Impaired IFN-γ Production Research Articles

Microbial translocation in liver fibrosis induces chronic IFNAR signaling that directly affects innate immune responses to systemic bacterial infection

A common clinical complication in patients with liver fibrosis or cirrhosis is exacerbations of bacterial infections, which often persist in these patients. In order to study the underlying pathophysiological cellular and molecular mechanisms in mice, we investigated the immune responses to Listeria infections in the context of liver fibrosis. Using the bile-duct ligation model for the induction of liver fibrosis in mice we found an enhanced susceptibility to Listeria infections leading to persistence of infection, recapitulating clinical situations in humans. While the innate clearance of Listeria in the spleen of those mice was unaltered, bactericidal activity of intrahepatic macrophages and bacterial clearance in the liver were strongly impaired. This reduced local clearance of Listeria was correlated to impaired IFNγ, IL-12 and ROS production by the myeloid cells in the liver. Mechanistically, we identified IFNAR signaling in the myeloid cells as the basis for impaired anti-bacterial immune responses in mice with liver fibrosis. Using germ free mice we could show that IFNAR signaling is induced by translocated gut microbiota during liver fibrosis. This chronic Type I IFN signals on myeloid cells in turn lead to IL-10 release, which directly inhibited IFNγ, IL-12 and ROS production. Strikingly, we could rescue mice form chronic infection by either blocking IFN signaling in myeloid cells or systemic blockade of IL10 signaling, opening an exciting new therapeutic avenue for bacterial infections in patients suffering from liver fibrosis or cirrhosis. Corresponding author: Abdullah, Zeinab; Knolle, Percy E-Mail:zeinab.abdullah@ukb.uni-bonn.de, percy.knolle@tum.de

B7-H6-mediated downregulation of NKp30 in NK cells contributes to ovarian carcinoma immune escape

In this study the phenotype and function of tumor-associated NK cells from perito- neal fluids of a selected cohort of patients with seropapillary ovarian carcinoma were analyzed. In >50% of these patients the expression of the activating receptor NKp30 (1) in tumor-associated NK cells was substantially reduced as compared to autolo- gous peripheral blood NK cells. The impaired expression of this receptor was associ- ated with the presence of one of its cellular ligands (B7-H6) (2), which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the peri- toneal fluid. NK cells from patients expressing this NKp30low phenotype displayed an impaired IFNγ production and cytolytic function when tested against target cells expressing surface B7-H6. Our data also suggest that in these patients the defective expression and function of NKp30 may be induced by the chronic engagement of this receptor by soluble B7-H6 or by tumor cells expressing this ligand. The impairment of NK cell functions described herein could represent a novel mechanism by which the tumor microenvironment may contribute to the escape from immune surveillance. This work was supported by grants awarded by Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC)-Special Project 5x1000 no. 9962 and IG 2014 Id. 15704 (Alessandro Moretta); PRIN 2010 (Alessandro Moretta); Progetto di Ricerca Fondazione Carige 2013 (Emanuela Marcenaro); Progetto di Ricerca di Ateneo 2014 (Emanuela Marcenaro). Eric Vivier laboratory is supported by the European Research Council (THINK Advanced Grant), by Equipe Labellisee La Ligue and by institutional grants from INSERM, CNRS, and Aix-Marseille University to CIML.

Abstract 2549: Defective STAT1 activation associated with impaired IFN-γ production in lymphocytes from metastatic melanoma patients treated with HD IL-2

Abstract High dose (HD) IL-2 therapy is one of the most effective immunotherapies against melanoma, but clinical responses occur only in a fraction (10-15%) of patients. HD IL-2 is known to activate and promote the proliferation of various lymphocyte subsets including T and NK cell subsets via STAT signaling. However, evidence on how different lymphocyte subsets from metastatic melanoma patients respond to HD IL-2 treatment and whether any defects exist in different lymphocyte subsets from metastatic melanoma patients that could possibly reduce the efficacy of treatment are still lacking. To address this question, we investigated the extent of HD IL-2 induced STAT 1, 3, and 5 tyrosine phosphorylation (activation) in different T cell and NK cell subsets from the melanoma patients during the IL-2 exposure in comparison to normal donors (ND). Phosphoflow cytometry analysis revealed that HD IL-2 treatment induced a dramatic activation of STAT5 in all T-cell and NK cell subsets when PBMCs were stimulated with HD IL-2 in vitro or after the first bolus of HD IL-2 infusion. In all cases, STAT3 activation by IL-2 was never detected. Similar levels of STAT5 activation was observed in the T and NK cell compartments in both ND and patients. Furthermore, HD IL-2 treatment induced a substantial increase in the proportion of Ki67-expressing cells in CD4+, Tregs, CD8+ T-cells, CD56hi and CD56low NK cell subsets in both melanoma patients and ND. No apparent defects in the proliferation capacity of all T and NK cell subsets was noted when comparing the proportion of Ki67+ cells between melanoma patients and ND. In contrast to STAT5, STAT1 activation was found to be defective in all lymphocyte subsets after HD IL-2 exposure and showed a dramatic age-related decline not found in ND. This blunted STAT1 activation was associated with a reduced IFN-γ production by PBMCs from melanoma patients, while TNF-α, IL-1β, IL-8, IL-6 and GM-CSF release was unaffected. Intracellular cytokine staining found that the aberrant IFN-γ response was mainly in the CD56hi and CD56low NK cell subsets, which were the major IFN-γ producers in response to HD IL-2. The aberrant IL-2-induced IFN-γ secretion by NK cell subsets not only reduced STAT1 activation in these NK cell populations but also led to a defective STAT1 activation in CD4+ T and CD8+ T cells. Taken together, these findings indicate that metastatic melanoma patients have a defective IFN-γ secretion by NK cells and STAT1 activation in response to HD IL-2 that may reflect a generalized suppression of this pathway that may be involved in the lack of therapeutic benefit of IL-2 in most patients. Citation Format: Geok Choo Sim, Sheng Wu, Lei Jin, Patrick Hwu, Laszlo Radvanyi. Defective STAT1 activation associated with impaired IFN-γ production in lymphocytes from metastatic melanoma patients treated with HD IL-2. [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 2549. doi:10.1158/1538-7445.AM2014-2549

Histone deacetylase inhibitors impair the elimination of HIV-infected cells by cytotoxic T-lymphocytes.

Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.

Decreased expression of NKG2D, NKp46, DNAM-1 receptors, and intracellular perforin and STAT-1 effector molecules in NK cells and their dim and bright subsets in metastatic melanoma patients.

Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.

Disruption of CD8-Coreceptor Binding Abrogates Tolerance Induction Via Liver-Directed Expression of Donor MHC Class I - A Role for PD-L1?

Introduction. We previously used an adeno-associated viral (rAAV) vector to express donor MHC (H-2Kb) in recipient livers and induce donor-specific tolerance in a mouse skin transplant model. Kb expression in B10.BR mice led to impaired production of IFN-γ in response to Kb, but no substantial deletion of CD8 T cells, suggesting that tolerance was associated with functional silencing. Methods. To determine the role of direct recognition of class I by CD8-dependent T cells in tolerance induction, we generated rAAV encoding a mutant Kb (rAAV-D227K), where Asp at position 227 of the α3 domain is replaced with Lys, abrogating CD8 binding. Expression of Kb, D227K-Kb and PD-L1 on hepatocytes was assessed by FACS and IHC. Kb-bearing 178.3 skin was grafted onto uninjected recipients and mice injected with rAAV-D227K or rAAV-Kb ± PD-L1 blockade. Results. Uninjected B10.BR mice rejected 178.3 skin (MST=16 d, n=6), while grafts onto rAAV-Kb-injected mice survived long term (MST>250, n=5). Survival of 178.3 grafts onto mice injected with rAAV-D227K was only slightly prolonged (MST=27, n=6), suggesting that CD8 coreceptor engagement is needed for tolerance induction. Kb expression in B10.BR livers resulted in increased PD-L1, but this was not seen following Kb-D227K expression, or with Kb expression in C57BL/6 livers, suggesting that alloreactive CD8 T cell activation results in PD-L1 upregulation. Antibody blockade of PD-L1 plus rAAV-Kb treatment resulted in increased ALT levels (721±73) compared to treatment with rAAV-Kb (33±4) or anti-PD-L1 (35±2) alone. Experiments to determine the effect of PD-L1 blockade on skin graft survival are ongoing. Conclusion. Disruption of CD8 coreceptor engagement abolishes tolerance induction via liver-directed expression of donor MHC class I, indicating that direct recognition by CD8-dependent T cells is essential for the process. PD-L1 upregulation accompanied alloreactive CD8 T cell activation and blockade of PD-L1 resulted in hepatitis, suggesting that this molecule might play a role in the functional silencing of CD8 T cells in this model.

The receptor for advanced glycation end products (RAGE) affects T cell differentiation in OVA induced asthma.

The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ

A, Genealogical tree for the kindred. Arrow highlights index case. Asterisks denote family members who died within the first 2 months of life with febrile illnesses. B, Histology showing granuloma in skin by hematoxylin and eosin staining.

IL-15 trans-presentation regulates homeostasis of CD4+ T lymphocytes

Interleukin-15 (IL-15) is essential for the survival of memory CD8(+) and CD4(+) T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4(+) T cells. Adoptive transfer of splenocytes from non-obese diabetic (NOD) mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.Il15(-/-) mice when compared to NOD.scid recipients. The increased accumulation of CD4(+) T cells is also observed in NOD.Il15(-/-) mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the IL-15Rα chain, but not those lacking the common gamma chain, also show increased accumulation of CD4(+) T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4(+) T cells and requires trans-presentation of IL-15. CD4(+) T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4(+) T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-γ production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4(+) T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.

Immunodominance and functional alterations of tumor‐associated antigen‐specific CD8+T‐cell responses in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8(+) T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire alpha-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8(+) T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon-γ (IFN-γ)-producing CD8(+) T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8(+) T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8(+) T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8(+) T-cell proliferation but did not restore IFN-γ-production. Naturally occurring TAA-specific CD8(+) T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8(+) T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8(+) T-cell responses.

Reduced Macrophages IL-12 Production After Stimulation By BCG/INF-Gamma Suggestive Of Impaired INF-Gamma Pathway Signalling In a Child With Disseminated Atypical Mycobacterial Infection and History Of Chemotherapy For Langerhans Cell Histiocytosis

Impaired INF-γ mediated immunity is characterised by increased susceptibility to poorly virulent mycobacteria. Here, we present a case of a patient with disseminated atypical mycobacterial infection poorly responding to antimicrobial treatment and INF-γ. This in a setting of previous chemotherapy for LCH resulting in profound T-cell lymphopaenia and impaired Th1/Th17 function. Functional studies of INF-γ pathway together with sequencing of INFGR1, INFGR2 and STAT1 genes were performed. A 21 month old boy was diagnosed with a disseminated Mycobacterium haemophilum infection after previous chemotherapy for LCH following LCH III, LCH III Salvage and HLH 2004 protocols, completed by Alemtuzumab, resulting in severe T-cell lymphopaenia (<200/mm3). Despite treatment with multiple anti-microbial drugs and high dose INF-γ, the infection continued to progress. Functional in vitro studies showed impaired macrophage IL-12 production after stimulation by BCG/INF-γ and severely impaired T-cell IFN-γ and IL-17 production in response to PHA. Sequencing of INFGR1, INGR2 and STAT1 cDNA showed no pathogenic mutations. Progressive disseminated atypical mycobacterial infection despite multiple anti-microbial treatment and high-dose INF-γ is very suggestive of an underlying Interferon Gamma Pathway immunodeficiency. Despite no mutations found in previously described associated genes (INFGR1, INFGR2 and STAT1), we believe that a secondary immunodeficiency induced by chemotherapy alone is not a sufficient explanation to the character of the infection and that a yet not identified defect of the Interferon Gamma Pathway might be causative. This unique case also raises questions about the character of LCH, a condition which remains to be idiopathic.

A disturbed interaction with accessory cells upon opportunistic infection with Pseudomonas aeruginosa contributes to an impaired IFN-γ production of NK cells in the lung during sepsis-induced immunosuppression

Impaired resistance to Pseudomonas aeruginosa-induced pneumonia after cecal ligation and puncture (CLP), a mouse model for human polymicrobial sepsis, is associated with decreased IFN-γ, but increased IL-10, levels in the lung. We investigated the so far unknown mechanisms underlying this reduced IFN-γ synthesis in CLP mice. CD11b(+) NK cells, but not T or NKT cells in the lung were impaired in IFN-γ synthesis upon challenge with Pseudomonas in vitro and in vivo after CLP. The inhibition of NK cells was independent of IL-10. IFN-γ synthesis of NK cells was only partly restored by addition of recombinant IL-12. Accessory cells including dendritic cells and alveolar macrophages were required for maximal IFN-γ secretion. But accessory cells of CLP mice suppressed the IFN-γ secretion from naive lung leukocytes. In turn, naive accessory cells were unable to restore the IFN-γ production from lung leukocytes of CLP mice. Thus, a disturbed interaction of accessory cells and NK cells is involved in the impaired IFN-γ release in response to Pseudomonas in the lung of CLP mice. Considering the importance of IFN-γ in the immune defense against bacteria the dysfunction of accessory cells and NK cells might contribute to the enhanced susceptibility to Pseudomonas after CLP.

T-Cell Dysfunction In CLL Is Mediated Not Only By PD-1/PD-L1 But Also By PD-1/PD-L2 Interactions - Partial Functionality Is Maintained In PD-1 Defined CD8 Subsets and This Can Be Further Promoted By Ibrutinib Treatment

BackgroundCLL-induced severe T-cell dysfunction and ineffective anti-tumor immune-responses are hallmarks of the disease, but the specific interactions remain poorly understood. The PD-1/PD-L1 axis is an important mediator of T-cell dysfunction in solid tumors, and we have previously demonstrated that T cells from CLL patients exhibit impaired immunological synapse (IS) formation, predominantly mediated by PD-L1 (CD274) expression on CLL cells. We have also shown that the corresponding T-cell ligand PD-1 (CD279) is also upregulated, probably as a result of chronic antigenic stimulation, and that T cells have similarities to exhausted T cells observed in the context of chronic viral infection. Recent studies demonstrated that ibrutinib has impressive clinical activity in CLL, and mechanisms of action include irreversible binding of essential components of both B-cell- and T-cell-receptor signaling and interactions with the tumor microenvironment. Modulation of PD-1/PD-L1 interactions might therefore be an additional potential mode of action of this drug. Using the well-established Eμ-TCL1 (TCL1) mouse model of CLL, our aims were to demonstrate that (1) altered expression of PD-L1 on CLL cells and PD-1 on T cells and CLL are causally related, (2) the second ligand of PD-1, PD-L2, is also involved in mediating T-cell dysfunction, (3) T-cell effector function and IS formation are directly linked to PD-1 expression and (4) PD-1 associated in vivo T-cell responses can be modulated by treatment with ibrutinib. MethodsAs we have previously demonstrated that the spleen is the major organ of disease and representative of T-cell changes in peripheral blood and lymph nodes, experiments were performed on spleens from young TCL1 and wild-type (WT) C57Bl/6 mice with established CLL after adoptive transfer (AT) of syngeneic CLL cells (n=10), and on matched litter-mates after AT of healthy mouse B cells (n=10). An additional 12 mice were randomized to treatment with 25 mg/kg/d ibrutinib in 10% HP-β-CD, vehicle control, or sterile water, all administered by gavage, three weeks after AT of syngeneic CLL cells, and sacrificed 20 days later at a pre-defined endpoint. Multicolor flow cytometry was used to characterize T-cell subsets, expression of PD-1, PD-L1 and PD-L2 and T-cell effector function. Entire population CD8 T cells, PD-1+ve and PD-1-ve CD8 T cells were flow-sorted and used in IS formation assays with healthy murine B cells as antigen-presenting cells. ResultsOur previous studies using aged TCL1 mice and age-matched WT controls indicated that CLL-related PD-1 upregulation on antigen-experienced CD44+ CD8 T cells is masked by aging. However, PD-1 expression could also be induced in young TCL1 and WT mice by AT of CLL cells but not healthy B cells, suggesting a causal relationship with disease. Both PD-L1 and PD-L2 surface expression on CLL B cells were significantly increased compared to healthy B cells. Using TCL1 mice at early stages of CLL development when a healthy CD19+ B-cell population is still present, we were able to confirm that PD-L2 expression is a unique feature of CLL cells, with PD-L2 being virtually absent on healthy B cells. We next compared effector function and the ability to form IS of PD-1+ve and PD-1-ve antigen-experienced CD44+ T-cell subsets in mice with CLL. While proliferation was equally impaired in these subsets, they were both able to degranulate but generally failed to localize granzyme B to the IS. Although subsets produced some IL2/TNFα/IFNγ cytokine responses, PD-1+ve cells had significantly impaired TNFα and slightly impaired IL2 and IFNγ production, and a highly significant impaired ability to form IS compared to PD-1-ve cells. Treatment with ibrutinib reduced PD-1 expression on antigen-experienced CD44+ CD8 T cells and promoted stronger IFNγ production of entire population CD8 T cells, but failed to restore proliferation and granzyme B relocation to the IS. ConclusionOur in vivo data suggests that CLL and PD-1/PD-L1-mediated T-cell dysfunction are causally related, but that phenotypic and functional T-cell changes are not absolute and might be at least partly reversible by ibrutinib treatment. We also show that the second ligand of PD-1, PD-L2, is also a critical mediator of PD-1 associated T-cell dysfunction in CLL. Disclosures:Riches:Celgene: Research Funding. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Protective role of adenylate cyclase in the context of a live pertussis vaccine candidate

Despite high vaccination coverage, pertussis remains an important respiratory infectious disease and the least-controlled vaccine-preventable infectious disease in children. Natural infection with Bordetella pertussis is known to induce strong and long-lasting immunity that wanes later than vaccine-mediated immunity. Therefore, a live attenuated B. pertussis vaccine, named BPZE1, has been developed and has recently completed a phase I clinical trial in adult human volunteers. In this study, we investigated the contribution of adenylate cyclase (CyaA) in BPZE1-mediated protection against pertussis. A CyaA-deficient BPZE1 mutant was thus constructed. Absence of CyaA did not compromise the adherence properties of the bacteria onto mammalian cells. However, the CyaA-deficient mutant displayed a slight impairment in the ability to survive within macrophages compared to the parental BPZE1 strain. In vivo, whereas the protective efficacy of the CyaA-deficient mutant was comparable to the parental strain at a vaccine dose of 5 × 105 colony forming units (CFU), it was significantly impaired at a vaccine dose of 5 × 103 CFU. This impairment correlated with impaired lung colonization ability, and impaired IFN-γ production in the animal immunized with the CyaA-deficient BPZE1 mutant while the pertussis-specific antibody profile and Th17 response were comparable to those observed in BPZE1-immunized mice. Our findings thus support a role of CyaA in BPZE1-mediated protection through induction of cellular mediated immunity.

Innate immune responses in hepatitis C virus-exposed healthcare workers who do not develop acute infection

Hepatitis C virus (HCV) infection typically results in chronic disease with HCV outpacing antiviral immune responses. Here we asked whether innate immune responses are induced in healthcare workers who are exposed to small amounts of HCV, but do not develop systemic infection and acute liver disease. Twelve healthcare workers with accidental percutaneous exposure to HCV-infected blood were prospectively studied for up to 6 months for phenotype and function of natural killer T (NKT) and NK cells, kinetics of serum chemokines, and vigor and specificity of HCV-specific T-cell responses. Eleven healthcare workers tested negative for HCV RNA and HCV antibodies. All but one of these aviremic cases displayed NKT cell activation, increased serum chemokines levels, and NK cell responses with increased CD122, NKp44, NKp46, and NKG2A expression, cytotoxicity (as determined by TRAIL and CD107a expression), and interferon-gamma (IFN-γ) production. This multifunctional NK cell response appeared a month earlier than in the one healthcare worker who developed high-level viremia, and it differed from the impaired IFN-γ production, which is typical for NK cells in chronic HCV infection. The magnitude of NKT cell activation and NK cell cytotoxicity correlated with the magnitude of the subsequent HCV-specific T-cell response. T-cell responses targeted nonstructural HCV sequences that require translation of viral RNA, which suggests that transient or locally contained HCV replication occurred without detectable systemic viremia. Exposure to small amounts of HCV induces innate immune responses, which correlate with the subsequent HCV-specific T-cell response and may contribute to antiviral immunity.

KLRG1 Negatively Regulates Natural Killer Cell Functions through the Akt Pathway in Individuals with Chronic Hepatitis C Virus Infection

In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56(+) NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV in vitro. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-γ) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1(+) NK cells, including CD56(bright) and CD56(dim) subsets, exhibit impaired cell activation and IFN-γ production but increased apoptosis compared to KLRG1(-) NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-γ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.

Programmed death-1 (PD-1)-dependent functional impairment of CD4+ T cells in recurrent genital papilloma

Genital papilloma is caused by human papilloma virus (HPV) infection and recurs frequently. Although T cells are known to play a critical role in the control of HPV infection and papilloma development, the function and phenotype of these cells in the lesion remain to be elucidated. In the present study, we examined the function and phenotype of CD4(+) T cells isolated from the lesions of primary (n = 9) and recurrent (n = 11) genital papillomas. In recurrent papillomas, the frequency of proliferating (Ki-67(+)) CD4(+) T cells was significantly reduced compared with primary papillomas. Cytokine production was evaluated by intracellular cytokine staining in anti-CD3/anti-CD28-stimulated CD4(+) T cells. CD4(+) T cells from recurrent lesions showed impaired production of IL-2, IFN-γ, and TNF-α. Of interest, the frequency of cytokine-producing CD4(+) T cells significantly correlated with the frequency of Ki-67(+)CD4(+) T cells. We also studied expression of programmed death-1 (PD-1), a T-cell exhaustion marker. The frequency of PD-1(+)CD4(+) T cells was significantly increased in recurrent lesions and inversely correlated with the frequency of cytokine-producing CD4(+) T cells. The functional significance of PD-1 expression was determined in blocking assays with anti-PD-L1, which restored cytokine production of CD4(+) T cells from recurrent lesions. Taken together, in recurrent genital papilloma lesions, proliferation, and cytokine production by CD4(+) T cells are impaired and the PD-1/PD-L1 interaction is responsible for the functional impairment of CD4(+) T cells.

Parasite-Derived Arginase Influences Secondary Anti-LeishmaniaImmunity by Regulating Programmed Cell Death-1–Mediated CD4+ T Cell Exhaustion

The breakdown of L-arginine to ornithine and urea by host arginase supports Leishmania proliferation in macrophages. Studies using arginase-null mutants show that Leishmania-derived arginase plays an important role in disease pathogenesis. We investigated the role of parasite-derived arginase in secondary (memory) anti-Leishmania immunity in the resistant C57BL/6 mice. We found that C57BL/6 mice infected with arginase-deficient (arg(-)) L. major failed to completely resolve their lesion and maintained chronic pathology after 16 wk, a time when the lesion induced by wild-type L. major is completely resolved. This chronic disease was associated with impaired Ag-specific proliferation and IFN-γ production, a concomitant increase in programmed cell death-1 (PD-1) expression on CD4(+) T cells, and failure to induce protection against secondary L. major challenge. Treatment with anti-PD-1 mAb restored T cell proliferation and IFN-γ production in vitro and led to complete resolution of chronic lesion in arg(-) L. major-infected mice. These results show that infection with arg(-) L. major results in chronic disease due in part to PD-1-mediated clonal exhaustion of T cells, suggesting that parasite-derived arginase contributes to the overall quality of the host immune response and subsequent disease outcome in L. major-infected mice. They also indicate that persistent parasites alone do not regulate the quality of secondary anti-Leishmania immunity in mice and that the quality of the primary immune response may be playing a hitherto unrecognized dominant role in this process.

Modulation of T cell function through L-arginine metabolism: a new therapy from an old enemy

Recent studies have suggested the relevance of different energy metabolic pathways in the balance between protective T cell immunity and T cell anergy in tumors. We and others have suggested the role of the depletion of the non-essential amino acid L-arginine as a mechanism for the induction of T cell suppression in tumors. Therefore, we hypothesize that it is possible to metabolically regulate T cell responses simply through the modulation of L-arginine. In this study, we aimed to determine the effect of a pegylated form of the human L-arginine-metabolizing enzyme arginase I (peg-Arg I) in T cell responses. Activation of antigen-specific CD4+ and CD8+ T cells in the presence of peg-Arg I prevented cell proliferation and production of IFNγ in vitro and in vivo. Similarly, peg-Arg I impaired proliferation and IFNγ production in T cells activated with PMA/Ionomycin, suggesting that the effect of peg-Arg I was independent of T cell receptor (TCR) signaling. In fact, the anti-proliferative effect induced by peg-Arg I correlated with an arrest of T cells in the G0-G1 phase of the cell cycle, a decreased expression of cyclin D3 and cdk4, and a major inhibition of de novo translation. Interestingly, treatment of T cells with peg-Arg I did not impair the expression of activation markers CD25, CD69, and the production of IL-2, which correlated with an intact mitochondrial biogenesis. As a result, peg-Arg I did not have an effect in oxygen consumption (OCR) by mitochondrial respiration, but significantly blocked glycolytic pathways in activated T cells. Furthermore, peg-Arg I treated T cells increased the expression of genes associated with integrated stress responses (IRS) and arrest in translation including GCN2, Chop, and Atf4. In fact, GCN2 was a major mediator of the effects induced by peg-Arg I. Then, we tested the effect of peg-Arg I in mouse models of graft versus host disease (GVHD) and inflammatory bowel disease (IBD), both mediated through activated T cells. Peg-Arg I significantly extended the survival of mice in these 2 disease models, which associated with a decreased production of IFNγ. Altogether the results suggest the potential effect of the modulation of the metabolism of L-arginine as a mean to modulate T cell responses. Continuation of this study will advance in the understanding of the metabolic effects of L-arginine in T cell function, which could enable the development of therapies to modulate T cell responses in transplantation or autoimmunity.

Involvement of NFAT Signaling in Function and Anti-Tumor Reactivity of NK Cells

AbstractAbstract 1887NK cells play an important role in anti-tumor immunity. They significantly contribute to the clinical success of allogeneic stem cell transplantation (SCT). Their reactivity as a consequence of an integrative response mediated by various activating and inhibitory surface receptors results in the induction of yet only partially defined signal transduction pathways. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells). While its role in T cell development and function is meanwhile well defined, surprisingly little is known on its function in NK cells. NFAT seems to be dispensable for NK cell development, but several lines of evidence clearly point to its involvement in NK reactivity and function. Cyclosporin A (CsA) and tacrolimus are immunosuppressive drugs that are widely used in transplant medicine. They mediate their immunosuppressive effects through inhibition of the serine/threonine phosphatase calci-neurin, which dephosphorylates and thereby activates NFAT. Here we studied the role of NFAT in NK cells and found that all five NFAT family members are expressed in NK cells with their levels being dependent on NK cell activation state. CsA and tacrolimus, but not mycophenolic acid which mediates its immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase, reduced activation and degranulation of NK cells, resulting in impaired cytotoxicity and IFN-γ production in response to leukemia targets. NK reactivity was also suppressed by the specific NFAT inhibitors VIVIT and INCA-6, indicating that the calcineurin inhibitors CsA and tacrolimus in fact modulate NK reactivity by inhibition of NFAT proteins and not by potential “off target”-effects. These results provide evidence for the critical involvement of the transcription factor NFAT in NK cell reactivity and also indicate that potential effects on NK cell immunosurveillance should be considered upon choice and dosing of immunosuppressive treatment regimens after SCT. Disclosures:No relevant conflicts of interest to declare.