Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein–Barr virus-specific T cells as targets of interest in immunotherapeutic approaches

Tischer, Sabine; Dieks, Daria; Sukdolak, Cinja; Bunse, Carola E.; Figueiredo, Constança; Immenschuh, Stephan; Borchers, Sylvia; Stripecke, Renata; Maecker‐Kolhoff, Britta; Blasczyk, Rainer; Eiz‐Vesper, Britta

doi:10.1016/j.jim.2014.05.011

Cited by 42 publications

(38 citation statements)

References 51 publications

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“…The current study used FluoroSpot assays to measure IFN-␥ production by CD4 ϩ T cells; FluoroSpot is an enzyme-linked immunosorbent spot (ELISPOT) assay which enables measurement of multiple cytokines simultaneously (59). Experience from our own studies and other groups suggests that the ELISPOT assay is more sensitive than other methods for detecting T cell responses to HCMV antigens (60), so the contrasting observations obtained in this study compared to those obtained in previously published work are not explained by the use of different techniques to measure IFN-␥ responses. The studies demonstrating an increased frequency of CMV-specific CD4 ϩ T cells in older donors (19,21,22) did not investigate the absolute size of the T cell compartment in peripheral blood.…”

Section: Discussioncontrasting

confidence: 58%

Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Jackson

Sedikides

Mason

et al. 2017

J Virol

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Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8 ϩ T cell response to HCMV has been extensively studied, the HCMVspecific CD4 ϩ T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-␥) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4 ϩ T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-␥ response was still dominant. CD4 ϩ T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1␤ secretion) effector responses. Importantly, when we measured the CD4 ϩ T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro, we observed that the CD4 ϩ T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4 ϩ T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4 ϩ T cell responses, even those from elderly individuals, are highly functional and are directly antiviral.IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the host's lifetime. The dysfunction of immune cells associated with the long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when people are older. In this study, we investigated the response of a subset of immune cells (CD4 ϩ T cells) to HCMV proteins in healthy donors of all ages, and we demonstrate that the functionality of CD4 ϩ T cells is maintained. We also show that CD4 ϩ T cells produce effector functions in response to HCMV-infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

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Section: Discussioncontrasting

confidence: 58%

Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Jackson

Sedikides

Mason

et al. 2017

J Virol

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“…ELISpot represents the most sensitive read-out system and thus is most appropriate for the detection of low-level responses [23, 24]. In particular, ELISpot is more sensitive than ICS for the detection of antigen-specific cells, such as in vitro-reactivated memory T cells, which produce only low amounts of cytokines [15, 25].…”

Section: Introductionmentioning

confidence: 99%

“…Conventional CMV-specific ELISpot assays are based on the detection of IFN-γ-producing CD4 + and/or CD8 + T cells, depending on the antigens used for PBMC stimulation [23, 24, 29, 30]. The careful selection and formulation of antigens is crucial to ensure specificity, sensitivity and thus a diagnostic value to the assay.…”

Section: Introductionmentioning

confidence: 99%

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

et al. 2017

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BackgroundIn healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.MethodsObjective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.ResultsOptimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3−CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.ConclusionThe combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0195-y) contains supplementary material, which is available to authorized users.

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“…Recent technical advancements of multimers facilitate detection of low affinity T cells due to higher binding avidity to surface TCR [70,71] and allow reversible pMHClabeling of antigen-specific T cells without affecting T-cell function [72,73]. Multimer staining achieves high sensitivity comparable to ELISPOT [74]. The major limitation is that requires knowledge of the patient's HLA-type and the relevant peptide antigens.…”

Section: Pmhc Multimer Stainingmentioning

confidence: 99%

“…EBVspecific T-cell responses can be quantified by ELISPOT [101][102][103][104], ICS [105,106] and pMHC multimer staining [101,107] or by combining pMHC multimer staining and ICS techniques [106] in SOT recipients. A recent study compared five immunoassays [the functional EBV-specific IFN-␥ ELISPOT, ICS, ELISA and cytokine secretion assays (CSA) and the detection of EBV-specific T cells by pMHC multimer staining] in healthy subjects and identified the peptidebased IFN-␥ ELISPOT as the most accurate technique for immune monitoring and CSA for a more detailed phenotypic and functional analysis of T cells [74]. EBV-specific CD8 + T-cell responses have been studied in detail showing that epitopes from the EBNA3 family of proteins induce the strongest response [108].…”

Section: Ebvmentioning

confidence: 99%

Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications

Calarota

Aberle

Puchhammer‐Stöckl

et al. 2015

Journal of Clinical Virology

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Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein–Barr virus-specific T cells as targets of interest in immunotherapeutic approaches

Cited by 42 publications

References 51 publications

Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications

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Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein–Barr virus-specific T cells as targets of interest in immunotherapeutic approaches

Cited by 42 publications

References 51 publications

Human Cytomegalovirus (HCMV)-Specific CD4 + T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Human Cytomegalovirus (HCMV)-Specific CD4 + T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

Approaches for monitoring of non virus-specific and virus-specific T-cell response in solid organ transplantation and their clinical applications

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Product

Resources

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Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro

Human Cytomegalovirus (HCMV)-Specific CD4 ⁺ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro