Magnitude Of IFN-γ Responses Research Articles

Elevated antigen-specific IFN-γ responses in bronchoalveolar lavage fluid impervious to clinical comorbidities improve the pulmonary tuberculosis diagnosis

The extremely slow growth rate of Mycobacterium tuberculosis (Mtb) challenges traditional methods for tuberculosis (TB) diagnosis. Here, we assessed the efficacy of a previously developed Mtb antigen-specific gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) performed on bronchoalveolar lavage fluid (BALF) from a cohort of 414 patients including 333 PTB patients (202/333 were sputum culture positive) for the diagnosis of PTB. We could confirm that antigen-specific IFN-γ-producing CD4+ T cells were concentrated significantly in BALF mononuclear cells (BALMC) compared with that in peripheral blood mononuclear cells (PBMC) assayed in parallel, but not those of CD8+ T cells both in sputum culture-negative and positive PTB. The magnitude of IFN-γ responses in the BALF was associated with bacterial load, and 9/202 of PTB with endobronchial TB (EBTB) were slightly reduced by the anti-TB treatment. Moreover, antigen-specific IFN-γ ELISPOT performed on BALMC showed higher sensitivity than PBMC ELISPOT. In addition, the differences of the BALMC ELISPOT between PTB and PTB with diabetes were not found, whereas PBMC IFN-γ responses were decreased in PTB with diabetes. Combined with the microbiological detection in BALF, such as microscopy and culture, the BALMC ELISPOT offers the opportunity for the more accurate diagnosis of PTB, especially those with clinical comorbidities.

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory Tcells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

Development of IFN-γ secretory ELISPOT based assay for screening of ADCC responses

Antibody dependent cell mediated cytotoxicity has been established as one of the important protective immune mechanisms against HIV making it essential to evaluate it while testing immunogenicity of emerging vaccine candidates. IFN-γ secretory ELISPOT assay, widely used for evaluation of CTL response in HIV vaccine trials, was adapted for measuring ADCC responses and the results were compared with the standard ICS based assays. IFN-γ responses elicited by plasma samples of 23 HIV infected individuals against Env and Gag peptides using granulocytes as antigen presenting cells were assessed by both the methods. Supernatants of the activated cells in ELISPOT assay were also assessed for cytokine/chemokine estimation. ELISPOT assays detected significantly more ADCC responders against HIV-Env and Gag peptide pools than ICS assay. The magnitude of IFN-γ response in both the assay correlated significantly (p=0.002). NK cells were found to be the predominant cell type secreting IFN-γ in the assay. Although IFN-γ and IL-6 levels were significantly higher in supernatants of Env peptides stimulated cells, IP-10 and MCP-1α levels were found to be more against Gag peptides. Thus, IFN-γ secretory ELISPOT assay was found to be more sensitive in detecting ADCC responders than ICS assay making it a valuable tool for screening of ADCC responses in future vaccine trials. Differences in cytokine pattern of Env versus Gag stimulated cells warrants a need for investigating their role in protection against HIV infection.

Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy

The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO₂) were stimulated with: 10 μg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 μg/ml), PHA (1 μg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.

Consistency ofMycobacterium tuberculosis-Specific Interferon-Gamma Responses in HIV-1-Infected Women during Pregnancy and Postpartum

Background. We determined the consistency of positive interferon-gamma (IFN-γ) release assays (IGRAs) to detect latent TB infection (LTBI) over one-year postpartum in HIV-1-infected women. Methods. Women with positive IGRAs during pregnancy had four 3-monthly postpartum IGRAs. Postpartum change in magnitude of IFN-γ response was determined using linear mixed models. Results. Among 18 women with positive pregnancy IGRA, 15 (83%) had a subsequent positive IGRA; 9 (50%) were always positive, 3 (17%) were always negative, and 6 (33%) fluctuated between positive and negative IGRAs. Women with pregnancy IGRA IFN-γ>8 spot forming cells (SFCs)/well were more likely to have consistent postpartum IGRA response (odds ratio: 10.0; 95% confidence interval (CI): 0.9–117.0). Change in IFN-γ response over postpartum was 10.2 SFCs/well (95% CI: −1.5–21.8 SFCs/well). Conclusion. Pregnancy positive IGRAs were often maintained postpartum with increased consistency in women with higher baseline responses. There were modest increases in magnitude of IGRA responses postpartum.

Age-Related Differences in Naturally Acquired T Cell Memory to Plasmodium falciparum Merozoite Surface Protein 1

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP142), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP142 driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5–1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP142. However, adults had higher proportions of MSP142 driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA− CD62L−) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP142 driven IFN-γ secreting naïve-like, transitional (CD45RA+ CD62L+) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.

Short-Term Reproducibility of a Commercial Interferon Gamma Release Assay

Interferon gamma release assays (IGRAs) have been shown to be sensitive and highly specific for the detection of immune memory against Mycobacterium tuberculosis. Little is known about the reproducibility and within-person variability of these assays. Various aspects of short-term reproducibility of a commercial IGRA, the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, were assessed. The QFT-IT assay was performed twice within 3 days in 27 health care workers in Cape Town, South Africa. Two sets of tests were performed by different operators on day 1, and one set was performed on day 3. Aspects such as interoperator, intraoperator, day-to-day variability, and test-retest variability as well as different the storage methods of plasma were investigated. Seventeen of 27 (63%) of participants had at least one positive QFT-IT text; six had discordant results. The agreement of all aspects studied was high, with kappa values between 0.82 and 1.00 for dichotomous measures, and interclass correlations (ICC) of 0.809 to 0.965 were observed for continuous gamma interferon (IFN-gamma) measures. The variability of the magnitude of response was highest comparing measures obtained from individuals on different days (ICC of 0.809). The magnitude of the IFN-gamma responses between assays performed for individual participants was variable, with ranges from 0.03 to 11 IU/ml, resulting is discordant results for five participants. The results indicate that the QFT-IT assay is a robust and highly reproducible assay. Considerable intraindividual variability occurs in the magnitude of IFN-gamma responses, which may influence the interpretation of serial measures.