDK4/6) axis is one of the most frequently dysregulated pathways in human cancers, and CDK4/6 inhibitors have shown significant activity against a number of solid tumors, including breast cancer. Analogous to clinical experience, we found that the CDK4/6 inhibitor, abemaciclib, caused significant tumor regression in the MMTV-rtTA/tetO-HER2 mouse model of luminal breast cancer. However, as CDK4/6 inhibitors are known to block tumor cell proliferation, but not directly induce tumor cell apoptosis, as we confirmed in our study, the mechanisms by which CDK4/6 inhibition caused tumor regression were not clear. Notably, abemaciclib therapy increased total CD3+ T cells in these tumors, while decreasing the immunosuppressive CD4+ regulatory T cell population. Further investigation revealed that CDK4/6 inhibition directly suppresses regulatory T cells by inhibiting their proliferation without impacting natural regulatory T cell formation in the thymus, the differentiation of inducible regulatory T cells, or regulatory T cell apoptosis. We are currently exploring potential effects of CDK4/6 inhibition on regulatory T cell function and lineage stability. Analysis of CD8+ and CD4+ T cells in abemaciclib-treated tumors revealed a marked reduction in the expression of the inhibitory immune checkpoint receptors PD-1, Tim-3, CTLA-4, and LAG3. Specifically, the fraction of CD8+ T cells expressing known markers of T cell exhaustion (PD-1/Tim-3 double-positivity or high expression of PD-1) was decreased in abemaciclib-treated tumors. Importantly, antibody-mediated depletion of CD8+ T cells established that response of MMTV-rtTA/tetO-HER2 tumors to abemaciclib is dependent on CD8+ T cells. Finally, given the observed immunomodulatory effects of abemaciclib, we sought to determine if abemaciclib treatment would sensitize MMTV-rtTA/tetO-HER2 tumors to immune checkpoint blockade. Pretreatment with abemaciclib followed by combination abemaciclib and α-PDL1 significantly enhanced tumor regression compared to abemaciclib or α-PDL1 alone. Our studies reveal a novel mechanism by which CDK4/6 inhibitors directly elicit an anti-tumor immune response. These results provide strong rationale for further investigations into combining CDK4/6 inhibitors with immune checkpoint blockade in breast cancer. Citation Format: Molly J. DeCristo*, Shom Goel*, April C. Watt, Haley BrinJones, Jaclyn Sceneay, Ben Li, Jessalyn M. Ubellacker, Shaozhen Xie, Susanne Ramm, Hye-Jung Kim, Sandra S. McAllister, Jean J. Zhao. CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2017-1634

CD14+ CD15− HLA-DR− myeloid-derived suppressor cells impair antimicrobial responses in patients with acute-on-chronic liver failure

ObjectiveImmune paresis in patients with acute-on-chronic liver failure (ACLF) accounts for infection susceptibility and increased mortality. Immunosuppressive mononuclear CD14+HLA-DR− myeloid-derived suppressor cells (M-MDSCs) have recently been identified to quell antimicrobial...

Effect of high-dose corticosteroids on CD4+ or CD8+ lymphocyte proliferation or IL-2 production after stimulation with pembrolizumab.

e14587 Background: Immune checkpoint inhibitor (ICI) therapy has improved survival of patients with melanoma and other types of cancers. The effect of immunosuppresive corticosteroids on ICI efficacy is unknown, however treatment of immune-related adverse events with high-dose steroid therapy impairs neither efficacy nor time to failure of ICI. In certain clinical circumstances (for instance in the treatment of patients with symptomatic brain metastases) it may be advantageous to begin ICI therapy despite a requirement for steroid therapy. This study examined the effect of high-dose steroid therapy on lymphocyte proliferation/functionality after stimulation with anti-CD3 in the presence of pembrolizumab (PD-1 inhibitor) ex vivo. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from patients with either chronic hepatitis C infection or active malignant melanoma. CD4+ and CD8+ T cells were isolated and subjected to flow cytometry stating for the expression of PD-1. Isolated PBMCs were then pre-treated with prednisone (1 nM - 1 mM) before anti-CD3 stimulation. Anti-CD3-stimulated cells were then incubated with pembrolizumab (5 µg/mL) and their proliferative capacity and IL-2 secretion ability were measured by carboxyfluorescein succinimidyl ester (CFSE) staining and ELISA, respectively. Results: A dose-dependent inhibition of both lymphocyte proliferation and IL-2 production was observed, but at physiologically relevant nanomolar prednisone concentration the steroid effect was negligible. Millimolar prednisone concentrations resulted in > 50% relative reduction in lymphocyte proliferation, however even at this steroid concentration treatment with pembrolizumab was able to further stimulate proliferation and IL-2 secretion versus anti-CD3stimulation alone. Conclusions: Despite heavily immunosuppressive treatment, CD4+ and CD8+ lymphocytes demonstrate proliferative capacity and immunological activity following treatment with pembrolizumab. This suggests that treatment with pembrolizumab may be efficacious despite a requirement for steroid therapy, a scenario often encountered in patients with brain metastases.

A phase I trial of ALKS 4230, an engineered cytokine activator of NK and effector T cells, in patients with advanced solid tumors.

TPS3111 Background: ALKS 4230 is an engineered fusion protein comprised of a circularly permuted interleukin-2 (IL-2) and IL-2 Receptor (IL-2R) α designed to selectively activate the intermediate-affinity (ia) IL-2R, comprised of IL-2Rβ and γc. The iaIL-2R is expressed predominantly on effector lymphocytes, which play an important role in driving antitumor immune responses. In contrast, unmodified IL-2 activates high-affinity (ha) IL-2R, driving the expansion of haIL-2R-expressing cell types including immunosuppressive CD4+ regulatory T (Treg) cells at concentrations below those at which iaIL-2R bearing effector cells are activated. Also, the haIL-2R is expressed on endothelial cells and may contribute to IL-2 mediated toxicity via capillary leak syndrome. Thus, selective activation of the iaIL-2R by ALKS 4230 has the potential to provide enhanced tumor killing as well as improved tolerability. Methods: ALKS 4230 is being studied in a phase 1 first-in-human trial in patients with advanced solid tumors. Key study objectives are to determine a recommended phase 2 dose and characterize the safety profile, pharmacokinetics (PK), pharmacodynamics (PD) and evidence of antitumor activity. A dose-escalation phase in patients with refractory solid tumors (Part A) will be followed by expansion cohorts in defined populations (Part B). ALKS 4230 is administered as a 30-minute intravenous infusion once daily for five days each cycle. Eligibility requires age 18, ECOG PS 0-1 and adequate bone marrow, liver and kidney function. The dose will be escalated until reaching MTD or an Optimal Biologic Dose. The first two dose cohorts will use a 3+3 design. Subsequent cohorts in Part A will enroll a minimum of 6 subjects. In Part B up to 21 patients will be enrolled into each of four tumor-specific cohorts. Peripheral blood samples will be collected for PK, immunogenicity and PD assessments. The primary PD endpoint is change-from-baseline in CD8+ T, NK and Treg cell counts. Other PD measures include serum concentrations of multiple proinflammatory cytokines and immunohistochemical assessment of markers of immune activation in tumor tissue from selected patients. Recruitment for Part A is ongoing. Clinical trial information: NCT02799095.

Improving immunotherapeutic efficacy against metastatic tumors in a pre-clinical model of murine renal cell carcinoma.

Abstract Renal and pelvic cancers are the ninth most common cancer in the U.S., and although five-year survival rates for localized renal tumors are >90%, those for metastasized tumors are <12%. Metastatic renal cell carcinoma (RCC), the most common type of renal neoplasm, is resistant to radio- and chemotherapies. Immune-based therapies, including checkpoint blockade, are being pursued in an attempt to amplify protective adaptive immunity but overall response rates remain at 20–30%. We hypothesized that a therapeutic strategy employing T cell priming plus checkpoint blockade would result in improved clearance of renal tumors verses checkpoint alone. Utilizing our previously described in vivo T cell priming therapy, adenovirus-encoded tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (AdTRAIL) with class B oligonucleotides (CpG), we evaluated therapeutic effects on primary tumor outgrowth and metastatic burden in a pre-clinical model of orthotopic murine RCC. We report here that administration of intra-renal AdTRAIL/CpG in combination with α-CTLA-4, but not α-PD-1, causes significant decreases in primary tumor burdens. Strikingly, we observed that this therapeutic strategy caused significant reductions in metastatic lung burdens – equating to a 98% reduction over no therapy controls. Flow cytometric analysis revealed increases in CD8+ T cells in AdTRAIL-treated groups over no therapy/checkpoint alone; results were further supported by immunosuppressive CD4+ to CD8+ T cell ratios that were in favor of anti-tumoral responses. Our results suggest AdTRAIL/CpG can be used as an effective upstream therapeutic approach to enhance the efficacy of α-CTLA-4 checkpoint blockade in a pre-clinical model of murine RCC.

Attenuated in vitro effects of IFN-α, IL-2 and IL-12 on functional and receptor characteristics of peripheral blood lymphocytes in metastatic melanoma patients

Considering tumor-induced suppression of lymphocytes the aim of this study was to investigate in vitro effects of IFN-α, IL-2, IL-12 and IL-18 as immunomodulating agents on the functional and receptor characteristics of peripheral blood lymphocytes (PBL) in metastatic melanoma (MM) patients compared to healthy controls (HC). In HC IFN-α, IL-2 and IL-12 enhanced mRNA level of perforin by inducing pSTAT-1 and pSTAT-5 signaling molecules. Additionally, the expression of NKG2D activating receptor and its DAP10 signaling molecule was upregulated by IL-2. Contrary to this, in MM patients only IL-2 by upregulating pSTAT-5 increased perforin-mediated cytotoxicity of lymphocytes. Furthermore, there was significantly negative correlation between the percentage of CD4+CD25bright+CD27+ regulatory T (Treg) cells and NK cell cytotoxicity, as well as the expression of NKG2D receptor on PBL in HC and MM patients. Therefore, the absence of IL-2 effect on the increase of NKG2D/DAP10 level in MM patients could be the consequence of the increased percentage of immunosuppressive CD4+CD25bright+CD27+ cells after this cytokine treatment in patients. However, in MM IL-12 significantly decreases the percentage of these inhibitory cells. Although IL-2 as a single agent has numerous side effects, it remains the important cytokine for PBL activation in melanoma immunotherapy. Additionally, the removal of Treg cells from patient PBL by IL-12 before in vitro stimulation with IL-2, may lead to the generation of more potent cytotoxic lymphocytes against tumor cells. Therefore, lymphocyte based therapy for MM patients should integrate not only the choice of appropriate immunostimulatory cytokine, but also the removal of inhibitory cells from tumor microenvironment.

THU-063 - A novel population of immunosuppressive CD8 T cells is expanded in patients with decompensated liver disease

THU-063 - A novel population of immunosuppressive CD8 T cells is expanded in patients with decompensated liver disease

FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival

ABSTRACTGenome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFβ, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFβ levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFβ accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFβ and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression.

Signal-Regulatory Protein-α (SIRP- α) Expression Delineates Distinct Subsets in Monocytes/Macrophages in Normal Tissue and in B-Cell Non-Hodgkin Lymphoma

BACKGROUNDMonocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function.METHODSHuman mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry.RESULTSUsing a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues.CONCLUSIONSWe have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. DisclosuresNo relevant conflicts of interest to declare.

Vitamin D Blocks CLL Cell-Mediated MDSC Induction

Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence amongst adults. CLL-associated immune defects promote tumor immune escape and antagonize immune-based therapies. We and others have reported the accumulation of so-called myeloid-derived suppressor cells (MDSCs) in CLL. Accumulation of immunosuppressive CD14+HLA-DRlow monocytic MDSCs is associated with advanced disease and poor prognosis. The mechanisms re-polarizing CLL-monocytes remain unknown. Here, we describe that CLL-cell-derived exosomes elicit a phenotypical and functional skewing of regular monocytes towards MDSCs. However, pre-treating CLL-cells with vitamin D led to a loss of the exosomes' MDSC-promoting capability. In fact, higher vitamin 25D serum concentrations were linked to lower levels of circulating MDSCs in CLL. A plethora of bioactive molecules including microRNAs (miRs) is shuttled in exosomes. MiR-155 is found abundantly in CLL-exosomes and using antagomir against miR-155 prevented exosomal MDSC-induction. Furthermore, exosomes from patients with lower vitamin D levels contained more miR-155 copies. Accordingly, vitamin D application reduced miR-155 production in CLL-cells Overall, we identified that exosomal miR-155 transfer promotes MDSCs in CLL. Vitamin D interferes with this interplay and could thereby represent a mean for enhancing immune responses in CLL. DisclosuresNo relevant conflicts of interest to declare.

Immunosuppressive CD14+HLA-DRlo/neg monocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

ABSTRACTImmunological strategies to treat pancreatic cancer offer new therapeutic approaches to improve patient outcomes. Understanding alterations in the immune systems of pancreatic cancer patients will likely lead to advances in immunotherapy for the disease. We profiled peripheral blood leukocytes from pancreatic cancer patients (n = 22) and age-matched controls (n = 20) using flow cytometry. Immune profiling of pancreatic cancer patients identified phenotypic changes in various immune cell populations, including a population of immunosuppressive monocytes (CD14+HLA-DRlo/neg), which were shown to be increased in these patients. There was a correlation between the levels of CD14+ monocytes and the levels of CD14+HLA-DRlo/neg monocytes in peripheral blood from pancreatic cancer patients. HLA-DR downregulation of monocytes was shown to occur through pancreatic cancer-derived exosome interactions with monocytes. In an in vitro model, exosomes from patient-derived xenograft cell lines and patient plasma decreased HLA-DR expression on CD14+ monocytes. Additionally, tumor-derived exosomes caused immune suppression in monocytes through altered STAT3 signaling, induction of arginase expression, and reactive oxygen species. These findings provide novel insights into the mechanisms that govern immunosuppression in pancreatic cancer. Understanding monocyte–exosome interactions could lead to novel immunotherapies for this disease.

Correlation Between Immunosuppressive Therapy and CD4(+) T-Cell Intracellular Adenosine Triphosphate Levels in Liver Transplant Recipients.

The aim of this work was to analyze the correlation between immunosuppressive therapy and CD4(+) T-cell intracellular adenosine triphosphate (iATP) levels after liver transplantation and to describe the distribution characteristics of iATP in CD4(+) T cells among liver transplant recipients. We studied 172 patients who were followed regularly after liver transplantation with long-term stable conditions from July 2010 to October 2012. CD4(+) T-cell iATP levels were detected with the use of the Cylex Immuknow Assay method and analyzed retrospectively according to immunosuppressive therapy protocol. There was a significant difference in CD4(+) T-cell iATP level among the recipients receiving different immunosuppressive therapy protocols after liver transplantation. CD4(+) T-cell iATP level in the FK506 group and FK506+ prednisone (Pred) groups was higher than in the FK506+ mycophenolate mofetil (MMF), FK506+ MMF+ Pred, and rapamycin (Rapa) groups. CD4(+) T-cell iATP level in patients receiving an MMF protocol was lower than in the group without MMF. There is a relationship between distribution immunosuppressive therapy protocol and CD4(+) T-cell iATP level after liver transplantation. MMF and Rapa lower the CD4(+) T-cell iATP level significantly.

CD147 handles lipid: a new role for anti-cancer target

The future of anti-cancer therapy is promising as immunotherapy strategies have been revolutionary in treatment of a number of cancers. However, some organs such as the liver are characterised by immunosuppression of CD4 + and CD8 + activity, necessitating alternative strategies for cancers in such tissues. This occurs at least partly through aberrant expression of alpha-fetoprotein that in turn dampens immune activation (1). Hepatocellular carcinoma (HCC) is the second leading cause of death from cancer worldwide (2). Complementary strategies to CD4 + and CD8 + T cell-mediated immunotherapies that specifically target HCC are needed for the future of effective and comprehensive cancer therapy. On the short-list for a molecular target for such a therapy is CD147/EMMPRIN.