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 Control Infection
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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-2Kb-Restricted
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
Citable link
http://hdl.handle.net/1721.1/91544
Terms of Use
Creative Commons Attribution
Cell Reports
Report
CD8
+
T Cells from Mice Transnuclear for a TCR that
Recognizes a Single H-2K
b
-Restricted MHV68 Epitope
Derived from gB-ORF8 Help Control Infection
Sharvan Sehrawat,1,5Oktay Kirak,4,5Paul-Albert Koenig,1Marisa K. Isaacson,1Sofia Marques,2,3Gunes Bozkurt,1 J. Pedro Simas,2,3Rudolph Jaenisch,1and Hidde L. Ploegh1,*
1Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA 2Instituto de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal 3Instituto Gulbenkian de Cieˆncia, 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 DOI10.1016/j.celrep.2012.03.009
SUMMARY
To study the CD8
+T cell response against a mouse
g-herpes virus, we generated K
b-MHV-68-ORF8
604–612RAG
/CD8
+T cell receptor transnuclear (TN) mice
as a source of virus-specific CD8
+T cells. K
b-ORF8-Tet
+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
func-tionality 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 K
b-ORF8-Tet
+cells. When compared to
OT-1 cells, TN cells displayed distinct properties in
response to lymphopenia and cognate antigen
stimu-lation, 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
inter-actions unique to g-herpes viruses.
INTRODUCTION
g-herpes viruses (g-HVs) are species-specific lymphotropic viruses associated with cellular transformation and malignan-cies, 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. Conse-quently, 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 (Flan˜o 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; Gredmark-Russ 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 require-ments 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 ( Gred-mark-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 Kb -ORF8-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 prolifer-ated more rapidly than transgenic OT-1 cells that are restricted by the same MHC element. ORF8 TN mice most closely approx-imate 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
MH V-68 infection of (BALB/ c x C57BL/6) F1 mice Sort Kb-ORF8- Tet + cells Transfer nucleus into enucleated oocyte ESC derivation ESC injection of Blastocyst Birth of chimeric mice Identify TCR and characterize cells Breeding with Rag1-/-C57BL/6 mice A TCR alpha TCR beta V gene/allele TRAV10N*01 TRBV31*01 D gene/allele --- TRBD1*01 J gene/allele TRAJ33*01 TRBJ2 -2*01 C gene/ allele TRAC ( n.d.) TRBC2*03 Peptide recognized KNYIFEEKL
B 1.72 0.0662 68.4 29.8 CD8 CD4 Kb-ORF8-tet CD3 B220 0.919 52.2 0.722 46.1 CD8 Kb-ORF8-tet H-2Kb 0.0358 0.538 93 6.38 H-2Kd V 14 5.23 71.6 12.5 10.6 3.77 0.87 85.2 10.2 1.37 0.99 81.8 15.9 24.8 27.4 30.9 16.9
Thymus Pooled LN and Spleen
CD8 CD4 OT1T g ORF8 TN D KNYIFEEKL ANYIFEEKL KA YIFEEKL KN A IFEEKL KNY A FEEKL KNYI A EEKL KNYIF A EKL KNYIFE A KL KNYIFEE A L KNYIFEEK A KNY V FEEKL K Q YIFEEKL KN F IFEEKL KNY L FEEKL KNYIF D EKL KNYIFE D KL KNYIFEE R L 0 1 2 3 4 4 6 8 10 0 1 2 3 4 2000 4000 6000 8000 10000 H-2Kb IFN- + APL EC 50 H-2K b (F old c hange) EC 50 IFN -g (F old c hange) K NY
I
F
E
E
KL
F KNYIFEEKL ANYIFEEKL KA YIFEEKL KN A IFEEKL KNY A FEEKL KNYI A EEKL KNYIF A EKL KNYIFE A KL KNYIFEE A L KNYIFEEK A KNY V FEEKL K Q YIFEEKL KNYIF D EKL KNYIFE D KL KNYIFEE R L 0 100 200 300 400 APLIL
-2
c
o
n
c
.
(p
g
/m
l)
G C EFigure 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
homo-geneous staining with Kb
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 MHV-68 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 Kb
-ORF8-Tet+CD8+T cells after 21 days of MHV-68 infection. We as-certained 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 -MHV-68-ORF8 tetramers. Because TCR a and TCR b segregate inde-pendently, 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 genera-tions, 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 50rapid amplification of cDNA ends (50RACE)-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 inFigures 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 inFigure S1C. The nucleotide and predicted amino acid sequence for the complementarity-determining 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 inFigure 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 distribu-tion 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 trans-genesis, 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+CD8cells 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 Kb -ORF8-Tet+(Figure S2B), albeit at lower staining intensity,
consis-tent with a contribution of class I MHC-CD8 interactions in deter-mining 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-2Kbrestricted 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-2Kbexpression on the surface of TAP-deficient RMA/S cells at 37C as an indicator of their binding to H-2Kb(Figure 1F). The class I MHC anchor
resi-dues 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 EC50of 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.
Days 0 CD45.2+TN cells into naive CD45.1 B6 mice 1 MHV-68 infection (1x106pfu ip) Analysis (4-35) A 0.097 11 17.2 71.7 CD8 CD45.2 CFSE 0.047 99.5
Input cells 5.5dpi
CD44 73.6 15.1 B C D 50,000 CD45.2 ORF8 TN cells transferred 0 10 21 0 20 40 60 50,000 CD45.2+ORF8 TN cells transferred 0 500 1500 15000 50000100000500000 0 10 20 30 40 50 no of transferred CD45.2+ TN cells % CD45.2 + of total CD8 + cells
days post infection
0 12.8 87.2 0 0 1.18 98.8 0 0 0.905 99.1 0 CD8 Kb-ORF8-T et Uninfected MHV-68 (A9) MHV-68 (WT) 0.905 1.18 12.8 0 52.2 98.6 CFSE 1.09 0.52 22.9 75.5 1.01 8.31 7.43 83.2 CD45.2 IFN-γ Unstimulated KNYIFEEKL 1.48 6.27 15.1 77.2 1.26 27.5 0.909 70.3 7.54e-3 97.4 2.62 0.0226 CD8 CD45.2 CD8 CD45.2 Kb-ORF8-T et Kb-ORF8-T et Host cells Donor cells 6.1 92.4 1.33 0.149 0.213 98.5 1.24 6.46e-3 99.2 0.0959 0.0113 0.666 79.8 19.2 0.353 0.674 CD45.2 Control PD1 CD44 CD62L Kb-ORF8-Tet+gate Host cells Donor cells 0.0903 1.35 18.9 79.6 0.0455 12 83.9 4.06 0 64.6 35.4 0 0 96.7 3.28 0 CD8 18.9999 3.28888 CD8 CD8 CD44 0 Kb-ORF8-T et CD127 0 CD62L 35.4444 CD45.2 0 0.209 99.8 0 0 1.4 98.6 0 0 30.1 69.9 0 3.05 31.6 30.8 34.6 CD127 CD45.2 CD8 CD45.2 CD8 Control CD8 0 Kb-ORF8-T et Host cells Donor cells Kb-ORF8-Tet+ gate CD45.2+ gate F G H I E % CD45.2 + of total CD8 + cells
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 13 106pfu 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.
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 exper-iments we identified APLs that were equipotent in stabilization of (binding to) H-2Kbbut with inferior T cell stimulatory capacity (e.g., KNYAFEEKL). Other peptides were far worse at stabilizing H-2Kbbut 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 carboxy-fluorescein succinimidyl ester (CFSE)-labeled or unlabeled CD8+T cells from naive ORF8 TN mice (CD45.2+) were trans-ferred 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 trans-ferred 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 ( Fig-ure 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 ( Fig-ure 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 andS2E). As a convincing demonstration of the fine specificity of ORF8 TN cells, we infected mice that had received 5 3 105 of CFSE-labeled 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-68-ORF8 (L9A) mutant virus, endogenous MHV-68-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 post-infection (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 alterna-tive 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 ( Fig-ure 2H). In the early memory phase of the response (35 dpi),
(B) A total of 503 103
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 503 103
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 (53 105
) 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 53 105
pfu of MHV-68 (WT) or 53 105
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 503 103
ORF8 TN cells were transferred in congenic CD45.1+
mice 1 day prior to MHV-68 infection with 13 106
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.
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).
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
Days 0 Transfer ORF8 TN Rag-/- cells 1 Infected with MHV-68 6-7 5x10 6 CFSE hiKNYIFEEKL + 5x10 6 CFSE lo ) 2hr post transfer (ip or in) 500 CD8 TN cells No transfer uninfected CFSE 44.3 55.7 57.2 42.8 60.7 39.3 89 11 infected with MHV68 50x103 CD8 TN cells A B no tra nsfer 5x10 2 TN cells 10x10 3 TN cells 50x10 3 TN cell s 0 20 40 60 80 100 * P = 0.03 *** P = 0.003 0.54 1.16 37.6 60.7 2.74 25.1 23.7 48.5 1.04 7.98 26.3 64.7 CD44 Kb-ORF8-T et
No Transfer 1x106 TN cells 4x106 TN cells
No Transfer 1x10 6 TN cells 4x10 6 TN cells 0 5000 10000 200000 400000 600000 *** (p = 0.0004) ** (p = 0.006) no of Kb-ORF8-T et + CD8 + cells C D E F %specific lysis no of ORF8 TN cells no of ORF8 TN cells Contro l 1x10 6TN c ells 4x10 6 TN cells 0 1 2 5 10 15 ** (p = 0.005) * (p = 0.04)
pfu/g of lung tissue (log
10
)
transferred 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 13 106
pfu of MHV-68 i.p. or 13 105
pfu i.n. At 6.5 dpi, 53 106
peptide-pulsed CD45.1+
splenocytes labeled with high concentrations of
CFSE and 53 106
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 inExperimental Proceduressection.
(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 13 105
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.
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 13 106and 43 106ORF8 TN cells into C57BL/6 mice 1 day prior to intra-nasal (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 ( Fig-ure 3F). ORF8 TN cells thus confer a measure of protection against MHV-68 infection, even though only a single CD8+
T cell H-2Kbrestricted epitope out of the40 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), 23 106CFSE-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, andFigure 4C). Because homeostatic expansion in response to lymphopenia is believed to be driven by cross-reactivity with self-peptide-MHC 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 lymphopenia-induced 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, andFigure 4D). These findings underscore the rele-vance 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 intrinsi-cally 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-2Kbon 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 concen-tration 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-2Kbwere 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 versus60% 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 responsive-ness 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 dissoci-ation assays as a surrogate measure for TCR affinities. As shown inFigure 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 prop-erties because inherently different methodologies were used for obtaining them.
DISCUSSION
We generated Kb-MHV-68-ORF8604–612TCR 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
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 23 106
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.
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 physi-ological 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 develop-ment 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, prolifer-ation, 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 than40 H-2Kband 20 H-2Dbrestricted 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 endog-enous 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 approxi-mation 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 respec-tive 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 infec-tion 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 anti-bodies, 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 13 106
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.
EXPERIMENTAL PROCEDURES
Detailed information on mice, virus, cell lines, reagents used, and methodology
can be found in theExtended 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
iden-tified 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 antibody-labeled cells was performed using a FACSaria II instrument.
Measurement of Responsiveness of ORF8 TN CD8+T Cells
The responsiveness of ORF8 TN cells was measured using ICCS assays as
described bySehrawat et al. (2010). Various concentrations of index peptide,
i.e., KNYIFEEKL and APLs that were used to stimulate ORF8 TN cells isolated from infected mice that received naive ORF8 TN cells 1 day before infection. IFN-g production by ORF8 TN cells was measured by flow cytometry using fluorochrome-labeled antibodies. In order to explore the fine specificity of TN cells using epitope variants, all measurements were expressed as fold
change of EC50values relative to those measured for the index peptide
KNYIFEEKL. To measure the ex vivo stimulation of naive ORF8 TN cells by
index peptide and different APLs, 13 105
of purified ORF8 TN cells isolated
from lymphoid organs of TN mice were cocultured with 23 104
of ORF8
(APLs)-pulsed BMDCs for 72 hr at 37C in a humidified incubator. IL-2 levels
were measured in the culture supernatants at the end of incubation period by ELISA using a kit from BD Biosciences.
Measuring the Functionality of ORF8 TN Cells upon Viral Infection
Graded numbers of purified cells obtained from pooled LN and spleens of ORF8 TN mice were transferred into C57BL/6 or congenic CD45.1 mice
1 day prior to infection i.p. or IN with 13 106
pfu or 13 105
of MHV-68 in 200 and 50 ml vol, respectively. Cells were isolated from infected mice, stained, and analyzed by flow cytometry. To measure frequencies of IFN-g producing cells, ICCS and in vivo CTL assays were performed as described elsewhere
(Sehrawat et al., 2010). The viral load in extracted lung tissue samples from
different groups of mice was measured by standard plaque assays as
described earlier (Marques et al., 2008).
Statistical Analysis
Statistical analysis to compare responses between groups was done by Student’s t test and ANOVA test as indicated in the figure legends. The results
are presented as mean± SEM or SD as indicated in figure legends. The
p values are shown in the figures or figure legends and are represented as
*p% 0.05, **p % 0.01, or ***p % 0.001.
ACCESSION NUMBERS
The NCBI accession numbers for the ORF8 TCR a and b chain sequences are JQ929654 and JQ969253, respectively.
SUPPLEMENTAL INFORMATION
Supplemental Information includes an Extended Discussion, Extended Exper-imental Procedures, and two figures and can be found with this article online at
doi:10.1016/j.celrep.2012.03.009.
LICENSING INFORMATION
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported
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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