INF-γ Production Research Articles

Novel imiquimod nanovesicles for topical vaccination.

Development of needle and pain free noninvasive immunization procedures is a top priority for public health agencies. In this work the topical adjuvant activity of the immunomodulator imiquimod (IMQ) carried by ultradeformable archaeosomes (UDA2) (nanovesicles containing sn-2,3 ether linked phytanyl saturated archaeolipids) was surveyed and compared with that of ultradeformable liposomes lacking archaeolipids (UDL2) and free IMQ, using the model antigen ovalbumin and a seasonal influenza vaccine in Balb/c mice. UDA2 (250 ± 94 nm, -26 ± 4 mV Z potential) induced higher IMQ accumulation in human skin and higher production of TNF-α and IL-6 by macrophages and keratinocytes than free IMQ and UDL2. Mixed with ovalbumin, UDA2 was more efficient at generating cellular response, as measured by an increase in serum IgG2a and INF-γ production by splenocytes, compared with free IMQ and UDL2. Moreover, mixed with a seasonal influenza vaccine UDA2 produced same IgG titers and IgG2a/IgG1 isotypes ratio (≈1) than the subcutaneously administered influenza vaccine. Topical UDA2 however, induced highest stimulation index and INF-γ levels by splenocytes. UDA2 might be a promising adjuvant for topical immunization, since it produced cell-biased systemic response with ≈ 13-fold lower IMQ dose than the delivered as the commercial IMQ cream, Aldara.

Chimeric Antigen Receptors Based on Low Affinity Mutants of FcεRI Re-direct T Cell Specificity to Cells Expressing Membrane IgE.

IgE is the key mediator of allergic responses. Omalizumab, an IgE-specific monoclonal antibody that depletes IgE, is effective for treating severe allergic asthma. The need for frequent administration of the expensive drug, however, limits its applications. Taking advantage of T cell memory, adoptive T cell therapy (ACT) targeting IgE-producing cells has the potential to achieve long-term suppression of IgE and relief of symptoms for severe allergic diseases. The transmembrane form of IgE (mIgE), which is present on all IgE-producing cells, serves as an excellent molecular target for ACT that employs chimeric antigen receptors (CARs). Here, we designed and tested CARs that use the extracellular domain of high affinity IgE receptor, FcεRIα, for mIgE recognition. When expressed on Jurkat T cells, FcεRIα-based CARs mediated robust responses in terms of CD69 upregulation to U266 myeloma cells expressing low levels of mIgE. FcεRIα-based CARs specifically recognized cells expressing mIgE, but not cells with secreted IgE captured through Fcε receptors. CAR+ Jurkat cells did not respond to LAD2 mast cells with secreted IgE bound through FcεRI or Ramos cells with secreted IgE bound through FcεRII. Co-culture of CAR+ Jurkat cells and LAD2 mast cells with IgE bound did not trigger LAD2 cell degranulation. The activity of CAR using wild type FcεRIα for mIgE binding was inhibited by the presence secreted IgE, which likely blocked CAR-mIgE interaction. The activities of CARs using low affinity mutants of FcεRIα, however, tolerated secreted IgE at relatively high concentrations. Moreover, primary human CD8+ T cells expressing a low affinity mutant CAR responded to U266 cells with INFγ production and cytotoxicity despite the presence of secreted IgE. The potency, specificity, and robustness of our CAR design, combined with repaid advances in the safety of ACT, hold promise for novel and highly effective cell-based therapies against severe allergic diseases.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

A Lactate Fermentation Mutant of Toxoplasma Stimulates Protective Immunity Against Acute and Chronic Toxoplasmosis.

Toxoplasma gondii is an important zoonotic pathogen infecting one-third of the world’s population and numerous animals, causing significant healthcare burden and socioeconomic problems. Vaccination is an efficient way to reduce global sero-prevalence, however, ideal vaccines are not yet available. We recently discovered that the Toxoplasma mutant lacking both lactate dehydrogenases LDH1 and LDH2 (Δldh) grew well in vitro but was unable to propagate in mice, making it a good live vaccine candidate. Here, we tested the protection efficacy of ME49 Δldh using a mouse model. Vaccinated mice were efficiently protected from the lethal challenge of a variety of wild-type strains, including type 1 strain RH, type 2 strain ME49, type 3 strain VEG, and a field isolate of Chinese 1. The protection efficacies of a single vaccination were nearly 100% for most cases and it worked well against the challenges of both tachyzoites and tissue cysts. Re-challenging parasites were unable to propagate in vaccinated mice, nor did they make tissue cysts. High levels of Toxoplasma-specific IgG were produced 30 days after immunization and stayed high during the whole tests (at least 125 days). However, passive immunization of naïve mice with sera from vaccinated mice did reduce parasite propagation, but the overall protection against parasite infections was rather limited. On the other hand, Δldh immunization evoked elevated levels of Th1 cytokines like INF-γ and IL-12, at early time points. In addition, splenocytes extracted from immunized mice were able to induce quick and robust INF-γ and other pro-inflammatory cytokine production upon T. gondii antigen stimulation. Together these results suggest that cellular immune responses are the main contributors to the protective immunity elicited by Δldh vaccination, and humoral immunity also contributes partially. We also generated uracil auxotrophic mutants in ME49 and compared their immune protection efficiencies to the Δldh mutants. The results showed that these two types of mutants have similar properties as live vaccine candidates. Taken together, these results suggest that mutants lacking LDH were severely attenuated in virulence but were able to induce strong anti-toxoplasma immune responses, therefore are good candidates for live vaccines.

Abstract 3806: The pattern of NKG2DL expressions determines immunogenicity and antitumor effect of protein-bound polysaccharide-K in tumor-bearing mouse models

Abstract Background and Aim: Natural killer group 2, member D ligands (NKG2DLs) have paradoxical immunological characteristics, exerting both immune-stimulatory and immune-suppressive effects on T cell activation. First, we attempted to determine the relationship between the antitumor effect of protein-bound polysaccharide-K (PSK) in a tumor-bearing mouse model and the expression profile of NKG2DLs on a tumor. Next, we investigated whether NKG2DL expression on tumor cells affects T cell activation by examining the expression of NKG2D on and IFN-γ production in CD8+ T cells in mice bearing NKG2DL–expressing and NKG2DL–nonexpressing tumors.Materials and Methods: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in the cell lines. Results: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60.Conclusion: we demonstrated that the expression pattern of NKG2DLs affects tumor immunity and the efficacy of immunotherapy in a tumor-bearing mouse model. In particular, the expression of H60 and Rae-1 family members may be an immunosuppressive factor. Further analysis is required to gain a better understanding of the mechanism of interaction between NKG2D expressed on immune effector cells and H60/Rae-1 expression on tumors. Although NKG2DL expression may represent a marker predicting response to immunotherapy, further studies in humans are required to validate this finding in the clinical setting. Citation Format: Tomohiro Takehara, Kenzo Soejima, Tomoko Betsuyaku, Kiyoshi Yoshimura. The pattern of NKG2DL expressions determines immunogenicity and antitumor effect of protein-bound polysaccharide-K in tumor-bearing mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3806.

Abstract 4553: A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint

Abstract PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired anticancer T cells. However, their efficacy is potentially reduced by ‘on-target/off-tumor' binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity potentially induces a generalized activation of all antigen-experienced T cells as is evidenced by frequent autoimmune-related adverse events during and after treatment. To address these issues, we constructed a tetravalent bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and thus reactivate anticancer T cells more selectively. The IC50 of PD-L1xEGFR for binding to EGFR+ cancer cells was ~140 fold lower compared to an analogous PD-L1-blocking bsAb of irrelevant target antigen specificity (PD-L1xMock). Importantly, treatment with PD-L1xEGFR selectively enhanced activation status, INF-γ production, and activity of anti-CD3xanti-EpCAM-redirected T cells in presence of EGFR+/EpCAM+, but not EGFR-/EpCAM+ carcinoma cells. Similarly, capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-specific CD8+ effector T cells was enhanced in the presence of EGFR+/CMVpp65+ cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in tumor-bearing mice 111In-PD-L1xEGFR showed a significantly higher tumor uptake compared to 111In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, which promotes a more selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies. Citation Format: Iris Koopmans, Douwe F. Samplonius, Robert J. van Ginkel, Peter J. Wierstra, Sandra Heskamp, Edwin Bremer, Wijnand Helfrich. A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4553.

Sulfasalazine alters microglia phenotype by competing endogenous RNA effect of miR-136-5p and long non-coding RNA HOTAIR in cuprizone-induced demyelination

Sulfasalazine (SF) promotes remyelination and improves the outcome of multiple sclerosis (MS) patients. However, the underlining mechanism remains elusive. Here, we examined whether SF blocks microglia switching to a pro-inflammatory M1-like phenotype through a competing endogenous RNA (ceRNA) effects in cuprizone-induced demyelination. The microglia reprogramming effects of SF in the mice model of cuprizone-induced demyelination was measured by histological, immunohistochemical and molecular biological methods. We also measured the effects of the condition media from SF-treated microglia on the differentiation of OLN-93 cells. Insights of the mechanism of ceRNAs of miR-136-5p and long non-coding RNA (lncRNA) HOTAIR were gained from bioinformatic analysis, luciferase assays and RNA binding protein immunoprecipitation. Microglia switched to a pro-inflammatory M1-like phenotype in cuprizone induced-demyelination. Conversely, SF inhibited the M1-like polarization with the increased remyelination which was attenuated by microglia depletion. SF inhibited production of M1-like factors TNF-α and INF-γ in microglia, and thereby promoted the differentiation of OLN-93 oligodendrocytes. SF down-regulated lncRNA HOTAIR but up-regulated miR-136-5p, and thus inactivated AKT2-NF-κB in cuprizone-treated microglia. Importantly, lncRNA HOTAIR overexpression reversed the increased miR-136-5p expression by SF and thereby attenuated the inhibition of AKT2-mediated NF-κB activation. Mimic of miR-136-5p inhibited cuprizone-induced activation of AKT2-NF-κB in the microglia. In summary, SF blocks microglia switching to a pro-inflammatory M1-like phenotype by ceRNA effect of miR-136-5p and lncRNA HOTAIR in cuprizone-induced demyelination. Our findings show the therapeutic potential of SF for human MS probably by targeting epigenetic regulation in microglia.

Effect of the blocking by monoclonal antibodies of TLR2 and CD14 receptors on the expression of intracellular signaling molecules and the production of cytokines in THP-1 macrophages infected with Mycobacterium tuberculosis

Abstract INTRODUCTION There are several reports that indicate the importance of TLR2 and CD14 in the response against Mycobacterium tuberculosis since they activate the cascade of intracellular signaling and the production of inflammatory cytokines necessary for the infiltration of neutrophils and macrophages in the focus of the infection, however it can remain the live bacillus inside the granuloma. MATERIAL AND METHODS In this study, monoclonal antibodies (mAbs) against TLR2 and CD14 were used to block these receptors in the macrophages of the THP-1 cell line infected with Mycobacterium tuberculosis. Molecules of the intracellular signaling cascade were measured: MYD88 and TRAF6 and the transcription factor NFKB by RT-PCR, preinflammatory and anti-inflammatory cytosines by ELISA, also CFU and nitric oxide were measured. RESULTS An increase in the expression of Myd88, TRAF6 and NFKB was found; a decrease in CFU, increased production of TNF-α and INF-γ and low production of nitric oxide. CONCLUSION The results suggest that the mechanisms of intracellular signaling were modified by helping macrophages kill the intracellular bacillus, so we suggest that the use of these mAbs as an immunotherapy that together with the antimicrobial can help to resolve the infection in the patients with tuberculosis. Blockade of TLR2 and CD14 favored the expression of MyD88, TRAF6 and NFkB. Cytosine was also produced, such as TNF-α, which helps the confinement of bacilli in the granuloma and INF-γ, which probably also helped the intracellular death of Mycobacterium tuberculosis.

Unique Cytokine Profile of Novel Innate Lymphoid Cells in Human Decidua

Abstract Immune cells at the maternal-fetal interface play a complex role in regulation of vascular remodeling, fetal tolerance and protection from infection. We have previously presented the identification of two novel CD56highNKs based on Eomes expression (Eomeslo/hi). Activation of these subsets revealed preferential production of IL-8 and IL-17A by decidual innate lymphoid cells (ILCs). Decidual specimens were dissected from term placentas and mononuclear cells (MCs) were isolated by mechanical (GentleMACS) and enzymatic (Collagenase, DNAse) disruption. MCs were then simulated with PMA/ionomycin or IL-23, then labeled by flurochrome-conjugate antibodies against surface CD3, 14, 16, 19, 34, 45, 49a, 56, 94, 117, 127, 335 and intracellular Granzyme B, IL-8, IL-17A, IL-22, INFγ, TNFα. Data acquisition was performed using BD Fortessa flow cytometer in a 5 laser, 18 detector configuration and data analysis was performed using FlowJo 10.2. Activation of decidual ILCs revealed a unique milieu of cytokine production. The novel subsets, CD56high Eomeslo/hi, preferentially produced IL-8 and IL-17A, while decreasing production of INFγ, Granzyme B, and TNFα. In addition, as expected, ILC3s and LTi-like cells produced IL-17A and IL-22. Furthermore, differences in response were observed between decidual basalis and decidual parietalis ILCs, with decidual parietalis ILCs being more responsive to stimulation. Preliminary results suggest that both CD56high Eomeslo/hi subsets demonstrate a similar altered cytokine profile that is conducive to decidual remodeling. Further gene expression analysis will reveal if these novel subsets represent a truly unique population or are members of the expanding tissue-resident NK cell family.

Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3+ T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA+/CD197+ naive T cells, CD45RA−/CD197+ central memory T cells, CD45RA−/CD197− effector memory T cells and CD45RA+/CD197− terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114+ T cells in central memory CD4+ T cell compartment. A dilution series of G-CSF (range, 0.1–10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38+ T cells in CD4+ naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4− central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells.

Cellular immune response in MDR-TB patients to different protein expression of MDR and susceptible Mycobacterium tuberculosis: Rv0147, a novel MDR-TB biomarker.

Tuberculosis (TB) is a crucial public health problem with prevalence of multidrug resistant (MDR) rising. An accurate TB biomarker is urgently needed to monitor the response to treatment in patients with MDR tuberculosis. To analyze interaction between selected MDR-TB purified protein and immune cells, dendritic cells from MDR-TB patients and healthy subjects were stimulated by 55KDa protein fractions (Rv0147). The purified proteins identified by proteomic techniques (two-dimensional gel electrophoresis, mass spectrometry) and peptide sequences are known to bind a MHC class I alleles which are extracted from the Immune Epitope Database and Analysis Resource database ( www.iedb.org ). T cells were isolated from PBMC by negative selection and cells were cultured in RPMI-1640 at 37°C and 5% CO2. Cell culture was assayed for cytokine IL-10 and INF-γ by ELISA. We found that INF-γ production was significantly (335 ± 35.5pg/ml, P ˂0.05) upregulated after protein candidate (Rv0147) stimulation by dendritic cells from MDR-TB patients, whereas IL-10 production was greatly reduced compared with production in healthy subjects (212 ± 9.94pg/ml, P ˂0.05). In fact, the purified protein, Rv0147, stimulated dendritic cells from MDR-TB patients, failed to produce IL-10 and directly stimulates INF-γ production by T cells. These results suggest that the purified protein, Rv0147, may stimulate Th1 type protective cytokine response in MDR-TB patients but not in normal subjects. The production of INF-γ but not IL-10 in the presence of purified protein, Rv0147, may be shifted to Th1 responses in MDR-TB patients and supports its potential as protein vaccine candidates against TB.

Immunogenicity and Efficacy of Polytope DNA Vaccine Against Cutaneous Leishmaniosis In Vivo

Background: About 12 million people are affected by les leishmaniosis worldwide. It is an important public health problem due to the difficulty in its control and causing epidemic outbreaks. An effective anti-leishmanial vaccine remains elusive. Isolated dominant epitopes arranged as polytope DNA vaccine represent a promising approach. Methods: Balb/c mice were vaccinated subcutaneously with pCDNA3.1/LAKJB93 DNA and challenged with Leishmania major promastigotes; then, the level of interferon (INF)-γ and interleukin (IL)-4 were measured by the enzyme-linked immunosorbent assay (ELISA) technique. Production of immunoglobulin (Ig)-G1 and IgG2a antigen specific antibodies were measured against LAKJB93. Results: The enhanced production of antigen-specific INF-γ and IgG2a in serum showed the stimulation of T helper type 1 (Th1) response. This protective immune response was induced by the expression of pcDNA3.1/ LAKJB93 plasmid against leishmaniosis and therefore, pcDNA3.1/ LAKJB93 can be considered an appropriate vaccine for leishmaniosis. Conclusions: Immunization with DNA vaccine may offer an effective route to prevent leishmaniosis.

Reduced CD200 expression is associated with altered Th1/Th2 cytokine production in placental trophoblasts from preeclampsia.

To determine if altered trophoblast CD200 and CD200R expressions promote inflammatory cytokine production in preeclamptic placentas. Placental tissue CD200 and CD200R expressions were determined by immunostaining. Tissue sections from first-, second-, and third-trimester, normal term, and preeclamptic placentas were used. CD200 and CD200R expressions and cytokine production of TNFα, sTNFR1, INFγ, IL-4, IL-6, IL-8, and IL-10 were determined in trophoblasts from normal and preeclamptic placentas and in normal trophoblasts transfected with CD200 siRNA. CD200, but not CD200R, expression was significantly reduced in trophoblasts from preeclamptic compared to normal placentas. Trophoblast from preeclamptic placentas and trophoblast transfected with CD200 siRNA produced significantly more TNFα, sTNFR1, IL-6, and IL-8, but significantly less IL-10, than trophoblasts from normal control placentas. Downregulation of CD200 expression resulted in an imbalance of increased Th1 cytokine and decreased Th2 cytokine production in placental trophoblasts in preeclampsia.

Correlation Between NKG2DL Expression and Antitumor Effect of Protein-bound Polysaccharide-K in Tumor-bearing Mouse Models.

We investigated the relationship between the expression of natural killer group 2, member D ligands (NKG2DLs) and the antitumor effects of protein-bound polysaccharide-K (PSK). PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in multiple cell lines. PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8+ T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60. We demonstrated that the expression of NKG2DLs affects tumor immunity and the efficacy of immuno therapy in tumor-bearing mouse model.

Polysaccharides from Epimedium koreanum Nakai with immunomodulatory activity and inhibitory effect on tumor growth in LLC-bearing mice

Ethnopharmacological relevanceEpimedium koreanum Nakai is documented as tonic herbal in China for over a thousand years and has the potential to enhance the body's immunity according to the theory of traditional Chinese medicine. Polysaccharides are one of the most important effective compounds in Epimedium koreanum Nakai. Accumulating evidence indicated polysaccharides derived from traditional Chinese medicine have potent immune-enhancing properties and relatively nontoxic effects in cancer treatment. However, information about immunological regulation in tumor of Epimedium koreanum Nakai polysaccharides is limited and the reports of purification, characterization of polysaccharides have remained less. The purpose of our study was to further investigate the active polysaccharides from Epimedium koreanum Nakai by evaluating the immune-regulation activities in tumor-bearing mice and provide reasonable explanation for traditional application. Materials and methodsWe firstly purified Epimedium koreanum polysaccharide (EPS) from crude extracts and evaluated EPS in vitro using immunological experiments including maturation and Ag presentation function of DCs, CD4 T-cell differentiation and secretion of anti-cancer cytokines. In LLC-bearing mice model, we investigated its antitumor activities through evaluation of tumor cell proliferative activity, calculation of immune organ indexes and relative host immune system function tests. ResultsResults showed that EPS (180 × 104Da) was composed of mannose (Man), rhamnose (Rha), glucuronic acid (GlcUA), galactosamine (GalN), glucose (Glc), galactose (Gal), arabinose (Ara) and fructose (Fuc). Chemical composition assay indicated EPS was a fraction with 28.20% uronic acid content. FT-IR suggested the presence of pyraoid ring in EPS and SEM displayed smooth surface embedded by several pores. Moreover, Our study suggested EPS could remarkably stimulate macrophages to secrete substantial anti-cancer cytokines and promote maturation as well as Ag presentation function of DCs. Strikingly, CD4 T-cell differentiation and increased INF-γ production stimulated by EPS–activated macrophages were observed in the research. Furthermore, EPS exhibited prominent antitumor activities through regulating host immune system function in LLC-bearing mice. Taken together, experimental findings suggested EPS could be regarded as a potential immune-stimulating modifier for cancer therapy. ConclusionOur studies demonstrated the polysaccharide (180 × 104Da) purified from Epimedium koreanum Nakai could promote maturation and Ag presentation function of DCs, increase the level of immunomodulatory cytokines and activate CD4 T-cell differentiation. Furthermore, it may inhibit the tumor growth in LLC-bearing mice through regulating host immune system function

EMPD-specific chimeric antigen receptor mediates T cell cytotoxicity to B cells expressing membrane IgE

Abstract IgE is the key mediator of allergic immune responses. Targeting the source of IgE, the IgE-expressing B cells, using adoptive T cell therapy (ACT) represents an attractive therapeutic approach for severe allergic diseases such as severe allergic asthma. To develop ACT for allergy, we designed chimeric antigen receptors (CARs) that recognize the extracellular membrane-proximal domain (EMPD) of membrane IgE (mIgE). EMPD serves as an ideal marker for IgE-expressing B ells since it exists only on mIgE but not on secreted IgE, and all IgE-expressing B cells express mIgE, although at varying levels. To construct EMPD-specific CARs, variable regions of four EMPD-specific monoclonal antibodies were cloned to assemble single chain variable fragments (scFvs) for EMPD binding. EMPC-specific CARs were efficiently expressed on the surface of primary human T cells. One of the CARs, 2E3E10, mediated potent cytotoxicity and INFγ production in response to U266 cells and Daudi cells expressing mIgE. The responses to U266 cells are especially encouraging since the cells express very low levels of mIgE. Our study demonstrates the proof of concept for using T cells expressing EMPD-specific CARs to eliminate IgE-expressing B cells. The rapid advances in the efficacy and safety of ACT should facilitate the development of CAR T cell immunotherapy for severe allergic diseases. Taking advantage of the ability of CAR T cells to persist and continuously eliminate IgE-expressing B cells, the approach may relieve symptoms for an extended period of time with a single treatment.

Cold-induced stress modulates the maturation of immune cells of mice during Chlamydia muridarum genital infection

Abstract Chlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease (STD) in the United States. Results in a cold-induced stress mouse model established in our laboratory show that cold-induced stress increases the intensity of chlamydia genital infection. This study was to survey the production of T helper cell type 1 (Th1) and (Th2) cytokines in the stress mouse model. Cells harvested from bone marrow or T cells from spleens of stressed and non-stressed mice during Chlamydia muridarum genital infection were allowed to proliferate in vitro. Cytokine production in culture supernatants was tested by ELISA. Gene expression of GATA 3, T-bet and INF-γ in splenic T cells were determined by QPCR. Production of IL-12 by dendritic cells of stressed mice was 0μg compared to 41.06 μg in non stressed mice. However, C. muridarum infection induced increased IL-12 production (97.27 μg and 123 μg) in stressed and non-stressed mice, respectively. Stress induced a 2-fold decrease in T-bet gene expression in T-cells compared to non-stressed group. In contrast, gene expression of GATA 3 in T cells of stressed mice was significantly increased compared to all other groups tested. INF-γ gene expression in T cells of stressed mice showed a 4-fold decrease compared to T cells of non stressed mice. Stress resulted in decreased IL-12 production in dendritic cells and macrophages, INF-γ production and gene expression of T-bet by Th1 cells. Furthermore, the increased gene expression of GATA 3 only in stressed chlamydia infected T cell suggests that cold-induced stress leads to a skewed immune response toward marked increase in production of Th2 cytokines that favor immunosuppression during chlamydia genital infection.

Glycemic control and the production of cytokines in diabetic patients with chronic periodontal disease

ABSTRACT Objective: To evaluate the association of glycemic control and cytokine production in type 2 diabetic subjects with chronic periodontitis Methods: Gingival biopsies were performed in 40 patients, divided into four groups: systemically healthy subjects without periodontal disease (S); systemically healthy patients with chronic periodontitis (P); patients with well-controlled type 2 diabetes mellitus (DM) with chronic periodontitis (C); poorly controlled type 2 diabetes mellitus with chronic periodontitis (D). The production of interleukin (IL) -4, -6, -10, -17 and interferon (IFN) -γ was quantified by ELISA. Results: The production of IL-4, IL-10, IL-17 and INF-γ was higher on group D when compared to other groups (p <0.05), which in turn were similar (p ≥0.05). In addition, there was no difference in the production of IL-6 in any of the evaluated groups (p≥0.05). Conclusion: Were observed significantly elevated levels of pro-inflammatory and anti-inflammatory cytokines in patients with poorly controlled type 2 diabetes and chronic periodontitis, demonstrating that glycemic control may be associated to the immune inflammatory response of sites with chronic periodontitis.

Haploidentical Natural Killer Cell Infusion Induces Remission in Non-Hodgkin Lymphoma and Overcomes Resistance to Rituximab

Patients with chemotherapy refractory non-Hodgkin lymphoma (NHL) have poor prognosis. We report the results of a phase 2 clinical trial testing the efficacy of haploidentical donor NK cell infusion with rituximab for refractory NHL. Sixteen patients were enrolled, and 14 were evaluable.Treatment included IV lymphodepleting chemotherapy (pentostatin 3.75mg x 2 days; n=6 or fludarabine 25mg/m2 x 4 days; n=10) with cyclophosphamide (60mg/kg IV day -5; n=14). To enhance NK cell function and deplete regulatory T cells(Treg), we used the IL-2 receptor-targeting agent denileukin diftitox (18 μg/kg/day IV day -1; n=5) or methylprednisolone (1 mg/kg day -2 through day +9; n=9). Donor lymphocytes were CD3- and CD19-depleted and overnight stimulated with 1000 U/ml IL-2. The cellular product at a dose of 1.5-8 x 107 NK cells/kg was infused IV on day 0 followed by subcutaneous infusion of IL-2 at 9 million IU x 6 doses. Peripheral blood (PB) donor NK cells were evaluated by short tandem repeats and/or flow cytometry analyzing donor-specific HLA alleles.Patients characteristics included median age of 52 years (range 48-67); diffuse large B cell lymphoma was the most common diagnosis (n=11); all patients were rituximab and chemotherapy refractory.Of 14 evaluable patients at 2 months post-infusion, 4 achieved remissions (2 complete, 2 partial; 28%). Three patients in continuous remission proceeded to allogeneic donor transplantation at 2,3 and 9 months after NK cell therapy.Compared to healthy controls, NK cells in patients prior to therapy had reduced function (CD107a, INFγ and TNFα production), lower expression of natural cytotoxic receptors NKp46, NKp44 and DNAM (but higher NKp30) and reduced expression of suppressor receptors TIGIT, PD1 and TIM3.In all evaluable patients (n=9), donor NK cells persisted in the PB for at least 7 days after infusion with median 18% (range 0.5-97%) of the NK cells were of donor origin. In one responding patient, donor NK cells sustained beyond day 28. Responding patients (R) had significantly lower levels of absolute number of Treg (Foxp3+CD25+CD127-) at baselineand 14 days after therapy (Figure) as compared to non-responders (NR), however neither denileukin diftitox nor methylprednisolone resulted in PB Treg depletion. Expectedly, lower PB Tregs correlated with low serum levels of IL-10 (R2=0.7; p<0.001; n=12), suggestive of attenuated immuno-suppressive milieu. In addition, frequencies of PB myeloid derived suppressor cells (MDSC) were remarkably low in responders as compared to non-responders at baseline (4.5%±0.05% vs. 11%±6%) and after therapy (day 14: 3.8%±1%; vs. 9.4%±5%).Endogenous Inteleukin-15 (IL-15) serum levels peaked at the day of NK cell infusion (range 11-33pg/dl). Responders had higher IL-15 levels compared to non-responders at day 0 (Figure). IL-15 levels also correlated with functional NK cell killing at day 14 as measured by expression of CD107a (R2=0.8; p=0.008; n=5), suggesting that higher endogenous IL-15 improved NK cell cytotoxic capacity.We observed expected, but transient hematologic toxicity. Therapy was associated with grade 3 adverse effects (tumor lysis syndrome, Pneumocystis pneumonia; Clostridium difficile colitis, neutropenic fever; thrombotic microangiopathy). One unevaluable patient developed sepsis prior to NK cell infusion, and the second died of sepsis a week after therapy.In summary, our observation supports further investigation of donor NK cellular therapies for advanced NHL as a strategy to overcome chemoresistance. NK cell expansion and therapeutic efficacy might be further improved by administration of IL-15. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees.

Abstract 4253: Modeling the effects of inflammatory stress on human intestinal epithelial cells in 3D enteroid co-culture system

Abstract The molecular pathogenesis of colitis-associated colorectal cancer (CAC) has been suggested to involve oxidative stress-induced DNA damage, resulting in mutations of tumor-suppressor genes and activation of pro-oncogenic pathways. However, the exact relationship between inflammation and colorectal cancer is not well understood due to the lack of a human model system that not only recapitulates in vivo growth/differentiation patterns, but also can be easily manipulated. In order to gain insight into the mechanism of colitis-associated colorectal cancer, we explored human intestinal enteroids as an ex vivo model to study the interaction of intestinal epithelial cells with immune cells under inflammatory conditions. Human intestinal crypts were isolated from adult intestinal tissue and grown in Matrigel culture to form enteroids. To model inflammation, we incorporated MHC-mismatched human peripheral blood mononuclear cells (PBMCs) into the Matrigel. TH1 immune response was induced by a cocktail of cytokines including IL-2, IL-7 and IL-15. A TH1 response was confirmed by the production of pro-inflammatory cytokines INFγ and TNFα over the course of 6-day treatment with the cytokine cocktail. Several cytokines related to TH1 and TH2 responses were induced. Interestingly, co-culture with human intestinal enteroids significantly enhanced the level of pro-inflammatory cytokines INFγ, IL-6 and TNFα, anti-inflammatory cytokine IL-10, as well as TH2 cytokine IL-13. Immunophenotyping of PBMCs shows that both CD4+ T cells (10-30%) and CD8+ T cells (1-10%) were activated upon TH1 induction. In addition, the percentage of activated CD8+ T cells was much higher in co-culture than in PBMC alone controls. Moreover, the cytokine cocktail dramatically induced the production of INFγ by purified CD4+ or CD8+ T cells but only when in co-culture with enteroids and not when cultured alone. In the enteroids, the TH1 response significantly inhibited cell proliferation and increased the apoptosis of epithelial cells when in co-culture only. Increased γH2AX (DNA damage marker) and impaired integrity of intestinal enteroids were also observed when in co-culture with the TH1 response. Cell-cell contact between enteroids and immune cells is required for these effects. Moreover, the TH1 response increases the mRNA levels of GRP78 (master regulator of ER stress) and Nrf2 (regulator of oxidative stress); decreases the mRNA level of Lgr5 [marker of active intestinal stem cells (ISCs)], but has no obvious effect on Bmi1 (marker of quiescent ISCs) in co-culture. Taken together, our data provide insight into the interaction of intestinal epithelial cells with immune cells at the molecular and cellular levels and establish this approach as a viable model for exploring the mechanism of CAC. Future studies will examine specifically for oxidative DNA damage and the effects of specific tumor suppressor inactivation on epithelial cell responses. Citation Format: Fang Wang, Dorottya Laczkó, Mary Ann Crissey, Gary Falk, Bradley Johnson, Anil Rustgi, John Lynch. Modeling the effects of inflammatory stress on human intestinal epithelial cells in 3D enteroid co-culture system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4253.