Interferon-gamma Enzyme-linked Immunosorbent Spot Assays Research Articles

Utility of the Interferon-Gamma Enzyme-Linked Immunosorbent Spot Assay to Predict Risk of Cytomegalovirus Infection in Kidney Transplant Recipients.

Non-specific interferon-gamma (IFN-γ) enzyme-linked immunosorbent (ELISpot) responses after solid organ transplant (SOT) and their relationship with cytomegalovirus (CMV) reactivation have hardly been investigated. Adult kidney transplant (KT) recipients underwent measurement of IFN-γ-producing T cells using the ELISpot assay before and 1 month after transplantation. Data for CMV infection episodes were collected. Risk factors for post-transplant CMV infection, based on IFN-γ responses, were analyzed using a Cox proportional hazards model. A total of 93 KT recipients were enrolled in the study and 84 evaluable participants remained at 1 month post KT. Thirty-three (39%) recipients developed subsequent CMV infection within 6 months post-transplant. At 1-month post-transplant, IFN-γ-producing T cells with <250 spot-forming units (SFUs)/2.5 × 105 peripheral blood mononuclear cells (PBMCs) were significantly associated with CMV infection (HR 3.1, 95% CI 1.4-7.1, p = 0.007). On multivariable analysis, posttransplant IFN-γ-producing T cells with <250 SFUs/2.5 × 105 PBMCs remained independently associated with CMV infection (HR 3.1, 95% CI 1.2-7.8, p = 0.019). Conclusions: KT recipients with low IFN-γ-producing T cells measured by the ELISpot assay are more likely to develop CMV infection after transplantation. Therefore, measurement of nonspecific cell-mediated immunity ELISpot responses could potentially stratify recipients at risk of CMV infection (Thai Clinical Trials Registry, TCTR20210216004).

Occupational Exposure to Hepatitis C Virus: Early T-Cell Responses in the Absence of Seroconversion in a Longitudinal Cohort Study

T-cell responses have been described in seronegative patients who test negative for hepatitis C virus (HCV) RNA despite frequent HCV exposure. However, the cross-sectional design of those studies did not clarify whether T cells were indeed induced by low-level HCV exposure without seroconversion or whether they resulted from regular acute infection with subsequent antibody loss. Over a 10-year period, our longitudinal study recruited 72 healthcare workers with documented HCV exposure. We studied viremia and antibody and T-cell responses longitudinally for 6 months. All healthcare workers remained negative for HCV RNA and antibodies. However, 48% developed proliferative T-cell response and 42% developed responses in interferon-gamma enzyme-linked immunosorbent spot assays, with 29 healthy HCV-unexposed controls used to define assay cutoffs. The response prevalence was associated with the transmission risk score. T-cell responses peaked at week 4 and returned to baseline by week 12 after exposure. They predominantly targeted nonstructural HCV proteins, which are not part of the HCV particle and thus must have been synthesized in infected cells. Subclinical transmission of HCV occurs frequently, resulting in infection and synthesis of nonstructural proteins despite undetectable systemic viremia. T-cell responses are more sensitive indicators of this low-level HCV exposure than antibodies.

T-Cell Responses in Children to Internal Influenza Antigens, 1 Year After Immunization With Pandemic H1N1 Influenza Vaccine, and Response to Revaccination With Seasonal Trivalent–inactivated Influenza Vaccine

During seasonal influenza epidemics, 5-15% of the population are affected with an illness having a nontrivial mortality, morbidity and economic burden. Inactivated influenza vaccines are routinely used to prevent influenza infection, primarily by inducing humoral immunity. In addition, trivalent-inactivated influenza vaccines have previously been shown to boost influenza-specific T-cell responses in a small percentage of adults. We investigate here the influenza-specific T-cell response, in children, 1 year after pandemic H1N1 vaccination and the ability to boost the T-cell response with trivalent-inactivated influenza immunization. Peripheral blood mononuclear cells (PBMCs) were isolated from children previously vaccinated with pandemic H1N1 vaccine, pre- and postseasonal 2010-2011 trivalent influenza vaccine (TIV) vaccination. Samples were analyzed by interferon-gamma enzyme-linked immunosorbent spot for reactogenicity toward internal influenza antigens (nucleoprotein, matrix protein 1 and nonstructural protein 1). Basal ex vivo T-cell responses to nucleoprotein, matrix protein 1 and nonstructural protein 1 measured by interferon-gamma enzyme-linked immunosorbent spot assay were significantly higher in those children who had previously received an AS03B-adjuvanted split virion pandemic vaccine 12 months earlier rather than a nonadjuvanted whole virion vaccine. Boosting of these responses, 21 days after 2010/2011 seasonal TIV vaccination was observed regardless of age or prior pandemic vaccination regime, although boosting was greater in those groups with the lowest initial response. We show here that children previously vaccinated with the 2009 pandemic H1N1 vaccine have measurable T-cell responses 1 year after vaccination. The magnitudes of these responses are dependent on both age of vaccine and type of pandemic H1N1 vaccine used. After 2010/2011 seasonal TIV vaccination, these T-cell responses undergo a small but significant boost.

Clinical and Immunologic Responses of HLA-A3 + Breast Cancer Patients Vaccinated with the HER2/ neu-Derived Peptide Vaccine, E75, in a Phase I/II Clinical Trial

We have treated disease-free breast cancer patients with an HER2/neu-derived peptide, E75, as an adjuvant vaccine. E75 was originally described as HLA-A2-restricted and has been previously tested in this population. Based on computer modeling, E75 is predicted to bind to HLA-A3, and preclinical data support this. We conducted a clinical trial of E75 in HLA-A3(+), A2(-) (A3) patients. Disease-free breast cancer patients were enrolled after standard therapy in phase I/II trials. A3 patients were enrolled in parallel with A2 patients and vaccinated with E75 and granulocyte-macrophage colony-stimulating factor immunoadjuvant. A2(-), A3(-) patients were followed as controls. Toxicities were graded. Immunologic responses were assessed by delayed-type hypersensitivity reactions and E75-specific interferon-gamma enzyme-linked immunosorbent spot assay. Clinical recurrences were documented. Thirteen A3 patients completed the vaccine schedule. Clinicopathologic features were similar between A3, A2, and control patients, except for more HER2/neu-overexpressing tumors in the A2 group and more estrogen-receptor/progesterone-receptor-negative tumors in A2 and A3 groups. Toxicity profiles and postvaccination delayed-type hypersensitivity were similar in A3 and A2 patients. Enzyme-linked immunosorbent spot assay results varied, but A3 patients' median spots increased pre- to postvaccination (p = 0.2). Recurrences were lower in the A3 group (7.7%) at 30-month median follow-up compared with published recurrence in A2-vaccinated (8.3%) and control groups (14.8%) at 26-month median follow-up. HLA restriction limits potential use of peptide-based cancer vaccines. This trial demonstrates that HLA-A3 patients respond similarly to E75 vaccination as HLA-A2 patients, suggesting the potential use of the E75 vaccine in up to 76% of the population.

Pretransplantation Cellular Alloreactivity Is Predictive of Acute Graft Rejection and 1-Year Graft Function in Kidney Transplant Recipients

To study cellular alloimmunity in kidney allograft recipients using an interferon-gamma enzyme-linked immunosorbent spot assay (ELISPOT). Donor splenocyte peripheral blood mononuclear cells were obtained during kidney recovery in 53 kidney recipients including 11 with positive panel-reactive antibodies pretransplantation. For ELISPOT data analysis, the spot number, size, and intensity were calculated, reflecting the volume of cytokine secretion at the single-cell level. Results were recalculated as the ratio of the values observed for donor-stimulated to unstimulated recipient cells corrected for residual donor activity. Significantly greater pretransplantation donor-stimulated activity was observed in recipients who experienced an acute rejection episode (ARE) within 1 year (P < .05). Mean change in spot number, size, and intensity in patients without or with AREs was 0.99 vs 3.33, 1.60 vs 6.05, and 1.40 vs 6.31, respectively. The assessed parameters were prognostic of high risk of ARE: 1.5-fold increase in spot number (ARE incidence, 52% vs 9%), 2.5-fold increase in spot size (ARE incidence, 53% vs 13%), and 2.7-fold increase in spot intensity (ARE incidence, 52% vs 9%). The 3 parameters correlated with 1-year serum creatinine concentration (P < .05). In 14 recipients, AREs could have been predicted in 11 using pretransplantation ELISPOT results, and in only 2 on the basis of panel-reactive antibodies. The ELISPOT-determined capacity of donor-induced reactivity observed in recipient cells obtained just before transplantation is predictive of risk of graft rejection and 1-year allograft function.

T-Cell Recognition of Prostatic Peptides in Men With Chronic Prostatitis/Chronic Pelvic Pain Syndrome

A potential etiology of chronic prostatitis/chronic pelvic pain syndrome is autoimmunity. We determined whether T cells from men with chronic prostatitis/chronic pelvic pain syndrome would recognize peptides derived from the normal self-prostatic proteins prostate specific antigen and prostatic acid phosphatase. CD4 T cells purified from peripheral blood of 31 patients with chronic prostatitis/chronic pelvic pain syndrome and from the buffy coat preparation of 27 normal male blood donors were stimulated in vitro with a panel of immunogenic peptides from prostate specific antigen and prostatic acid phosphatase, and assayed for reactivity with the peptides by interferon-gamma enzyme-linked immunosorbent spot assay. Intermediate resolution HLA typing was done by polymerase chain reaction. Peptides were also tested by binding assay against different class II alleles. Peptide PAP(173-192) was recognized more frequently by CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome than from healthy donors. The recognition of prostate specific antigen peptides was not statistically different when comparing cases to normal male blood donors individually. Peptide reactivity was more common in patients than in normal male blood donors for any prostate specific antigen peptide or any tested peptide. All peptides showed high promiscuity on binding assays. There was no association of cases with any specific HLA class II phenotype at intermediate resolution. CD4 T cells from patients with chronic prostatitis/chronic pelvic pain syndrome have a higher rate of recognizing the self-prostatic proteins prostatic acid phosphatase and prostate specific antigen compared to those from normal male blood donors. Data provide further evidence to support the role of autoimmunity in some men with chronic prostatitis/chronic pelvic pain syndrome.

Using Dendritic Cell Maturation and IL-12 Producing Capacity as Markers of Function: A Cautionary Tale

Effective dendritic cell (DC) function depends on sufficient expression of antigen and costimulatory molecules, and secretion of interleukin (IL)-12. We sought to augment DC stimulatory capacity by optimizing DC phenotype and IL-12 production. DCs, obtained by CD14-selection, were matured using 8 different cytokine cocktails, and expression of costimulatory/major histocompatibility complex molecules and IL-12 production at the end of maturation was assessed. DC stimulatory capacity was determined after pulsing with immunogenic adenoviral CD8 peptide epitopes or after transduction with an Ad5f35-null vector. Resultant T-cell cultures were analyzed using pentamer and interferon-gamma enzyme-linked immunosorbent spot assays. On the basis of DC expression of maturation markers and IL-12 production, we defined prototype "minimal" [tumor necrosis factor-alpha (TNF-alpha), prostaglandin E2], "standard" (IL-1, IL-6, TNF-alpha, prostaglandin E2), and "optimal" (IL-1, IL-6, TNF-alpha, interferon-alpha, CD40 ligand) DC cocktails. Optimal DCs were functionally superior when pulsed with CD8 peptides, but when transduced with Ad5f35, functioned poorly as antigen-presenting cells. We investigated the mechanisms underlying this discrepancy and suggest that prolonged stimulation with potent cytokines (optimal cocktail) in combination with adenoviral transduction alters the kinetics of DC maturation such that the DCs are functionally exhausted by the traditional 48-hour maturation time point. Shortening the DC maturation period posttransduction restored optimal DC stimulatory capacity. Thus, maturation stimuli and viral transduction affects DC phenotype, IL-12 producing capacity, and kinetics of maturation, and all must be considered before designing protocols to generate the optimal DC for cytotoxic T lymphocyte generation.

Monitoring of Cellular Immunity by Interferon-Gamma Enzyme-Linked Immunosorbent Spot Assay in Kidney Allograft Recipients: Preliminary Results of a Longitudinal Study

Several efforts have been made in past years to identify markers for patients at heightened risk of acute and chronic immune-mediated allograft rejection. The ex vivo monitoring of cellular immunity by the enzyme-linked immunosorbent spot (ELISPOT) assay has recently emerged as a primary tool in predicting short- and long-term outcomes in kidney allograft recipients. Therefore, we started the systematic application of interferon-gamma (IFN-γ) ELISPOT assay to measure the frequency of producing IFN-γ in recipient peripheral blood lymphocytes (PBLs) stimulated with donor lymphocytes before and 7, 14, 21, 28, and 60 days after transplantation. Preliminary results in eight kidney transplant patients indicated that the number of HLA mismatches never correlated with the number of IFN-γ spots. The frequencies of pretransplantation IFN-γ spots were positively and significantly correlated with the number of posttransplantation IFN-γ spots. Clinical outcomes were better among recipients with lower frequencies than those with higher frequencies of pre- and/or posttransplantation IFN-γ spots. The highest pre- and posttransplantation number of IFN-γ spots was observed in a patient who developed early acute rejection. Significant increases in the number of IFN-γ spots preceded the onset of acute rejection events and were decreased by supplemental IV steroid administration. Considering the low number of observations, these preliminary results must be considered cautiously; nevertheless, we are encouraged to extend the systematic application of serial IFN-γ ELISPOT assay measurements in a more consistent cohort of patients.

Alloreactivity in renal transplant recipients with and without chronic allograft nephropathy.

The pathogenesis of chronic allograft nephropathy (CAN) involves both immunologic (antigen-dependent) and nonimmunologic (antigen-independent) mechanisms. In order to provide further insight into the immunologic basis of this disease, a cross-sectional analysis of cellular and humoral immunity in human renal allograft recipients with or without deteriorating renal function and biopsy proven CAN was performed. Interferon-gamma enzyme-linked immunosorbent spot assays were used to assess cellular immunity to donor, or fully mismatched third-party stimulator cells (direct pathway), and to synthetic peptides derived from donor HLA molecules (indirect pathway). Anti-HLA antibodies were evaluated by flow cytometry using HLA-coated beads. Both the mean frequencies of donor-reactive peripheral blood lymphocytes and the number of individuals who responded to donor antigens per group were statistically higher in CAN patients versus control subjects (P < 0.02). Calculated ratios of donor/third-party enzyme-linked immunosorbent spot responses showed mean values of 2.61 +/- 3.0 in the CAN group, with ratios of 0.50 to 0.72 +/- 0.42 in control subjects (P < 0.001), confirming that direct, donor-specific cellular immunity predominated in patients with CAN. Fifty percent of CAN patients studied exhibited donor peptide reactivity compared with only 28.6% in control subjects. Finally, 33% of patients in the CAN group developed new posttransplantation anti-HLA antibodies compared with only 4% in the control group (P < 0.05). Overall, the results suggest that persistent cell-mediated and humoral alloimmunity contribute to the development of CAN and further demonstrate that anti-donor immunity in patients with CAN is heterogeneous. Immune monitoring to predict long-term outcome should include multiple measures of cellular and humoral immunity.