IFN-γ Secretion In Response Research Articles

DAP12 deficiency in liver allografts results in enhanced donor DC migration, augmented effector T cell responses and abrogation of transplant tolerance (TRAN3P.883)

Abstract Liver interstitial dendritic cells (DC) have been implicated in immune regulation and tolerance induction. We found that the transmembrane immuno-adaptor DNAX-activating protein of 12kDa (DAP12) negatively regulated conventional liver DC maturation and their in vivo migratory and T cell allostimulatory ability. Livers were transplanted from B6 wild-type (wt) or DAP12-/- mice into wt C3H recipients. Donor DC (H2-Kb+CD11c+) were quantified in spleens by flow cytometry. Anti-donor T cell reactivity was evaluated by ex vivo CFSE-MLR and in vivo delayed-type hypersensitivity responses, while T effector and regulatory T cells were determined by flow analysis. A 3-4-fold increase in donor-derived DC was detected in the spleens of DAP12-/- liver recipients compared with those given wt grafts. Moreover, pro-inflammatory cytokine gene expression in the graft, IFNγ production by graft-infiltrating CD8+ T cells, and systemic levels of IFNγ were all elevated significantly in hosts of DAP12-/- liver recipients. DAP12-/- grafts also exhibited reduced incidences of CD4+Foxp3+ cells and enhanced CD8+ T cell IFNγ secretion in response to donor antigen challenge. Unlike wt grafts, DAP12-/- livers failed to induce tolerance and were rejected acutely. Thus, DAP12 expression in liver grafts regulates donor DC migration to host lymphoid tissue, alloreactive T cell responses and transplant tolerance.

Targeting high‐grade B cell lymphoma with CD19‐specific T cells

Adoptive T cell therapy is an important additional treatment option for malignant diseases resistant to chemotherapy. Using a murine high-grade B cell lymphoma model, we have addressed the question whether the B cell differentiation antigen CD19 can act as rejection antigen. CD19(-/-) mice inoculated with CD19(+) B cell lymphoma cells showed higher survival rates than WT mice and were protected against additional tumor challenge. T cell depletion prior to tumor transfer completely abolished the protective response. By heterotypic vaccination of CD19(-/-) mice against murine CD19, survival after tumor challenge was significantly increased. To define protective epitopes within the CD19 molecule, T cells collected from mice that had survived the tumor transfer were analyzed for IFNγ secretion in response to CD19-derived peptides. The majority of mice exhibited a CD4(+) T cell response to CD19 peptide 27, which was the most dominant epitope after CD19 vaccination. A peptide 27-specific CD4(+) T cell line protected CD19(-/-) mice against challenge with CD19(+) lymphoma and also cured a significant proportion of WT mice from recurrent disease in a model of minimal residual disease after chemotherapy. In conclusion, our data highlight CD19-specific CD4(+) T cells for adoptive T cell therapy of B cell lymphomas.

Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin

Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

Suppression of Allergic Reactions in Ovalbumin-Sensitized Mice by Yam Storage Proteins Dioscorins

To study the biomedical functions of dioscorins isolated from various species of Dioscorea , we investigated their antiallergic potential using an OVA-induced allergy mouse model. All the dioscorins suppressed allergic reactions by decreasing the serum IgE and histamine levels. The serum IFN-γ and IgG2a levels increased in all the dioscorin-treated mice. The spleen cells from the dioscorin-treated mice also exhibited an up-regulation of IFN-γ secretion in response to ConA stimulation. Although dioscorins did not affect the IgG1 levels, the IL-5 levels decreased to basal levels in mice treated with dioscorins of D. alata or D. japonica and in most of the lymphoid cells of the dioscorin-treated mice in response to ConA stimulation. The decrease of IgE and histamine levels was concomitant with an increase in IFN-γ and IgG2a levels and with a decrease in IL-5 levels, suggesting that dioscorins suppressed the OVA-induced allergic reactions, possibly through modulating an imbalanced Th1/Th2 immune response.

Ex Vivo Innate Immune Cytokine Signature of Enhanced Risk of Relapsing Brucellosis

BackgroundBrucellosis, a zoonotic infection caused by one of the Gram-negative intracellular bacteria of the Brucella genus, is an ongoing public health problem in Perú. While most patients who receive standard antibiotic treatment recover, 5–40% suffer a brucellosis relapse. In this study, we examined the ex vivo immune cytokine profiles of recovered patients with a history of acute and relapsing brucellosis.Methodology/Principal FindingsBlood was taken from healthy control donors, patients with a history of acute brucellosis, or patients with a history of relapsing brucellosis. Peripheral blood mononuclear cells were isolated and remained in culture without stimulation or were stimulated with a panel of toll-like receptor agonists or heat-killed Brucella melitensis (HKBM) isolates. Innate immune cytokine gene expression and protein secretion were measured by quantitative real-time polymerase chain reaction and a multiplex bead-based immunoassay, respectively.Acute and relapse patients demonstrated consistently elevated cytokine gene expression and secretion levels compared to controls. Notably, these include: basal and stimulus-induced expression of GM-CSF, TNF-α, and IFN-γ in response to LPS and HKBM; basal secretion of IL-6, IL-8, and TNF-α; and HKBM or Rev1-induced secretion of IL-1β, IL-2, GM-CSF, IFN-Υ, and TNF-α. Although acute and relapse patients were largely indistinguishable by their cytokine gene expression profiles, we identified a robust cytokine secretion signature that accurately discriminates acute from relapse patients. This signature consists of basal IL-6 secretion, IL-1β, IL-2, and TNF-α secretion in response to LPS and HKBM, and IFN-γ secretion in response to HKBM.Conclusions/SignificanceThis work demonstrates that informative cytokine variations in brucellosis patients can be detected using an ex vivo assay system and used to identify patients with differing infection histories. Targeted diagnosis of this signature may allow for better follow-up care of brucellosis patients through improved identification of patients at risk for relapse.

The CXCR3(+)CD56Bright Phenotype Characterizes a Distinct NK Cell Subset with Anti-Fibrotic Potential That Shows Dys-Regulated Activity in Hepatitis C

BackgroundIn mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C.MethodsCirculating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(−) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15).ResultsWe show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis.ConclusionWe show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis.

STAT4 regulates antiviral gamma interferon responses and recurrent disease during herpes simplex virus 2 infection.

STAT4 is an important transcription factor that contributes to the incidence and severity of different autoimmune diseases and is implicated in the antiviral immune responses in mice. In this study, we evaluated the role of STAT4 in human and murine herpes simplex virus 2 (HSV-2) infections. We show that STAT4 regulates antiviral gamma interferon (IFN-γ) responses and disease severity during chronic HSV-2 infections in humans and vaccine-induced IFN-γ-mediated protection against HSV-2 infection in mice. In a cohort of 228 HSV-2-infected individuals, representing both patients with recurrent disease and asymptomatic HSV-2 carriers, we found that genetic variations in the STAT4 gene were associated with asymptomatic HSV-2 infection, as well as with increased in vitro secretion of IFN-γ in response to the virus. Mice that lacked STAT4 had impaired HSV-2-specific IFN-γ production and delayed-type hypersensitivity responses following vaccination, which led to impaired viral clearance in the genital tract of vaccinated animals after a genital HSV-2 challenge. We conclude that STAT4 plays an important role in IFN-γ-mediated HSV-2-specific immunity, affecting the severity of genital HSV-2 infection.

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

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.

Cellular immune responses in human immunodeficiency virus (HIV)-1-infected children: is immune restoration by highly active anti-retroviral therapy comparable to non-progression?

The objective of this study was to investigate whether the restored immune functions of vertically human immunodeficiency virus (HIV)-infected children who were severely immunodeficient before the initiation of highly active anti-retroviral therapy (HAART) are comparable to those of untreated slow progressors. We therefore assessed T cell proliferation and cytokine [interferon (IFN)-γ, interleukin (IL)-5 and IL-13] secretions after mitogen, recall antigens and HIV-1-specific stimulation in 12 untreated slow progressors, 16 untreated progressors and 18 treated patients. Treated children were profoundly immunodeficient before the initiation of HAART and had long-lasting suppression of viral replication on treatment. We demonstrated that slow progressors are characterized not only by the preservation of HIV-1-specific lymphoproliferative responses but also by the fact that these responses are clearly T helper type 1 (Th1)-polarized. Children on HAART had proliferative responses to HIV-1 p24 antigen, purified protein derivative (PPD) and tetanus antigen similar to slow progressors and higher than those of progressors. However, in contrast to slow progressors, most treated children exhibited a release of Th2 cytokines accompanying the IFN-γ secretion in response to the HIV-1 p24 antigen. Moreover, despite higher proliferative responses to phytohaemagglutinin (PHA) than the two groups of untreated children, treated children had lower levels of IFN-γ secretion in response to PHA than slow progressors. These data show that in severely immunodeficient vertically HIV-infected children, a long-lasting HAART allows recovering lymphoproliferative responses similar to untreated slow progressors. However, alterations in IFN-γ secretion in response to the mitogen PHA persisted, and their cytokine release after HIV-specific stimulation was biased towards a Th2 response.

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.

Severity of the compensatory anti-inflammatory response determined by monocyte HLA-DR expression may assist outcome prediction in cirrhosis

To investigate variations in expression of the monocyte antigen presentation molecule HLA-DR in cirrhosis. HLA-DR expression was measured by flow cytometry in 100 patients within 48 h of admission and repeated a week later in 21 patients admitted to ICU. IL-10, TNF-α and IFN-γ secretion in response to lipopolysaccharide and recall antigens were measured by enzyme-linked immunosorbent spot (ELISPOT) assay in 12 patients (7 with clinical immunoparesis, 5 stable). HLA-DR level was 71% in stable patients, 53% with single organ dysfunction and 34% with multiple organ failure (p < 0.02). Within these groups, no significant differences in admission HLA-DR were seen between survivors and non-survivors. HLA-DR expression less than 40% predicted 90-day mortality with a specificity of 80% and sensitivity of 59% [area under the receiver operator curve (AUROC) 0.76]. HLA-DR less than 40% was an independent predictor of prognosis in multivariate analysis with a relative risk of 2.35 (p = 0.04), although sequential organ failure assessment score (SOFA) score displaced HLA-DR when included. In those admitted to intensive care failure to increase HLA-DR expression was predictive of death within 30 days (risk ratio 6.9, p = 0.007). Follow-up values predicted outcome with similar accuracy to acute physiology and chronic health evaluation II (APACHE II)/SOFA scores (AUROC 0.88). Response to endotoxin and recall antigen was characterised by an anti-inflammatory cytokine secretion profile, and was associated with impairment in recall antigen presentation capacity. HLA-DR expression less than 40% and a failure of recovery predict poor outcome in decompensated cirrhosis, but overall prognostic power remains inferior to conventional markers. Ex vivo experiments demonstrate reduced Th1 response to antigenic stimulation and an exaggerated counter-inflammatory cytokine secretion profile.

Memory B cells from a subset of treatment‐naïve relapsing‐remitting multiple sclerosis patients elicit CD4+ T‐cell proliferation and IFN‐γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.

A novel immunoregulatory function for IL‐23: Inhibition of IL‐12‐dependent IFN‐γ production

Most studies investigating the function of IL-23 have concluded that it promotes IL-17-secreting T cells. Although some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 antagonizes IL-12-induced secretion of IFN-γ. When splenocytes or purified populations of T cells were cultured with IL-23, IFN-γ secretion in response to IL-12 was dramatically reduced. The impact of IL-23 was most prominent in CD8(+) T cells, but was also observed in NK and CD4(+) T cells. Mechanistically, the IL-23 receptor was not required for this phenomenon, and IL-23 inhibited signaling through the IL-12 receptor by reducing IL-12-induced signal transducer and activator of transcription 4 (STAT4) phosphorylation. IL-23 was also able to reduce IFN-γ secretion by antagonizing endogenously produced IL-12 from Listeria monocytogenes (LM)-infected macrophages. In vivo, LM infection induced higher serum IFN-γ levels and a greater percentage of IFN-γ(+)CD8(+) T cells in IL-23p19-deficient mice as compared with WT mice. This increase in IFN-γ production coincided with increased LM clearance at days 2 and 3 post-infection. Our data suggest that IL-23 may be a key factor in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ.

Gemcitabine directly inhibits myeloid derived suppressor cells in BALB/c mice bearing 4T1 mammary carcinoma and augments expansion of T cells from tumor-bearing mice

Myeloid derived suppressor cells (MDSCs) accumulate in 4T1 mammary carcinoma bearing mice and present a barrier to the success of adoptive immunotherapy (AIT) by suppressing T cell immunity. In this study, we investigated the inhibition of MDSCs by gemcitabine (GEM), a chemotherapy agent that may have favorable immunologic effects. BALB/c mice were inoculated with 4T1 mammary carcinoma cells and treated with GEM either once a week starting 5 days after tumor inoculation (EARLY GEM) or as a single dose at days 20–25 (LATE GEM). Splenic mononuclear cells were isolated, activated in vitro, expanded, and stimulated with tumor antigen. T cells were then used for AIT to treat tumor-bearing mice. EARLY GEM treatment of 4T1 tumor-bearing mice significantly inhibited tumor growth, reduced splenomegaly, and significantly decreased MDSC proportion in the spleen. Support for a direct effect was demonstrated through suppression of MDSCs in spleens, bone marrow, and blood harvested 24 and 48 h after LATE GEM treatment, despite no significant decrease in tumor burden. Interestingly, treatment of tumor-bearing mice with GEM augmented in vitro expansion of splenic T cells and boosted IFN-γ secretion in response to stimulation by tumor antigen. However, despite GEM-mediated inhibition of MDSC suppression, splenic T cells from mice with advanced tumors were ineffective in vivo against established tumors. This study provides support for direct inhibition of MDSCs and direct reduction of tumor burden by GEM in 4T1 tumor-bearing mice. GEM treatment of mice with advanced tumors improves T cell function and growth in vitro.

Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells

Persistent immune activation is a hallmark of chronic viremic HIV-1 infection. Activation of cells of the innate immune system, such as NK cells, occurs rapidly upon infection, and is sustained throughout the course of the disease. However, the precise underlying mechanism accounting for the persistent HIV-1-induced activation of NK cells is poorly understood. In this study, we assessed the role of uridine-rich ssRNA derived from the HIV-1 long terminal repeat (ssRNA40) on activation of NK cells via TLR7/8. Although dramatic activation of NK cells was observed following stimulation of PBMC with ssRNA40, negligible activation was observed following stimulation of purified NK cells despite their expression of TLR8 mRNA and protein. The functional activation of NK cells by this HIV-1-encoded TLR7/8 ligand could not be reconstituted with exogenous IL-12, IFN-alpha, or TNF-alpha, but was critically dependent on the direct contact of NK cells with plasmacytoid dendritic cells or CD14(+) monocytes, indicating an important level of NK cell cross-talk and regulation by accessory cells during TLR-mediated activation. Coincubation of monocyte/plasmacytoid dendritic cells, NK cells, and ssRNA40 potentiated NK cell IFN-gamma secretion in response to MHC-devoid target cells. Studies using NK cells derived from individuals with chronic HIV-1 infection demonstrated a reduction of NK cell responsiveness following stimulation with TLR ligands in viremic HIV-1 infection. These data demonstrate that HIV-1-derived TLR ligands can contribute to the immune activation of NK cells and may play an important role in HIV-1-associated immunopathogenesis and NK cell dysfunction observed during acute and chronic viremic HIV-1 infection.

IL-23 acts in a novel fashion by antagonizing IL-12 induced IFN-γ secretion (95.16)

Abstract Most studies have concluded that interleukin-23 (IL-23) plays a unique role in promoting IL-17 secreting T cells. While some reports have also characterized IL-23 as having redundant pro-inflammatory effects with IL-12, we have instead found that IL-23 can regulate the innate immune response in a novel manner, by antagonizing IL-12 induced secretion of interferon-γ (IFN-γ). Our recent data documents the rapid, innate production of IFN-γ from memory CD8 T cells, as well as NK cells, stimulated with IL-12 and IL-18. When splenocytes are cultured with IL-23, IFN-γ secretion in response to IL-12 is dramatically reduced, as measured by ELISA and intracellular cytokine staining. The impact of IL-23 is most prominent in CD8 T cells, but can also be seen in NK, NK-T, and CD4 T cells. We also show that IL-23 can regulate the induction of IFN-γ by endogenously produced IL-12 from Listeria monocytogenes infected macrophages. Furthermore, IL-23 appears to act directly on purified CD8 T cells to negatively regulate IFN-γ induced by IL-12. We are currently investigating the mechanism by which IL-23 inhibits IFN-γ secretion. In addition, experiments to determine if IL-23 can inhibit other effector functions are ongoing. Our data suggest that IL-23 may be a key player in determining the responsiveness of lymphocytes to IL-12 and their subsequent secretion of IFN-γ. This research is supported by NIH AI064592.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes.It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

Varicella-Zoster-Virus-Specific CD4+ Cytotoxic T Cells as Tumor-Specific Effector Cells in Cancer Immunotherapy.

Adoptive transfer of gene-engineered T cells expressing tumor antigen-specific chimeric receptors (chRec) is a promising tool in cancer immunotherapy. A major limitation is the failure of chRec to induce proliferative T cell responses, resulting in a rapid loss of function. To provide a strategy for reexpansion of tumor-reactive T cells in vivo, we generated dual-specific T cells that respond to varicella zoster virus while also possessing chRec-mediated tumor reactivity. We expanded VZV-specific cytotoxic T cell lines (VZV-CTL) from four seropositive donors by culturing peripheral blood-derived T cells with lysates extracted from VZV-infected fibroblasts. Repeated stimulation with VZV lysates resulted in efficient and continued expansion for 10–12 weeks. >1x109 T cells were routinely obtained from a starting number of 1x106 peripheral blood T cells. The T cells displayed a mainly CD3+CD4+ (90±5%) phenotype. ELISPOT assays showed specific, MHC class II-restricted IFN-γ release in response to CD40-activated B cells expressing the viral glycoproteins gE and IE62. VZV-CTL belong to a non-regulatory effector T cell subset, shown by their failure to exert antiproliferative effects against cocultured autologous T cells and lack of Foxp3 expression. Retroviral transduction with chRec recognizing the tumor ganglioside antigen GD2 (14.G2a-ζ) and the B cell lineage antigen CD19 (CD19-ζ) resulted in receptor surface expression on 29–74% and 39–45% of cells, respectively. Gene-modified VZV-CTL efficiently recognized antigen-expressing tumor targets in an MHC-independent manner, as demonstrated by antigen-specific secretion of IFN-γ in response to coincubation with GD2-expressing tumor targets. Furthermore, chRec-transduced VZV-CTL performed potent and antigen-specific tumor cytolysis. Antibody blocking experiments revealed that tumor cells were lysed in a granulysin-dependent manner. ChRec-transduced CD3+CD4+ cytolytic VZV-CTL may provide a source of highly potent tumor-reactive cells for adoptive immunotherapy cancer. Endogenous viral reactivations or administration of booster doses of varicella vaccine may lead to survival of these tumor-reactive T cells for prolonged periods of time in vivo.

Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum

IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN-gamma secretion in response to erythrocytes infected with Plasmodium falciparum (Pf), a causative agent of human malaria. In addition, direct sensing of Pf infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are individually dispensable for NK cell activation induced by Pf-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages, and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection.