Intracellular IFN-γ Production Research Articles

Dual roles of scavenger receptor MARCO in innate versus adaptive immune responses during Cryptococcus neoformans infection

Abstract Resistance to the opportunistic fungal pathogen Cryptococcus neoformans (Cn) is associated with development of Th1/Th17 immune bias while a Th2 immune response results in increased susceptibility. Macrophage Receptor with Collagenous Structure (MARCO) is important for recognition of some pathogens and subsequent activation and polarization of the immune responses by DC and macrophages, however, its role during Cn infection remains unknown. Here we found that in spite of diminished early (innate) control of Cn in the infected lungs, Cn-infected MARCO−/− mice showed improved fungal clearance during the adaptive phase (wk 5) compared to MARCO+/+ mice. MARCO−/− mice showed: 1) improved accumulation of exudate macrophages and CD11b+ DC and their improved activation phenotype (CD80+, CD86+ and MHCIIhigh); 2) increased intracellular IFN-γ and IL-17 production by pulmonary CD4+ and CD8+ T cells; 3) decreased IL-4, IL-5, IL-10 and increased IFN-γ and IL-17 production by splenocytes treated with Cn antigen; 5) diminished systemic level of IgE. Interestingly, in spite of improved clearance, MARCO−/− mice showed decreased frequency of lung resident CD44highCD62LlowCD69+ CD103+CD11a+CCR7− effector memory T cells. We conclude that while important for the innate control of Cn in the lungs, MARCO can be exploited by Cn to interfere with immune polarization leading to an altered, non-protective efferent immune bias, which outweighs potentially beneficial effects of MARCO.

Significant IFNγ responses of CD8+ T cells in CMV-seropositive individuals with autoimmune arthritis

BackgroundLatent Cytomegalovirus (CMV) infection accelerates immunosenescence in elderly with reactivations reported in Rheumatoid Arthritis (RA) and abnormal responses towards CMV in Juvenile Idiopathic Arthritis (JIA). ObjectivesConsidering the signs of premature T-cell immunosenescence in arthritis patients, the known effect of CMV latency on speeding up many of these signs in an age-dependent manner and the role of CMV on IFNγ-mediated inflammation in healthy elderly and RA, we hypothesized that latent CMV infection accelerates TCR repertoire restriction, loss of CD28, peripheral T-cell proliferation and aberrant IFNγ responses in arthritis patients. Study designUnspecific and CMVpp65-specific IFNγ responses were investigated in peripheral CD8+ T-cells in RA or JIA patients and healthy, age-matched controls. ResultsDespite higher prevalence and concentrations of IgG-anti-CMV, arthritis patients showed lower unspecific IFNγ production, lower CD69-mediated activation and lower CD8+ T-cell proliferation. CMV-seropositive RA patients showed higher intracellular IFNγ production and increased proportions of CD28-CD8+ T-cells after specific CMVpp65 long-term stimulation which was not altered by in vitro blockade of TNFα or IL-6. A skewed TCR repertoire towards oligoclonality and less polyclonality was found in JIA. DiscussionCMVpp65-specific IFNγ production with expansion of CD28-CD8+ T-cells suggests an efficient control of latent CMV regardless of immunosuppressive therapy or in vitro blockade of TNFα or IL-6 in CMV-seropositive arthritis patients. Increased IgG-anti-CMV antibody concentrations and increased proportions of intracellular IFNγ-producing CMVpp65-specific CD8+ T-cells in long-term cultures propose a possibly role of endogenous CMV reactivations boosting antibody levels and a higher possibly CMV-driven IFNγ-mediated inflammatory potential of CD8+ T-cells in arthritis patients.

Th1 Polarization of Cytokine and Cytokine Receptor Polymorphisms Affect the Susceptibility and Severity of ITP

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the production of platelet antibodies, resulting in the destruction of platelets and inhibition of their production. Many cytokine profile study have revealed a clear T helper type1 (Th1) cytokine polarization in chronic ITP patients, using quantitative RT-PCR and flow cytometry method. However, it remains unclear whether genetic factors of Th1/Th2 cytokine and cytokine receptor affect chronic ITP. We investigated the impact of IFN-γ+874T/A, IFN-γ receptor (R) -611G/A, IL-4 -590C/T, and IL-4 receptor (R) a Q576R polymorphisms on the susceptibility and clinical feature of chronic ITP and Th1/Th2 ratio in peripheral blood of ITP patients,Patients and methods: Genotyping was determined by PCR based technique and direct sequencing. The diagnosis and response criteria of the ITP were defined according to International Working Group criteria. We evaluated Th1/Th2 ratio in peripheral blood of 15 normal donors and 25 ITP patients by intracellular flow cytometry. Intracellular IL-4 (Th2 cytokine) and IFN-γ (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin by flow cytometry. This study was approved by the IRB of our hospital.Results: The platelet count ranged from 1x109/L to 98x109/L with a mean count of 32x109/L at the initial diagnosis. Eighty-three patients (56.1%) had bleeding tendency and 24 patients (16.2%) had severe thrombocytopenia (< 10 x109/L). Steroid treatment was given to 86 patients (58.1%), and eradication of Helicobacter pylori was performed in 38 patients (25.7%), while splenectomy was performed in only 18 patients (12.2%). As compared to control group, chronic ITP patients had significantly higher frequency of the IL-4R#576 non-QQ (low function type) than QQ (high function type) (29.7% vs 15.2%, P<0.05). ITP patients with IL-4-590 CC genotype (low expression type) showed lower platelet counts than those with IL-4-590 non-CC genotype (high expression type) (21±17 X109/mL vs 33±27 X109/mL, p<0.05). ITP patients with IFN-γ+874 non-AA genotype (high expression type) showed lower response rate to steroid treatment than those with IFN-γ+874 AA genotype (low expression type) (76.9% vs 97.5%, p<0.05). We examined the association between Th1/Th2 polymorphisms and Th1/Th2 ratio in both normal donors and ITP patients. Th1/Th2 ratio was not significantly different among IFN-γ+874T/A, IFN-γR-611G/A, IL-4-590C/T, and IL-4Ra Q576R polymorphisms in normal donors. In contrast, ITP patients with IL-4Ra576 non-QQ genotype (low function type) had higher tendency of Th1/Th2 ratio compared to IL-4Ra576 QQ genotype (high function type) (111.2±216.1 vs. 20.8±21.6, p = 0.12).Conclusion: These findings suggested that Th1 polarization of Th1/Th2 cytokine and cytokine receptor polymorphisms affect the susceptibility and severity of chronic ITP. Especially, IL-4Ra576 QQ genotype may be closely associated with the risk of ITP and Th1 polarization. DisclosuresNo relevant conflicts of interest to declare.

Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency

Abstract 653: NKTT320, a novel monoclonal antibody activates the immuno-stimulatory functions of human invariant natural killer T cells

Abstract Invariant natural killer T cells (iNKTs) are innate-type lipid-reactive T lymphocytes that directly kill tumor cells as well as exhibit robust capacity to trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), T and B cells. Based on their dual functions as potent cytokine producers and tumor killers, iNKTs serve as a powerful tool for use in cell-based immunotherapies for cancer. In most cases, iNKT cell functions are T cell receptor (TCR)-dependent and require engagement of the invariant T cell receptor (iTCR) by lipids such as the potent iNKT agonist α-galactosyl ceramide (αGC). However, transformed cells often down-regulate CD1d expression and as a result, they become invisible to iNKT cell attack. To circumvent this critical barrier, which limits our ability to capitalize on the therapeutic potential of iNKTs, we recently characterized the iNKT stimulatory properties of a novel monoclonal antibody (NKTT320), which is specific for the human iTCR. NKTT320 is a recombinant DNA derived humanized monoclonal antibody that binds selectively and with high affinity to the CDR3 loop of the alpha chain within the α-β hetero-dimer of the extracellular domain of the human iTCR. For these studies, we have developed methods to successfully expand human iNKTs up to 1000-2000 fold and purify them from human peripheral blood mononuclear cells. To assess whether the NKTT320 antibody induces human iNKT cell functions, highly purified human iNKTs were cultured with or without varying concentrations (0.01-10.0 μg/ml) of plate-bound NKTT320 antibody. At varying times, culture supernatants were collected and cells were harvested, counted and viability assessed. iNKT cell activation (by examining for upregulation of activation markers [CD25, CD69], degranulation (measured by exposure of CD107 on the cell surface) and proliferation (by measuring CFSE dilution) was assessed by flow cytometry. Cell culture supernatants were evaluated for the secretion of a wide array of cytokines and chemokines using Luminex. Strikingly, immobilized NKTT320 antibody induced a robust dose-dependent iNKT cell activation, proliferation and degranulation. Additionally, iNKTs stimulated by the plate-bound antibody secreted increased levels of Th1 (IL-1β, IFN-γ, TNF-α, IL-2, IL-6) and Th2 (IL-4, IL-5, IL-10) cytokines as well as chemokines in a dose-dependent manner. Our in vitro studies are consistent with in vivo data in Vα24 transgenic mice, which express the human iTCR alpha chain. Dosing of NKTT320 in these animals led to iNKT cell activation (upregulation of activation markers and intracellular IFN-γ production), as well as incorporation of BRDU indicating in vivo iNKT cell proliferation. These studies provide a framework by which human iNKT cell functions could be enhanced for therapeutic purposes in cancer. Citation Format: Rupali Das, Felix Scheuplein, Nishant P. Patel, Peng Guan, Robert G. Schaub, Kim E. Nichols. NKTT320, a novel monoclonal antibody activates the immuno-stimulatory functions of human invariant natural killer T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2014-653

IFNGamma Priming Protects Fetal and Embryonic MSC from NK Cell-Mediated Killing and Improves their Immunosuppressive Properties: Role of Activating and Inhibitory Receptors

The enhancement of Mesenchymal Stem Cells (MSC) immunosuppressing function can favour the engraftment of MSC employed in tissue repair. IFN-γ is a pro-inflammatory cytokine, which induces tolerogenic molecules in bone marrow (BM) MSC. We have investigated the interaction of IFN-γ primed MSC from fetal (FL-MSC) and embryonic (ES-MSC) origin with natural killer (NK) cells. IFN-γ -primed FL-/ES-MSC were less susceptible to NK cell-mediated killing, where a major role was played by the IFN-γ-induced up-regulation of HLA-ABC and HLA-E. Monoclonal antibody-mediated blocking of CD94/NKG2A on NK cells increased killing of IFN-γ-primed MSC, suggesting a role of this NK cell inhibitory receptor in MSC protection. NKG2D ligands (such as MICA), LFA-1 and ICAM1 expressed on MSC were also involved in NK cell-mediated killing of un-primed, but not of IFN-γ-primed, FL-/ES-MSC. Importantly, NK cells from IFN-γ-primed FL-/ES-MSC-NK cell co-cultures displayed a reduced intracellular free calcium increase, pERK activation, degranulation, cytolysis and IFN-γ production upon interaction with the NK sensitive target cell K562 compared to NK cells from un-primed FL/ES-MSC-NK cell co-cultures. Finally, PGE-2, increased during NK/MSC co-cultures, appeared to be a key soluble factor responsible for FL-/ES-MSC-mediated immunosuppressive effect. These results suggest that surface molecules such as MICA, HLA-E and ICAM1 can play a role in recognizing un-primed FL/ES-MSC but not IFN-γ-primed MSC where HLA-I is a key molecule for NK cellmediated recognition. Further, the strong immunosuppressive effect of IFN-γ-primed FL-/ES-MSC on NK cells could be exploited in cellular therapy protocols.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood

Is menstrual blood a suitable source of endometrial derived lymphocytes? Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-γ, granzyme B and perforin after culture in the presence of IL-2 and IL-15. In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages of T cells were found in MMC when compared with PBMC from the same female. The MMC-derived NK cells were pre-dominantly CD56(bright)/CD16(-), in contrast to the primarily CD56(dim)/CD16(+) peripheral blood NK cells. MMC-derived NK cells expressed CD103, indicating their mucosal origin. In addition, the pattern of natural cytotoxicity receptor (NCR) expression in MMC-derived NK cells was comparable with that in endometrial biopsy-derived NK cells. Compared with PBMC, the NKp30 expression was decreased, while the percentage of NKp44 positive cells was increased in MMC samples. CXCR3 and CXCR4 were hardly expressed by MMC-derived NK cells, indicating that these cells are not of PBMC origin. NK cells from MMC samples were functional as shown by their capacity to produce IFN-γ, granzyme B and perforin, upon stimulation with IL-2 and IL-15. MMC-derived T cells revealed an increased expression of CD103, CD69 and CXCR4 compared with PBMC-derived T cells. Importantly, MMC collection using a menstrual cup proved highly reliable and reproducible between women and between cycles. Based on the parameters we studied, MMC appear similar to biopsy-derived endometrial mononuclear cells. However, sampling is not done at the exact same time in the menstrual cycle, and thus we cannot exclude some, as yet undetected, differences. Also, it should be considered that for some women, the use of the menstrual cup may be unpleasant. Menstrual blood may be a source of endometrial cells and may create new opportunities to study uterine immunological cells in fertility issues. No external funding was obtained for the present study. None of the authors have any conflict of interest to declare. NA.

AB0063 Gammadelta t cells and their intracellular cytokine profile in peripheral blood of patients with systemic lupus erythematosus

BackgroundGammadelta T cells represent a minor population of human peripheral blood T lymphocytes. As very rapid cytokine-producing cells, gammadelta T cells can regulate other lymphocytes activation and assist their local...

Invariant NKT cells increase lipopolysacchride-induced pregnancy loss by a mechanism involving Th1 and Th17 responses

Objective: To determine the role of invariant natural killer T (iNKT) cells in infection-associated pregnancy loss.Methods: B6 and iNKT cell-deficient Jα18−/− mice were injected i.p. with lipopolysaccharide (LPS) or vehicle, and pregnancy outcomes were examined. Decidual iNKT cell expression of CD69 and intracellular cytokine production were analyzed. Mouse decidual iNKT cells were co-cultured with LPS or PBS-treated dendritic cells (DCs), and iNKT cell CD69 expression and intracellular and extracellular cytokine production were assessed.Results: The embryo resorption rate was notably lessened for Jα18−/− mice treated with LPS on day 6 or day 9 gestation in comparison with B6 mice treated with LPS. Decidual iNKT cell CD69 expression and intracellular IFN-γ and IL-17 production for B6 mice injected with LPS on day 6 or day 9 gestation were significantly up-regulated compared with PBS-treated mice. Levels of IFN-γ and IL-17 in the supernatants of the co-culture of decidual iNKT cells and LPS-sensitized DCs were strikingly increased in comparison with the co-culture of iNKT cells and PBS-treated DCs. CD69 expression and intracellular IFN-γ and IL-17 production of iNKT cells co-cultured with LPS-sensitized DCs were remarkably up-regulated compared with iNKT cells co-cultured with PBS-treated DCs.Conclusions: Our results suggest that iNKT cells may play a role in LPS-induced pregnancy loss by Th1 and Th17 cytokine-dependent manner.

Leptin modulates dose‐dependently the metabolic and cytolytic activities of NK‐92 cells

Leptin, a hormone-cytokine produced primarily in the adipose tissue, has pleiotropic effects on many biological systems and in several cell types, including immune cells. Hyperleptinemia is associated with immune dysfunction and carcinogenesis. Natural killer (NK) cells are critical mediators of anti-tumor immunity, and leptin receptor deficiency in mice leads to impaired NK function. It was thus decided to explore the in vitro effects of leptin on human NK cell function. NK-92 cells were cultured during 48 h with different leptin concentrations [absence, 10 (physiological), 100 (obesity), or 200 ng/ml (pharmacology)]. Their metabolic activity was assessed using the resazurin test. NK-92 cell cytotoxicity and intracellular IFN-γ production were analyzed by flow cytometry. NK-92 cell mRNA and protein expression levels of cytotoxic effectors were determined by RT-qPCR and Western blot. In our conditions, leptin exerted a dose-dependent stimulatory effect on NK-92 cell metabolic activity. In addition, high leptin concentrations enhanced NK-92 cell cytotoxicity against K562-EGFP and MDA-MB-231-EGFP target cells and inversely reduced cytotoxicity against the MCF-7-EGFP target. At 100 ng/ml, leptin up-regulated both NK cell granzyme B and TRAIL protein expressions and concomitantly down-regulated perforin expression without affecting Fas-L expression. In response to PMA/ionomycin stimulation, the proportion of IFN-γ expressing NK-92 cells increased with 100 and 200 ng/ml of leptin. In conclusion, leptin concentration, at obesity level, variably increased NK-92 cell metabolic activity and modulated NK cell cytotoxicity according to the target cells. The underlying mechanisms are partly due to an up-regulation of TRAIL and IFN-γ expression and a down-regulation of perforin.

Cytotoxic function of CD8+ T lymphocytes isolated from patients with acute severe cerebral infarction: an assessment of stroke-induced immunosuppression

BackgroundThere is increasing evidence on complex interaction between the nervous and immune systems in patients with cerebral infarction. This study was conducted to evaluate cytotoxic function of CD8+ T lymphocytes isolated from patients with acute severe cerebral infarction. In order to determine role of immune system in stroke, peripheral blood mononuclear cells (PBMCs) were taken and cytotoxic function of CD8+ T lymphocytes were induced by virus peptides and cells were analyzed on a four-color flow cytometer. Expression of CD107a, intracellular expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and cell proliferation assay were analyzed by using carboxyl fluorescein diacetate succinimidyl ester (CFSE).ResultsA total of 30 patients with cerebral infarction and 30 healthy volunteers with an average age 57 (range, 49 to 71) years, were evaluated. The PBMCs were separated from blood samples of both, patients with cerebral infarction 6 hours after onset of stroke and healthy volunteers. After stimulation with virus peptides, CD107a expression and intracellular production of IFN-γ and TNF-α was decreased in patients with cerebral infarction as compared to healthy volunteers (p < 0.01). Degranulation analysis reported decreased expression of CD107a + in patient group as compared to healthy group, p <0.01. A mild decrease in intracellular expression of IFN-γ and TNF-α was also shown in patients without stimulation of virus peptides (p < 0.05). However, proliferation of CD8+ T lymphocytes in patients with acute severe cerebral infarction was not decreased.ConclusionsThe study results indicated that cytotoxic function of CD8+ T lymphocytes were suppressed in patients with acute severe cerebral infarction. This could possibly be associated with complicated infectious diseases and neuroprotective mechanism.

Oral coinfection can stress peripheral lymphocyte to inflammatory activity in leprosy

This study evaluated the intracellular profile of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from leprosy patients based on oral infections presence to determine whether these coinfections could be associated with pro-inflammatory activity in leprosy. Leprosy patients regardless of clinical form and specific leprosy treatment (n=38) were divided into two groups: Group I - leprosy patients with oral infections (n=19), and Group II - leprosy patients without oral infections (n=19). Non-leprosy patients presenting oral infections were assigned to the control Group (n=10). Intracellular IL-2, IL-4, IL-10 and IFN-γ production was evaluated by flow cytometry (FACS) before and 7 days after controlling the oral infection in the Group I, before and 7 days after dental prophylaxis in the Group II, and during oral infection process in control Group. Low percentages of CD3+ lymphocytes bearing IL-2, IL-10 and IFN-γ were observed in the Group I and Group II at baseline and 7 days after therapy or prophylaxis compared to controls. Group I showed reduced percentages of IL-4 at baseline and 7 days after therapy compared to controls, or at baseline of Group II, and the Group II showed reduced percentages of CD3+ cells bearing IL-4 compared to control. An increase of the percentages of CD3+cells bearing IL-4 was observed in the Group I after the oral infections treatment. The occurrence of oral infections favors the intracellular cytokines expression and, probably, the inflammatory reaction operating as a stimulatory signal triggering the leprosy reactions.

Function of ssDNA aptamer and aptamer pool against Mycobacterium tuberculosis in a mouse model

Novel antibacterial agents against Mycobacterium tuberculosis (MTB) are crucial due to the high infection and mortality rates associated with the disease. Our previous study confirmed that aptamers from a whole bacterium obtained by the Systematic Evolution of Ligands by Exponential Enrichment method specifically bound to the MTB virulent strain (H37Rv). In the present study, the function of aptamers against MTB in a mouse model was further determined. It was demonstrated that the NK2 aptamer has marked inhibitory effects on the adhesion/invasion of H37Rv to macrophages in vitro and stimulates intracellular IFN-γ production in CD4+ T-cells. The aptamer pool exhibited the strongest inhibitory effect on H37Rv adhesion/invasion to CD8+ T-cells in vitro compared with all aptamers-treated and control groups. Histopathological examination of lung biopsy specimens revealed a correlation between aptamer presence and lower pulmonary alveoli fusion, swelling and more prominent air spaces. Acid-fast staining of biopsy specimens from the lungs of the mice demonstrated parallel treatment effects. Results of the present study indicate that the 10th pool aptamers and NK2 may play an active role against H37Rv, however, the effect was different in vivo and in vitro. The treatment effect of 10th pool aptamers was found to be better in comparison to NK2 in vivo. Additional target sites involved in pathogenicity of H37Rv were also revealed and the NK2 binding site and aptamers, including the 10th pool aptamers, may antagonize these sites. Further studies are required to screen for other valuable aptamers which may be used as therapeutic drugs in combination with NK2.

Altered functions of natural killer cells in response to L-Arginine availability

L-Arginine (L-Arg) availability is crucial in the regulation of immune response. Indeed, L-Arg deficiency induces T-cell dysfunction and could modulate the properties of natural killer (NK) cells involved in the early host defense against infections and tumors. We explored the impact of L-Arg depletion on NK cell functions using two models – an NK-92 cell line and isolated human blood NK cells. Below 5mg/L of L-Arg, NK-92 cell proliferation was decreased and a total L-Arg depletion reduced NK-92 cell viability. NK cell cytotoxicity was significantly inhibited in presence of low L-Arg concentration (2.5 mg/L). L-Arg depletion reduced the expression of NK-92 activating receptors, NKp46 and NKp30, the expression of NK ζ chain and the NK-92 intracellular production of IFN-γ. Whatever the L-Arg concentrations tested, no significant variation in the gene expression of transporters and enzymes involved in L-Arg metabolism was found. Thus, L-Arg availability modulates the phenotypic and functional properties of NK cells.

HIV gag‐specific immune response mediated by double negative (CD3+CD4−CD8−) T cells in HIV‐exposed seronegative individuals

Double negative (DN) T cells are CD3(+), CD4(-), CD8(-) cells with either T-cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV-exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV-serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV-specific immune responses mediated by DN T-cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV-Gag peptides. Thirty-five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag-specific DN T-cell responses were found in 3/13 (23%) HIV-exposed seronegative individuals (Group A), involving both DN/αβ(+) and DN/γδ(+) T-cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag-specific responses but were mediated exclusively by DN/γδ(+) T-cells, mainly through IFNγ production. DN T-cells in Group A individuals can display differential HIV-specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A.

Ublituximab, an Optimized Anti-CD20 Monoclonal Antibody, Demonstrates Greater NK-Mediated ADCC Than Rituximab in Waldenstrom's Macroglobulinemia Patients Supporting a Therapeutic Strategy with Ublituximab

AbstractAbstract 1654 Background:Anti-CD20 monoclonal antibody (mAb) therapy is an important therapeutic option in the treatment of Waldenström’s Macroglobulinemia (WM), exhibiting an ORR up to 55% when used as monotherapy. NK cells are involved in mAb therapy by an antibody-dependent cellular cytotoxicity (ADCC) mechanism through their FcγRIIIa (CD16) receptor. Ublituximab (TGTX-1101 or LFB-R603) is a novel, glycoengineered chimeric anti-CD20 mAb that has a high affinity for FcγRIIIa (CD16) receptors. In this study, we evaluate the ADCC functional capacities of NK cells in the presence of ublituximab compared to rituximab. Patients and Methods:Blood samples from 37 un-treated, or without ongoing treatment WM patients and from 30 age-matched healthy donors (Ctl) were collected to quantify CD16 expression (clone 3G8, Quantibrite) on NK cells and/or to measure their functional capacities. Patients were divided in two groups relative to the presence (WM clone+) or absence (WM clone-) of blood clonal B cells. NK cell degranulation was assessed by the surface expression of CD107a on NK cells after incubation of PBMC with or without Raji CD20+ target cells in the presence of anti-CD20 mAbs at 10 and 1,000 ng/ml. ADCC experiments were performed using a chromium assay with purified NK cells and autologous B cells or Raji target cells, in the presence of anti-CD20 mAbs at 1 and 100 ng/ml. Statistical analyses were performed with Prism 5 software. Intergroup comparisons were assessed with the nonparametric Krustal–Walis test, with the Dunns postanalysis test. Results:In the presence of Raji cells, at low concentration, a significantly greater amount of CD107a expression was observed with ublituximab compared to rituximab (P<0.01), regardless of patient’s groups. In contrast, at the highest concentration, similar effects were obtained with both anti-CD20 mAbs. These results were confirmed by ADCC. In the presence of Raji cells, a high level of ADCC (>40%) was detected at low concentration of ublituximab and remained stable at 100 ng/ml. In contrast, with rituximab the highest concentration was necessary to reach similar efficacy. In the presence of autologous B cells, degranulation assays revealed that none of the NK cells from WM clone- patients exhibited degranulation, irrespectively of the anti-CD20 mAb or its concentration. More importantly, NK cells of 3/8 WM clone+ patients exhibited CD107a+ NK cells in the presence of both concentrations of ublituximab. In contrast, with rituximab only 1/8 patients expressed CD107a+ NK cells, and only at the highest concentration. Of note, similar frequency and cell-surface expression level of CD16 on NK cells were detected in both patient groups. Importantly, these data were confirmed by ADCC. In the presence of autologous purified B cells from WM clone- patients, absence or low levels of ADCC were detected, irrespective of the concentration and the anti-CD20 mAb used. Interestingly, in WM clone+ patients, ADCC was detected in all of the 5 tested patients with ublituximab, but only in 2/5 patients with rituximab, and at the highest concentration. These results are confirmed by polyfunctionality assays of NK cells against Raji cells (assessment of CD107a degranulation and intracellular production of IFN-γ and TNF-α). Conclusion:These results show that ublituximab is more efficient than rituximab in inducing ADCC at low doses, in the presence of Raji cells. More importantly, our results suggest that ublituximab could be more efficient than rituximab both to induce NK cell degranulation and ADCC in the presence of autologous peripheral tumor cells. These findings highlight a new putative role of this optimized anti-CD20 mAb in the control of WM, and prompt further investigations in a large cohort of WM patients. A Phase I/II trial with single agent ublituximab in patients with rituximab relapsed/refractory NHL, including WM patients is currently ongoing. Disclosures:Le Garff-Tavernier:LFB: 2012 filed patent application Other, Research Funding. Herbi:LFB: Employment, Research Funding. de Romeuf:LFB: 2012 filed patent application Other, Employment. Prost:LFB: Employment. Urbain:LFB: Employment. Leblond:LFB: 2012 filed patent application Other. Vieillard:LFB: 2012 filed patent application Other. Merle-Béral:LFB: 2012 filed patent application Other, Research Funding.

Expanded Natural Killer (NK) Cells Transfected with Anti-CD20 Chimeric Antigen Receptor (CAR) mRNA Have Significant Cytotoxicity Against Poor Risk B-Cell (CD20+) Leukemia/Lymphoma (B-L/L).

AbstractAbstract 3007 Background:The outcome for children and adolescents with B-L/L has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically engineered and expanded NK cells with an anti-CD19 CAR have been previously reported by Campana et al (Li L et al, Cancer Gene Ther. 2010). Anti-CD20 CAR transduced primary NK cells by retrovirus were reported by our group (Chu & Cairo, et al, ASH, 2011). Objective:To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-virus method, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR+) modified PBNK cells following mRNA nucleofection against CD20+ B-L/L. Methods:PBMC were expanded with mitomycin C treated K562-mbIL15–41BBL cells for 7 or 14 days. CD56+CD3− expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ was subcloned into a pcDNA3 vector. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit from T7 promoter. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab’)2 fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay at different E:T ratios against CD20+ B-L/L. CD107a degranulation and intracellular IFNgƒnproduction in exPBNK were measured by flow cytometry after stimulation with medium, K562-mbIL15–41BBL, CD20+ Ramos, CD20+Daudi, or CD20− RS4;11 for 4–6 hrs. Results:CD56+CD3− exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15–41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56+CD3−. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection. CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors (CD16, CD69, NKG2D, CD244, NKp30, NKp44, NKp46) or inhibitory receptors (NKG2A, KIR2DS4, CD94, CD158a, CD158b, CD158e). exPBNK in vitro cytotoxicity was significantly enhanced by CAR+ exPBNK compared to CAR− exPBNK against CD20+ B-L/L at 10:1 (n>3): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p<0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%, p<0.001), Raji (21.45+ 1.98% vs 6.94+ 5.64%, p<0.05), Raji-2R (a Rituximab resistant cell line) (96.39+ 1.03% vs 86.3+ 1.52%, p<0.01), and U-698-M (82.84+ 1.17% vs 26.2+ 0.776%, p<0.001). However, there was no significant difference against CD20− RS4;11 or Jurkat cells. Consistently, CD107a degranulation was enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos (31.47+ 1.74% vs 15.2+ 0.26%, p<0.001, n=3) and Daudi (38.9+ 2.7% vs 19.73+ 0.58%, p<0.001, n=3) stimulation, however, there was no significant difference in response to RS4;11 or medium. Intracellular IFNγ production was also enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos and Daudi specific stimulation. We also observed that the expression of exPBNK activating receptors (CD69, NKp44 and NKG2D) were enhanced similarly and inhibitory receptors (CD94 and CD158b) were unchanged in mock exPBNK and CAR+ exPBNK cells after incubation with U-698-M compared to medium, implying CAR+PBNK directed enhanced cytotoxicity is mainly mediated by engineered CAR but not by endogenous NK receptors. Conclusion:Anti-CD20 CAR expression in exPBNK cells by mRNA nucleofection was associated with a significant increase in CD107 degradulation and INF-g production after stimulated with CD20+ BL/L compared to mock exPBNK cells. Consequently, Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20+ B-L/L. Future directions include examining CAR+ exPBNK cytotoxic activity against CD20+ primary B-L/L tumor cells isolated from patients and testing the anti-tumor activity of CAR+ exPBNK against B-L/L and animal survival in xenograft mice. Disclosures:No relevant conflicts of interest to declare.

Kinetics of peripheral hepatitis B virus-specific CD8+ T cells in patients with onset of viral reactivation

Patients with resolved hepatitis B virus (HBV) infection undergoing chemotherapy or immunosuppressive therapy are potentially at risk of HBV reactivation. However, it remains unclear how liver disease develops after HBV reactivation. To compare the host immune response against HBV, we performed immunological analyses of six HBV reactivation patients. The numbers of peripheral HBV-specific CD8+ T cells were investigated longitudinally in six HLA-A2- and/or A24-positive patients with HBV reactivation. In addition, 34 patients with resolved HBV, 17 patients with inactive chronic hepatitis B (ICHB), 17 patients with chronic hepatitis B (CHB) and 12 healthy controls were analyzed. The number and function of HBV-specific CD8+ T cells were assessed by flow cytometry using tetramer staining and intracellular IFN-γ production. Furthermore, the numbers of CD4+ CD25+ or CD4+ Foxp3+ T cells and serum inflammatory cytokine levels were analyzed. The frequency of HBV-specific CD8+ T cells was significantly increased in HBV reactivation patients compared with ICHB and CHB patients. In addition, the number of HBV-specific CD8+ T cells was increased in resolved HBV patients compared with ICHB patients. PD-1 expression was decreased in HBV reactivation patients compared with ICHB and CHB patients. The numbers of HBV-specific CD8+ T cells and CD4+ CD25+ or CD4+ Foxp3+ T cells were negatively correlated following onset of HBV reactivation. During HBV reactivation, the frequency of HBV-specific CD8+ T cells increased even though the administration of immunosuppressive drugs and interactions with CD4+ regulatory T cells may be important for the onset of liver disease.

Recognition of heat shock protein 60 epitopes in children with type 1 diabetes

Treatment with a specific HSP60 epitope in new onset of type 1 diabetes (T1D) patients has been shown to preserve endogenous insulin production. Previously, recognition of pan HLA-DR-binding HSP60 epitopes in various autoimmune diseases was found; this study investigated recognition of these epitopes in newly diagnosed T1D patients and correlated findings to the occurrence of a partial remission. Peripheral blood mononuclear cells of 18 children with T1D were prospectively collected at disease onset and a few months after diagnosis. Epitope-specific T-cell proliferation and cytokine production (intracellular and in culture supernatants) were measured. Results were compared with 31 longstanding T1D patients and ten healthy controls. Although HSP60 epitope-specific T-cell proliferative responses were detected, overall proliferative responses were low. At onset, epitope-specific intracellular IFN-γ production was higher in T1D patients compared with healthy controls (p < 0.05). At follow-up, both IL-10 and IFN-γ production were higher in those without a partial remission than in those with a partial remission (both p < 0.05). Also, IL-10 and IFN-γ production were higher compared with onset for patients without a PR (both p < 0.01). In supernatants of HSP60 epitope-specific T-cell cultures, no substantial differences in cytokine production were found between T1D patients with and without a partial remission, either at onset or a few months after onset. As patient numbers were small, results should be interpreted with caution. Pan-DR-binding HSP60 peptides induced low peptide-specific proliferative responses and peptide-specific production of some, mainly intracellular, cytokines in T1D patients. Recognition did not differ significantly between patient groups and various time points.