IFN-γ In Supernatants Research Articles

Effect of the bone marrow mesenchymal stromal cells cytokine-producing activity on mobilization of hematopoietic stem cells

Hematopoietic stem cell transplantation is used to treat hemoblastoses and some other diseases. Depending on the diagnosis, autologous cells of the patient or allogeneic cells obtained from a healthy related or unrelated donor are used. A sufficient count of harvested hematopoietic progenitor cells is necessary for successful transplantation. Currently, stimulation of their egress from the bone marrow into the peripheral blood by a granulocyte colony-stimulating factor, followed by collection by leukapheresis, is widely used for their preparation. Hematopoietic stem cells leave the bone marrow niche formed by the stromal microenvironment when their holding bonds are interrupted. Mesenchymal stromal cells regulate the release of hematopoietic precursors by producing various cytokines and other biologically active substances. Therefore, the study of the functional properties of bone marrow mesenchymal cells can help in solving the problem of “poor” mobilizations that occur in patients with multiple myeloma.The aim of the study was to estimate the effectiveness of mobilization of hematopoietic stem cells depending on the cytokine-producing ability of mesenchymal stromal cells in the bone marrow of donors and patients with multiple myeloma.The yields of hematopoietic progenitors were studied in 10 donors (median age 27 years) and 18 patients with multiple myeloma (median age 57 years). In donors, the release of hematopoietic stem cells into the peripheral blood was stimulated by subcutaneous administration of G-CSF at a dose of 10 μg/kg/day. Patients with multiple myeloma received vinorelbine at a dose of 35 mg/m2 of body surface, followed by administration of G-CSF (10 μg/kg/day subcutaneously). A culture of mesenchymal stromal cells was obtained from bone marrow taken prior to mobilization and the content of interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α and IFN-γ in supernatants was studied by enzyme-linked immunosorbent assay using kits of reagents produced by Vector-Best (Novosibirsk).Patients with multiple myeloma were characterized by high secretion of the pro-inflammatory cytokine IL-2 (4.70 pg/ml vs 3.55 pg/ml in healthy individuals, p=0.003) and low IFN-γ (0.41 pg/ml vs 2.14 pg/ml in healthy, p<0.001), but no relationship was found between these cytokines and the hematopoietic stem cells yield. The present study showed that in patients with ineffective harvesting of hematopoietic precursors, secretion of IL-8 by stromal cells was low (308.08 pg/ml vs 561.29 pg/ml in healthy individuals, p=0.04).The obtained results are consistent with the literature data on the important role of IL-8 in the mobilization of hematopoietic stem cells, which allows to consider IL-8 as an informative marker for predicting the insufficient yields of hematopoietic precursors.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

Perforin Is Dispensable for Anti-CD19 CAR-T Cell Cytotoxicity but Contributes to CAR-T Cell Contraction and Regulates Host Inflammatory Response

Background: Chimeric antigen receptor transduced T cell (CAR-T) therapy against B-cell malignancies is highly effective, but is frequently associated with cytokine release syndrome (CRS). In severe cases, CRS can be associated with a prolonged life-threatening systemic inflammatory response mimicking manifestations of hemophagocytic lymphohistiocytosis (HLH). Primary HLH occurs among individuals harboring genetic defects in granule-mediated cytotoxicity pathway, such as perforin gene mutations. In a murine primary HLH model, IFNγ derived from antigen-specific CD8+ T cells is implicated as a major mediator of HLH pathology. We explored the role of granule-mediated cytotoxicity and IFNγ in CAR-T response and toxicity, using an established syngeneic murine model of anti-CD19-CAR-T therapy against B-cell malignancy.Methods: Splenic T cells derived from perforin knockout (Prf-/-), wild-type (WT), and IFNγ adenylate-uridylate-rich element-deleted (ARE-/-: a strain with stable IFNγ mRNA transcript) mice on the B6 background were transduced with second-generation murine anti-CD19 CAR (ScFV-CD28-CD3z). In in vivo experiments, mice received cyclophosphamide (200 mg/kg) intraperitoneally 24 hours prior to CAR-T adoptive transfer as lymphodepletion.Results: Both WT and Prf-/- T cells were successfully transduced with a mean transduction efficiency of 80%, and resulted in similar immunophenotypic composition (CD8/CD4, TCM/TEM) at baseline. Compared to WT CAR-T, Prf-/- CAR-T produced a higher amount of IFNγ when co-cultured with CD19+ leukemia (12-hour coculture supernatant IFNγ, WT 7822 pg/mL vs. Prf-/- 14743 pg/mL, p<0.0001). In vitro cytotoxicity assays (Incucyte) demonstrated that Prf-/- CAR-T were less efficient in clearing leukemia compared to WT. The differential cytotoxic activity between WT and Prf-/- CAR-T became apparent in vivo when CAR-T were given at low dosage (1E5, which is a curative dosage for WT CAR-T). However, at higher dosage (1E6 or higher), Prf-/- CAR-T eradicated CD19+ leukemia in vivo with the same leukemia clearance kinetics as WT CAR-T and achieved long-term complete remission. During maximal in vivo CAR-T expansion, mice treated with high-dose Prf-/- CAR-T (7E6) had elevated serum IFNγ level, while IFNγ in WT CAR-T group was barely detectable (Fig. 1). Such an increase in IFNγ serum levels did not occur at the lower cell dosage (1E5) in either Prf-/- or WT CAR-T group. Maximal in vivo CAR-T expansion occurred between day 3-4 in both WT and Prf-/- groups. With WT cells, CAR-T contracted by day 8 as leukemia cleared (Fig. 2), and the surface CAR expression dropped and CAR-T phenotype transitioned from TEM to TCM. Surprisingly, Prf-/- CAR-T had a secondary proliferation between day 13-20 (Fig. 2) despite no measurable leukemia or endogenous B-cells at that time. Prf-/- CAR-T had a sustained high level of surface CAR expression and the majority remained in a TEM phenotype. Host-derived CD8+ T-cell phenotype also skewed towards TEM (instead of Tnaive or TCM) in Prf-/- CAR-T group compared to WT CAR-T group. Additionally, spleens derived from Prf-/- CAR-T-treated mice demonstrated increased phagocytosis. These changes in host-derived immune-associated cells suggest that the lack of perforin not only affects CAR-T but may also induce a pro-inflammatory microenvironment through soluble mediators. Among cytokines that we have tested so far (IFNγ, IL-6, TNFα, IL-2, IL-4, IL-17A, IL-10, IL-12p70, MCP-1, GM-CSF), IFNγ has been the only cytokine differentially elevated in vitro and in vivo, comparing Prf-/- to WT CAR-T. IFNγ levels during initial CAR-T expansion phase may be playing a critical role in the persistence of CAR-T. Preliminary results from our ARE-/- CAR-T model also demonstrated that significantly higher serum IFNγ levels during initial CAR-T expansion phase was associated with higher CAR-T in vivo persistence, regardless of antigen availability.Conclusion: Collectively, these data suggest that perforin is dispensable in murine CD19-CAR-T cytotoxic activity, but contributes to CAR-T contraction, and possibly to the regulation of a secondary inflammatory response. We hypothesize that IFNγ may be the major mediator of the observed differences. Experiments are underway to further elucidate the interplay of perforin and INFγ in CAR-mediated cytotoxicity and to determine whether targeting IFNγ may be a therapeutic option in HLH-like CAR toxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD137 ligand feedback upregulates PD-L1 expression on lung cancer via T cell production of IFN-γ.

BackgroundThe expression of PD‐L1 and its regulation in tumors remains unclear. The importance of IFN‐γ in upregulating the PD‐L1 expression in various tumors, and the effects of other essential cytokines in the tumor microenvironment (TME), need to be further elucidated.MethodsConstitutive expression of PD‐L1 and CD137L in all 13 lung cancer cell lines were tested by flow cytometry. CD137L mRNA of lung cancer cell lines was detected by RT‐PCR. PD‐L1 expression rates following stimulation with these cytokines (IFN‐γ, TNFα and IL2) were measured. After coculture of cells expressing CD137L (lung cancer cells or 293FT cells transfected with CD137L plasmid) with T cells, the PDL1 expression of lung cancer cells and IFN‐γ in supernatant was detected.ResultsOur data revealed that adenocarcinoma and squamous cell carcinoma cells had the highest positive expression rate. IFN‐γ was the core‐inducing factor for enhancing the PD‐L1 expression. CD137L was also widely expressed in the lung cancer cell lines at the mRNA level, whereas its expression was generally low at the protein level. However, the low expression of CD137L protein was still enough to induce T cells to produce IFN‐γ, which subsequently increased the PD‐L1 expression by lung cancer cells. The CD137 signal induces IFN‐γ secretion by T cells, which stimulates high‐level of PD‐L1 expression in cancer cells; this negative immune regulation may represent a mechanism of immune escape regulation.ConclusionsCD137L mRNA was widely expressed in lung cancer cell lines whereas levels of protein expression were generally low. The low level of CD137L protein was still enough to induce T cells to produce IFN‐γ that subsequently increased PD‐L1 expression. The CD137L‐induced negative immune regulation may represent a mechanism of immune escape.

Dihydromyricetin inhibits the M1 polarization of macrophage induced by oxidized low-density lipoprotein through JAK3/STAT5 signaling pathway

Objective To observe the effect of dihydromyricetin(DMY) on the M1 polarization of macrophages induced by oxidized low-density lipoprotein(Ox-LDL) and to explore its mechanism. Methods RAW 264.7 cells were incubated in RPMI1640 medium containing Ox-LDL(50 mg/L) and DMY(40 μmol/L) for 24 h. The effect of JAK/STAT signaling pathway inhibitor AG-490 on DMY was observed.The levels of tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), transforming growth factor-β(TGF-β) and interleukin-10(IL-10) in the supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA). The protein expressions of inducible nitric oxide synthase(iNOS) and arginase 1(Arg1) were tested by Western blot. Results Compared with the control group, the Ox-LDL group showed that the protein expression in macrophage of iNOS and levels of TNF-α and IFN-γ in the supernatant of culture medium were significantly increased(t=4.12, 2.89 and 3.44, P=0.023, 0.036 and 0.031), while the protein expression of Arg1 in cells and levels of TGF-β and IL-10 in the supernatant of culture medium were significantly decreased(t=3.71, 2.31 and 2.67, P=0.034, 0.038 and 0.034), and the ratios of p-JAK3/JAK3 and p-STAT5/STAT5 in cells were significantly increased(t=3.93 and 2.65 and P=0.007 and 0.021). Compared with the Ox-LDL group, the Ox-LDL+ DMY group showed that the protein expression of iNOS in cells and levels of TNF-α and IFN-γ in the supernatant of culture medium were significantly decreased(t=5.74, 2.37 and 3.07, P=0.022, 0.031 and 0.033), while the protein expression of Arg1 in cells and levels of TGF-β and IL-10 in the supernatant of culture medium were significantly increased(t=3.94, 2.19 and 2.44, P=0.025, 0.047 and 0.035), and the ratios of p-JAK3/JAK3 and p-STAT5/STAT5 in cells were significantly decreased(t=3.41 and 2.32, P=0.015 and 0.023). Compared with the Ox-LDL+ DMY group, the Ox-LDL+ DMY+ AG-490 group showed that the protein expression of iNOS and levels of TNF-α and IFN-γ in supernatant of culture medium were significantly increased(t=3.14, 3.41 and 3.74, P=0.017, 0.028 and 0.023), while the protein expression of Arg1 and levels of TGF-β and IL-10 were significantly decreased(t=2.87, 2.85 and 3.47, P=0.025, 0.035 and 0.028). Conclusions Dihydromyricetin inhibits the M1 polarization of macrophages induced by Ox-LDL, and the mechanism may be related with the activation of JAK3/STAT5 signaling pathway. Key words: Atherosclerosis; Lipoprteins, LDL; Macrophage activation

The distinct role and regulatory mechanism of IL-17 and IFN-γ in the initiation and development of plaque vs guttate psoriasis

BackgroundFew studies have explored the differences of immunopathogenesis in plaque vs guttate psoriasis, especially on the inhibitory role of regulatory T cells (Tregs) on IL-17/ IFN-γ production and the impact of CD4+T cells on keratinocytes. ObjectiveTo investigate the percentage and inhibitory function of CD4+CD25highTreg and differential expressions of IL-17/ IFN-γ in plaque vs guttate psoriasis, and the effects of CD4+T cells on the proliferation of keratinocytes. MethodsPeripheral blood mononuclear cells (PBMCs) were prepared from patients with the plaque and guttate psoriasis. The percentage of CD4+CD25high Tregs, IL-17/IFN-γ- producing CD4+ or CD8+T cells, and apoptosis and cell cycle of Hacat cells were determined by flow cytometry. The level of IFN-γ in supernatants was analyzed by ELISA. ResultsThe percentage of CD4+CD25highTregs in plaque psoriasis was significantly increased, and they can inhibit IFN-γ production from CD4+CD25- effector T cells. The percentage of CD8+IFN-γ+cells was also significantly increased in plaque psoriasis, and these cells positively correlated with disease severity. The percentage of CD4+CD25highTregs was decreased and CD4+IFN-γ+/IFN-γ+IL-17+ cells were predominantly increased in guttate psoriasis. CD4+T cells from guttate psoriasis induce apoptosis of keratinocytes while they promote the proliferation of keratinocytes in plaque psoriasis by decreasing late apoptosis and increasing the percentage of G1 phase. ConclusionThere was considerable discrepancy of the phenotype and function of T cells between plaque vs guttate psoriasis. IFN-γ and IL-17 from CD4+T cells play a crucial role in guttate psoriasis, however, IFN-γ and IL-17 from CD8+T cells are more important in the immunopathogenesis of plaque psoriasis.

Potential Immunological Biomarkers for Detection of Mycobacterium tuberculosis Infection in a Setting Where M. tuberculosis Is Endemic, Ethiopia.

Accurate diagnosis and early treatment of tuberculosis (TB) and latent TB infection (LTBI) are vital to prevent and control TB. The lack of specific biomarkers hinders these efforts. This study's purpose was to screen immunological markers that discriminate Mycobacterium tuberculosis infection outcomes in a setting where it is endemic, Ethiopia. Whole blood from 90 participants was stimulated using the ESAT-6/CFP-10 antigen cocktail. The interferon gamma (IFN-γ)-based QuantiFERON diagnostic test was used to distinguish between LTBI and uninfected control cases. Forty cytokines/chemokines were detected from antigen-stimulated plasma supernatants (SPSs) and unstimulated plasma samples (UPSs) using human cytokine/chemokine antibody microarrays. Statistical tests allowed us to identify potential biomarkers that distinguish the TB, LTBI, and healthy control groups. As expected, the levels of IFN-γ in SPSs returned a high area under the receiver operating characteristic curve (AUC) value comparing healthy controls and LTBI cases (Z = 0.911; P < 0.001). The SPS data also indicated that interleukin 17 (IL-17) abundance discriminates LTBI from healthy controls (Z = 0.763; P = 0.001). RANTES and MIP-1β were significantly elevated in SPSs of TB-infected compared to healthy controls (P < 0.05), while IL-12p40 and soluble tumor necrosis factor receptor II (sTNF-RII) were significantly increased in active TB cases compared to the combined LTBI and control groups (P < 0.05). Interestingly, quantitative changes for RANTES were observed using both SPSs and UPSs, with P values of 0.013 and 0.012, respectively, in active TB versus LTBI cases and 0.001 and 0.002, respectively, in active TB versus healthy controls. These results encourage biomarker verification studies for IL-17 and RANTES. Combinations of these cytokines may complement IFN-γ measurements to diagnose LTBI and distinguish active TB from LTBI cases.

Characterization of the immune response elicited by the vaccinia virus L3 protein delivered as naked DNA

Poxviruses are complex dsDNA viruses with over 200 genes, many of them with unknown role in the stimulation of immune responses. Among these, the vaccinia virus (VACV) L3L ORF encodes an essential protein for the transcription of the VACV early genes. To the best of our knowledge, the immune response elicited by L3 has not been characterized. In this regard, our data describes a DNA L3-coding plasmid (pL3L) that stimulates both, humoral- and cell-mediated immune responses in a mouse model. Cell-mediated immune responses were measured by IFN-γ and IL-4 ELISPOT assays. We performed CD8+ cells depletion and flow cytometry analysis to account for the contribution of cytotoxic T lymphocytes in the IFN-γ production. Moreover, results from ELISPOT were confirmed by measuring the concentration of IL-4 and IFN-γ in supernatant of antigen-stimulated splenocytes by cytokine ELISA. Additionally, dominant antigenic regions of L3 protein were identified by epitope mapping analysis. Humoral immune responses were assessed by ELISA. Specifically, the production of total IgG, IgG1 (TH-2) and IgG2a (TH-1) were determined one week after the final immunization. Our ELISPOT data shows pL3L-immunized animals to produce significantly higher frequencies of IFN-γ Spot-Forming Cells (SFC) versus controls. IL-4 levels remained unchanged in all three groups, demonstrating the increase in antigen-specific IFN-γ releasing cells. Flow cytometry assay results showed that CD8+ T cells are a major contributor to the production of IFN-γ. Moreover, our formulation enhances the production of total IgG, predominantly IgG2a isotype. Immunization with pL3L promotes a robust cytotoxic immune response, crucial against viral pathogens. In addition, our vaccine candidate promotes an increase in IgG levels, especially IgG2a (TH-1 type). Our data encourages further studies of L3 as a novel antigen in vaccine development against poxviruses.

NKG2D modulates aggravation of liver inflammation by activating NK cells in HBV infection

Hepatitis B virus (HBV) infection is thought to be an immune-mediated liver disease. The mechanisms underlying natural killer (NK) cell group 2D receptor (NKG2D) that activates NK cells and participates in anti-HBV immunity and immunopathology has not been thoroughly elucidated. Peripheral NKG2D+ and IFN-γ+ NK cells frequencies and intrahepatic NKG2D and IFN-γ mRNA and protein expressions were determined in HBV-infected patients. Levels of NKG2D and IFN-γ mRNA and protein in NK cells, co-cultured with HBV-replicating HepG2 cells with or without NKG2D blockade, were analyzed. Serum and supernatant IFN-γ, TNF-α, perforin and granzyme B were measured. In results, peripheral NKG2D+ and IFN-γ+ NK cells frequencies, intrahepatic NKG2D and IFN-γ mRNA and protein levels, and serum IFN-γ, TNF-α, perforin and granzyme B levels were all highest in HBV-related acute-on-chronic liver failure group, followed by chronic hepatitis B and chronic HBV carrier groups. In vitro, NKG2D and IFN-γ mRNA and protein levels were higher in NK cells with IFN-α stimulation than without stimulation. Supernatant IFN-γ, TNF-α, perforin and granzyme B levels were increased under co-culture or IFN-α stimulating conditions, but were partially blocked by NKG2DmAb. In conclusion, NKG2D regulates immune inflammation and anti-viral response partly through activation of NK cells during HBV infection.

Cytokine profile of sipuleucel-T (sip-T) in differentiating reactivation of latent immunity from de novo immune responses.

163 Background: Sip-T is an FDA-approved immunotherapy for treating patients (pts) with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). It is manufactured from autologous peripheral blood mononuclear cells (PBMCs) cultured with PA2024, a fusion of prostatic acid phosphatase (PAP) and granulocyte macrophage colony-stimulating factor. Survival of sip-T–treated mCRPC pts correlates with immune responses to PA2024 and/or PAP. PA2024- or PAP-specific CD4+ and CD8+ T cell proliferation and cytokine production and release were assessed to better understand sip-T–induced T cell responses. Methods: Pts with biochemical recurrence or mCRPC were from sip-T trials (NCT01431391, NCT01981122). PBMCs collected at baseline through 6 mo post–sip-T were cultured in vitro and stimulated with PA2024 or PAP. CD4+ and CD8+ T cells were assessed (n=19) for proliferation and intracellular IL-2 and IFN-γ. The cytokine profile was confirmed in supernatant with a meso scale discovery assay. P&lt;0.10 was statistically significant. Results: Compared with baseline, PA2024-specific proliferating CD4+ and CD8+ T cells had increased intracellular IL-2 and IFN-γ levels at wk 6 and mo 6, with a similar trend for PAP-specific proliferating T cells (Table 1). Compared with unstimulated controls, a significant &gt;2-fold increase in PA2024-stimulated IL-2 and IFN-γ in supernatant was observed at wk 6 and mo 6 over baseline (p&lt;0.001). PAP-stimulated IL-2 and IFN-γ supernatant levels increased over baseline and were significantly elevated for IFN-γ at wk 6 (p&lt;0.10). Conclusions: Sip-T therapy generated a de novo PA2024-specific T cell response, as indicated by the cytokine release profile. The PAP-stimulated cytokine profile suggests that pre-existing immunity with terminally differentiated T cells are expanded. Thus, sip-T reactivated an anti-PAP response in memory T cells, thereby overcoming immunosuppressive mechanisms in PC. Clinical trial information: NCT01431391; NCT01981122. [Table: see text]

Rhizopus arrihizus swollen spores induce mice dendritic cells to Th1 and Th17 differentiation

Objective To study the mechanism of adaptive immunity against Rhizopus arrihizus (R.arrihizus) infections. Methods Bone marrow derived dendritic cells (BMDCs) were separated from C57BL/6 mice and Card9-/- mice and then were cultured in vitro. Resting spores and swollen spores of R. arrihizus were in vitro co-cultured with BMDCs with or without Syk inhibition. Secretion of cytokines (IL-23, IL-1β and IL-12) was analyzed by ELISA after 24 hours of culture. Naive T cells derived from C57BL/6 mice were in vitro co-cultured with spore-stimulated BMDCs for four days. Levels of IL-17A and IFN-γ in supernatants of cell culture were analyzed by ELISA. Flow cytometry was performed to analyze T cell differentiation. Confocal microscopy was used to observe the images of stained β-glucan on the surface of resting and swollen spores. Swollen spores were co-cultured with Dectin-1, Dectin-2, TLR2 and mannose receptor (MMR), and the binding results were analyzed by flow cytometry. Results Swollen spores but resting spores could induce the maturation of BMDCs and promote the secretion of cytokines (IL-23, IL-1β and IL-12). Co-culturing T cells with swollen spore-stimulated BMDCs enhanced their differentiation to Th17 and Th1. In addition, swollen spores promoted the secretion of Th1-related cytokine (IFN-γ) and Th17-related cytokine (IL-17A). Adding Syk inhibitor to Card9-/- BMDCs or wild type BMDCs significantly inhibited the secretion of cytokines and T cell differentiation, especially in the Card9-/- group. β-glucan was overserved on the surface of swollen spores, but not on resting spores. On the surface of swollen spores existed pathogen associated molecular patterns (PAMPs) that could bind with Dectin-1 and TLR2. Conclusion Swollen spores of R. arrihizus could active BMDCs to secrete cytokines of IL-23, IL-1β and IL-12 and trigger T cell responses in vitro. The possible mechanism might be associated with β-glucan exposed on the surface of swollen spores that binds with Dectin-1. The responses between BMDCs and R. arrihizus are Syk-Card9-dependent. Key words: BMDCs; Rhizopus arrihizus; Syk-Card9

The role of microRNA-146a in the modulation of CD4+ T lymphocyte in children with psoriasis vulgaris

Objective To investigate the effect of miRNA-146a expression on the differentiations and functions of CD4+ T lymphocyte in children with psoriasis vulgaris. Methods Thirty children with psoriasis vulgaris and 30 normal controls were enrolled in this study.Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of miRNA-146a in CD4+ T lymphocytes of the peripheral blood.The CD4+ T lymphocytes were isolated from mononuclear cells prepared with Ficoll-Hypaque density-gradients centrifugation from human peripheral blood by magnetic cells sorting system.The CD4+ T lymphocytes were divided into 3 groups for the experiment: control group, miRNA-146a group, and miRNA-146a inhibitor group.The number of Th1 cells was mea-sured by flow cytometry analysis (FACS). The protein levels and expressions of IFN-γRα were measured by using Western blot and RT-PCR, respectively.The levels of IFN-γ in culture supernatants of CD4+ T lymphocytes were detected by using enzyme-linked immunosorbent assay(ELISA). Results Compared with the normal control group, there was significant increase in the expression of miRNA-146a and serum level of IFN-γ in the psoriasis vulgaris group[2.43±0.94 vs 1.05±0.23, (27.69±7.64) ng/L vs (9.75±2.81) ng/L] (t=6.651, 4.237, all P<0.01). There was a positive correlation between miRNA-146a and serum IFN-γ (r=0.837, P<0.01). However, in vitro, the number of Th1, and supernatant IFN-γ increased, and the expression of IFN-γRα protein decreased in miRNA-146a group compared with that of the control group(all P<0.01). And the miRNA-146a inhibitor could attenuate the effect of miRNA-146a effectively. Conclusions The miRNA-146a can promote differentiation and improve the function of Th1, It may play an important role in the pathogenesis of psoriasis vulgaris in children. Key words: Psoriasis vulgaris; MicroRNA; T lymphocyte; Cytokine; Child

Dynamic evaluation of cell-secreted interferon gamma in response to drug stimulation via a sensitive electro-chemiluminescence immunosensor based on a glassy carbon electrode modified with graphene oxide, polyaniline nanofibers, magnetic beads, and gold nanoparticles

A sensitive electrochemiluminescent (ECL) immunosensor was developed for real-time and dynamic evaluation of the concentration of interferon gamma (IFN-γ) that is secreted by peripheral blood mononuclear cells. IFN-γ is a diagnostic marker for latent infections with Mycobacterium tuberculosis (MTB), and its levels vary in response to drugs stimulation. The ECL immunosensor was fabricated by attaching gold nanoparticle-coated magnetic beads (AuNP@MB) on a nanofiber prepared from graphene oxide and polyaniline (GO-PANI-NF). The sensor possesses a three-dimensional structure nanocomposite that is beneficial for immobilization of primary antibodies (Ab1), and this enhances the intensity of ECL. The assay is based on a sandwich-type immunoreaction in which secondary antibodies (Ab2) labeled with CdS quantum dots (QDs) are used to generate ECL. The assay has a wide linear range (0.1–500 pg⋅mL−1) and a low detection limit (30 fg∙mL−1). The method was applied to real-time and dynamic determination of IFN-γ in supernatants of peripheral blood mononuclear cells in response to stimulation by chlorogenic acid. Conceivably, it may be used for monitoring the dynamic variation of biomarkers in cells supernatants in response to drug stimulation and to diagnose other infectious diseases.

Impaired M. tuberculosis Antigen-Specific IFN-γ Response without IL-17 Enhancement in Patients with Severe Cavitary Pulmonary Tuberculosis.

BackgroundTh1 cells play an essential role in immune protection against tuberculosis. Th17 cells might be involved with immune pathology in active human tuberculosis (TB). The balance between Th1 and Th17 cells in patients with cavitary tuberculosis needs to be clarified which might help understanding the immunological basis of pathologic pathogenesis in TB.MethodInitially treated pulmonary TB (PTB) patients with or without cavities were recruited before chemotherapy. We isolated peripheral blood mononuclear cells, stimulated with phytohemagglutinin (PHA), PPD, or ESAT-6 antigens, and assayed supernatant IFN-γ and IL-17 by ELISA after 24 or 72 hours incubation, respectively. Cells were also stained with antibodies to CD3, CD4, CD8, IFN-γ or IL-17 and the proportion of stained cells was measured by flow cytometry.ResultsWe found wide variation of IFN-γ response in active PTB patients, but less subject-to-subject variation of IL-17 was observed as we previously reported. There were no significant differences in IFN-γ and IL-17 between cavitary and non-cavitary PTB; however, we found decreased IFN-γ secretion in severe cavitary PTB compared to mild lesion non-cavitary PTB (p < 0.05). We also found a decrease in the proportion of CD3+CD4+ T cells in the blood of severe cavitary PTB patients (p < 0.05).ConclusionsIL-17 seemed to have no association with the formation of cavities in active PTB from the study of PBMC. Impaired IFN-γ without IL-17 enhancement occurs in peripheral blood during severe cavitary PTB. Our results demonstrate that M. tuberculosis antigen-specific Th1 response is decreased when PTB lesions develop to severe cavities.

Differential T-Helper Cell Polarization after Allergen-Specific Stimulation of Autologous Dendritic Cells in Polysensitized Allergic Patients

Background: Dendritic cells (DCs) play an important role in the induction and regulation of adaptive immune responses by polarizing T-helper (Th) cells. In allergic disease this response is dominated by Th2 cells. It is still unclear whether the activation of Th cells by DCs in atopic individuals is allergen specific. Methods: Monocyte-derived DCs (MoDCs) obtained from polysensitized patients were stimulated with purified Bet v 1, Phl p 5 and Act d 10, and the surface marker expression was analysed. Proliferation and cytokine profiles of autologous naïve CD4+ T cells co-cultured with allergen-pulsed MoDCs were assessed. Results: The addition of either Bet v 1 or Phl p 5 did not further increase the expression of surface markers from matured MoDCs in all study groups. In co-cultures, autologous naïve CD4+ T cells proliferated when DCs obtained from individuals allergic to birch and grass pollen were stimulated with Bet v 1 and Phl p 5, respectively. In the co-culture supernatants, significantly increased levels of IL-5 and IL-13 were detected. This effect correlated with the sensitization background and was absent when applying an unspecific allergen, Act d 10. The levels of IL-10 in supernatants of MoDCs and the levels of IL-10 and IFN-γ in supernatants of T cells remained unchanged upon stimulation with allergens. Conclusions: In this study we observed that allergen-specific stimulation of MoDCs induces T-cell proliferation and upregulation of Th2-type cytokines. Interestingly, this Th2 polarization was only observed in cells stimulated with the allergen to which the patients were sensitized.

Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome.

In spite of extensive research, immunologic control mechanisms against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) remain poorly understood. Cytokine responses have been exhaustively studied in nursery pigs and show contradictory results. Since no detailed reports on cytokine responses to PRRSv in pregnant females exist, the objectives of this study were to compare host cytokine responses between PRRSv-infected and non-infected pregnant gilts, and to investigate relationships between cytokine levels in infected gilts and viral load or fetal mortality rate. Serum samples and supernatants of peripheral blood mononuclear cells (PBMC) either stimulated with PRRSv or phorbol myristate acetate/Ionomycin (PMA/Iono) were analyzed for cytokines/chemokines: interleukins (IL) 1-beta (IL1β), IL4, IL8, IL10, IL12, chemokine ligand 2 (CCL2), interferon alpha (IFNα) and interferon gamma (IFNγ). Three cytokines (IFNα, CCL2, IFNγ) in gilt serum differed significantly in inoculated versus control gilts over time. In supernatants of PRRSv stimulated PBMC from PRRSv-infected gilts, levels of IFNα were significantly decreased, while IL8 secretion was significantly increased. PRRSv infection altered the secretion of all measured cytokines, with the exception of IFNα, from PBMC after mitogen stimulation, indicating a possible immunomodulatory effect of PRRSv. IFNα, CCL2, and IFNγ in serum, and IFNγ in supernatants of PMA/Iono stimulated PBMC were significantly associated with viral load in tissues, serum or both. However, only IFNα in supernatants of PRRSv stimulated PBMC was significantly associated with fetal mortality rate. We conclude that of the eight cytokines tested in this study IFNα was the best indicator of viral load and severity of reproductive PRRSv infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0113-8) contains supplementary material, which is available to authorized users.

Enhancement of antigen presenting ability in the leukemic plasmacytoid dendritic cell line (PMDC05) by lentiviral vector-mediated transduction of CD80 gene (P5059)

Abstract [Aim] PMDC05, a leukemic plasmacytoid dendritic cell (pDC) line which was established from CD4+CD56+ leukemia cells in our laboratory, showed a capacity of generating antigen-specific cytotoxic T lymphocytes (CTLs). In order to enhance an antigen presenting ability of PMDC05, PMDC05 was transduced with CD80 gene by lentiviral vector, which was named as PMDC11. We investigated whether PMDC11 could enhance allogeneic T cell response. [Materials and Methods] Surface antigen presentation-associated molecules and antigen presenting ability were analyzed in PMDC05 and PMDC11 with or without stimulation by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (CI) by flow cytometry or mixed leukocyte culture (MLC). [Results] CD80 gene transduction induced a definite up-regulation of CD1a and HLA-DR in addition to CD80. In original PMDC05 cells, which had antigen presenting ability without any stimulation, PMA/CI could enhance the ability significantly. PMDC11 cells without stimulation had an antigen presenting ability equivalent to PMA/CI-stimulated PMDC05. Measurement of IFN-γ in supernatant of MLC demonstrated that PMDC11 stimulated allogeneic lymphocyte to produce much more IFN-γ compared with PMDC05. [Conclusion] Our data suggested PMDC11 cells, which were established by transducing CD80 gene into PMDC05 cells, could be efficiently applied as antigen presenting cells in adoptive cellular immunotherapy for tumors and severe infections.

Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.

Event Abstract Back to Event Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth. Claudia Mongini1*, Julieta De Toro1, Leticia Hershlik1, María J. Gravisaco2, Claudia Waldner1 and Paula Di Sciullo1 1 National Research Council Of Argentina, Argentina 2 Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina Our aim was to study the immunomodulating and proapoptotic properties of the TLR7 agonist Imiquimod to be used as adjuvant in a tumor vaccine. Imiquimod showed an antiproliferative effect on LBC T-cell lymphoma in vitro at 48 and 72 h with doses between 0.5 and 200 μg/ml, inducing apoptosis in a dose dependent manner. BALB/c mice inoculated intraperitoneally twice with LBC cells lysates with or without 100 μg/mouse of Imiquimod and challenged one week later with LBC cells intraperitoneally, showed a significant increase in survival compared to un-immunized controls (LBC-lysate<0.05, LBC-lysate + IMQ<0.0005) and to mice receiving Imiquimod (p<0.0002; Logrank).Tumor incidence was only 5±5% in LBC-lysate+IMQ group whereas in LBC-lysate group was 52.5±2.5%. Although the percentages of specific cytotoxicity were above normal in both immunized groups (p<0.05, ANOVA), LBC-lysate+IMQ immunization induced the highest levels. A significant enhance in peritoneal cavity CD8+, CD4+ T and B cells (p<0.0001, ANOVA) with an increase IFNγ in supernatant from peritoneal cavity cells and splenocytes was demonstrated in all vaccinated groups compared to normal (p<0.001, ANOVA). Our vaccine also generated circulating antibodies in all immunized mice that rejected the tumor up to 75 days after challenge. Finally, mice that rejected the first tumor challenge were re-challenged with LBC tumor with an increased in survival in all immunized groups over the control (p<0.01, Logrank) with rejection rates close to 100%. We demonstrated that Imiquimod not only modulates immune response efficiently to reject tumor generating a long-lasting memory, but also is capable of inducing tumor apoptosis. Keywords: imiquimod, T-cell lymphoma, TLR7 agonist, Apoptosis, tumor cell lysate vaccine Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Mongini C, De Toro J, Hershlik L, Gravisaco MJ, Waldner C and Di Sciullo P (2013). Imiquimod enhances anti-lymphoma response in a tumor cell lysate vaccine and inhibits tumor cells growth.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01162 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Claudia Mongini, National Research Council Of Argentina, Buenos Aires, Buenos Aires, 1121, Argentina, cmongini@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Google Scholar Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo PubMed Claudia Mongini Julieta De Toro Leticia Hershlik María J Gravisaco Claudia Waldner Paula Di Sciullo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Assessment of Cytokine Release After in Vitro Stimulation of Whole Blood with Legionella Pneumophila in Immunocompromised Patients

This study we examined ex vivo potential of the immune response after stimulation of whole blood with L. pneumophila SG 1, SG 2-14 and L. pneumophila standard strain ATCC 33152 in immunocompromised patients, such as: hemodialysis patients and patients after renal transplantation. The levels of TNF-α and IFN-γ in supernatants were measured with the use of commercial ELISA kits. The synthesis of TNF-α and IFN-γ after stimulation with L. pneumophila were analyzed in two aspects: differentiated stimulatory activity in relation to SG 1, SG 2-14 and ATCC 33152 L. pneumophila and differentiated response of the hemodialysis patients and patients after renal transplantation in relation to the control group. The positive and negative results of anti-L. pneumophila antibodies of two groups of our patients were found for the analysis of the stimulatory activity of L.pneumophila as a primary or secondary response. In patients with immunosuppression the response in the secretion of cytokines (TNF-α and IFN-γ) was reduced after stimulation of L. pneumophila SG 1 but in varying degrees after stimulation of L. pneumophila SG 2-14, which indicates that the risk of the infection is varied.

Bioimmunological characteristics of mature or immature murine dendritic cells

To explore the biological characteristics of immature and mature murine bone marrow-derived dendritic cells (DCs). The murine bone marrow cells were cultured and induced in vitro into immature DCs (imDCs). Then ImDCs were incubated with TNF (tumor necrosis factor)-α to obtain mature DCs (mDCs). The DCs were purified by murine CD11c microbeads. The morphologies of DCs were observed by electron microscopy (EM). Such surface markers as MHC-II, CD80 and CD86 were tested by flow cytometry (FCM). The proliferation capacity of allogeneic T cells stimulated by DCs was examined by Cell Counting Kit-8 (CCK-8) in mixed lymphocyte reaction (MLR). The expressions of cell factors in mRNA of DCs were tested by real-time polymerase chain reaction (QPCR). The cytokines in supernatant were measured by ELISA (enzyme-linked immunosorbent assay). As compared with mDCs, fewer, shorter spines and more phagocytic vesicles and lysosome were observed in imDCs under EM. The expressions of cell surface molecules in imDCs were significantly lower than those of mDC by FCM, [MHC-II (27.2%) vs (97.7%); CD80 (27.6%) vs (97.2%); CD86 (29.5%) vs (96.4%)]. In MLR, the capacity of same-reaction ratio imDCs group for stimulating the proliferation of T-cells was remarkably lower than that of mDCs group,[1:5 (1.63 ± 0.04) vs (2.21 ± 0.09); 1:10 (1.50 ± 0.08) vs (1.92 ± 0.02); 1:20 (1.28 ± 0.07) vs (1.64 ± 0.01); 1:40 (1.19 ± 0.04) vs (1.45 ± 0.06), P < 0.01]. In imDCs, the relative mRNA expressions of IL12p35 (0.66 ± 0.13), IL12p40 (0.57 ± 0.10) and IFN-γ (0.74 ± 0.08) were lower than those of mDCs (1.00 ± 0.00), (P < 0.05), but the expression of TGF-β (1.35 ± 0.09) was higher than that of mDCs by QPCR (1.00 ± 0.00), (P < 0.05). The expressions of IL12p70 and IFN-γ in supernatants from imDCs were lower than those of mDCs [IL12p70: (6 ± 4) vs (120 ± 22); IFN-γ: (56 ± 15) vs (90 ± 15), P < 0.05] while the expression of TGF (transforming growth factor)-β was higher than that of mDCs by ELISA [TGF-β: (176 ± 23) vs (55 ± 18), P < 0.05]. ImDCs can induce anergic T cells and immune tolerance through a down-regulation of co-stimulatory molecules, MHC-II and Th1 cytokines and an up-regulation of Th2 cytokine (TGF-β). And MDCs may activate naive T cells and stimulate immune responses by an up-regulation of co-stimulatory molecules, MHC-II and Th1 cytokines.