The rapid antigen-reactive T cell enrichment procedure ... · The rapid antigen-reactive T cell...
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The rapid antigen-reactive T cell enrichment procedure enables direct ex vivo BKV-specific CD4 + T cell characterization Michaela Niemöller, Mario Assenmacher, and Anne Richter Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Introduction BK virus (BKV) is a ubiquitous polyomavirus, which persists in a latent and asymptomatic state in healthy individuals after primary infection. In immunocompromised patients BKV reactivation may occur, which can result in, e.g., hemorrhagic cystitis after allogeneic stem cell transplantation or loss of the allograft after renal transplantation. The detailed characterization of BKV-specific T cells is the basis for a better understanding of the BKV immune response. This in turn provides the foundation for the development of strategies towards the prevention or treatment of infections in immunocompromised patients, for example, the adoptive transfer of virus-specific T cells. The Rapid ARTE approach allowed us to process larger cell numbers compared to detection by flow cytometry alone. Therefore, the new approach enabled the reliable characterization of functional subsets of the entire BKV-reactive CD154 + CD4 + T cell pool, i.e., TNF-α-, IFN-γ-, IL - 10–, and IL-2–producing T cells. PBMC (2.5×10⁷) from six healthy donors were processed as described in figure 2. Subsequently, the cytokine profile of the BKV-reactive CD4 + T cells was analyzed (A). TNF-α and IL-2 were expressed by the majority of BKV-specific T cells in all samples analyzed for these cytokines. In contrast, the frequency of IFN-γ + cells among BKV-reactive CD4 + T cells varied between 27 and 74% in the different donor samples (B). Furthermore, IL-10– producing T cells could be identified at very low frequencies, which could not have been detected by flow cytometry without pre-enrichment. In blood samples BKV-specific T cells are present only at very low frequencies, in the range of the detection limit of flow cytometry analysis. Therefore, we used the Rapid ARTE protocol for enumeration of BKV-specific T cells. Up to 2.5×10⁷ PBMC from 14 healthy donors were left untreated or stimulated with BKV peptide pools covering the complete sequence of the large T antigen (LT) and virion protein VP1. Both proteins are immunodominant targets for T cell immunity. After six hours, cells were treated as described in figure 2, and the absolute numbers of enriched CD154 + CD4 + T cells were determined. In blood samples of each donor, BKV- reactive CD4 + T cells were found. Absolute numbers ranged between 87 and 2340 per 1×10⁷ PBMC. Enumeration of BKV-specific CD154 + CD4 + T cells 3 Figure 3 Characterization of BKV-specific T cells using Rapid ARTE 4 Results PBMC from six randomly selected healthy donors were stimulated with various peptide pools for immunodominant antigens of BKV, JCV, CMV, influenza, EBV, and AdV, and for positive control with a mixture of CMV/EBV/influenza MHC class I–restricted peptides. After 6 hours, IFN-γ production within the CD4 + and CD8 + T cell compartments was analyzed by intracellular staining using the Rapid Cytokine Inspector. CMV-, EBV-, and AdV-specific IFN-γ + T cells were clearly detectable in several donor samples. In contrast, IFN-γ + BKV- and JCV-specific T cells were detectable only at very low frequencies, between 0.01 and 0.03%, in five out of six donor samples. BKV-specific T cells are rare in healthy donors 1 Figure 1 Recently, a protocol for the sensitive functional analysis of different rare antigen-specific CD4 + T cell repertoires directly ex vivo was developed. This so-called antigen-reactive T cell enrichment (ARTE) approach involves the magnetic enrichment of antigen-activated CD154 + CD4 + T cells to subsequently uncover their phenotypic and functional profile by flow cytometry¹². We further streamlined the ARTE protocol with regard to incubation times and hands- on time (Rapid ARTE). This new protocol allows the stimulation of up to 2.5×10⁷ PBMC. After six hours cells are fluorescently labeled for detection of various cell surface markers, such as CD4. At the same time cells are magnetically labeled for CD154. All steps are performed directly in the cell culture plate without cell harvesting and washing, which simplifies the procedure and reduces hands-on time. After surface labeling, the cells are fixed, harvested, and directly applied onto a column for magnetic enrichment of antigen-activated CD154 + T cells. The cells retained in the column are permeabilized, stained intracellularly for cytokines and CD154, and washed directly on the column to avoid centrifugation steps. Finally, CD154 + T cells are eluted from the column for subsequent flow cytometry analysis. Procedure for the rapid antigen-reactive T cell enrichment (Rapid ARTE) 2 A B Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 w/o JCV LT JCV VP1 BKV LT H1N1 MP1 H1N1 NP EBV BZLF1 AdV5 Penton CEF MHC Class 1 BKV VP1 CMV IE-1 CMV pp65 EBV EBNA1 EBV LMP2A AdV5 Hexon % IFN-γ + among CD4 + T cells 0.14 0.20 0.22 0.12 0.18 0.10 0.16 0.08 0.06 0.04 0.02 0.00 CD4 + T cells Number of CD154 + CD4 + T cells enriched from 1×10⁷ PBMC 2500 2400 2300 800 700 600 300 500 200 400 100 0 w/o antigen BKV LT + VP 1 Cell stimulation Stimulate 1–2.5×10⁷ PBMC/mL/well with peptide pools in a 24-well plate. 6 h Cell surface labeling and fixation Remove 800 μL cell culture supernatant. Add antibodies for cell surface labeling (incl. CD154-Biotin). Add Anti-Biotin MicroBeads. Add 300 μL 3.7% Inside Fix. 2 min shaking + 3 min incubation 2 min shaking + 8 min incubation 2 min shaking + 13 min incubation Enrichment of labeled CD154 + cells, permeabilization, and intracellular staining Apply the fixed cell suspension onto the column placed in a separator. Wash with 500 μL of PEB buffer. Wash with 500 μL Inside Perm. Apply CD154 and Anti-Cytokine antibodies for intracellular staining. Wash with 500 μL Inside Perm. Wash with 500 μL PEB buffer. 10 min incubation Elution of CD154 + CD4 + T cells Remove column from the separator and place it on a suitable collection tube. Pipette 500 μL PEB buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. Flow cytometric analysis Automated sample acquisition. Enumeration and characterization of antigen-activated T cells. Hands-on time: 30 min Total procedure: ≈ 7 h Figure 2 A w/o Antigen CD4 BKV LT BKV VP1 CD8 IFN-γ * among CD4 + T cells ** among CD8 + T cells 0.03%* 0.02%** 0.02%* 0.02%** Conclusion • We developed a rapid (7 h) and convenient protocol to detect and characterize very rare antigen-specific CD4 + T cell subpopulations in blood samples. • The process does not involve any centrifugation step and requires only minimal hands-on time. • High-resolution characterization of rare T cell repertoires is achieved combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154 + T cells. • In contrast to conventional flow cytometry analysis, this approach enabled us to identify BKV-specific T cells and specify their function. References 1. Bacher, P. et al. (2013) J. Immunol. 190: 3967–3976. 2. Bacher, P. et al. (2013) Cytometry A 83: 692–701. Figure 4 B w/o antigen Before enrichment BKV LT and VP1 After enrichment CD154 IL-2 TNF-α IFN-γ IFN-γ TNF-α IFN-γ IL-2 % Cytokine + among CD154 + CD4 + T cells 0 10 20 30 40 50 60 70 80 90 Donor A Donor B Donor C Donor D Donor E Donor F not determined IL-10 IFN-γ TNF-α * * * * * * * w/o JCV LT JCV VP1 BKV LT H1N1 MP1 H1N1 NP EBV BZLF1 AdV5 Penton CEF MHC Class 1 BKV VP1 CMV IE-1 CMV pp65 EBV EBNA1 EBV LMP2A AdV5 Hexon % IFN-γ + among CD8 + T cells 0.06 0.04 0.02 0.00 1.2 1.0 0.8 0.6 0.4 0.2 0.14 0.12 0.10 0.08 1.4 CD8 + T cells * * * * * Gated on CD4 + T cells
Transcript of The rapid antigen-reactive T cell enrichment procedure ... · The rapid antigen-reactive T cell...
The rapid antigen-reactive T cell enrichment procedure enables direct ex vivo BKV-specific CD4+ T cell characterization
Michaela Niemöller, Mario Assenmacher, and Anne RichterMiltenyi Biotec GmbH, Bergisch Gladbach, Germany
IntroductionBK virus (BKV) is a ubiquitous polyomavirus, which persists in a latent and asymptomatic state in healthy individuals after primary infection. In immunocompromised patients BKV reactivation may occur, which can result in, e.g., hemorrhagic cystitis after allogeneic stem cell transplantation or loss of the allograft after renal transplantation. The
detailed characterization of BKV-specific T cells is the basis for a better understanding of the BKV immune response. This in turn provides the foundation for the development of strategies towards the prevention or treatment of infections in immunocompromised patients, for example, the adoptive transfer of virus-specific T cells.
The Rapid ARTE approach allowed us to process larger cell numbers compared to detection by flow cytometry alone. Therefore, the new approach enabled the reliable characterization of functional subsets of the entire BKV-reactive CD154+CD4+ T cell pool, i.e., TNF-α−, IFN-γ−, IL-10–, and IL-2–producing T cells. PBMC (2.5×10⁷) from six healthy donors were processed as described in figure 2. Subsequently, the cytokine profile of the BKV-reactive CD4+
T cells was analyzed (A). TNF-α and IL-2 were expressed by the majority of BKV-specific T cells in all samples analyzed for these cytokines. In contrast, the frequency of IFN-γ+ cells among BKV-reactive CD4+ T cells varied between 27 and 74% in the different donor samples (B). Furthermore, IL-10–producing T cells could be identified at very low frequencies, which could not have been detected by flow cytometry without pre-enrichment.
In blood samples BKV-specific T cells are present only at very low frequencies, in the range of the detection limit of flow cytometry analysis. Therefore, we used the Rapid ARTE protocol for enumeration of BKV-specific T cells. Up to 2.5×10⁷ PBMC from 14 healthy donors were left untreated or stimulated with BKV peptide pools covering the complete sequence of the large T antigen (LT) and virion protein VP1. Both proteins are immunodominant targets for T cell immunity. After six hours, cells were treated as described in figure 2, and the absolute numbers of enriched CD154+CD4+ T cells were determined. In blood samples of each donor, BKV-reactive CD4+ T cells were found. Absolute numbers ranged between 87 and 2340 per 1×10⁷ PBMC.
Enumeration of BKV-specific CD154+CD4+ T cells 3
Figure 3
Characterization of BKV-specific T cells using Rapid ARTE4
Results
PBMC from six randomly selected healthy donors were stimulated with various peptide pools for immunodominant antigens of BKV, JCV, CMV, influenza, EBV, and AdV, and for positive control with a mixture of CMV/EBV/influenza MHC class I–restricted peptides. After 6 hours, IFN-γ production within the CD4+ and CD8+ T cell compartments was analyzed
by intracellular staining using the Rapid Cytokine Inspector. CMV-, EBV-, and AdV-specific IFN-γ+ T cells were clearly detectable in several donor samples. In contrast, IFN-γ+ BKV- and JCV-specific T cells were detectable only at very low frequencies, between 0.01 and 0.03%, in five out of six donor samples.
BKV-specific T cells are rare in healthy donors1
Figure 1
Recently, a protocol for the sensitive functional analysis of different rare antigen-specific CD4+ T cell repertoires directly ex vivo was developed. This so-called antigen-reactive T cell enrichment (ARTE) approach involves the magnetic enrichment of antigen-activated CD154+CD4+ T cells to subsequently uncover their phenotypic and functional profile by flow cytometry¹². We further streamlined the ARTE protocol with regard to incubation times and hands-on time (Rapid ARTE). This new protocol allows the stimulation of up to 2.5×10⁷ PBMC. After six hours cells are fluorescently labeled for detection of various cell surface markers, such
as CD4. At the same time cells are magnetically labeled for CD154. All steps are performed directly in the cell culture plate without cell harvesting and washing, which simplifies the procedure and reduces hands-on time. After surface labeling, the cells are fixed, harvested, and directly applied onto a column for magnetic enrichment of antigen-activated CD154+ T cells. The cells retained in the column are permeabilized, stained intracellularly for cytokines and CD154, and washed directly on the column to avoid centrifugation steps. Finally, CD154+ T cells are eluted from the column for subsequent flow cytometry analysis.
Procedure for the rapid antigen-reactive T cell enrichment (Rapid ARTE)2
A
B
Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Donor 6
w/o
JCV
LT
JCV
VP1
BK
V L
T
H1N
1 M
P1
H1N
1 N
P
EBV
BZL
F1
Ad
V5
Pent
on
CEF
MH
C C
lass
1
BK
V V
P1
CM
V IE
-1
CM
V p
p65
EBV
EB
NA1
EBV
LM
P2A
Ad
V5
Hex
on
% IF
N-γ
+ a
mon
g C
D4+
T c
ells
0.14
0.20
0.22
0.12
0.18
0.10
0.16
0.08
0.06
0.04
0.02
0.00
CD4+ T cells
Num
ber
of C
D15
4+C
D4+
T c
ells
enr
iche
d
from
1×1
0⁷ P
BM
C
2500
2400
2300800
700
600
300
500
200
400
100
0w/o antigen BKV LT + VP 1
Cell stimulation
Stimulate 1–2.5×10⁷ PBMC/mL/well with peptide pools in a 24-well plate. 6 h
Cell surface labeling and fixation
Remove 800 µL cell culture supernatant. Add antibodies for cell surface labeling (incl. CD154-Biotin).
Add Anti-Biotin MicroBeads.
Add 300 µL 3.7% Inside Fix.
2 min shaking + 3 min incubation
2 min shaking + 8 min incubation
2 min shaking + 13 min incubation
Enrichment of labeled CD154+ cells, permeabilization, and intracellular staining
Apply the fixed cell suspension onto the column placed in a separator. Wash with 500 μL of PEB buffer.
Wash with 500 μL Inside Perm. Apply CD154 and Anti-Cytokine antibodies for intracellular staining. Wash with 500 μL Inside Perm. Wash with 500 μL PEB buffer. 10 min incubation
Elution of CD154+CD4+ T cells
Remove column from the separator and place it on a suitable collection tube. Pipette 500 μL PEB buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
Flow cytometric analysis
Automated sample acquisition.Enumeration and characterization of antigen-activated T cells.
Hands-on time: 30 minTotal procedure: ≈ 7 h
Figure 2
A
w/o Antigen
CD
4
BKV LT BKV VP1
CD
8
IFN-γ
* among CD4+ T cells
** among CD8+ T cells
0.03%*
0.02%**
0.02%*
0.02%**
Conclusion• We developed a rapid (7 h) and convenient protocol
to detect and characterize very rare antigen-specific CD4+ T cell subpopulations in blood samples.
• The process does not involve any centrifugation step and requires only minimal hands-on time.
• High-resolution characterization of rare T cell repertoires is achieved combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154+ T cells.
• In contrast to conventional flow cytometry analysis, this approach enabled us to identify BKV-specific T cells and specify their function.
References1. Bacher, P. et al. (2013) J. Immunol. 190: 3967–3976.2. Bacher, P. et al. (2013) Cytometry A 83: 692–701.
Figure 4
B
w/o antigen
Bef
ore
enri
chm
ent
BKV LT and VP1
Aft
er e
nric
hmen
t
CD
154
IL-2
TNF-
α
IFN-γ IFN-γ TNF-α IFN-γ
IL-2
% C
ytok
ine+
am
ong
CD
154+
CD
4+ T
cel
ls
0
10
20
30
40
50
60
70
80
90
Donor A Donor B Donor C Donor D Donor E Donor F
not determined
IL-10
IFN-γ
TNF-α
* * * * * * *
w/o
JCV
LT
JCV
VP1
BK
V L
T
H1N
1 M
P1
H1N
1 N
P
EBV
BZL
F1
Ad
V5
Pent
on
CEF
MH
C C
lass
1
BK
V V
P1
CM
V IE
-1
CM
V p
p65
EBV
EB
NA1
EBV
LM
P2A
Ad
V5
Hex
on
% IF
N-γ
+ a
mon
g C
D8+
T c
ells
0.06
0.04
0.02
0.00
1.2
1.0
0.8
0.6
0.4
0.20.14
0.12
0.10
0.08
1.4
CD8+ T cells
*
*
*
*
* Gated on CD4+ T cells
