IFN-γ Secretion In Response Research Articles

T cell reactivity to P0, P2, PMP-22, and myelin basic protein in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy

Objectives: It has been suggested that autoimmunity to peripheral myelin proteins is involved in the pathogenesis of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). We aimed to compare...

Costimulation through NKG2D Enhances Murine CD8+ CTL Function: Similarities and Differences between NKG2D and CD28 Costimulation

Multiple studies have demonstrated that the NK cell activating receptor NKG2D can function as a costimulatory receptor for both mouse and human CD8+ T cells. However, it has recently been suggested that stimulation through NKG2D is insufficient for costimulation of CD8+ T cells. To aid in the delineation of NKG2D function in CTL responses, we investigated whether stimulation of NKG2D by the natural ligand RAE1epsilon was able to costimulate effector functions of a murine CTL line generated from DUC18 TCR transgenic mice. We found that NKG2D was able to costimulate DUC CTL responses and did so in a manner similar to CD28 costimulation. The T cells exhibited increased proliferation, IFN-gamma release, and cytotoxicity when presented antigenic peptide by P815 cells expressing RAE1epsilon or B7-1 compared with untransfected P815. In addition, both RAE1epsilon and B7-1 enhanced Ag-independent IFN-gamma secretion in response to IL-12 and IL-18 by DUC CTL. However, only costimulation through CD28 allowed for DUC CTL survival upon secondary stimulation, whereas ligation of NKG2D, but not CD28, induced DUC CTL to form an immune synapse with target cells in the absence of TCR stimulation. Understanding the outcomes of these differences may allow for a better understanding of T cell costimulation in general.

Continuous Presence of Th1 Conditions Is Necessary for Longer Lasting Tumor-Specific CTL Activity in Stimulation Cultures With PBL

The generation of tumor-associated, but self-antigen specific cytotoxic T lymphocytes (CTL) response is possible by vaccination even in patients at the advanced stages of the disease. The in vivo expansion of such CTLs is now the most important objective of the immunotherapy. In human melanoma, we show here that MART-1 27–35-specific CTLs generated with purified CD8 + cells survive and maintain their activity longer in culture than those CTLs generated by using total peripheral blood lymphocytes (PBL) taken either from patients or from normal donors. When PBL are grown under Th1 conditioning the quantity and quality of CTL with total PBL are comparable with that of the CTLs generated with purified CD8+ cells. For patients either autologous melanoma tumor cells or MART-1 27–35 peptide pulsed autologous DC were used to generate CTL responses. For normal donors MART-1 27–35 peptide pulsed autologous DC were used. For both normal donors or patients, polarization of PBL with Th1 conditioning with interleukin (IL)-12 (250 U/ml) and anti IL-4 antibody 1 μg/ml for 7 days before CTL generation, induced better and longer living CTL response and prevented the expansion of CD4+ T cells that have downregulatory activity. We show that continuous presence of Th1 conditioning in cultures with total PBL generated significantly higher number of antigen-specific CTLs as detected by MART-1 HLA-A2 tetramer staining. The antigen specificity of such CTLs were determined by IFN-γ secretion in response to target cells bearing the specific antigen. Our observations indicate that Th1 conditioning results in a longer lasting CTL response in vitro and points toward a newer approach for vaccine strategy.

Interferon-Gamma (IFN-γ) Secretion Defect in Peripheral Blood Mononuclear Cells (PBMCs) from Severe Hemophilia A (HA) Patients Prior to Therapeutic Exposure To Factor VIII (FVIII) Concentrates.

The recent availability of highly purified recombinant coagulation factor concentrates to treat hemorrhages in hemophilic patients is thought to circumvent perturbations in immune function by preventing chronic intravenous exposure to multiple foreign protein antigens. We previously reported a PHA-induced IFN-γ secretion defect in severe hemophilia A patients who had been exposed exclusively to highly purified coagulation factor VIII concentrates. We now report the results of cytokine secretion in response to PHA in cultured PBMCs from severe hemophilia A patients never exposed to FVIII concentrates. PBMCs were isolated from whole blood from three severe HA patients, 3 age-matched normal boys, 3 boys with severe hemophilia B and an adult control. Cells were stained with PKH26 (Sigma), a fluorescent dye with aliphatic tails that intercalate into membrane lipids; cells were than washed and cultured in complete media in wells of microtiter plates with and without PHA. Cells were harvested at 5 days and proliferation detected by loss of membrane PKH26 staining detected by flow cytometry. Cytokines secreted into the supernatants of 48 hour and 5 day cultures were measured by ELISA assays (BD Pharmingen). PBMCs from severe HA patients proliferated normally in response to PHA but failed to secrete IFN-γ when compared to normal boys and boys with severe HB:PBMC Proliferation and Secretion of IFN-γ in Response to PHASubject GroupHAHBNormal BoysAdult ControlPHA Proliferation (% unstained cells) M± 1SD79.3 ±3.778.2±13.395.4±1.589.3±5.3INF-γ Secretion (pg/mL) M± 1 SD6.7±4.7429.0±46469.1±32.4770.1±148.7The severe HA patients did not have improvement in INF-γ secretion following therapeutic exposure to FVIII concentrates. Addition of physiologic concentrations of FVIII or FIX to cultures with or without PHA did not alter the results of proliferation or cytokine secretion regardless of the inhibitor history of the patients. (1 HA and 1 HB patient developed high responder inhibitors.) Secretion of IL-4, IL-5 and IL-10 were moderately decreased in some HA patients as well. In summary, we have confirmed an INF-γ secretion defect in PBMCs from patients with HA in spite of normal proliferation in response to PHA. The defect is not present in HB or normal age-matched control subjects and is not corrected by addition of FVIII to cultures. We conclude that the defect is not due to exposure to intravenous foreign antigens. The precise clinical significance and any possible relation to the higher risk of inhibitor development in HA will require further investigation.PBMC Proliferation and Secretion of IFN-γ in Response to PHASubject GroupHAHBNormal BoysAdult ControlPHA Proliferation (% unstained cells) M± 1SD79.3 ±3.778.2±13.395.4±1.589.3±5.3INF-γ Secretion (pg/mL) M± 1 SD6.7±4.7429.0±46469.1±32.4770.1±148.7

1002. Immune Responses to AAV and to Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed Gene Transfer for Hemophilia B

Studies in mice, hemophilic dogs, and non-human primates have shown long-term expression of Factor IX (FIX) after administration of a recombinant AAV-2 vector expressing FIX into the portal vein or hepatic artery. Specifically, infusion of ~1×1012 vg/kg AAV vector expressing canine FIX in hemophilic dogs showed sustained (>3 years) circulating levels of FIX in the range of 4-12% of normal (Blood 99:2670). In a Phase I dose escalation study of hepatic artery infusion of AAV-2-F.IX in humans with severe hemophilia B, two subjects receiving a dose of 2×1012 vg/kg (titered against a supercoiled standard) showed expression of detectable levels of FIX, but expression was transient, with peak levels of 3% in one subject and 11.8% in the other. FIX levels had returned to baseline by 4 weeks and 10 weeks post vector infusion respectively. In the latter subject, the decline in circulating FIX levels was accompanied by a transient rise in serum transaminase levels. This subject had a low baseline neutralizing antibody titer (NAB) to AAV-2 (1:2), which rose to >1:1000 two weeks later. The other subject experienced no change in serum transaminases; his pre-treatment NAB to AAV-2 was 1:17, and increased to 1:96,000 two weeks later. To further assess the role of the immune response, PBMCs from these two subjects were incubated with peptides derived from the AAV-2 capsid sequence and from the wild-type hF.IX sequence. IFN-γ secretion was measured in ELISpot assays, which showed, in the latter subject, a 10-fold elevation in IFN-γ compared to media control at the 4 wk time point following incubation with one of the AAV peptide pools and with the F.IX peptide pool. When normalized to levels of maximal IFN-γ secretion in response to a positive control, IFN-γ response was >10-fold that of normal controls for the F.IX peptide pool, and >2-fold that of normal controls for the reactive AAV-2 peptide pool. We conclude that: 1) AAV-F.IX can transduce human hepatocytes in vivo, resulting in therapeutic circulating factor levels; 2) the vector dose required to achieve a therapeutic factor level was accurately predicted by animal models; 3) the transient nature of expression seen in subjects treated at 2×1012 vg/kg was not predicted by earlier studies in animal models; 4) T cell responses to AAV-2 and to F.IX may determine the outcome of AAV-2-mediated gene transfer in human subjects. More recently the clinical study has resumed with a planned target dose of 1.2×1012 vg/kg. The goal is to determine whether subjects can achieve therapeutic levels of expression at this dose, and whether specific immune responses to either AAV or F.IX will limit duration of expression in humans.

Tetramer binding and secretion of interferon-γ in response to antigenic stimulation are compatible with a range of affinities of MHC:TCR interaction and distinct programs of cytotoxic T-lymphocyte activation

Tetramer staining and detection of IFN-γ secretion in response to specific stimulation are widely used to quantify and isolate specific T-cells. However, it remains unclear how these assays reflect different functional outcomes of T-cell triggering with MHC:peptide ligands. An immunogenic EBV-derived A11-restricted CTL peptide epitope and its partially agonistic analogue trigger different programs of activation induced cell death (AICD) in specific CTLs. In this study we analysed a panel of CTL clones, bulk CTL cultures and PBMCs isolated from HLA A11-positive EBV-infected individuals for their ability to bind tetrameric complexes assembled with either of the two peptides and correlated tetramer binding with the activity of the peptides in functional assays. This analysis demonstrates that specific tetramer staining and secretion of IFN-γ are compatible with at least two activation programs in CTLs. One of these programs corresponds to full-scale CTL activation and death of a proportion of activated T-cells in a Fas-dependent manner. In contrast, the alternative program is characterized by selective induction of IFN-γ and TNF-α, absence of proliferative response and Fas-independent cell death. These findings may have important implications for the evaluation of data obtained with MHC:peptide tetramers and IFN-γ secretion assays, especially in experimental systems with extensive antigenic variability.

IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking.

IFN-gamma-inducible protein 10 (IP-10, CXCL10), a chemokine secreted from cells stimulated with type I and II IFNs and LPS, is a chemoattractant for activated T cells. Expression of IP-10 is seen in many Th1-type inflammatory diseases, where it is thought to play an important role in recruiting activated T cells into sites of tissue inflammation. To determine the in vivo function of IP-10, we constructed an IP-10-deficient mouse (IP-10(-/-)) by targeted gene disruption. Immunological analysis revealed that IP-10(-/-) mice had impaired T cell responses. T cell proliferation to allogeneic and antigenic stimulation and IFN-gamma secretion in response to antigenic challenge were impaired in IP-10(-/-) mice. In addition, IP-10(-/-) mice exhibited an impaired contact hypersensitivity response, characterized by decreased ear swelling and reduced inflammatory cell infiltrates. T cells recovered from draining lymph nodes also had a decreased proliferative response to Ag restimulation. Furthermore, IP-10(-/-) mice infected with a neurotropic mouse hepatitis virus had an impaired ability to control viral replication in the brain. This was associated with decreased recruitment of CD4(+) and CD8(+) lymphocytes into the brain, reduced levels of IFN-gamma and the IFN-gamma-induced chemokines monokine induced by IFN-gamma (Mig, CXCL9) and IFN-inducible T cell alpha chemoattractant (I-TAC, CXCL11) in the brain, decreased numbers of virus-specific IFN-gamma-secreting CD8(+) cells in the spleen, and reduced levels of demyelination in the CNS. Taken together, our data suggest a role for IP-10 in both effector T cell generation and trafficking in vivo.

The Role of T Cell Subsets and Cytokines in the Pathogenesis ofHelicobacter pyloriGastritis in Mice

Gastritis due to Helicobacter pylori in mice and humans is considered a Th1-mediated disease, but the specific cell subsets and cytokines involved are still not well understood. The goal of this study was to investigate the immunopathogenesis of H. pylori-induced gastritis and delayed-type hypersensitivity (DTH) in mice. C57BL/6-Prkdc(scid) mice were infected with H. pylori and reconstituted with CD4+, CD4-depleted, CD4+CD45RB(high), or CD4+CD45RB(low) splenocytes from wild-type C57BL/6 mice or with splenocytes from C57BL/6(IFN-gamma-/-) or C57BL/6(IL-10-/-) mice. Four or eight weeks after transfer, DTH to H. pylori Ags was determined by footpad injection; gastritis and bacterial colonization were quantified; and IFN-gamma secretion by splenocytes in response to H. pylori Ag was determined. Gastritis and DTH were present in recipients of unfractionated splenocytes, CD4+ splenocytes, and CD4+CD45RB(high) splenocytes, but absent in the other groups. IFN-gamma secretion in response to H. pylori Ags was correlated with gastritis, although splenocytes from all groups of mice secreted some IFN-gamma. Gastritis was most severe in recipients of splenocytes from IL-10-deficient mice, and least severe in those given IFN-gamma-deficient splenocytes. Bacterial colonization in all groups was inversely correlated with gastritis. These data indicate that 1) CD4+ T cells are both necessary and sufficient for gastritis and DTH due to H. pylori in mice; 2) high expression of CD45RB is a marker for gastritis-inducing CD4+ cells; and 3) IFN-gamma contributes to gastritis and IL-10 suppresses it, but IFN-gamma secretion alone is not sufficient to induce gastritis. The results support the assertion that H. pylori is mediated by a Th1-biased cellular immune response.

ELISPOT assay for detection of peptide specific Interferon-γ secreting cells in rhesus macaques

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51Cr-release cytotoxic T cell (CTL) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-γ (IFN-γ) ELISPOT assay capable of detecting IFN-γ secretion in response to stimulation with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/10 6 PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-γ response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4 +, but not CD8 +, T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10 6 PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-γ assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 antigen are recognized by antigen-specific human T cell lines.

A synthetic-peptide approach was used to map epitope regions of the Mycobacterium tuberculosis 6-kDa early secreted antigen target (ESAT-6) by testing human CD4(+) T cell lines for secretion of IFN-gamma in response to recombinant ESAT-6 (rESAT-6) and overlapping 20-mer peptides covering the antigen sequence. The results demonstrate that all of the ESAT-6 peptides screened were able to induce IFN-gamma secretion from one or more of the T cell lines tested. Some of the individual T cell lines showed the capacity to respond to all peptides. Human leukocyte antigen (HLA-DR) typing of the donors showed that rESAT-6 was presented to T cells in association with multiple HLA-DR molecules. The results suggest that frequent recognition of the M. tuberculosis ESAT-6 antigen by T cells from patients with tuberculosis is due to the presence of multiple epitopes scattered throughout the ESAT-6 sequence.

CD40-CD40 Ligand Interaction Is Central to Cell-Mediated Immunity Against Toxoplasma gondii: Patients with Hyper IgM Syndrome Have a Defective Type 1 Immune Response That Can Be Restored by Soluble CD40 Ligand Trimer

Abstract Cell-mediated immunity that results in IL-12/IFN-γ production is essential to control infections by intracellular organisms. Studies in animal models revealed contrasting results in regard to the importance of CD40-CD40 ligand (CD40L) signaling for induction of a type 1 cytokine response against these pathogens. We demonstrate that CD40-CD40L interaction in humans is critical for generation of the IL-12/IFN-γ immune response against Toxoplasma gondii. Infection of monocytes with T. gondii resulted in up-regulation of CD40. CD40-CD40L signaling was required for optimal T cell production of IFN-γ in response to T. gondii. Moreover, patients with hyper IgM (HIGM) syndrome exhibited a defect in IFN-γ secretion in response to the parasite and evidence compatible with impaired in vivo T cell priming after T. gondii infection. Not only was IL-12 production in response to T. gondii dependent on CD40-CD40L signaling, but also, patients with HIGM syndrome exhibited deficient in vitro secretion of this cytokine in response to the parasite. Finally, in vitro incubation with agonistic soluble CD40L trimer enhanced T. gondii-triggered production of IFN-γ and, through induction of IL-12 secretion, corrected the defect in IFN-γ production observed in HIGM patients. Our results are likely to explain the susceptibility of patients with HIGM syndrome to infections by opportunistic pathogens.

Increased systemic B- and T-lymphocyte responses in hereditary motor and sensory neuropathy (HMSN I)

Immune mechanisms of possible importance for the development and maintenance of peripheral nerve myelin breakdown in HMSN I were analysed by measuring B- and T-cell activation in blood, bone marrow and cerebrospinal fluid. Patients with polyneuropathies of other etiologies served as one control group and patients with tension headache as another. Flow cytometry of blood and bone marrow mononuclear cells revealed that an increased number of CD3 +, CD4 + and CD4 − CD8 − T-cells expressed a late stage activation marker (Ta1). Analysis of T-cells primed for myelin antigens, by studies of IFN-γ secretion in response to antigen in vitro, showed that both HMSN I and other polyneuropathy patients had low (but significant) numbers of T-cells recognizing whole PNS-myelin. Increased numbers of IgG- and IgM-producing cells were found in blood and bone marrow in the HMSN I patients. Patients with both HMSN I and the other polyneuropathies had few cells in peripheral blood and in bone marrow producing antibodies binding to P 2, MAG and MBP in a solid phase immunospot assay. Many cells in the cerebrospinal fluid produced antibodies against MAG. Thus, there was a strong general activation of B- and T-cells in HMSN I while the immunity directed toward peripheral nerve was only slightly elevated. It is an open question if this immune activation is related to the primary gene defect or a secondary event to the nerve damage. The pathogenetic importance of the immune response in maintaining the nerve damage in HMSN I is unclear.