14964
2015
eng
56-70
1
4
article
1
2017-06-02
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ELISPOTs produced by CD8 and CD4 cells follow Log Normal size distribution permitting objective counting
Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
Cells
10.3390/cells4010056
PMC4381209
urn:nbn:de:bvb:20-opus-149648
Cells 2015, 4(1), 56-70. DOI: 10.3390/cells4010056
CC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International
Alexey Y. Karulin
Kinga Karacsony
Wenji Zhang
Oleg S. Targoni
Ioana Moldova
Marcus Dittrich
Srividya Sundararaman
Paul V. Lehmann
eng
uncontrolled
ELISPOT
eng
uncontrolled
software
eng
uncontrolled
IFN-γ
eng
uncontrolled
IL-17
eng
uncontrolled
T cells
eng
uncontrolled
Normal Distribution
eng
uncontrolled
spot size
eng
uncontrolled
gating
eng
uncontrolled
cytokines
eng
uncontrolled
IL-2
eng
uncontrolled
IL-4
eng
uncontrolled
IL-5
eng
uncontrolled
CD8
eng
uncontrolled
CD4
Biowissenschaften; Biologie
open_access
Theodor-Boveri-Institut für Biowissenschaften
Universität Würzburg
https://opus.bibliothek.uni-wuerzburg.de/files/14964/094_Karulin_Cells.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
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