Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Wunsch, Marie; Zhang, Wenji; Hanson, Jodi; Caspell, Richard; Karulin, Alexey Y.; Recks, Mascha S.; Küerten, Stefanie; Sundararaman, Srividya; Lehmann, Paul

doi:10.3390/v7082828

Cited by 19 publications

(29 citation statements)

References 67 publications

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“…Th2 and Th17 cells [ 17 ] specific for CPI were seen only in a subset of donors ( Figure 5 ): Seven of these 17 donors (41%) displayed a CPI-induced IL-4 and IL-17 recall response, and three out of 17 (17%) responded with IL-5. This outcome is not surprising, as we have obtained similar results in a systematic dissection of the HCMV-specific CD4 T cell response in healthy donors [ 12 ].…”

Section: Resultssupporting

confidence: 67%

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A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Schiller¹,

Zhang²,

Li³

et al. 2017

Cells

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Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the ‘CPI pool’), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

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Section: Resultssupporting

confidence: 67%

“…The human interferon-γ ELISPOT assay was performed as previously described using the ImmunoSpot ® Test Kit (CTL, Cat # hIFNG-1M) following the manufacturer’s protocol, and as described in [ 12 ]. In brief, the PVDF membrane was coated with a capture antibody overnight.…”

Section: Methodsmentioning

confidence: 99%

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Schiller¹,

Zhang²,

Li³

et al. 2017

Cells

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“…IFN-γ recall responses have been commonly observed. In contrast, only occasional donors have been reported to generate IL-4, IL-5 and IL-17 recall responses to select antigens and they showed a delayed kinetic [ 18 ]. We therefore set out to identify antigen/donor combinations suitable for the detection of Th2 and Th17 memory cells.…”

Section: Resultsmentioning

confidence: 99%

“…In mice induction of IL-4, IL-5 and IL-17 by the corresponding T cell subset also peaks within 24 h [ 17 ]. In humans, however, there have been occasional observations of delayed IL-4, IL-5 and IL-17 secretion kinetics by memory T cells [ 18 ], but the detection of these effector T cell classes in PBMC has mostly been unsuccessful, likely because of the low frequency of such T cells, and possibly because too short antigen-stimulation cultures have been used. A systematic study to this extent is missing so far, and is presented in this report.…”

Section: Introductionmentioning

confidence: 99%

Delayed Activation Kinetics of Th2- and Th17 Cells Compared to Th1 Cells

Duechting

Przybyla

Küerten

et al. 2017

Cells

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During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.

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“…The human interferon-γ (IFN-γ) ELISPOT assay was performed as we previously described [ 13 ]. Briefly, the PVDF membrane was coated with capture antibody overnight.…”

Section: Methodsmentioning

confidence: 99%

How frequently are predicted peptides actually recognized by CD8 cells?

Moldovan¹,

Targoni²,

Zhang³

et al. 2016

Cancer Immunol Immunother

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Detection of antigen-specific CD8 cells frequently relies on the use of peptides that are predicted to bind to HLA Class I molecules or have been shown to induce immune responses. There is extensive knowledge on individual HLA alleles’ peptide-binding requirements, and immunogenic peptides for many antigens have been defined. The 32 individual peptides that comprise the CEF peptide pool represent such well-defined peptide determinants for Cytomegalo-, Epstein–barr-, and Influenza virus. We tested the accuracy of these peptide recognition predictions on 42 healthy human donors that have been high-resolution HLA-typed. According to the predictions, 241 recall responses should have been detected in these donors. Actual testing showed that 36 (15 %) of the predicted CD8 cell responses occurred in the high frequency range, 41 (17 %) in mid-frequencies, and 45 (19 %) were at the detection limit. In 119 instances (49 %), the predicted peptides were not targeted by CD8 cells detectably. The individual CEF peptides were recognized in an unpredicted fashion in 57 test cases. Moreover, the frequency of CD8 cells responding to a single peptide did not reflect on the number of CD8 cells targeting other determinants on the same antigen. Thus, reliance on one or a few predicted peptides provides a rather inaccurate assessment of antigen-specific CD8 cell immunity, strongly arguing for the use of peptide pools for immune monitoring.

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Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Cited by 19 publications

References 67 publications

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Delayed Activation Kinetics of Th2- and Th17 Cells Compared to Th1 Cells

How frequently are predicted peptides actually recognized by CD8 cells?

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