15146
2015
eng
4414
4437
7
article
1
2017-07-13
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Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity
Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot\(^{®}\) assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-\({\gamma}\) and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-\({\gamma}\) and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.
Viruses
10.3390/v7082828
urn:nbn:de:bvb:20-opus-151462
Viruses 2015, 7, 4414-4437. DOI: 10.3390/v7082828
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Marie Wunsch
Wenji Zhang
Jodi Hanson
Richard Caspell
Alexey Y. Karulin
Mascha S. Recks
Stefanie Kuerten
Srividya Sundararaman
Paul V. Lehmann
eng
uncontrolled
memory cells
eng
uncontrolled
hcv infection
eng
uncontrolled
signature
eng
uncontrolled
Enzyme-Linked Immunospot assay (ELISPOT)
eng
uncontrolled
cytokine secretion kinetics
eng
uncontrolled
chronic viral infection
eng
uncontrolled
HCMV infection
eng
uncontrolled
CD4 T cells
eng
uncontrolled
exhaustion
eng
uncontrolled
activation
eng
uncontrolled
human cytomegalovirus (HCMV)
eng
uncontrolled
B cells
eng
uncontrolled
cytomegalovirus
eng
uncontrolled
elispot
Krankheiten
open_access
Institut für Anatomie und Zellbiologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15146/012_Wunsch_Viruses.pdf
15021
2015
eng
40-55
1
4
article
1
2017-06-09
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Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay
As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.
Cells
10.3390/cells4010040
urn:nbn:de:bvb:20-opus-150213
Cells 2015, 4:1, 40-55. DOI: 10.3390/cells4010040
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Marie Wunsch
Richard Caspell
Stefanie Kuerten
Paul V. Lehmann
Srividya Sundararaman
eng
uncontrolled
T cell assay
eng
uncontrolled
apoptosis
eng
uncontrolled
acceptance
eng
uncontrolled
viability
eng
uncontrolled
ELISPOT
Medizin und Gesundheit
open_access
Institut für Anatomie und Zellbiologie
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/15021/129_Wunsch_Cells.pdf
14996
2015
eng
96-111
1
4
article
1
2017-06-08
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Normal distribution of CD8+ T-cell-derived ELISPOT counts within replicates justifies the reliance on parametric statistics for identifying positive responses
Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 – 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.
Cells
10.3390/cells4010096
PMC4381212
urn:nbn:de:bvb:20-opus-149968
Cells 2015, 4(1), 96-111. DOI: 10.3390/cells4010096
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Richard Caspell
Marcus Dittrich
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
statistics
eng
uncontrolled
t-Test
eng
uncontrolled
ANOVA
eng
uncontrolled
T-cells
eng
uncontrolled
normal distribution
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14996/108_Karulin_Cells.pdf