Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality in allogeneic hematopoietic cell transplant (allo-HCT) recipients. CMV cell-mediated immunity (CMV-CMI) as ...determined by a peptide-based enzyme-linked immunospot (ELISPOT) CMV assay may identify patients at risk for clinically significant CMV infection (CS-CMVi).
The CS-CMVi was defined as CMV viremia and/or disease necessitating antiviral therapy. CMV-CMI was characterized as high when the intermediate-early 1 (IE-1) antigen spot counts (SPCs) were >100 (cutoff 1) or when the IE-1 and phosphoprotein 65 antigen SPCs were both >100 SPCs per 250 000 cells (cutoff 2), and a low CMV-CMI when SPCs were below these thresholds. In this prospective multicenter study, we evaluated CMV-CMI every 2 weeks from the pretransplant period until 6 months posttransplantation in 241 allo-HCT recipients with positive CMV serostatus. The primary endpoint was CS-CMVi occurring within 2 weeks of the last measurement of CMV-CMI.
CS-CMVi occurred in 70 allo-HCT recipients (29%). CMV-CMI was low in patients who experienced CS-CMVi (94%), whereas those who had a high CMV-CMI were less likely to have CS-CMVi (P < .0001). Patients with CS-CMVi had higher all-cause mortality (P = .007), especially those with low CMV-CMI (P = .035). On multivariable analysis, CMV-CMI, sex, race, antithymocyte globulin, and steroid use were independent predictors of CS-CMVi, and the time from transplant to engraftment was the only predictor of mortality.
Measurement of CMV-CMI using a novel ELISPOT assay would be useful clinically to monitor allo-HCT recipients and distinguish between those at risk of developing CS-CMVi and requiring antiviral prophylaxis or therapy and those who are protected.
Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and ...functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.
Summary
Impaired cytomegalovirus (CMV)‐specific cell‐mediated immunity (CMV‐CMI) is a major cause of CMV reactivation and associated complications in solid‐organ transplantation. Reliably assessing ...CMV‐CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T‐Track® CMV, a novel IFN‐γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE‐I CMV proteins, to monitor CMV‐CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate‐risk renal transplant recipients. CMV‐CMI, CMV viral load, and clinical complications were monitored over 6 months post‐transplantation. Ninety‐five percent and 88–92% ELISpot assays were positive pre‐ and post‐transplantation, respectively. CMV‐specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65‐specific response was ninefold higher in patients with self‐clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T‐Track® CMV is a highly sensitive IFN‐γ ELISpot assay, suitable for the immunomonitoring of CMV‐seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV‐related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).
The number of immunotherapeutic clinical trials in type 1 diabetes currently being conducted is expanding, and thus there is a need for robust immune-monitoring assays which are capable of detecting ...and characterizing islet specific immune responses in peripheral blood. Islet- specific T cells can serve as biomarkers and as such can guide drug selection, dosing regimens and immunological efficacy. Furthermore, these biomarkers can be utilized in patient stratification which can then benchmark suitability for participation in future clinical trials. This review focusses on the commonly used immune-monitoring techniques including multimer and antigen induced marker assays and the potential to combine these with single cell transcriptional profiling which may provide a greater understanding of the mechanisms underlying immuno-intervention. Although challenges remain around some key areas such as the need for harmonizing assays, technological advances mean that multiparametric information derived from a single sample can be used in coordinated efforts to harmonize biomarker discovery and validation. Moreover, the technologies discussed here have the potential to provide a unique insight on the effect of therapies on key players in the pathogenesis of T1D that cannot be obtained using antigen agnostic approaches.
We evaluated the effect of specific HLA alleles and haplotypes on cytomegalovirus (CMV)‐specific cell mediated immunity (CMI) in kidney transplant (KT) candidates. CMV‐specific ELISPOT against pp65 ...and IE‐1 antigens (hereafter referred to as pp65 and IE‐1, respectively) was performed in 229 seropositive KT candidates. We analyzed the results related to 44 selected HLA alleles (9 HLA‐A, 15 HLA‐B, 9 HLA‐C, and 11 HLA‐DR) and 13 HLA haplotypes commonly found in study participants. The pp65 and IE‐1 results in 229 seropositive candidates were 227.5 (114.5–471.5) and 41.0 (8.8–185.8) (median interquartile range) spots/2 × 10
5
PBMCs, respectively. The pp65 and IE‐1 results showed significant differences between candidates with different HLA alleles (
A*02
vs.
A*26
p
= 0.016,
A*24
vs.
A*30
p
= 0.031,
B*07
vs.
B*46
p
= 0.005,
B*54
vs.
B*35
p
= 0.041,
B*54
vs.
B*44
p
= 0.018,
B*54
vs.
B*51
p
= 0.025, and
C*06
vs.
C*14
p
= 0.034).
HLA‐A*02
and
B*54
were associated with increased pp65 and IE‐1 results, respectively (
p
= 0.005 and
p
< 0.001, respectively). In contrast, the
HLA‐A*26
and
B*46
alleles were associated with a decreased pp65 response, whereas the
A*30
allele was associated with a decreased IE‐1 response (
p
< 0.05). The pp65 results correlated with the HLA‐A allele frequencies (
R
= 0.7546,
p
= 0.019) and the IE‐1 results correlated with the HLA‐C allele frequencies of the study participants (
R
= 0.7882,
p
= 0.012). Among 13 haplotypes, HLA‐
A*30
~
B*13
~
C*06
~
DRB1*07
showed decreased CMV‐CMIs compared to the other HLA haplotypes, probably due to a combination of HLA alleles associated with lower CMV‐CMIs. Our results demonstrated that CMV‐specific CMIs may be influenced by the HLA allele as well as the HLA haplotype. To better predict CMV reactivation, it is important to estimate risk in the context of HLA allele and haplotype information.
Synthetic peptides derived from antigen sequences are essential reagents for the detection of CD8+ cytotoxic T lymphocytes (CTLs), in assays such as ELISPOT/ImmunoSpot®. Indeed, the combination of ...peptides and ImmunoSpot® has been widely used for immune monitoring in numerous vaccine trials. Target antigens in pathogens or cancers may be large in size and multiple in number, often seemingly necessitating in silico peptide epitope predictions using algorithms and programs for certain HLA alleles to narrow down the numbers of required peptides. In this commentary, we discuss our data in the context of immune responses to viral and cancer antigens, concluding that systematic high-throughput immune monitoring of CD8+ T cells will provide more reliable insights on the host’s response to cancer than the reliance on select CD8+ T cell epitopes, no matter whether these are in silico predicted or even if they had been empirically established. We show the feasibility of large scale, high-throughput systematic CD8+ T cell epitope testing towards this goal.
Since 2010, Tembusu virus (TMUV) has spread widely in China, causing huge economic losses to the poultry industry. Due to the infectious and zoonotic nature of flaviviruses, their potential threat to ...public health is of great concern. Cellular immune responses usually play a critical role in combating viral infections. To study the molecular basis of cell immunity induced by TMUV, 14 cytotoxic T lymphocyte (CTL) epitope peptides of TMUV antigen E, NS1 and NS3 were predicted by bioinformatics tools. Their abilities to induce cellular immune responses were determined by IFN-γ ELISpot assay, and 4 peptides were found to exhibit highly significant responses upon stimulation. In addition, the cytotoxic activity induced by the epitope peptides was assessed by lactate dehydrogenase (LDH) release assay. Finally, among these peptides, we identified two murine TMUV NS3-derived H-2d-restricted CTL epitopes in BALB/c mice, which could be used to further study of epitope vaccines against TMUV infection.
•Intracerebral inoculation with TMUV in mice can induce a specific cellular immune response.•Two novel TMUV NS3-derived H-2d-restricted CTL epitopes were identified in BALB/c mice.•The sequence of the peptide NS3299–307(GYISTKVEL) is highly conserved in flaviviruses.
•Tool to identify the risk of acute rejection in kidney transplantation is needed.•Patients with positive donor-specific ELISPOT are at risk for acute rejection.•Donor-specific ELISPOT assay could ...aid the personalization of immunosuppression.
Acute rejection remains an important problem after kidney transplantation. Enzyme-linked immunosorbent spot (ELISPOT) assay has been investigated extensively and has shown promising results as a predictor of allograft rejection. The objective of this study was to systematically review and analyze the predictive value of the donor-specific ELISPOT assay to identify recipients at risk for acute rejection. Electronic databases were searched for studies reporting donor-specific ELISPOT and kidney transplantation outcomes. Odds ratio (OR) for acute rejection was calculated, along with standardized mean difference (SMD) of cytokine producing-cells between recipients with and without acute rejection. Pooled estimates were calculated using random-effect models. The positive ELISPOT cutoff frequencies were extracted from each study. From 665 articles found, 32 studies were included in the meta-analysis. IFN-γ was the most investigated cytokine (30 out of 32 studies). Patients with positive pre-transplantation donor-reactive IFN-γ ELISPOT had an OR of 3.3 for acute rejection (95%-CI 2.1 to 5.1), and OR of 6.8 (95%-CI 2.5 to 18.9) for post-transplantation ELISPOT. Recipients with rejection had significantly higher frequencies of pre- and post-transplantation cytokine producing-cells (SMD 0.47, 95%-CI 0.07 to 0.87 and SMD 3.68, 95%-CI 1.04 to 6.32, respectively). Pre-transplantation ELISPOT had a positive predictive value of 43% and a negative predictive value of 81% for acute rejection. A positive ELISPOT result was associated with a lower estimated glomerular filtration rate (SMD −0.59, 95%-CI −0.83 to −0.34). In conclusion, patients with a high frequency of donor-reactive IFN-γ ELISPOT are at higher risk for acute rejection. The donor-specific IFN-γ ELISPOT assay can serve as an immune-monitoring tool in kidney transplantation.
Autologous and allogeneic adult mesenchymal stem/stromal cells (MSCs) are increasingly being investigated for treating a wide range of clinical diseases. Allogeneic MSCs are especially attractive due ...to their potential to provide immediate care at the time of tissue injury or disease diagnosis. The prevailing dogma has been that allogeneic MSCs are immune privileged, but there have been very few studies that control for matched or mismatched major histocompatibility complex (MHC) molecule expression and that examine immunogenicity in vivo. Studies that control for MHC expression have reported both cell-mediated and humoral immune responses to MHC-mismatched MSCs. The clinical implications of immune responses to MHC-mismatched MSCs are still unknown. Pre-clinical and clinical studies that document the MHC haplotype of donors and recipients and measure immune responses following MSC treatment are necessary to answer this critical question.
This review details what is currently known about the immunogenicity of allogeneic MSCs and suggests contemporary assays that could be utilized in future studies to appropriately identify and measure immune responses to MHC-mismatched MSCs.
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