IFN-γ Release In Response Research Articles

Myeloid-derived suppressor cells and their association with vaccine immunogenicity in South African infants.

The role of Myeloid-Derived Suppressor Cells (MDSC) in infant immune ontogeny is unknown. Here, we evaluated MDSC frequency and relationship with infant vaccine responses throughout the first year of life in a prospective cohort study. Ninety-one South African infant-mother pairs were enrolled at delivery, and blood samples were collected at 0, 6, 10, and 14weeks, 6 months, 9 months, and 1 year. MDSC frequencies were quantified, and immune responses to the childhood vaccines Bacillus Calmette-Guérin (BCG), hepatitis B (HepB), and combination diphtheria, tetanus, and pertussis (dTaP) were measured by Ag-specific CD4+ T cell proliferation and interferon gamma (IFN-γ) production. Vaccine-specific Ab responses to HepB, dTaP, and Haemophilus influenzae type b (Hib) were quantified via Enzyme-Linked Immunosorbent assay (ELISA). MDSC frequency in mother-infant pairs was strongly correlated; the frequency of MDSC decreased in both mothers and infants during the months after delivery/birth; and by 1 year, infant MDSC frequencies rebounded to birth levels. Higher MDSC frequency at vaccination was associated with a lack of subsequent IFN-γ release in response to vaccine Ags, with the exception of BCG. With the exception of a weak, positive correlation between MDSC frequency at 6 weeks (time of initial vaccination) and peak Hepatitis B surface antigen Ab titer, Polymorphonuclear Myeloid-Derived Suppressor Cells (PMN-MDSC) was not correlated with T cell proliferation or Ab responses in this study. The potential for MDSC-mediated suppression of vaccine Ag-specific IFN-γ responses should be explored further, and considered when evaluating candidate infant vaccines.

CGMP Mass Production of FT538, a First-of-Kind, Off-the-Shelf, Multiplexed Engineered Natural Killer Cell Cancer Immunotherapy Derived from a Clonal Master Induced Pluripotent Stem Cell Line

FT538 is an investigational, off-the-shelf, multiplexed engineered natural killer (NK) cell cancer immunotherapy that is derived from a clonal master induced pluripotent stem cell (iPSC) line engineered with three functional components to enhance innate immunity: a novel high-affinity, non-cleavable CD16 (hnCD16) Fc receptor; an IL-15/IL-15 receptor fusion (IL-15RF); and the elimination of CD38 expression. The use of a clonal master engineered iPSC line as a starting cell source enables routine mass production of FT538 and supports off-the-shelf product availability to reach many patients. The clonal master iPSC line for the manufacture of FT538 was made by reprogramming and engineering donor-consented human fibroblasts to induce pluripotency using a proprietary non-integrating system and to integrate a bicistronic cassette containing hnCD16 and IL-15RF into the CD38 locus, which resulted in complete disruption of the CD38 gene. The engineered iPSC population was then sorted to isolate single clones, and each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, and off-target effects of engineering. Accordingly, a single engineered iPSC clone was selected as the FT538 clonal master iPSC line. The clonal master engineered iPSC line serves as a renewable source for the routine cGMP mass production of FT538 drug product. Routine cGMP manufacture of FT538 drug product consists of three stages: 1) differentiation of the clonal master engineered iPSC line to CD34-expressing hematopoietic progenitor cells; 2) further differentiation to and expansion of NK cells; and 3) fill/finish and cryopreservation of the FT538 drug product. A cGMP manufacturing campaign yielded sufficient CD34-expressing hematopoietic progenitor cells to support at least 15 batches of NK cell differentiation, the first batch of which was used to produce a total of 3 × 1011 FT538 NK cells filled into over 300 units of cryopreserved drug product. The cGMP campaign had a theoretical yield of 4.5 x 1012 FT538 NK cells. The FT538 drug product was characterized and found to be comprised of uniformly engineered CD56+ NK cells with homogeneous expression of hnCD16 and lacking expression of CD38. Importantly, there were no residual iPSCs detected in the FT538 drug product. Additionally, the FT538 drug product exhibited potent effector function in a candidate potency assay measuring IFN-γ release in response to RPMI 8226 target cells in the presence of daratumumab. An Investigational New Drug application for FT538 has been cleared by the U.S. Food and Drug Administration (FDA) for the conduct of a multicenter, multi-dose Phase I clinical trial for the treatment of patients with relapsed/refractory (r/r) acute myelogenous leukemia (AML) and multiple myeloma (MM). The dose-escalation utilizes a 3+3 design to identify the maximum tolerated dose of three doses of FT538 on Days 1, 8, and 15 as a monotherapy in r/r AML (Regimen A) and in combination with daratumumab (Regimen B) or elotuzumab (Regimen C) in r/r MM. The trial will test up to five FT538 dose levels ranging from 50 million to 1.5 billion cells, and up to 105 patients will be enrolled. The trial is expected to begin patient enrollment in 2020. Disclosures Rezner: Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Solchaga:Fate Therapeutics, Inc: Current Employment. Reyes:Fate Therapeutics, Inc: Current Employment. Lin:Fate Therapeutics, Inc: Current Employment. Abujarour:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Wang:Fate Therapeutics, Inc: Current Employment.

Antigen-specific interferon-gamma release is decreased following the single intradermal comparative cervical skin test in African buffaloes (Syncerus caffer)

Effective disease management of wildlife relies on the strategic application of ante-mortem diagnostic tests for early identification and removal of M. bovis-infected animals. To improve diagnostic performance, interferon-gamma release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST). Since buffaloes are major maintenance hosts of M. bovis, optimal application of bovine TB diagnostic tests are especially important. We aimed to determine whether the timing of blood collection relative to the TST has an influence on IFN-γ production and diagnostic outcome in African buffaloes. Release of IFN-γ in response to bovine purified protein derivative (PPD), avian PPD and PC-HP® and PC-EC® peptides was measured by Bovigam® and an in-house IGRA in a group of Bovigam®-positive and – negative buffaloes at the time the TST was performed and three days later. There was significantly lower IFN-γ release in response to these antigens post-TST in Bovigam®-positive buffaloes, but no significant changes in Bovigam®-negative buffaloes. Also, a significantly greater proportion of buffaloes were Bovigam®-positive prior to the TST than three days later. We therefore recommend that blood samples for use in IGRAs be collected prior to or at the time the TST is performed to facilitate the correct identification of greater numbers of IGRA-positive buffaloes.

Monocyte-Derived Signals Activate Human Natural Killer Cells in Response to Leishmania Parasites.

Activated natural killer (NK) cells release interferon (IFN)-γ, which is crucial for the control of intracellular pathogens such as Leishmania. In contrast to experimental murine leishmaniasis, the human NK cell response to Leishmania is still poorly characterized. Here, we investigated the interaction of human blood NK cells with promastigotes of different Leishmania species (Leishmania major, Leishmania mexicana, Leishmania infantum, and Leishmania donovani). When peripheral blood mononuclear cells or purified NK cells and monocytes (all derived from healthy blood donors from Germany without a history of leishmaniasis) were exposed to promastigotes, NK cells showed increased surface expression of the activation marker CD69. The extent of this effect varied depending on the Leishmania species; differences between dermotropic and viscerotropic L. infantum strains were not observed. Upregulation of CD69 required direct contact between monocytes and Leishmania and was partly inhibitable by anti-interleukin (IL)-18. Unexpectedly, IL-18 was undetectable in most of the supernatants (SNs) of monocyte/parasite cocultures. Confocal fluorescence microscopy of non-permeabilized cells revealed that Leishmania-infected monocytes trans-presented IL-18 to NK cells. Native, but not heat-treated SNs of monocyte/Leishmania cocultures also induced CD69 on NK cells, indicating the involvement of a soluble heat-labile factor other than IL-18. A role for the NK cell-activating cytokines IL-1β, IL-2, IL-12, IL-15, IL-21, and IFN-α/β was excluded. The increase of CD69 was not paralleled by NK cell IFN-γ production or enhanced cytotoxicity. However, prior exposure of NK cells to Leishmania parasites synergistically increased their IFN-γ release in response to IL-12, which was dependent on endogenous IL-18. CD1c+ dendritic cells were identified as possible source of Leishmania-induced IL-12. Finally, we observed that direct contact between Leishmania and NK cells reduced the expression of CD56 mRNA and protein on NK cells. We conclude that Leishmania activate NK cells via trans-presentation of IL-18 by monocytes and by a monocyte-derived soluble factor. IL-12 is needed to elicit the IFN-γ-response of NK cells, which is likely to be an important component of the innate control of the parasite.

Use of an Interferon Gamma Release Assay (IGRA) to test T-cell responsiveness to soluble Leishmania infantum antigen in whole blood of dogs from endemic areas

Interferon-Gamma (IFN-γ) Release Assays (IGRAs) are easy tests that allow rapid screening of primed memory T-cells immunity in response to antigen. The aim of this study was to use IGRA to assess IFN-γ release in response to Soluble Leishmania infantum antigen (SLA) in whole blood of dogs living in endemic area of visceral leishmaniasis and to interpret IGRA results according to clinical examination, specific anti-Leishmania humoral response and presence of L. infantum DNA in blood.The study was carried out on 56 dogs living in greater Tunis area. Physical examination, quantitative serology and PCR on blood were used to characterize dogs’ status in relation to Leishmania infection and disease. IGRA consisted on testing by ELISA for IFN-γ-secretion in whole blood after a 20-h challenge with SLA. PBS and Phytohemagglutinin (PHA) stimulations were used as controls.Four groups of dogs were characterized: 31 were negative by both serology and PCR, two had doubtful serology, 10 presented no to mild clinical signs but low antibodies levels and 13 were affected by Canine Leishmaniasis (CanL). In seronegative dogs, IGRA was little contributory in 4 puppies (age <6months) and 5 old dogs (median age=72months, IQR: 45–84 months) that didn’t respond to PHA stimulation, IGRA was negative in 19 and positive in three animals with lymph node enlargement. In dogs with doubtful serology, IGRA was positive in one dog and negative in the other. In infected dogs with no to mild clinical signs, one dog exhibited high level of IFN-γ in absence of antigenic stimulation and all the other were positive by IGRA. CanL dogs showed variable IGRA results. Negative IGRAs (n=4) were shown in animals with the highest parasitic burden whereas positive IGRAs (n=5) were shown in dogs with negative PCR or low parasitic load. The 4 remaining dogs either didn’t respond to PHA (n=2) or showed non-specific secretion in PBS tube (n=2).The results of this study showed that IGRA is a useful new tool that can assess exposure to Leishmania in dogs with no to mild clinical signs in endemic area. Further comparative investigations using assays exploring cellular immunity are needed to determine its accuracy.

Human mesenchymal stromal/stem cells acquire immunostimulatory capacity upon cross-talk with natural killer cells and might improve the NK cell function of immunocompromised patients.

BackgroundThe suppressive effect of mesenchymal stromal/stem cells (MSCs) on diverse immune cells is well known, but it is unclear whether MSCs additionally possess immunostimulatory properties. We investigated the impact of human MSCs on the responsiveness of primary natural killer (NK) cells in terms of cytokine secretion.MethodsHuman MSCs were generated from bone marrow and nasal mucosa. NK cells were isolated from peripheral blood of healthy volunteers or of immunocompromised patients after severe injury. NK cells were cultured with MSCs or with MSC-derived conditioned media in the absence or presence of IL-12 and IL-18. C-C chemokine receptor (CCR) 2, C-C chemokine ligand (CCL) 2, and the interferon (IFN)-γ receptor was blocked by specific inhibitors or antibodies. The synthesis of IFN-γ and CCL2 was determined.ResultsIn the absence of exogenous cytokines, trace amounts of NK cell-derived IFN-γ licensed MSCs for enhanced synthesis of CCL2. In turn, MSCs primed NK cells for increased release of IFN-γ in response to IL-12 and IL-18. Priming of NK cells by MSCs occurred in a cell–cell contact-independent manner and was impaired by inhibition of the CCR2, the receptor of CCL2, on NK cells. CD56bright NK cells expressed higher levels of CCR2 and were more sensitive to CCL2-mediated priming by MSCs and by recombinant CCR2 ligands than cytotoxic CD56dim NK cells. NK cells from severely injured patients were impaired in cytokine-induced IFN-γ synthesis. Co-culture with MSCs or with conditioned media from MSCs and MSC/NK cell co-cultures from healthy donors improved the IFN-γ production of the patients’ NK cells in a CCR2-dependent manner.ConclusionsA positive feedback loop driven by NK cell-derived IFN-γ and MSC-derived CCL2 increases the inflammatory response of cytokine-stimulated NK cells not only from healthy donors but also from immunocompromised patients. Therapeutic application of MSCs or their soluble factors might thus improve the NK function after severe injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0353-9) contains supplementary material, which is available to authorized users.

Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

Strain-Dependent Cellular Immune Responses in Cattle following Escherichia coli O157:H7 Colonization

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective, and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, we studied the cellular immune responses of cattle during EHEC O157:H7 colonization. Calves were challenged either with a phage type 21/28 (PT21/28) strain possessing the Shiga toxin 2a (Stx2a) and Stx2c genes or with a PT32 strain possessing the Stx2c gene only. T-helper cell-associated transcripts at the terminal rectum were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Induction of gamma interferon (IFN-γ) and T-bet was observed with peak expression of both genes at 7 days in PT32-challenged calves, while upregulation was delayed, peaking at 21 days, in PT21/28-challenged calves. Cells isolated from gastrointestinal lymph nodes demonstrated antigen-specific proliferation and IFN-γ release in response to type III secreted proteins (T3SPs); however, responsiveness was suppressed in cells isolated from PT32-challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4(+) T cells from PT21/28-challenged calves, NK cells from PT32-challenged calves, and CD8(+) and γδ T cells from both PT21/28- and PT32-challenged calves following ex vivo restimulation with T3SPs. This study demonstrates that cattle mount cellular immune responses during colonization with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

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.

Rapid Interferon-Gamma Release from Natural Killer Cells Induced by a Streptococcal Commensal

Interferon-gamma (IFN-γ) is a critical cytokine for the initiation of immune responses against a variety of infectious agents and malignancies. We found that a range of Gram-positive and Gram-negative bacteria stimulated the rapid release (<24 h) of IFN-γ from murine leukocytes. Using fluorescence activated cell sorting and cd1d(-/-) and rag1(-/-) mice, we determined that dendritic cells (DCs) and natural killer (NK) cells were primarily responsible for IFN-γ release by Streptococcus salivarius, a Gram-positive commensal, previously noted to possess potent interleukin-12 (IL-12)-inducing potential. IFN-γ release from NK cells required DC:NK membrane contact and IL-12/IL-18 expression, but was independent of lymphocyte function-associated antigen-1-mediated interactions. IFN-γ release in response to bacteria was maintained in mice deficient for Toll-like receptor (TLR)-2 and TLR-4, suggesting that bacteria activate antigen-presenting cells via multiple, redundant pathways. Together, our results suggest that Gram-positive bacteria may be useful in driving NK cell activation and T helper 1 polarization and have the potential for development as effective adjuvants.

Lsr2 of Mycobacterium leprae and Its Synthetic Peptides Elicit Restitution of T Cell Responses in Erythema Nodosum Leprosum and Reversal Reactions in Patients with Lepromatous Leprosy

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.

Influence of exercise on NA- and Hsp72-induced release of IFNγ by the peritoneal suspension of macrophages and lymphocytes from genetically obese Zucker rats

Regular physical exercise is recognized as a nonpharmacological therapeutic strategy in the treatment of metabolic syndrome, and has been proposed for improving obesity, diabetic status, insulin resistance, and immune response. The aim of the present study was to evaluate the effect of a regular exercise program (treadmill running, 5days/week for 14weeks at 35cm/s for 35min in the last month) on the release of the pro-inflammatory cytokine interferon gamma (IFNγ) by peritoneal cells (macrophages and lymphocytes) from obese Zucker rats (fa/fa) in response to noradrenaline (NA) and heat shock proteins of 72kDa (Hsp72), and the possible adaptation due to training for a bout acute exercise (a single session of 25-35min at 35cm/s). In healthy (lean Fa/fa) and obese animals, peritoneal cells released greater concentrations of IFNγ in response to Hsp72 and lower concentrations in response to NA. The regular exercise training protocol, evaluated in the obese animals, produced a clear change in the regulation of the release of IFNγ. Peritoneal immune cells from trained animals released more IFNγ in response to NA, but there was a reduction in the release of IFNγ in response to Hsp72. In the obese animals, regular exercise caused a change in the inhibitory effect of NA (which now becomes stimulatory) and the stimulatory effect of Hsp72e (which now becomes inhibitory) in relation to the release of IFNγ. This reflects that Hsp72, induced by the prior release of NA following exercise-induced stress, plays a role in the homeostatic balance of release of IFNγ by peritoneal immune cells in obese animals during exercise.

Involvement of notch in activation of human γδ T cells (45.22)

Abstract γδ T cells recognize phosphorylated antigens and play an important role in tumor immunity. The aim of the present study was to investigate the role of Notch signaling pathway in antigen specific responses of γδ T cells. Expression of mRNA for Notch1 and Notch2 genes was predominantly observed in peripheral γδ T cells. The presence of active Notch intracellular domain in γδ T cells was confirmed by confocal microscopy and western blotting. Proliferation of γδ T cells and IFN-γ release in response to phosphoantigens was inhibited in the presence of γ-secretase inhibitor confirming involvement of Notch signal. Delta like ligands 1 and 4 activated γδ T cells while Jagged1 inhibited the γδ T cell response. Oral and breast cancer cells showed dominant expression of Jagged1 ligand which may exert inhibitory signal as a way to evade immune recognition by γδ T cells. Decreased expression of CD107a and diminished lysis of tumor targets in the presence of γ-secretase inhibitor confirmed that Notch was involved in cytotoxic effector functions of γδ T cells. Thus, our data for the first time demonstrates that Notch signal plays an indispensable role in antigen specific responses of γδ T cells. Future immunotherapeutic strategies can exploit Notch signaling in γδ T cells to achieve therapeutic benefits in cancer patients.

Antimycobacterial immune responses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed in the aetiology of sarcoidosis; however, studies still remain contradictory. We addressed the question of mycobacterial involvement in the pathogenesis of sarcoidosis by analysing cellular immune responses to mycobacterial antigens. We examined the interferon (IFN)-gamma production by enzyme-linked immunospot in response to purified protein derivate (PPD) mycobacterial-specific antigen early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 by peripheral blood mononuclear cells (PBMCs) and bronchoalveolar-lavage mononuclear cells (BALMCs) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-gamma in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC P = 0.2326; BALMC P = 0.1767) and were less frequently observed in both groups compared to patients with tuberculosis (BALMC P < 0.05; PBMC P < 0.0001). Within PBMC, the immunophenotype of sarcoidosis patients differed from that of patients with tuberculosis, as well as from that of controls, while within BALMC it resembled that of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immune responses is not elevated in patients with sarcoidosis when compared to controls. The immunophenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis while showing systemical difference. These observations do not support a role of an infection with M. tuberculosis in the pathogenesis of sarcoidosis.

Antimycobacterial immuneresponses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed, however, studies still remain contradictory. We examined the interferon (IFN)-γ production by enzyme-linked-immunospot in response to purified protein derivate (PPD) and mycobacterial specific antigens early-secretory-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 by peripheral blood mononuclear cells (PBMC) and bronchoalveolar-lavage mononuclear cells (BALMC) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-γ in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC p=0.1630; BALMC p=0.2199) and were less freuqently observed in both groups compared to patients with tuberculosis (BALMC p&lt;0,0131; PBMC p&lt;0.0001). Within PBMC the immunephenotype of sarcoidosis-patients differed from patients with tuberculosis, as well as from controls, while within BALMC it resembled the one of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immunresponses is not elevated in patients with sarcoidosis when compared to controls. The immunephenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis, while showing systemical difference. These observations do not support a role of a mycobacterial infection in the pathogenesis of sarcoidosis.

Evaluation of the diagnostic utility of a whole-blood interferon-γ assay for determining the risk of exposure to Mycobacterium tuberculosis in Bacille Calmette-Guerin (BCG)-vaccinated individuals

We evaluated the utility of the “QuantiFERON®-TB Gold In-Tube” (QuantiFERON®) test that uses tuberculosis (TB)-specific antigens for the diagnosis of latent infection in such individuals. We also examined the correlation between the interferon (IFN)-γ response to these antigens and the exposure risk to TB by evaluating antigen-specific IFN-γ release in comparison with IFN-γ release in response to purified protein derivative (PPD) in 3 groups: medical students, nurses in a TB hospital, and TB patients. All nurses and TB patients responded to PPD, whereas 52% ( P < 0.0001) and 79.2% ( P = 0.04) responded to QuantiFERON®, respectively. In the medical students, only 10.4% responded to QuantiFERON®, whereas 85.2% were positive to PPD ( P < 0.0001). There was also a significant correlation between the levels of IFN-γ production and the duration of employment in the group of nurses at the TB hospital, suggesting ongoing exposure in this high-risk group. Thus, these results demonstrate that Mycobacterium tuberculosis-specific IFN-γ release assay accurately discriminates low- and high-risk healthy subjects and might therefore be a useful diagnostic tool for the diagnosis of latent infection in Bacille Calmette-Guerin (BCG)-vaccinated individuals.

Gene Transfer of IL-7R alpha (IL-7Rα) on Antigen-Specific Cytotoxic T Cells (CTLs) Restores Their Ability To Respond to IL-7 Cytokine.

Systemic administration of IL-2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs. However, significant toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL-7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Nonetheless, the value of IL-7 for promoting proliferation of tumor-specific CTLs after adoptive transfer remains questionable, due to the lack of expression of the IL-7 receptor (IL-7Rα) on effector CTLs. Thus we have found IL-7Rα to be essentially undetectable in our model of EBV-specific CTL lines (EBV-CTLs) due to downregulation of IL-7Rα transcripts. To determine whether transgenic expression of IL-7Rα can restore the capacity of established EBV-CTLs to respond to IL-7, the full-length of IL-7Rα was cloned in the SFG retroviral vector (SFG/IL-7Rα). We transduced EBV-CTLs generated from 5 healthy donors and evaluated their growth kinetics and antigen specificity. As control, EBV-CTLs were transduced with the SFG retroviral vector encoding for a truncated form of the CD34 molecule (SFG.ΔCD34). After transduction with SFG/IL-7Rα, IL-7Rα was detectable in 58% to 76% of EBV-CTLs. The transgenic IL-7Rα was functional since addition of IL-7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL-7 sustained the expansion of EBV-CTLs/IL-7Rα+, transduced CTLs were stimulated weekly with autologous EBV-LCLs in the presence of IL-2 (50U/mL) or IL-7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL-7Rα+ expanded equally well with IL-2. However, only EBV-CTL/IL-7Rα+ significantly proliferated in the presence of IL-7 [from 1×106 cells to 103×106 cells (range, 38-286×106)] over a period of five weeks. CTL expansion remained antigen dependent, and antigen withdrawal led to cessation of CTL growth. EBV-CTLs/IL-7Rα+ showed the expected growth advantage in the presence of IL-7, increasing from 55%±15% to 79%±5% of the total within 5 weeks. In contrast, the proportion of IL-7Rα+ cells marginally declined when the cells were expanded with IL-2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Rα+ expanded with IL-7 retained their ability to respond to other common-γ-chain cytokines. EBV-CTLs/IL7Rα+ grown with IL-7 for more than 2 weeks, expanded further when exposed to both antigen and IL-2 or IL-15. Control CTLs grown with IL-2 and EBV-CTLs/IL-7Rα+ grown with IL-7 both remained polyclonal, as assessed by VβTCR repertoire analysis, and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL-7Rα+ also retained antigen specificity measured by tetramer staining, by IFNγ release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs 9±17% of allogeneic LCL); this killing was comparable to the activity of control CTLs cultured in IL-2 (51%±27%). Preliminary studies indicate that EBV-CTLs/IL-7Rα+ also expand in vivo in response to IL-7 and maintain their anti-tumor activity in a SCID mouse xenograft model.

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.

A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays

Vaccines in general and HIV vaccines in particular are focusing ever more on the induction of cellular immunity specifically the generation of cytotoxic T cells (CTL). As progress is made towards developing a safe and effective HIV vaccine, there is a need for a robust, sensitive and reproducible assay to evaluate vaccine-induced cellular immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable and amenable to high-through-put screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay. We selected a panel of 23 8–11 mer Influenza virus (Flu), Cytomegalovirus (CMV) and Epstein Barr virus (EBV) epitopes recognized by CD8 positive T cells and presented by 11 class I HLA-A and HLA-B alleles whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-γ) secretion in peripheral blood mononuclear cells (PBMC) incubated with the peptides using a modified ELISPOT assay. IFN-γ secretion was detected in 15/17 (88%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-γ in response to the pool of peptides was CD8+ T cell mediated and HLA restricted. In vitro stimulation of PBMC with individual peptides or the pool of peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen in a CTL assay. The size, shape and appearance of the spots produced using this peptide panel provided a standard for the establishment of acceptance criteria of spots for the evaluation of ELISPOT plates using an automated reader system.

IL-12/IL-18-Dependent IFN-γ Release by Murine Dendritic Cells

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.