Increased IFN-γ Secretion Research Articles

Toll like receptor 2 agonists lipoteichoic acid and peptidoglycan are able to enhance antigen specific IFNγ release in whole blood during recall antigen responses

BackgroundInterferon gamma release assays (IGRA) have been developed to support the diagnosis of diseases like tuberculosis, which lack robust serological test systems. IGRAs focus on cellular immunity especially memory T cells and thus complement serological testing. However, the low frequency of antigen-specific memory T cells in peripheral blood limits IFNγ production to minute amounts and constitutes a major challenge for downstream test systems. We hypothesized that certain toll like receptor (TLR) agonists might enhance IFNγ production in IGRAs after antigen challenge without inducing background cytokine production. In addition, we investigated the potential use of IL2 release after TLR agonist application as another surrogate marker in cytokine release assays. Methods176 healthy controls (HC) were tested for IFNγ- and IL2-secretion in whole blood in the presence of different TLR agonists with and without antigen challenge by ELISA. The selected TLR agonists were lipopolysaccharide (LPS ≙ TLR4), lipoteichoic acid (LTA ≙ TLR2), peptidoglycan (PGN ≙ TLR2), zymosan (Zym ≙ TLR2 and 6), polyinosinic-polycytidylic acid (Poly I:C ≙ TLR3), flagellin (Fla ≙ TLR5), R848 (≙TLR7 and 8), loxoribine (Lox ≙ TLR7) and bropirimine (Bro ≙ TLR7). ResultsTLR2 agonists LTA and PGN increased IFNγ secretion after antigen challenge nearly twofold (740 vs. 443pg/ml for LTA and 969 vs. 469pg/ml for PGN, respectively) without eliciting higher background expression. TLR3 agonist Poly(I:C) and TLR5 agonist Fla also induced a twofold increase in IFNγ synthesis (2.230 vs. 1.085pg/ml for Poly(I:C) and 518 vs. 278pg/ml for Fla, respectively), but background expression was slightly increased (114 vs. 7pg/ml for Poly(I:C) and 47 vs. 12pg/ml for Fla, respectively). IL2 production was not increased after antigen challenge in the presence of LTA, PGN, Poly(I:C) or Fla. The agonists LPS, Zym, R848, Lox and Bro did not raise cytokine synthesis after antigen challenge or they generated high levels of cytokines by themselves. ConclusionOf all tested agonists TLR2-specific LTA and PGN met the requirements to increase IFNγ synthesis in whole blood after challenge with recall antigens without heightening basal cytokine levels alone. Thus, they constitute a potential costimulating reagent for IGRAs. IL2 did not show any potential as a surrogate marker in cytokine release assays in combination with TLR agonists.

Neonatal Immunization with Respiratory Syncytial Virus Glycoprotein Fragment Induces Protective Immunity in the Presence of Maternal Antibodies in Mice

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFNγ secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period.

Somatostatin Expression in Human Hair Follicles and Its Potential Role in Immune Privilege

Immune privilege (IP) is believed to exist in the anagen hair follicle (HF). Studies have shown that downregulation of major histocompatibility complex Class I occurs and immunosuppressive factors are expressed in the HF bulb and bulge. However, demonstration and quantification of functional IP in HF cells are required. We examined the middle (sheath) and lower (bulb) portions of the human HF using quantitative real-time RT-PCR (qPCR), immunohistology, ELISA, in vitro coculture with peripheral blood mononuclear cells (PBMCs), and flow cytometry. We found that HF cells, relative to non-follicular epidermal cells, failed to promote allogeneic PBMC proliferation and CD4(+) and CD8(+) IFNγ production. By qPCR, we found significant downregulation of Class I and Class II HLA alleles in both the bulb and sheath, and upregulation of multiple immunoregulatory genes. It is noteworthy that somatostatin (SST) was significantly upregulated relative to epidermis. By immunohistochemistry, SST was most strongly expressed in the HF outer root sheath, and, by ELISA, cultured sheath cells secreted SST. PBMCs, cultured with stimulatory allogeneic epidermal cells and SST, secreted significantly less IFNγ than controls. Addition of SST antagonists to PBMCs cocultured with allogeneic HF cells increased IFNγ secretion. The data identify SST as a secretory factor potentially contributing to the HF IP repertoire.

Increased production of IFN-γ by natural killer cells triggered with bone marrow-derived dendritic cells cultured in the presence of retinoic acid

All-trans-retinoic acid (RA), a vitamin A metabolite, is beginning to be explored as an immunopharmacologic agent. While its effects on dendritic cells and the induction of regulatory T cells have been recognized, little is known about the effect of RA on dendritic cell–natural killer cell (DC–NK) crosstalk. DC–NK crosstalk is important in directing the innate immune response, as well as subsequent adaptive immune response during viral infection, cancer, pregnancy, as well as organ transplantation. Here we demonstrate with flow cytometry and cytokine bead array analysis, that bone marrow derived dendritic cells (BMDCs) cultured in the presence of ≥98% HPLC purified RA (RA-DCs) were suppressed in their ability to mature in response to TLR stimulation. 1µM of RA was found to be optimal without affecting the percentage of DCs in culture. Upregulation of MHCII and costimulatory molecule CD86, as well as IL-12 secretion were inhibited by RA treatment. RA-DCs differentially modulate NK cell function compared to BMDCs. In vitro co-culture of RA-DCs with NK cells reveal increased IFN-γ secretion. Increased production of IFN-γ in lung NK cells was also demonstrated when RA-DCs were injected into the tail vein. Our results suggest that RA-DCs exhibit a regulatory phenotype and function, which differentially modulates NK cell function. Furthermore, IFN-γ has various regulatory and immunological functions, depending on the immunological context. The effect of RA-DCs needs to be further explored in the context of a disease in order to understand the regulatory effects of retinoic acid.

Loss and aberrant function of NKp44+ ILCs in the SIV-infected gut are associated with RORγt suppression (P6175)

Abstract Recent studies have identified a novel NK cell-like population found in mucosal tissues and mucosae-draining lymph nodes distinguishable by high surface expression of NKp44 and referred to as innate lymphoid cells (ILCs). ILCs secrete IL-17/IL-22 which are critical for epithelial homeostasis and thus could play a role in gut integrity, a major factor in HIV/SIV pathogenesis. During chronic SIV infection, numbers of ILCs declined ~8-fold in gastrointestinal tissues, but remained comparable to naïve animals in distal sites such as mesenteric lymph nodes and lungs. Loss of ILCs was associated with up to 100-fold increases in apoptosis. Interestingly, gut ILCs expressed NFIL3, a classic NK cell-related transcription factor, as well as the IL-17 transcriptional regulator, RORγt, but during SIV infection RORγt was significantly downregulated associated with decreased IL-17 secretion. In these same animals increased IFN-γ secretion and acquired cytotoxicity were associated with an increase in ILC NFIL3. Loss and functional skewing of ILCs in vivo correlated with upregulated IDO-1 and IFN-α in the gut, and in vitro treatment of ILCs with IDO-1 catabolites and IFN-α also suppressed RORγt expression, ablated IL-17 production and induced apoptosis. SIV infection results in massive numerical and functional depletion of ILCs in the inflamed gut, but not distal sites. This phenomenon was due, at least in part, to IDO-1- and IFN-α-induced apoptosis and suppression of RORγt.

Anti-inflammatory effects of 27 selected terpenoid compounds tested through modulating Th1/Th2 cytokine secretion profiles using murine primary splenocytes

This study investigated 27 selected terpenoid compounds, including 8 monoterpenoids, 7 sesqui-terpenoids, 3 di-terpenoids, 8 tri-terpenoids, and 1 tetra-terpenoid, for their Th1/Th2 immunomodulatory potential using mouse primary splenocytes. Changes in Th1 cytokines, including interleukin (IL)-2 and interferon (IFN)-γ, and Th2 cytokines, including IL-4, IL-5 and IL-10, secreted by terpenoid-treated splenocytes were measured using the ELISA method. The results showed that triptolide, a diterpenoid, was most cytotoxic, reflecting an IC50 value of 46nM. Eucalyptol, limonene, linalool, thymol, parthenolide, andrographolide, 18β-glycyrrhetinic acid, lupeol, ursolic acid and β-sitosterol showed a strong Th2-inclination and anti-inflammation potential in vitro. In addition, (−)-trans-caryophyllene, oridonin, triptolide, diosgenin, betulinic acid, escin, and β-sitosterol treatments significantly inhibited both IL-2 (Th1) and IL-10 (Th2) cytokine production at the same time, suggesting that these terpenoid compounds have an anti-inflammation potential through the inhibition of T-cell immune responses. Diosgenin treatments significantly increased IFN-γ secretion levels using mouse splenocytes, suggesting that diosgenin may be useful in treating a viral infection through the stimulation of IFN-γ production. Menthone, farnesol and oridonin treatments did not markedly increase IL-10/IL-2 (Th2/Th1) cytokine secretion ratios, suggesting that menthone, farnesol and oridonin may have a relative Th1-inclination property, compared to the other selected terpenoid compounds. The relative Th1-inclination property of menthone, farnesol and oridonin may be applied to improve Th2-skewed allergic diseases.

Design of an Optimized Wilms’ Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo

Tumor antigen–encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K.

Enhancement of Tumor Cell Susceptibility to NK Cell Activity Through Inhibition of the PI3K Signaling Pathway.

Abstract 2152NK cells are the primary effectors of the innate immune response against cells that have undergone malignant transformation. Unlike T cells, NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the NK response against individual cells. Tumor cells have developed ways to escape killing by NK cells through a variety of mechanisms that are poorly understood. Using shRNA libraries, we previously demonstrated that several common signaling pathways modulate susceptibility of various tumor cells to NK cell activity. In this study we focused on one of the PI3K sub-units (PI3KCB, p110β isoform) that was identified in this genetic screen. The PI3K pathway has been linked to diverse cellular functions including cell growth, proliferation, survival and intracellular trafficking but PI3K signaling has not previously been associated with susceptibility to NK cells.To examine the mechanisms of PI3KCB involvement in tumor susceptibility to NK cell lysis, we tested 4 tumor cell lines representing different hematologic malignancies: IM9-multiple myeloma, U937-acute myeloid leukemia, K562-chronic myeloid leukemia and Jurkat-acute T cell leukemia. Stable cell lines were established expressing 4 independent shRNAs (P1, P2, P3, P4-shRNA) and 2 irrelevant shRNAs as controls. Western blot and RT-PCR showed different silencing activity of individual PI3KCB-shRNAs with efficient silencing of PI3KCB by 3 of 4 shRNAs in IM9 (P1, P2, P4-shRNA), 2 of 4 in K562 and U937 (P1, P2-shRNA) and 2 of 4 in Jurkat (P2, P4-shRNA). No PI3KCB gene silencing or protein down regulation was observed with P3-shRNA that was used as an additional negative control. PI3KCB silencing did not affect the proliferative capacity of the 4 tumor cell lines tested over a 5 day period. When compared with irrelevant shRNAs or P3-shRNA, effective silencing of PI3KCB in IM-9, K562 and U937 induced increased IFNγ secretion when target cells were incubated with primary purified NK effector cells and 2 different human NK cell lines (NKL and NK-92). Silencing of PI3KCB in Jurkat target cells did not affect IFNγ secretion by NK cells. To determine whether increased IFNγ secretion by NK cells correlated with increased lytic activity, we incubated target cells with NK effectors overnight and assessed cytotoxicity using AnnexinV/7AAD. Lysis of IM9-PI3KCB-P1, P2 and P4-shRNA was increased by 10%-15% compared to irrelevant controls (P=0.04, P=0.04 and P=0.03 respectively); lysis of U937-PI3KCB-P1 and P2-shRNA was increased by 20% and 22% (P=0.02 and P=0.04 respectively); and lysis of K562-PI3KCB-P1 and P2-shRNA was increased by 10% and 20% (P=0.04 and P=0.04, respectively). Correlating with IFNγ assays, increased lysis was not detected for the Jurkat-PI3KCB-silenced cell lines.To define mechanisms responsible for increased susceptibility to NK cells, we examined the expression of several activating/inhibitory ligands on the membrane of PI3KCB-silenced tumor cells. Flow cytometry demonstrated increased expression of MICB (NKG2D ligand) and CD48 (2B4 ligand) in IM9-PI3KCB-KO lines; increased expression of MICA, MICB, CD48 and CD112/CD115 (DNAM-1 ligands) in K562-PI3KCB-KO lines; and increased expression of CD48, CD112 in U937-PI3KCB-KO lines. Reduced expression of MHC class I was also found in IM9 and U937 lines after PI3KCB silencing. Importantly, none of the activating/inhibitory ligands tested were modulated in Jurkat cells after PI3KCB silencing. Co-culture experiments with NK cells and blocking antibodies targeting NKG2D significantly reduced NK IFNγ secretion against IM9-PI3KCB-KO and K562-PI3KCB-KO targets when compared to controls. In contrast, DNAM-1 blocking in U937-PI3KCB-KO produced a uniform decrease of IFNγ secretion in U937-PI3KCB-KO and controls suggesting that in this target, modulation of other NK ligands could also be involved. These data demonstrate that the beta sub-unit of PI3K (PI3KCB) can modulate tumor susceptibility in different hematologic malignancies and that this effect is, at least in part, due to modulation of several activating/inhibitory ligands. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies. Disclosures:No relevant conflicts of interest to declare.

In the presence of IL-21 human cord blood T cells differentiate to IL-10-producing Th1 but not Th17 or Th2 cells.

IL-21, a member of the IL-2 cytokine family, is mainly produced by activated CD4(+) T cells and controls the activity of immune and also non-immune cells. As a pleiotropic cytokine, IL-21 acts on both innate and adaptive immune responses, suggesting that IL-21 may be a master regulator of the T-cell-dependent adaptive immune response. Although IL-21 is described as mostly promoting inflammation, evidence also suggests inhibitory effects of IL-21. However, its role, particularly in the human neonatal immune system, has not been detailed so far. Here, we assessed the effect of IL-21 in the specific context of the neonatal immune response and delineated differences between the human newborn and adult immune response. In umbilical cord blood, we demonstrated that IL-21 polarized naive CD4(+) T cells into T(h)1 cells, producing IL-10, a key negative regulator during certain infections and autoimmunity. Furthermore, IL-21 stimulation increased IFNγ secretion and inhibited the development of T(h)2 and T(h)17 cells and molecules associated with their function. Thus, in neonates, known to show limitations in establishing T(h)1 responses, IL-21 played a clear role in supporting T(h)1 responses in vitro, while appearing irrelevant for the adult immune response. Overall, we demonstrated the capability of IL-21 to induce the immunosuppressive cytokine IL-10 and outlined its potential to compensate the restricted T(h)1 response in human newborns and consequently to reduce the susceptibility for infectious diseases in the first period of life.

Evaluation of γ-retroviral vectors that mediate the inducible expression of IL-12 for clinical application.

The clinical application of interleukin-12 (IL-12) has been hindered by the toxicity associated with its systemic administration. To potentially overcome this problem, we developed a promoter designed to direct IL-12 expression within the tumor environment using an inducible composite promoter containing binding motifs for the nuclear factor of activated T cells (NFAT) linked to a minimal IL-2 promoter. In this study, the NFAT promoter was coupled to a single-chain human IL-12 gene and inserted into 2 γ-retroviral self-inactivating vectors (SERS.NFAT.hIL12 and SERS.NFAT.hIL12.PA2) and 1 γ-retroviral vector (MSGV1.NFAT.hIL.12 PA2). Peripheral blood lymphocytes (PBLs) were double transduced with an antigen-specific T-cell receptor and the 3 NFAT.hIL12 vectors. Evaluation of inducible IL-12 expression, transduction efficiency, and vector production considerations led to the choice of the MSGV1.NFAT.hIL12.PA2 vector for clinical application. MSGV1.NFAT.hIL12.PA2 PG13 retroviral vector producer cell clones were screened by transduction of tumor antigen-specific PBLs. On the basis of expression studies in PBL, clone D3 was chosen to produce clinical-grade viral vector supernatant and was demonstrated to efficiently transduce young tumor-infiltrating lymphocytes (TIL). The vector-transduced young TIL with known tumor recognition demonstrated specific inducible IL-12 production after coculture with HLA-matched tumor targets and had augmented effector function as demonstrated by increased IFN-γ secretion. These results support the clinical application of adoptive transfer of young TIL engineered with the NFAT.hIL12 vector as a new approach for cancer immunotherapy.

Natural killer (NK) cell activation, cytokine production, and cytotoxicity in human PBMC/myeloma cell co-culture exposed to elotuzumab (Elo) alone or in combination with lenalidomide (Len).

8087 Background: Elo is a monoclonal IgG1 antibody targeting CS1, a cell surface glycoprotein highly expressed on >95% of myeloma cells. In preclinical models Elo exerts anti-myeloma activity via NK cell-mediated antibody-dependent cellular cytotoxicity. Len is an immunomodulatory agent that may activate NK cells. The combination of Elo + Len synergistically enhanced anti-tumor activity in myeloma xenograft models. We investigated the mechanism of enhancing NK cell activation and myeloma cell killing with Elo + Len. Methods: Human PBMC/OPM-2 co-cultures were treated for 24-72h with Elo, Len, or Elo + Len. Activation markers and adhesion receptors were evaluated by flow cytometry. Cytokines were measured by Luminex and ELISpot assays. Cytotoxicity was assessed by cell counting. Results: Elo + Len increased IFN-γ secretion significantly more than Elo or Len alone. IFN-γ elevates ICAM-1 expression, and ICAM-1 surface expression on OPM-2 target cells increased synergistically with Elo + Len. Elo, Elo + Len but not Len increased expression of CD25 (IL-2Rα) on NK cells. Len increased the levels of IL-2, but those were decreased in the presence of Elo due to increased consumption by CD25 expressing NK cells. Blocking uptake of IL-2 with anti-CD25 resulted in higher IL-2 levels than with Len. ELISpot assays confirmed that Elo + Len significantly increased the number of IL-2-producing cell colonies compared with Elo or Len. Elo induced NK dependent myeloma cell killing, and the effect was significantly higher with Elo + Len. Conclusions: Elo alone activated NK cells and mediated the killing of myeloma cells in PBMC/OPM-2 co-cultures. Elo + Len synergistically enhanced myeloma cell killing and increased expression/production of IFN-γ, ICAM-1, IL-2, and CD25. [Table: see text]

PD-1, IL-10 and IFN-γ form a network to regulate HIV-specific CD4 T cell dysfunction (105.2)

Abstract Blockade of the PD-1 or IL-10 pathways can restore antigen-specific T cell functions in chronic infections and cancer. PD-1 blockade is currently evaluated in oncology trials and is also considered in HIV infection. It is thus crucial to define the immune restoration elicited by these interventions, and to determine whether combined blockade of these molecules can synergistically revive immune function. We investigated 40 subjects at different stages of HIV infection. We measured proliferation and cytokine production by HIV-specific CD4 T cells stimulated with HIV Gag in the presence of blocking antibodies against PD-L1 and/or IL-10Rα, or IFN-γ. We also determined the modulatory impact of IFN-γ on PD-1 and its ligands on different cell subsets. In viremic subjects, combined blockade of PD-L1 and IL-10Rα resulted in a striking synergistic increase in IFN-γ secretion by HIV-specific CD4 T cells in contrast to only a moderate increase in IL-2 and IL-13 secretion, and proliferation. While IL-10Rα blockade preferentially increased IFN-γ, PD-L1 blockade had a balanced effect on IFN-γ, IL-2 and IL-13. Stimulation of HIV-specific CD4 T cells led to strong up-regulation of PD-L1 on monocytes that was further increased by anti-IL-10Rα. Blockade of IFN-γ abrogated PD-L1 induction and restored proliferation of HIV-specific CD4 T cells. These data provide a mechanistic insight on how the interplay between PD-1, IL-10 and IFN-γ regulates Thelper dysfunction in HIV infection.

The negative feedback mechanism between SLPI and IFNγ is impaired in tuberculosis patients (164.7)

Abstract Mycobacterium tuberculosis increases IFNγ secretion which plays an important role in granuloma formation and clearance of it. Also, the exposure of macrophages to M. tuberculosis increases secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor with antimycobacterial activity. Therefore, the aim of the present study was to determine the role of a putative interaction between IFNγ and SLPI in PBMC derived from healthy donors (HD) and tuberculosis patients (TB). Peripheral blood mononuclear cells (PBMC) from TB and HD were treated with Mtb antigen and IFNγ and SLPI was determined by ELISA. Our results showed that the stimulation with Mtb of PBMC from HD, it decreased SLPI (p<0.01) while increased IFNγ secretion. Furthermore, the blockage of IFNγ reversed the effect of the Mtb antigen on SLPI secretion in PBMC-derived HD. Surprisingly Mtb antigen did not modify the SLPI secretion in PBMC-derived from TB, although it increased IFNγ. Furthermore, the mAb against IFNγ did not affect the SLPI secretion. When plasma levels of SLPI and IFNγ were measured, we found a positive correlation between them in TB but not in HD. Moreover, tuberculosis patients showed more SLPI in plasma, being the levels higher with the severity of the disease. Overall, these results suggest that there is a negative cross talk between IFNγ and SLPI in HD that it is not present in TB patients. Therefore, the higher concentration of IFNγ and SLPI might contribute to control the infection.

Effect of Maternal Immune Status on Responsiveness of Bacillus Calmette-Guérin Vaccination in Mouse Neonates

ObjectivesBacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to Mycobacterium and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to M tuberculosis (M tb) can affect neonatal immunity to BCG using a mouse model. MethodsNeonates were obtained from mice that were previously exposed to live BCG, to live M avium, or to heat-killed M tb H37Rv, and from naïve control mothers. One week after birth, the neonates were divided into two subgroups: one group immunized with live BCG via the subcutaneous route and the other group of neonates sham-treated. Interferon-gamma (IFNγ) secretion in response to in vitro stimulation with heat-killed BCG or purified protein derivative (PPD) was examined. Protection against M tb infection was evaluated by challenging mice nasally with live M tb H37Rv followed by counting colonies from spleen and lung homogenates. ResultsBCG-immunized neonates showed increased IFNγ secretion in response to heat-killed BCG or PPD. All mice in BCG-immunized neonates subgroups showed reduced bacterial burden (colony forming unit) in the lungs when compared with control naive neonate mice. However, no statistically significant difference was observed when comparing BCG-immunized mice born from mothers previously exposed to M avium or immunized with either heat-killed H37Rv or live BCG and mice born from naïve mothers. ConclusionThe maternal immune status to M tb does not appear to impact on the immunogenicity of BCG vaccine in their progeny in our experimental conditions.

Short-term and long-term leptin exposure differentially affect human natural killer cell immune functions

Epidemiological studies have indicated that obesity is associated with a higher risk for certain cancers caused by elevated levels of adipocyte-derived hormones. Leptin, one such hormone produced by adipocytes, is a major regulator of metabolism and has also been shown to modulate immunity. However, its role in regulating human natural killer (NK) cell functions is largely unknown. Here, we show that the leptin receptor (Ob-R) is expressed on 5% of NK cells isolated from blood donors, as measured with flow cytometry, and expression of the signal-transducing long form of the leptin receptor Ob-Rb was confirmed with quantitative PCR. The Ob-R+ subpopulation displayed a lower expression of CD16, a cell surface receptor mediating antibody-dependent activation. Short-term stimulation with leptin increased IFNγ secretion, CD69 activation marker expression, and cytotoxic lysis of tumor cells; this was mediated by an improved conjugate forming between NK cells and tumor cells as well as higher expression of tumor necrosis factor-related apoptosis-inducing ligand. On the contrary, long-term incubation with leptin significantly impaired these NK cell immune functions and decreased cell proliferation. In addition, phosphorylation of Jak-2 after leptin stimulation was reduced in peripheral mononuclear blood cells from obese humans compared with normal-weight controls. NK cells represent an immune cell population that is crucial for an effective antitumor response. Here, we show that long-term exposure to leptin, similarly to the situation in obese individuals with elevated serum leptin levels, significantly impairs integral parts of NK cell immune functions, possibly linking leptin to increased cancer susceptibility in obesity.

Gut inflammation and indoleamine deoxygenase inhibit IL-17 production and promote cytotoxic potential in NKp44+ mucosal NK cells during SIV infection

AbstractNatural killer (NK) cells are classically viewed as effector cells that kill virus-infected and neoplastic cells, but recent studies have identified a rare mucosal NK- cell subpopulation secreting the TH17 cytokine IL-22. Here, we report identification of 2 distinct lineages of mucosal NK cells characterized as NKG2A+NFIL3+RORC– and NKp44+NFIL3+RORC+. NKG2A+ NK cells were systemically distributed, cytotoxic, and secreted IFN-γ, whereas NKp44+ NK cells were mucosae-restricted, noncytotoxic, and produced IL-22 and IL-17. During SIV infection, NKp44+ NK cells became apoptotic, were depleted, and had an altered functional profile characterized by decreased IL-17 secretion; increased IFN-γ secretion; and, surprisingly, increased potential for cytotoxicity. NKp44+ NK cells showed no evidence of direct SIV infection; rather, depletion and altered function were associated with SIV-induced up-regulation of inflammatory mediators in the gut, including indoleamine 2,3-dioxygenase 1. Furthermore, treatment of NKp44+ NK cells with indoleamine 2,3-dioxygenase 1 catabolites in vitro ablated IL-17 production in a dose-dependent manner, whereas other NK-cell functions were unaffected. Thus lentiviral infection both depletes and modifies the functional repertoire of mucosal NK cells involved in the maintenance of gut integrity, a finding that highlights the plasticity of this rare mucosal NK-cell population.

Functional Expression of NOD2 in Freshly Isolated Human Peripheral Blood γδ T Cells

γδ T cells play an important role in anti-infective immunity. The major subset of human γδ T cells selectively recognizes phosphorylated bacterial metabolites of the isoprenoid biosynthesis pathway, so-called phosphoantigens. The activation of γδ T cells is modulated by functionally expressed innate immune receptors, notably Toll-like receptor 2 and 3. It was also reported that in vitro expanded γδ T cells respond to muramyl dipeptide (MDP), the minimal peptidoglycan motif activating the nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, although it is unknown whether ex vivo isolated human γδ T cells express functional NOD2. Here, we report that freshly isolated, highly purified peripheral blood γδ T cells express NOD2 mRNA and detectable amounts of NOD2 protein. The biologically active MDP L-D isomer but not the inactive D-D isomer augmented the interferon-γ (IFN-γ) secretion in phosphoantigen-stimulated peripheral blood mononuclear cells. Moreover, a moderate but reproducible and statistically significant increase in IFN-γ secretion was also observed when highly purified peripheral blood γδ T cells were activated by T cell receptor cross-linking in the presence of MDP. Taken together, our results indicate that in addition to the T cell receptor and Toll-like receptors, circulating human γδ T cells express NOD2 as a third class of pattern recognition receptor for sensing bacterial products.

Restoration of Antitumor Immunity Through Selective Inhibition of Myeloid Derived Suppressor Cells by Anticancer Therapies

Accumulating evidence suggests that the success of some anticancer therapies not only relies on their direct cytotoxicity on tumor cells but also on their ability to promote anticancer immune responses. However, immunosuppressive cells such as Myeloid Derived Suppressor Cells (MDSC) that are generated during tumor progression blunt antitumor immune responses and thus represent a major obstacle to the clinical implementation of immunotherapy protocols. We have recently identified 5-Fluorouracil (5-FU) as an anticancer agent that selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5-FU increased IFNγ secretion by tumor specific CD8(+) T cells infiltrating the tumor and promoted T-cell dependent antitumor responses in vivo, suggesting that some anticancer therapies can reverse tumor-mediated immunosuppression. Here, we review the molecular pathways leading to the induction of MDSC in cancer and discuss how different anticancer agents successfully target these cells in vivo, thereby restoring potent anticancer immunity.

Individual heterogeneity determines the interaction between human dendritic cells and natural killer cells (147.23)

Abstract Previous research has shown that human dendritic cells (DC) can promote activation of natural killer cells (NK), including IFN-γ secretion and enhanced expression of activation markers. However, our recent research suggests that the heterogeneity of individual human donors determines the effect of DC on NK. In our experiments, DC, generated from peripheral blood monocytes and matured by LPS, were co-cultured with autologous NK cells purified from peripheral blood for 24 hours. In the 31 samples, only two donors’ mature DC significantly promoted the secretion of IFN-γ by NK. DC from an additional ten donors promoted the secretion of IFN-γ in the presence of a very low dosage IL-2 or IL-15 which by themselves had no effect. However, all DC could increase expression of CD69 on NK to varying degrees. Further, the DC, not the NK cells, were the key factor for increased IFN-γ secretion because those DC with higher ability to activate autologous NK could also activate allogeneic NK which had otherwise failed to be stimulated with their own autologous DC. Moreover, our current samples did not suggest that the age of the donor was significant for the interaction of NK and DC. Since both NK and DC are very important in infectious disease and cancer immunology, the heterogeneity of NK and DC crosstalk might be related to the susceptibility of some individuals to infection and cancer. However, the mechanisms that determine the effect of DC on NK activation remain to be investigated.

The two component adjuvant IC31 ® potentiates the protective immunity induced by a dengue 2 recombinant fusion protein in mice

Here we evaluated the suitability of the synthetic adjuvant IC31 ® to potentiate the protective capacity of PD5 protein (domain III of the envelope protein of dengue 2 virus fused to the carrier protein P64k). Unlike Alum, PD5 mixed with IC31 ® induced complete protection against virus challenge in mice and increased IFN-γ secretion after in vitro re-stimulation. The induced antibody response was highly specific to the homologous serotype and showed both IgG1 and IgG2a subtypes. IC31 ® is a promising adjuvant for PD5 recombinant protein based vaccination against dengue. Future work should address the suitability of PD5/IC31 ® formulations in non-human primate models.