CD8[superscript +] T Cells from Mice Transnuclear for a
TCR that Recognizes a Single H-2K[superscript b]Restricted MHV68 Epitope Derived from gB-ORF8 Help
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Citation
Sehrawat, Sharvan, Oktay Kirak, Paul-Albert Koenig, Marisa K.
Isaacson, Sofia Marques, Gunes Bozkurt, J. Pedro Simas,
Rudolph Jaenisch, and Hidde L. Ploegh. “CD8+ T Cells from
Mice Transnuclear for a TCR That Recognizes a Single H-2KbRestricted MHV68 Epitope Derived from gB-ORF8 Help Control
Infection.” Cell Reports 1, no. 5 (May 2012): 461–471.
As Published
http://dx.doi.org/10.1016/j.celrep.2012.03.009
Publisher
Elsevier
Version
Final published version
Accessed
Fri May 27 00:04:56 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/91544
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Creative Commons Attribution
Detailed Terms
http://creativecommons.org/licenses/by-nc-nd/3.0/
Cell Reports
Report
CD8+ T Cells from Mice Transnuclear for a TCR that
Recognizes a Single H-2Kb-Restricted MHV68 Epitope
Derived from gB-ORF8 Help Control Infection
Sharvan Sehrawat,1,5 Oktay Kirak,4,5 Paul-Albert Koenig,1 Marisa K. Isaacson,1 Sofia Marques,2,3 Gunes Bozkurt,1
J. Pedro Simas,2,3 Rudolph Jaenisch,1 and Hidde L. Ploegh1,*
1Whitehead
Institute for Biomedical Research, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA
de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
3Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal
4Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
5These authors contributed equally to this work
*Correspondence: ploegh@wi.mit.edu
DOI 10.1016/j.celrep.2012.03.009
2Instituto
SUMMARY
To study the CD8+ T cell response against a mouse gherpes virus, we generated Kb-MHV-68-ORF8604–612
RAG/ CD8+ T cell receptor transnuclear (TN) mice
as a source of virus-specific CD8+ T cells. Kb-ORF8Tet+ CD8+ T cells, expanded in the course of a
resolving MHV-68 infection, served as a source of
nucleus donors. Various in vivo and ex vivo assay
criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively
in response to viral infection, helped control viral
burden, and exhibited a phenotype similar to that of
endogenous Kb-ORF8-Tet+ cells. When compared to
OT-1 cells, TN cells displayed distinct properties in
response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the
TCR expressed by the TN cells. The availability of
MHV-68-specific CD8+ TCR TN mice provides a new
tool for investigating aspects of host-pathogen interactions unique to g-herpes viruses.
INTRODUCTION
g-herpes viruses (g-HVs) are species-specific lymphotropic
viruses associated with cellular transformation and malignancies, especially in immunocompromised hosts (Barton et al.,
2011). Their restricted host range and the paucity of virus strains
that can infect laboratory animals have hampered studies of their
pathogenesis and of host correlates of protection. Consequently, potential vaccine strategies have suffered as well (Belz
et al., 2000; Woodland et al., 2001). Infection of laboratory
mice with murine herpes virus 68 (MHV-68) serves as an animal
model for investigating the pathogenesis and immunity induced
by human g-HVs such as Epstein-Barr virus (EBV) and human
herpes virus 8 (HHV-8) (Long et al., 2011). These events
resemble those elicited by human g-HVs (Flaño et al., 2002;
Nash and Sunil-Chandra, 1994; Simas and Efstathiou, 1998;
Wu et al., 2010). Considerable efforts have been expended to
identify anti-MHV-68 CD8+ T cell epitopes, to explore the timing
of their recognition, and to examine various parameters related
to the quality as well as magnitude of the virus-specific T cell
responses (Freeman et al., 2010; Fuse et al., 2007; GredmarkRuss et al., 2008; Liu et al., 1999a, 1999b; Molloy et al., 2011;
Obar et al., 2004a, 2004b; Virgin et al., 1997), but there are no
T cell receptor (TCR) transgenic mice that can serve as a source
of naive MHV-68-specific CD8+ T cells. Such animals would
allow us to more precisely address questions on the requirements for induction and maintenance of g-HV-specific effector
and memory CD8+ T cell responses.
MHV-68 open reading frame 8 (ORF8), glycoprotein B (gB),
induces an intermediate level of CD8+ T cell response (Gredmark-Russ et al., 2008). To measure magnitude and quality of
MHV-68-specific CD8+ T cell responses, we used somatic cell
nuclear transfer (SCNT) to generate ORF8-specific transnuclear
(TN) CD8+ T cells. The donor nucleus was isolated from KbORF8-tetramer (Tet)+ cells, expanded in the course of resolution
of the infection.
Using an adoptive transfer approach and a variety of assay
criteria, we demonstrate that TN cells on a RAG1/ background
respond well to both antigenic stimulation and lymphopenia, and
curtailed viral growth when adoptively transferred into mice prior
to infection. The TN cells exhibited a phenotype similar to that of
endogenous Kb-ORF8-Tet+ cells in the acute phase of infection,
and a subset of initially expanded TN cells generated memory.
When compared head-to-head, ORF8-specific TN cells proliferated more rapidly than transgenic OT-1 cells that are restricted
by the same MHC element. ORF8 TN mice most closely approximate a component of the normal CD8+ T cell response against
MHV-68 and are thus a useful tool for exploring the unique
features of host defense against g-HVs.
RESULTS
Generation of ORF8 TN Mice and Identification
of TCR in ORF8 TN Cells
The generation of TN mice specific for the ORF8 epitope
(KNYIFEEKL) was done essentially as described by Kirak
et al. (2010), using the corresponding tetramers to isolate
Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors 461
A
B
Sort
Kb-ORF8- Tet +
cells
MH V-68 infection
of (BALB/ c x C57BL/6)
F1 mice
Transfer nucleus
into enucleated
oocyte
Identify TCR and
characterize cells
V gene/allele
ESC derivation
Birth of
chimeric mice
Breeding
with Rag1-/-C57BL/6
mice
TCR alpha
TCR beta
TRAV10N*01
TRBV31*01
D gene/allele
---
J gene/allele
TRAJ33*01
TRBJ2 -2*01
C gene/ allele
TRAC ( n.d.)
TRBC2*03
Peptide recognized
ESC injection of
Blastocyst
TRBD1*01
KNYIFEEKL
E
C
0.0662
1.72
Pooled LN and Spleen
CD8
CD3
0.87
10.6
12.5
ORF8 TN
68.4
29.8
71.6 3.77
CD4
B220
Thymus
5.23
Kb-ORF8-tet
10.2
85.2
24.8
27.4 1.37
0.99
16.9
30.9 15.9
81.8
0.538
CD4
0.0358
0.722
CD8
CD8
6.38
H-2Kb
93
V 14
G
K NY I F E E K L
F
10
6
4
4
3
2
1
0
KNYIFEEKL
ANYIFEEKL
KAYIFEEKL
KNAIFEEKL
KNYAFEEKL
KNYIAEEKL
KNYIFAEKL
KNYIFEAKL
KNYIFEEAL
KNYIFEEKA
KNYVFEEKL
KQYIFEEKL
KNFIFEEKL
KNYLFEEKL
KNYIFDEKL
KNYIFEDKL
KNYIFEERL
4
3
2
1
0
EC50 IFN-g
(Fold change)
10000
8000
6000
4000
2000
8
EC50 H-2Kb
(Fold change)
400
IFN- +
H-2Kb
IL-2 conc. (pg/ml)
46.1
300
200
100
0
KNYIFEEKL
ANYIFEEKL
KAYIFEEKL
KNAIFEEKL
KNYAFEEKL
KNYIAEEKL
KNYIFAEKL
KNYIFEAKL
KNYIFEEAL
KNYIFEEKA
KNYVFEEKL
KQYIFEEKL
KNYIFDEKL
KNYIFEDKL
KNYIFEERL
52.2
H-2Kd
Kb-ORF8-tet
0.919
OT1Tg
D
APL
APL
Figure 1. Generation of Kb-MHV-68-ORF8 TCR TN Mice and Defining the Fine Specificity of TCR
(A) Schematic for the generation of Kb-MHV-68-ORF8 CD8+ TCR TN mouse lines.
(B) The gene and allele usage by a and b chains of TCR of MHV-68-ORF8 CD8+ TN T cells is tabulated.
(C and D) Peripheral blood samples collected from Kb-MHV-68-ORF8 CD8+ TCR TN RAG/ mice were analyzed flow cytometrically using the indicated panel of
surface markers. FACS plots for gated lymphocyte populations are shown. The results indicate profound skewing toward CD8+ T cell subset and their homogeneous staining with Kb-ORF8 tetramer, H-2Kb, and Vb14 TCR.
462 Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors
ORF8-specific CD8+ T cells from MHV-68-infected (Balb/c 3
C57B6) F1 mice at 20 days postinfection (Figure 1A). F1 mice
were used for infection and isolation of Tet+ cells because
CD8+ T cells from such F1 mice are an efficient source of donor
nuclei to generate embryonic stem cells (ESCs) (Kirak et al.,
2010). We obtained ESCs from blastocysts and used them to
generate chimeric mice. Chimeric mice not exposed to MHV68 were then tested for the presence of Kb-ORF8-Tet+ cells as
an indicator for cells bearing the functionally rearranged TCR
a and b pair. In naive chimeric mice, around 3% of their total
CD8+ T cells were Kb-ORF8-Tet+, as compared to WT C57BL/6,
which showed less than 0.02% (naive), and 2.0% of KbORF8-Tet+CD8+ T cells after 21 days of MHV-68 infection. We ascertained germline transmission by backcrossing the chimeric
mice to C57BL/6 and checking for agouti coat color of the
offspring. The presence of the recombined TCR a and b in each
of the offspring was analyzed by flow cytometry using Kb-MHV68-ORF8 tetramers. Because TCR a and TCR b segregate independently, 25% of the offspring should inherit both of the
rearranged TCR alleles. By screening offspring from the chimera,
backcrossed onto the C57BL/6 background for multiple generations, we identified several animals with large numbers of
Kb-ORF8-Tet+ CD8+ T cell populations in peripheral blood
and lymphoid organs in the absence of virus infection, thus
establishing germline transmission of the TCR a and TCR b pair
(Figures S1A and S1B).
The identity of a and b chains of the TN TCR was established
by 50 rapid amplification of cDNA ends (50 RACE)-PCR, cloning,
and DNA sequence analysis. The gene segments used and the
genomic organization for the rearranged V and J regions (a chain)
and V, D, and J regions (b chain) are shown in Figures 1B and
S1C. The a chain uses TRAV10N*01, TRAJ33*01, and the b chain
uses TRBV31*01, D1*01, and TRBJ2-2*01 (Figure 1B). The
mechanism underlying recombination for the b chain is therefore
by inversional joining, as depicted in Figure S1C. The nucleotide
and predicted amino acid sequence for the complementaritydetermining regions (CDRs) 3 for a and b chains, as well as the
complete sequence for recombined segments of the ORF8 TN
cell TCR, are shown in Figure S1D.
Phenotypic Characterization of Kb-ORF8 TN RAG/
Mice
Mice positive for the rearranged ORF8-specific TCR were
crossed to obtain ORF8 TN RAG/ mice. Mice that inherited
both the rearranged a and b genes on a RAG/ background
had exclusively ORF8-specific CD8+ T cells, with only a small
population of CD4+ T cells remaining (Figure 1C, middle panel).
All ORF8-specific (Tet+) CD8+ T cells expressed Vb14 /TRBV31
(Figures 1C and 1D). We compared the cellular distribution in
the thymus and peripheral lymphoid organs of ORF8 TN and
OT-1 transgenic mice (Figure 1E). OT-1 Tg RAG/ mice showed
25% single-positive (SP) CD4+, 30% SP CD8+, 30%
double-positive (DP) CD4+CD8+ lymphocytes, whereas ORF8
TN RAG/ mice showed 5% SP CD4+, 12% SP CD8+,
70% DP CD4+CD8+ lymphocyte population (Figure 1D).
Thus, ORF8 TN RAG/ mice exhibited a thymic cellular distribution pattern distinct from that of OT-1 Tg RAG/ mice but similar
to that of WT mice (Figure S2A). This result might reflect
a different lymphoid developmental history imposed by transgenesis, possibly because of transgene copy number and site
of transgene integration. In the peripheral lymphoid organs of
OT-1 Tg RAG/ as well as ORF8 TN RAG/ mice, the majority
of lymphocytes were CD8+ T cells (Figure 1E). In peripheral
lymphoid organs, CD4+CD8 cells were about 1%–2% in OT-1
RAG/ mice and 2%–4% in ORF8 TN RAG/ mice. All of the
CD4+ in cells in ORF8 TN RAG/ mice were Vb14 and KbORF8-Tet+ (Figure S2B), albeit at lower staining intensity, consistent with a contribution of class I MHC-CD8 interactions in determining the strength of tetramer staining.
Identification of the Anchor Residues of the ORF8
Peptide for MHC Class I and TCR Binding
We modeled how the ORF8 peptide (KNYIFEEKL) binds to
H-2Kb, in comparison with two other H-2Kb restricted epitopes
(Figure S2C) (Mareeva et al., 2008). In order to identify the anchor
and contact residues of the ORF8 epitope (KNYIFEEKL) for
H-2Kb and the TCR, respectively, we examined a series of
peptide variants (altered peptide ligands [APLs]). These APLs
were tested for their ability to stabilize H-2Kb expression on the
surface of TAP-deficient RMA/S cells at 37 C as an indicator of
their binding to H-2Kb (Figure 1F). The class I MHC anchor residues were phenylalanine at position 5 (F5), leucine at position 9
(L9), and tyrosine at position 3 (Y3) in decreasing order of
strength. To assess the stimulatory ability of ORF8 APLs, TN
cells activated in vivo by MHV-68 infection were stimulated
with APLs ex vivo, and an intracellular cytokine staining (ICCS)
assay was used to measure IFN-g producing cells. Residues
important for TCR engagement in this setting included both
MHC anchor residues and TCR contact residues. Accordingly,
we identified Y3, F5, aspartic acid at position 6 (E6), isoleucine
at position 4 (I4), and L9. Positions 5 and 9 are major MHC anchor
residues and account for their involvement in stimulating TN
cells. The main TCR contacts are I4 and glutamic acid at position
6 and/or 7 (E6/E7). That I4 and E6/E7 likely contact the TCR was
confirmed by measuring the levels of IL-2 in culture supernatants
produced by TN cells stimulated either with index peptide or
(E) A comparison of distribution of CD4+ and CD8+ T cells in the thymus (left panel) and pooled lymphoid organs (right panel) of Kb-MHV-68-ORF8 TCR TN RAG/
mice and Kb-Ova TCR Tg (OT-1) RAG/ mice is shown.
(F) Upper panel is a representation depicting the involvement of various amino acid residues of KNYIFEEKL peptide in binding to H-2Kb molecule (red) and TCR
(green) as determined by class I stabilization on RMA/S cells and ability to induce IFN-g production by activated TN cells. The font size represents the influence on
strength of binding. Lower panel represents H-2Kb stabilizing ability on the surface of RMA/S cell (red bars) and ORF8 TN CD8+ T cell stimulatory capacity (green
bars) of ORF8 (APLs), shown by fold change in the EC50 of the respective APL as compared to the index peptide, KNYIFEEKL.
(G) The levels of IL-2 in the culture supernatant of in vivo-activated ORF8 TCR TN CD8+ T cells and BMDCs pulsed with index peptide and APLs after 3 days of
coculture are shown. All experiments were repeated three to four times. The results are represented as mean ± SEM.
See also Figure S1.
Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors 463
1
+
CD45.2 TN cells into
naive CD45.1 B6 mice
(4-35)
Analysis
MHV-68 infection
(1x10 6pfu ip)
B
C
00
00
0
00
00
Unstimulated
1.09
0.52
75.5
22.9
+
50,000 CD45.2 ORF8 TN
cells transferred
60
40
KNYIFEEKL
20
0
0
10
21
50
0
0
00
73.6
15.1
10
10
17.2
20
00
71.7
CD8
30
50
CFSE
40
0
0.047
00
99.5
11
15
CD45.2
0.097
50
15
50,000 CD45.2 ORF8 TN
cells transferred
0
+
+
% CD45.2 of total CD8 cells
5.5dpi
50
Input cells
E
D
IFN-γ
0
+
+
% CD45.2 of total CD8 cells
Days
A
no of transferred CD45.2+ TN cells
days post infection
1.01
8.31
83.2
7.43
CD45.2
CD44
G
99.1 0
1.48
12.8
98.8 0
87.2
52.2
77.2
1.26
15.1
CD8
CD8
0
6.27
98.6
70.3
0.909
CD45.2
+
Kb-ORF8-Tet gate
99.2
0.0959 6.1
7.54e-3
27.5
Donor cells
CD45.2
1.18 0
Kb-ORF8-Tet
Kb-ORF8-Tet
0.905 0
0
MHV-68 (WT)
MHV-68 (A9)
Uninfected
0
Kb-ORF8-Tet
F
97.4
Host cells
0.0226
2.62
CD8
92.4 0.213
98.5 79.8
19.2
1.33 6.46e-3
1.24 0.674
0.353
CFSE
Host cells
0.666
0.0113 0.149
PD1
Control
+
H
1.35
79.6
CD8
18.9
9
0
96.7
0
CD8
3.28
8
I
CD127
0.0903
Kb-ORF8-Tet
CD45.2
CD45.2
CD44
CD62L
gate
0
64.6
0
CD8
35.4
4
0.0455
CD62L
CD45.2
Donor cells
4.06
CD44
12
83.9
+
Kb-ORF8-Tet gate
0
0.209
1.4
0
30.1
3.05
31.6
34.6
30.8
CD8
99.8
0
CD8
98.6
Host cells
0
CD8
69.9
CD45.2
0
CD45.2
Donor cells
Control
Kb-ORF8-Tet
0
CD127
Figure 2. In Vivo Responsiveness of Adoptively Transferred MHV-68-ORF8-Specific TCR TN CD8+ T Cells upon MHV-68 Infection
Graded numbers of ORF8 TN cells were transferred into CD45.1 mice 1 day before i.p. infection with 1 3 106 pfu of MHV-68, and the fate and functionality of
transferred cells were analyzed at different time points after infection.
(A) A schematic of the experiments is shown.
464 Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors
ORF8 APLs (A4, A6/A7, or D6/D7)-pulsed bone marrow-derived
dendritic cells (BMDCs) for 3 days. Thus, lower levels of IL-2
were produced by TN cells stimulated by APL-pulsed BMDCs
compared to controls (Figure 1G). We conclude that residues
F5 and L9 act as strong anchors, whereas I4 and E6/E7 are
most critical in making contacts with the TCR. From these experiments we identified APLs that were equipotent in stabilization of
(binding to) H-2Kb but with inferior T cell stimulatory capacity
(e.g., KNYAFEEKL). Other peptides were far worse at stabilizing
H-2Kb but were still more potent at eliciting IFN-g production
(e.g., KQYIFEEKL).
In Vivo Responsiveness of ORF8 TN CD8+ T Cells
after MHV-68 Infection
To investigate the responsiveness of ORF8-specific CD8+ TN
cells to virus infection, graded numbers of purified carboxyfluorescein succinimidyl ester (CFSE)-labeled or unlabeled
CD8+ T cells from naive ORF8 TN mice (CD45.2+) were transferred into CD45.1+ C57BL/6 mice, which were then infected
with MHV-68 intraperitoneally (i.p.) (Figure 2A). After 5.5 days,
we measured the dilution of CFSE staining, an indicator of
proliferation and activation markers such as CD44 in the transferred TN CD8+ T cells (Figure 2B). At this time, infected mice
do not show significantly expanded populations of endogenous
ORF8-specific cells (Gredmark-Russ et al., 2008), and almost
all of the transferred cells homogeneously stained for
Kb-ORF8-Tet (Figure S2D). The transferred cells underwent
multiple rounds of division and upregulated the activation
marker CD44 (Figure 2B). Expansion of TN donor cells was
seen even when as few as 500 cells were transferred (Figure 2C). As expected, this expansion increased with the
number of cells transferred, the maximum occurring upon
transfer of 10,000–50,000 TN cells in different experiments (Figure 2C). A transfer of even greater numbers of TN cells resulted
in a decrease in the frequencies of expanded donor cells,
possibly because of limiting availability of antigen or cytokines,
an issue not investigated further. The expanded TN cells were
variably distributed over most of the lymphoid organs analyzed
(data not shown).
In a separate set of experiments, we examined the kinetics of
induction, expansion, as well as contraction of ORF8 TN cells in
response to infection. We transferred 50,000 ORF8 TN cells into
CD45.1+ mice, which expanded until day 6–7, and then rapidly
declined in numbers (Figure 2D). After 25 days, 2%–4% of
the transferred cells that responded to viral infection persisted
(Figure 2D). To investigate functional properties of ORF8 TN
cells, we assessed their ability to produce IFN-g in response to
infection. To this end, 50,000 naive TN cells were transferred
into CD45.1+ mice, which were then infected with MHV-68.
Five days later, spleen cells isolated from these mice were
analyzed by ICCS. Although very few of the unstimulated cells
scored positive for IFN-g production, up to 70%–75% of TN cells
produced IFN-g upon peptide stimulation (Figures 2E and S2E).
As a convincing demonstration of the fine specificity of ORF8 TN
cells, we infected mice that had received 5 3 105 of CFSElabeled ORF8 TN cells with a mutant strain of MHV-68, created
to carry a substitution in one of the anchor residues of the
ORF8-derived peptide (MHV-68-ORF8 [L9A]) (Figure 2F). This
mutation does not affect virus growth in vitro and in vivo
(J.P.S., unpublished data).
The transferred TN cells proliferated extensively upon infection
with WT MHV-68 (Figure 2F, right panel), but fewer cells divided
when mice were infected with the MHV-68-ORF8 (L9A) mutant
virus (Figure 2F, middle panel). After infection with MHV-68ORF8 (L9A) mutant virus, endogenous ORF8-specific CD8+
T cells did not expand significantly, whereas those specific for
other MHV-68 epitopes (derived from ORF61 and ORF75c)
expanded at similar levels (Figure S2F). The mutation in anchor
residue (L9A) therefore did not compromise viral growth in vivo
and, with the exception of the absence of ORF8-specific CD8
T cells, induced T cell responses indistinguishable from WT virus.
We also measured the phenotype of TN cells at the peak of
their response at day 7–8 postinfection, and in the contraction
as well as in the early memory phase, i.e., 18 and 35 days postinfection (dpi), respectively (Figures 2G–2I). At 8 dpi, similar to
the endogenous ORF8-specific cells, all TN cells were activated,
i.e., they upregulated PD1 and CD44 and downregulated
CD62L (Figure 2G), demonstrating that TN cells behave similar
to endogenous cells and therefore could be used as an alternative to investigate responsiveness of endogenous cells upon
virus infection. At 18 dpi, large numbers of donor TN cells were
CD44hi, CD62Llo, and of the surviving TN cells, more than 50%
cells expressed CD127 (IL-7Ra), a marker for memory cells (Figure 2H). In the early memory phase of the response (35 dpi),
(B) A total of 50 3 103 CFSE-labeled TN cells were transferred into CD45.1+ mice (n = 4) before MHV-68 infection and the proliferation as measured by the extent of
CFSE dilution (upper panel) and activation as measured by CD44 upregulation (lower panel) of gated CD45.2+ transferred TN cells at 5.5 dpi. Representative
FACS plots are shown.
(C) Expansion of transferred TN cells as a function of input cell numbers was measured at 5.5 dpi. Indicated numbers of TN cells were transferred into three mice of
each group.
(D) Kinetics of the expansion of TN cells is shown when 50 3 103 cells were transferred before infection. At each time point, spleens of three mice were analyzed.
(E) IFN-g producing ability of unstimulated and KNYIFEEKL peptide (1 mg/ml) stimulated ORF8 TN cells isolated from the spleens of MHV-68-infected mice at
5.5 dpi as measured by ICCS assays is shown.
(F) CFSE-labeled ORF8 TN cells (5 3 105) were transferred into B6 mice (n = 12), which were divided then into three groups. One group of mice served as
uninfected control; the other two groups of mice were infected with either 5 3 105 pfu of MHV-68 (WT) or 5 3 105 pfu of MHV-68-ORF8 (LA9). The frequencies and
the extent of proliferation of transferred TN cells were measured at 6.5 dpi.
(G) A total of 50 3 103 ORF8 TN cells were transferred in congenic CD45.1+ mice 1 day prior to MHV-68 infection with 1 3 106 pfu i.p. FACS plots show the
phenotype of transferred ORF8 TN and endogenous Kb-ORF8-Tet+ cells at 7 dpi.
(H and I) Phenotypic characterization of TN cells isolated from spleens of MHV-68-infected mice at18 dpi (H) and 35 dpi (I) as measured by the surface expression
of indicated activation and memory markers.
See also Figure S2.
Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors 465
Days
A
1
6-7
Infected with MHV-68
5x10 6 CFSE hiKNYIFEEKL
+
5x10 6 CFSE lo )
0
Transfer
ORF8 TN
Rag-/- cells
(ip or in)
B
2hr post
transfer
C
uninfected
infected with MHV68
44.3 57.2
42.8 60.7
89
39.3
%specific lysis
55.7
50x10 3 CD8 TN cells
500 CD8 TN cells
No transfer
* P = 0.03
100
11
*** P = 0.003
80
60
40
20
ll s
ce
03
TN
x1
x1
50
10
5
2
1
TN
6
10
4x
TN
6
10
ce
ce
tro
l
lls
lls
0
1x
10 6
TN
ce
lls
lls
ce
4x
1x
10 6
TN
sf
er
0
10
on
5000
** (p = 0.005)
* (p = 0.04)
15
C
10000
pfu/g of lung tissue (log10)
** (p = 0.006)
an
23.7
600000
400000
200000
Tr
26.3 48.5
25.1
o
37.6 64.7
4x10 6TN cells
7.98 2.74
*** (p = 0.0004)
N
CD44
1x10 6TN cells
1.16 1.04
no of Kb-ORF8-Tet+ CD8+cells
No Transfer
Kb-ORF8-Tet
F
E
60.7
03
TN
ce
TN
2
10
5x
no of ORF8 TN cells
D
0.54
ce
lls
r
fe
ns
tra
no
CFSE
lls
0
transferred ORF8 TN cells
no of ORF8 TN cells
Figure 3. MHV-68-ORF8-Specific TCR TN CD8+ T Cells Protect Mice from MHV-68 Infection
(A) Schematic for measuring the in vivo CTL activity of ORF8 TN cells is shown. Graded numbers of ORF8 TN CD8+ T cells were transferred into C57BL/6, which
were then infected with 1 3 106 pfu of MHV-68 i.p. or 1 3 105 pfu i.n. At 6.5 dpi, 5 3 106 peptide-pulsed CD45.1+ splenocytes labeled with high concentrations of
CFSE and 5 3 106 control CD45.1+ splenocytes labeled with low concentrations of CFSE were transferred intravenously. The proportion of CFSEhi and CFSElo
cells among CD45.1+ cells was measured 2 hr after transfer, and the percent-specific lysis was calculated as described in Experimental Procedures section.
(B) Representative FACS plots show the proportions of high and low CFSE-positive cells in indicated groups of mice.
(C) Bar diagram shows the percent-specific lysis in different groups of mice. Statistical significance was determined by unpaired Student’s t test. For each group
four to five mice were analyzed, and experiments were repeated three to four times.
(D–F) Graded numbers of ORF8 TN cells were transferred into C57BL/6 mice (n = 20) 1 day prior to IN infection with 1 3 105 pfu of MHV-68. At 6 dpi, draining
mediastinal LN and lungs were isolated from PBS-perfused mice and analyzed for the activation and expansion of transferred TN cells and viral burden,
respectively. (D) Representative FACS plots show the frequencies of expanded Kb-ORF8-Tet+ cells in the mediastinal LNs of control and recipient mice. (E) Total
number of expanded Kb-ORF8-Tet+ cells in the mediastinal LN of control and recipient mice is shown. Statistical significance was determined by ANOVA test. (F)
Virus load in the lung homogenates of control and TN cell recipient mice as measured by plaque assays is shown. From each group five mice were analyzed.
Statistical significance was determined by ANOVA test, and data are represented as mean ± SEM.
almost all of the TN cells expressed CD127, a well-recognized
marker of antigen-specific memory CD8+ T cells (Kaech et al.,
2003), but only about 50% of the host cells scored positive for
this marker (Figure 2I).
466 Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors
ORF8 TN Cells Exhibit Cytolytic Activity and Control Viral
Burden
To assess the protective ability of TN cells, we performed in vivo
cytolysis assays (Figure 3). Although few ORF8 peptide-loaded
cells were killed in mice that did not receive TN cells, the
disappearance of the target population was evident upon
transfer of as few as 500 TN cells, and was more prominent
with the transfer of greater numbers of cells (Figures 3B and
3C). These results demonstrate that ORF8 TN cells kill target
cells in vivo.
To investigate if TN cells contribute to the control of viral
growth in MHV-68-infected mice, we transferred 1 3 106 and
4 3 106 ORF8 TN cells into C57BL/6 mice 1 day prior to intranasal (IN) infection with MHV-68 (Figures 3D–3F). At 6 dpi, we
measured the frequencies as well as the absolute numbers
of Tet+ cells in the draining mediastinal LN. Viral titers in the
lungs were measured by plaque assay. Although only 1%–2%
of CD8+ T cells were ORF8 Tet+ in infected controls that
did not receive ORF8 TN cells (Gredmark-Russ et al., 2008),
high relative frequencies and numbers of ORF8 Tet+ CD8+
T cells were present in the mediastinal LN of mice that received
TN cells (Figures 3D and 3E). Most of the ORF8 Tet+ cells
expressed high levels of CD44, consistent with their activation
(Figure 3D). Viral titers were reduced by 100- to 1,000-fold in
mice that received ORF8 TN cells compared to controls (Figure 3F). ORF8 TN cells thus confer a measure of protection
against MHV-68 infection, even though only a single CD8+
T cell H-2Kb restricted epitope out of the 40 currently known
(Freeman et al., 2010; Gredmark-Russ et al., 2008) is being
targeted.
Comparison of the Responsiveness of Kb-ORF8 TCR TN
and Kb-OT-1 Tg CD8+ T Cells
To test and compare the proliferation of ORF8 TN cells and OT-1
TCR transgenic cells in response to a lymphopenic environment,
a setting that generates memory-like cells (Goldrath et al., 2000),
2 3 106 CFSE-labeled cells of each were transferred separately
into C57BL/6 RAG/ and CD45.1 WT mice (Figures 4A–4D).
After 10 days we assessed proliferation of the transferred cells,
isolated from the lymphoid organs of recipient mice. Although
in WT mice very few ORF8 TN or OT-1 Tg cells divided, a large
majority of both cell types underwent multiple rounds of division
in RAG/ mice (Figure S2G). Compared to OT-1 cells, fewer
ORF8 TN cells divided (Figure 4B, upper panel, and Figure 4C).
Because homeostatic expansion in response to lymphopenia is
believed to be driven by cross-reactivity with self-peptideMHC complexes (Murali-Krishna and Ahmed, 2000), ORF8 TN
cells are apparently less reactive to such self-antigens. Similar
results were obtained when 1 3 106 each of CFSE-labeled
ORF8 TN cells and OT-1 1 Tg cells were transferred into the
same RAG/ recipients, and proliferation of Kb-SIIN-Tet+ and
Kb-KNYI-Tet+ CD8+ T cells at 20 days post transfer was
measured (Figure 4D). However, as shown below, this is not
due to an intrinsic proliferative superiority of OT-1 Tg cells. To
further demonstrate that self-reactivity drives lymphopeniainduced proliferation of both ORF8 TN and OT-1 Tg cells, we
transferred equal numbers of ORF8 TN or OT-1 Tg cells into
TAP/ mice. TAP/ mice are largely defective in loading class
I MHC molecules with self-peptides, and consequently, upon
adoptive transfer, only few CD8+ T cells persist in the periphery
(Van Kaer et al., 1992). Even 20 days post-transfer in TAP/
mice, neither ORF8 TN nor OT-1 Tg cells divided (Figure 4B,
lower panel, and Figure 4D). These findings underscore the relevance of exploring the functional properties of CD8+ T cells for
more than a single TCR.
Our results show that ORF8 TN cells proliferated to a lesser
extent in a lymphopenic environment. Are ORF8 TN cells intrinsically hyporeactive when compared to OT-1 cells? To answer
this question, we compared their antigen-specific responses
under similar conditions of stimulation (Figures 4E–4I). We first
measured the relative abilities of the KNYIFEEKL and SIINFEKL
to stabilize surface H-2Kb on RMA/S cells. SIINFEKL peptide
stabilized surface H-2Kb more efficiently than KNYIFEEKL
peptide (Figures S2H and S2I). Thus, a 2- to 3-fold lower concentration of SIINFEEKL peptide was required to stabilize similar
levels of H-2Kb, compared to KNYIFEEKL peptide. At saturating
peptide concentrations (200 mg/ml = 169 mM of KNYIFEEKL and
208 mM of SIINFEKL), equivalent levels of H-2Kb were recorded.
Therefore, BMDCs pulsed with these concentrations of the
respective peptides were cocultured with CFSE-labeled naive
ORF8 TN or OT-1 Tg T cells. Although few if any of the ORF8
TN or OT-1 cells divided when cocultured with control BMDCs,
both ORF8 TN and OT-1 Tg CD8+ T cells underwent multiple
rounds of division when cultured with peptide-pulsed BMDCs
(Figures 4E–4G). The majority of OT-1 cells upregulated CD69
after 24 hr of coculture with BMDCs loaded with SIINFEKL
peptide, whereas about 50% of ORF8 TN cells upregulated
CD69 (Figure 4E). At later time points, both the ORF8 TN and
OT-1 Tg CD8+ T cells that were CD69+ started dividing. Thus,
at 36 hr post-coculture, 40% of ORF8 TN cells but only
25% of OT-1 Tg cells divided; these differences became
more pronounced at 48 hr (70% of ORF8 TN versus 35% of
OT-1 cells) and 72 hr (80%–90% of ORF8 TN versus 60% of
OT-1 cells). Both populations of dividing cells downregulated
CD69 and CD62L and upregulated CD44, with slower kinetics
for OT-1 Tg cells (Figures 4E–4G). The differences in responsiveness of ORF8 TN and OT-1 Tg cells were also evident from the
IL-2 levels in the culture supernatants (Figure 4H): only at 96 hr
did the OT-1 Tg T cells catch up with ORF8 TN T cells. Antigenic
stimulation thus produces subtly different outcomes for ORF8
TN and OT-1 Tg cells. If anything, ORF8 TN cells were superior
in their activation and proliferative potential as measured
ex vivo. Could such differences be attributable to differences in
affinity of the respective TCRs? We performed tetramer dissociation assays as a surrogate measure for TCR affinities. As shown
in Figure 4I, OT-1 Tg cells lost tetramer staining more rapidly and
to a greater extent than did ORF8 TN cells, showing that the
avidity with which ORF8 TN cells recognize peptide/class I
MHC tetramers is higher than for OT-1 Tg cells. However, TN
and transgenic CD8+ T cells may intrinsically differ in other properties because inherently different methodologies were used for
obtaining them.
DISCUSSION
We generated Kb-MHV-68-ORF8604–612 TCR TN mice by SCNT
to obtain a source of CD8+ T cells that recognizes a peptide
derived from the ORF8 protein of MHV-68 (Gredmark-Russ
et al., 2008). The nucleus from a virus-specific T cell is harvested
and then reprogrammed to generate ESCs. The antigen-specific
Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors 467
Figure 4. Comparison of the Responsiveness of Kb-ORF8-Tet+ TN and Kb-OT-1- Tg Cells
(A) A schematic of the experiments performed to measure lymphopenia-induced proliferation is shown. A total of 2 3 106 CFSE-labeled or unlabeled ORF8 TN or
OT-1 cells were transferred into RAG/ and TAP/ mice.
(B) The FACS plots show the frequencies of CD8+ T cells in pooled LNs of recipient mice at 20 days post-transfer.
(C) The quantitation of the frequencies of CD8+ T cells in the lymphoid organs of recipient RAG/ and TAP/ mice is shown. In each group three to four
mice were used, and experiments were repeated two times. Statistical significance was determined by Student’s t test. Values are represented as mean ± SEM.
**p % 0.01. ns, not significant.
468 Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors
T cells are isolated directly from the infected mouse, and no
in vitro culture or restimulation with antigen or coreceptors is
required. The resulting ESCs, which carry the rearranged TCR
a and b genes of the donor T cell, are then used to generate
mice that are TN for the TCR in question and express the
rearranged TCR genes of the donor cell (Kirak et al., 2010). These
rearranged TCR genes are expressed from the endogenous
loci under the control of their endogenous promoters, and
without genetic scars other than those resulting from the physiological TCR rearrangement process. Accordingly, in mature
T cells the pattern of TCR expression should resemble that
of normal T cells as closely as possible. Indeed, we note
that the distribution of the intermediate stages of T cell development found in the thymus of ORF8 TN mice more closely
approximates a normal profile than what we observed for the
OT-I transgenics. Nonetheless, precocious expression of the
TCR in the course of T cell development is an unavoidable
outcome for transgenic mice—and (presumably) for TN mice
as well.
The selection of the ORF8 epitope of MHV-68 was based on
the notion that CD8+ T cell responses against MHV-68 are
directed toward many epitopes in C57BL/6 mice (Freeman
et al., 2010; Gredmark-Russ et al., 2008; Liu et al., 1999a) and
that a focus on a more frequently recognized epitope might
bias the overall response against the pathogen in question. We
established functionality and protective ability of these TN cells
in vivo as well as using a panel of ex vivo assays, discussed
below. We could thus compare the responsiveness of ORF8
TN and OT-1 Tg cells in different settings. Compared to OT-1
cells, TN cells displayed different kinetics of activation, proliferation, and cytokine production, possibly due to the higher affinity
of their TCR as approximated in a tetramer dissociation assay.
CD8+ T cells obtained from ORF8 TN mice expanded upon viral
infection and decreased the viral burden. This is all the more
remarkable where the ORF8-derived epitope is only 1 of more
than 40 H-2Kb and 20 H-2Db restricted epitopes currently
known for MHV-68 (Freeman et al., 2010; Gredmark-Russ
et al., 2008). This also suggests that activated CD8+ T cells
specific for rare antigens, if expanded in a timely manner, might
help control infection. Recognition of a single viral epitope, as
might be achieved by an appropriate vaccination strategy, may
be sufficient to afford protection against virus infection and
perhaps subsequent latency. Human CD8+ T cells specific for
either dominant or subdominant epitopes were equally potent
in terms of their functional properties (Speiser et al., 2011). The
use of immunodominant epitopes for vaccination purposes
may not always be required, or even be the best approach,
because this may lead to T cell exhaustion or may sometimes
cause immunopathologies (Rickinson and Moss, 1997; Rouse
and Sehrawat, 2010; Ruckwardt et al., 2010; Virgin et al., 2009;
Welsh and Fujinami, 2007).
The activation profile of TN cells was similar to that of endogenous CD8+ T cells specific for the same pathogen-derived
epitope in the acute phase of resolving an MHV-68 infection.
TN cells may therefore represent the closest possible approximation of the endogenous responses. A pathogen that causes
a persistent infection, such as MHV-68, may continue to shed
low levels of antigen that can activate recent thymic emigrants.
These cells likely cover a spectrum of affinities for their respective antigen and cause them to respond to viral antigens with
different kinetics (Freeman et al., 2010; Gredmark-Russ et al.,
2008; Liu et al., 1999a). Moreover, the endogenous response
against persistent pathogens is usually polyclonal in nature
(Rickinson and Moss, 1997; Welsh and Fujinami, 2007). CD8+
T cells specific for viral antigens released during persistent infection may exhibit heterogeneity also in their surface molecules, as
was evident from our results with ORF8 TN cells at least at day 18
after infection (Figure 2H). Others have also reported similar
results with endogenous cells in the later stages of MHV-68
infection (Cush et al., 2007). Obviously, additional lines of TN
mice may be required to achieve more complete coverage of
the CD8+ T cell response.
The memory response against pathogens that persist and
release low levels of antigens intermittently is important because
lifelong control of the infection is needed for the survival of host
(Gray, 2002; Horst et al., 2011; Klenerman and Hill, 2005; Morens
et al., 2004; van der Burg et al., 2011). Our results show that
almost all of the TN cells exhibited a memory phenotype
(CD127+) when investigated early in the memory phase, whereas
only about 50% of endogenous cells expressed this marker. We
also observed that the peak expansion of ORF8 TN cells was
a few days earlier as compared to endogenous virus-specific
CD8+ T cells. The recall response of ORF8 TN memory cells is
not so easy to study because the natural immune response
against MHV-68 includes the generation of neutralizing antibodies, the presence of which precludes a simple challenge
experiment with the same virus (Andreansky et al., 2004). Our
TN models may provide a stepping stone to explore in greater
detail these and related issues in infection and vaccination
against persistent viral infections.
(D) A total of 1 3 106 CFSE-labeled ORF8 TN admixed with equal numbers of CFSE-labeled OT-1 Tg cells were transferred into same RAG/ mice. At 20 days
after transfer, CFSE dilution in Kb-ORF8-Tet+ and Kb-SIIN-Tet+ was analyzed. Thick and thin lines in FACS plots represent the CFSE staining in ORF8 TN cells and
OT-1 Tg cells, respectively.
(E–I) Comparison of activation and proliferative capacities of OT-1 Tg and ORF8-TN cells. MACS-purified OT-1 and ORF8 TN cells were cocultured for varying
time periods with BMDCs pulsed with 200 mg/ml of SIINFEKL or KNYIFEEKL peptide, respectively. The levels of CD69 expression and the extent of CFSE dilutions
were measured at indicated time points. (E) Representative FACS plots for ORF8 TN (upper panel) and OT-1 (lower panel) cells are shown. (F) FACS plots for the
levels of CD62L expression versus CFSE staining in ORF8 TN (upper panel) and OT-1 (lower panel) cells at 72 hr after coculture are shown. (G) FACS plots for the
levels of CD44 expression versus CFSE staining for ORF8 TN and OT-1 cells at 96 hr after coculture are shown. (H) IL-2 levels in the culture supernatants of ORF8
TN and OT-1 Tg cells are shown. Statistical significance was determined by Student’s t test. (I) The results obtained from representative tetramer dissociation
assay experiments for ORF8 and OT-1 cells are shown. The experiments were repeated three times, and statistical significance was determined by one-way
ANOVA test.
See also Figure S2.
Cell Reports 1, 461–471, May 31, 2012 ª2012 The Authors 469
EXPERIMENTAL PROCEDURES
Detailed information on mice, virus, cell lines, reagents used, and methodology
can be found in the Extended Experimental Procedures. This study was
carried out in strict accordance with the recommendations in the Guide for
the Institutional Animal Care and Use Committee (IACUC) of the National
Institutes of Health.
Generation of ORF8 TN Mice and Purification of T Cells
The schematic for the generation of Kb-MHV-68-ORF8 TN mice is shown in
Figure 1A. The sequences for the rearranged a and b chains of TCR were identified as described elsewhere (Kirak et al., 2010). CD8+ T cells were purified by
negative selection using a kit from Miltenyi Biotec as per the manufacturer’s
instructions. For some experiments cell sorting of fluorescent antibodylabeled cells was performed using a FACSaria II instrument.
License (CC-BY-NC-ND; http://creativecommons.org/licenses/by-nc-nd/3.0/
legalcode).
ACKNOWLEDGMENTS
We thank Barry T. Rouse of the University of Tennessee for critically reading
the manuscript. We acknowledge technical help provided by Patti Wisniewski
with FACS and Tom DiCesare for helping with figure drawing. The work was
supported by funding from NIH.
Received: December 15, 2011
Revised: March 7, 2012
Accepted: March 15, 2012
Published online: April 26, 2012
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The responsiveness of ORF8 TN cells was measured using ICCS assays as
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The NCBI accession numbers for the ORF8 TCR a and b chain sequences are
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