The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 1122 -1128
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iizuka, K.
Right arrow Articles by Kanagawa, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iizuka, K.
Right arrow Articles by Kanagawa, O.

Protection from Lethal Infection by Adoptive Transfer of CD8 T Cells Genetically Engineered to Express Virus-Specific Innate Immune Receptor1

Koho Iizuka2,3,*, Chigusa Nakajima3,{dagger}, Yoshie-Matsubayashi Iizuka*, Mitsuyo Takase{dagger}, Takako Kato{dagger}, Satoshi Noda{ddagger}, Kazuo Tanaka{ddagger} and Osami Kanagawa2,{dagger}

* Department of Medicine, Center for Immunology and Cancer Center, University of Minnesota, Minneapolis, MN 55455; {dagger} Laboratory for Autoimmune Regulation, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa, Japan; and {ddagger} Laboratory for Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3{zeta} cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Host defense against viral infection is mediated by two sets of immune responses, innate and adaptive immunity. Dendritic cells (DCs),4 the first line of defense of the innate immune system, use pattern recognition receptors, TLRs, which interact with various components of pathogens including protein, DNA, RNA, and polysaccharide (1). DCs rapidly secrete cytokines to trigger responses of other innate immunity cells, such as NK cells, and adaptive immunity cells (T and B cells). Both components of the immune system are required for effective protection of the host from pathogens. Lack of innate immunity leads to rapid replication of pathogens, resulting in death within a few days after infection (2). In contrast, in the absence of the adaptive immune system, pathogens escape from adaptive immune surveillance, making the host susceptible to chronic and recurrent infections, as is well documented in human immunodeficient patients (3).

CMV is a major opportunistic pathogen in immunocompromised hosts, such as organ transplant patients. Despite significant advancement in the treatment of CMV disease, it still poses a serious risk to immunocompromised patients. Murine CMV (MCMV) infection provides a useful model to study host-pathogen interaction and its immunity (4, 5). MCMV is a large dsDNA virus belonging to the beta-herpesvirus family. The viral genome contains a number of open reading frames (ORFs) that can neutralize, evade, and even stimulate robust host immune responses. Using the MCMV infection model, several receptors that initiate innate immune responses have been recently identified (6, 7, 8). One such receptor-ligand pair is the Ly49H NK cell activation receptor and m157, an MHC class I-like protein encoded by the MCMV genome (9, 10). Ly49H is a member of the Ly49 family of type II transmembrane lectin-type receptors localized in the NK gene complex (NKC) on mouse chromosome 6 (11). MCMV-infected cells express m157 on their surfaces and are recognized and killed by Ly49H-expressing NK cells through perforin- and IFN-{gamma}-mediated cytotoxicity. Genetic evidence from both pathogen and host sides support this mechanism. An MCMV mutant with m157 deletion gains high viral replication ability in an MCMV-resistant mouse strain (12). Consistent with this, mice lacking Ly49H gene are susceptible to MCMV infection (13, 14). Furthermore, the transgenic expression of Ly49H made MCMV-susceptible strains resistant to infection (15). Mice lacking the perforin or IFN-{gamma} gene in an MCMV-resistant strain are susceptible to MCMV infection (16). These data underscore the importance of NK cell activation by the virally encoded ligand to control acute-phase MCMV infection and subsequent mortality. However, NK cell activation is not determined simply by the status of the activation receptor; rather, it is determined by the balance between inhibitory and activation receptor signals (17). Interestingly, m157 has been shown to also bind to an inhibitory NK cell receptor, Ly49I129, an Ly49 family member expressed in the MCMV-susceptible mouse strain, 129/J (9). These data suggest that the association between viral protein and polymorphic NK receptors having allelic variations and opposing functions could lead to opposite outcome against viral protection.

Inhibitory mouse NK cell receptors, such as Ly49I129 and Ly49A, contain ITIMs in the cytoplasmic domain. In contrast, Ly49H does not have any signaling motifs in its cytoplasmic domain. Instead, Ly49H requires the signaling adaptor molecule, DAP12/KARAP, for surface expression and signaling. DAP12/KARAP is a transmembrane protein that contains ITAMs in the cytoplasmic region that recruit downstream activation kinases (18, 19).

We previously have demonstrated that T cells expressing chimeric receptors consisting of the orphan NK cell receptors extracellular and ITAMs-containing CD3{zeta} cytoplasmic domains can be activated by NK receptor-specific ligands to exhibit T cell effector mechanisms (20, 21). We also have demonstrated that either inhibitory or activation NK receptor ectodomains coupled with CD3{zeta} cytoplasmic domains can be used to detect the ligand for NK receptors with positive readouts (21). We postulated that a chimeric receptor composed of germline-encoded NK receptor ectodomain and CD3{zeta} cytoplasmic domain would direct the cytolytic function of CD8 T cells to target cells expressing viral Ags. To test this hypothesis in a viral infection setting, we introduced cytoplasmic CD3{zeta} chimeric receptor with the extracellular domain of Ly49H receptor into polyclonal CD8 T cells and tested these CD8 T cells for their ability to protect mice from lethal MCMV infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

BALB/c and B6 mice were purchased from Charles River Japan. RAG-2-deficient mice with B6 background were purchased from Taconic. Six- to 8-wk-old mice were used for the experiments. P815 (DBA/2-derived mast cell line), RMA (B6-derived thymoma cell line), A1 (anti-Ly49A Ab-producing hybridoma), and Plat-E (retrovirus packaging cell line provided by T. Kitamura, University of Tokyo, Tokyo, Japan), were maintained in vitro. Anti-Ly49H Ab-producing hybridoma, 3D10, was provided by W. Yokoyama (Washington University, St. Louis, MO). All experiments on mice were approved by the Animal Study Committees at the Research Center for Allergy and Immunology (RIKEN Yokohama Institute) and the Animal Experimentation Committee, Isehara Campus (Tokai University).

Retroviral vectors and transduction

Constructs of Ly49H-CD3{zeta} chimeric receptor, cytoplasmic-deleted Ly49H, Ly49A-CD3{zeta} chimeric receptor and cytoplasmic-deleted Ly49A were generated by PCR as described previously (20). In brief, Ly49H-CD3{zeta} chimeric receptor and cytoplasmic-deleted Ly49H constructs were produced by fusing the C-terminal extracellular domain of Ly49H to the transmembrane of Ly49A with or without the cytoplasmic portion of CD3{zeta}, respectively. These constructs were inserted into the first cistron of the pMX-IRES-GFP vector. m157 cDNA (provided by W. Yokoyama) and H2Dd were inserted into pMX-IRES-GFP and pMX-IRES-hCD4 vectors, respectively (20). Transduction of these cDNAs into target cells was conducted as previously described (20). GFP- or hCD4-positive target cells were sorted with MoFlow flow cytometer (DakoCytomation).

Generation of polyclonal CD8 T cells expressing chimeric receptors

Splenic CD8 T cells were purified with magnetic beads (Miltenyi Biotec) and stimulated with 5 µg/ml plate-bound anti-CD3 and 2 µg/ml monoclonal anti-CD28 Abs in medium containing 200 U/ml rIL-2. Cells were infected with retrovirus supernatant containing indicated cDNAs on days 1 and 2, similar to a method described previously (22). On day 3, cells were recovered and washed, and then cultured with 200 U/ml rIL-2 for another 24 h. Approximately 50–60% of CD8 T cells were GFP-positive on day 4, and these cells were used for in vivo virus protection assays. For in vitro killing assays, cells were purified by sorting GFP-positive cells according to Ly49H or Ly49A surface expression.

Cytotoxic assay

51Cr release assays with CD8 T cells were performed as described before (21). Polyclonal rat IgG serum (50 µg/ml) was added in all assays as control Ab.

MCMV preparation and infection

The Smith strain of MCMV (VR194) was obtained from the American Tissue Culture Collection. Stock solution was prepared from salivary glands of MCMV-infected BALB/c mice as previously described (23). Virus concentration of the MCMV stock solution was 2.0 x 109 pfu/ml as determined by plaque assay using subconfluent 3T3/Swiss albino cells as previously reported (24). Mice were infected with virus by i.p. injection of the virus stock solution.

PCR amplification and sequence of viral m157

Genomic DNA from salivary gland was prepared and viral m157 gene was PCR-amplified with primer as described by French et al. (25). Amplified PCR products were subcloned into TA cloning vector (Invitrogen Life Technologies and sequenced.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of chimeric receptor on polyclonal CD8 T cells

To test the function of the chimeric receptor in primary cells, we prepared an Ly49H-CD3{zeta} chimeric receptor construct having Ly49H extracellular, Ly49A transmembrane, and CD3{zeta} cytoplasmic domains (Ly49HZ) in a bicistronic retrovirus vector, pMX-IRES-GFP (pIG). To avoid difficulties in Ly49H expression and confounding association with other signaling molecules, we replaced the transmembrane region with that from Ly49A, a prototype inhibitory receptor that does not require coassociated molecules for expression. As a control, we prepared a cytoplasmic-deleted Ly49H construct having Ly49H extracellular and Ly49A transmembrane domains but no CD3{zeta} cytoplasmic domain (Ly49Hcyto-del). Using a retrovirus system, we transduced these chimeric receptors into CD8 T cells from an MCMV-susceptible BALB/c strain (H-2d haplotype). We obtained ~50–60% GFP-positive cells after the transduction, and most of the GFP-positive cells were positively stained by Ly49H-specific mAb. We sorted GFP-positive cells as effector cells and >90% of them were Ly49H positive (Fig. 1A). Then, we prepared DBA2-derived P815 (H-2d) as target cells. We transduced pMX-m157-IRES-GFP and pMX-IRES-GFP as control into P815 cells, P815-m157, and P815-GFP, respectively (Fig. 1B). Using these effector and target cells, we performed in vitro 51Cr release assays. CD8 T cells transduced with Ly49HZ receptor gained the capacity to kill P185-m157, whereas CD8 T cells transduced with Ly49Hcyto-del failed to do so. These data indicate that the engagement of Ly49H and m157 induces the activation of CD8 T cells through the CD3{zeta} portion of the chimeric receptor to induce target cell killing (Fig. 1C). To further support the specificity of the transduced chimeric receptor, we observed that anti-Ly49H-specific mAb blocked the killing of P185-m157 (Fig. 1C). Notably, these transduced CD8 T cells maintained tolerance to parental P185 and P185-GFP transduction control cells.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 1. Specific killing of m157-positive target cells by CD8 T cells expressing Ly49H-CD3{zeta} (Ly49HZ) chimeric receptor. A, BALB/c CD8 T cells were infected with retrovirus containing either Ly49Hcyto-del or Ly49HZ receptors. Cells were sorted for effector cells according to their GFP expression. CD8, GFP, and Ly49H expression on sorted cells is shown. B, P815 cells were transduced with either m157-IRES-GFP or IRES-GFP and sorted according to their GFP expression. GFP expression in each transductant is shown. C, 51Cr release assay was performed with P815 cells expressing m157-IRES-GFP ({circ}, •, {triangleup}) or IRES-GFP ({square}, {blacksquare}) as target cells and CD8 T cells expressing Ly49HZ ({circ}, {triangleup}, {square}) or Ly49Hcyto-del receptor (•, {blacksquare}) as effector cells. Cytolytic activity in the presence of Ly49H Ab (1/20 dilution of culture supernatant) is shown with open triangles ({triangleup}). Representative data from four independent experiments are shown.

 
Conversion of native receptor function

m157 protein has been shown to bind to the inhibitory NK cell receptor, Ly49I129, an Ly49 family member expressed in 129/J mice (9). Ly49A from C57BL/6 (B6) is the prototype inhibitory mouse NK cell receptor. Ly49A recognizes MHC class I H-2Dd, and its specificity is the most well characterized among mouse NK cell receptors (26, 27). Having changed the specificity of polyclonal CD8 T cells by transducing with the Ly49H-CD3{zeta} chimeric receptor, we next attempted to convert the native inhibitory NK cell receptor into an activating receptor in primary CD8 T cells by replacing cytoplasmic Ly49A with CD3{zeta}. We prepared the Ly49A-CD3{zeta} chimeric receptor construct having Ly49A extracellular and transmembrane domains fused with the CD3{zeta} cytoplasmic domain (Ly49AZ). As control, we also prepared cytoplasmic-deleted Ly49A, which was made with only Ly49A extracellular and transmembrane domains (Ly49Acyto-del). We prepared the B6-derived T cell line, RMA, as target cells. We transduced H-2Dd-IRES-human CD4 (hCD4) into RMA and IRES-hCD4 as the transduction control, RMA-H-2Dd and RMA-hCD4, respectively. We transduced the Ly49A chimeric receptor into CD8 T cells from B6 mice, which do not express endogenous ligands for Ly49A (Fig. 2A). Ly49Acyto-del receptor-transduced CD8 T cells failed to kill RMA-H-2Dd, whereas Ly49AZ receptor-transduced CD8 T cells effectively killed RMA-H-2Dd but not RMA or RMA-hCD4 (Fig. 2B). Furthermore, the killing of RMA-H-2Dd was blocked by Ly49A-specific mAb, A1 (Fig. 2C). These data indicate that polyclonal CD8 T cells transduced with Ly49AZ receptor specifically recognize its ligand and kill target cells. Taken together, these data indicate that CD8 T cells transduced with ligand recognition elements of an NK cell receptor fused with cytoplasmic CD3{zeta} gain specificities of the native NK cell receptors and kill target cells, regardless of inhibitory or activation native receptor function.


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 2. Conversion of native inhibitory receptor function into activation receptor by fusion with CD3{zeta} chimeric receptor. A, B6 CD8 T cells were infected with retrovirus containing either Ly49Acyto-del or Ly49AZ receptor. Cells were sorted for effector cells according to their GFP expression and analyzed for Ly49A surface expression. B, RMA cells were transduced with either IRES-hCD4 as control or H-2Dd-IRES-hCD4 and sorted according to hCD4 expression. H-2Dd and hCD4 expression of cells was determined before cytolytic assay. C, 51Cr release assays were performed with RMA-hCD4 ({square}, {blacksquare}) or RMA-H-2Dd ({circ}, •, {triangleup}) as target cells and CD8 T cells expressing Ly49AZ ({circ}, {triangleup}, {square}) or Ly49Acyto-del receptor (•, {blacksquare}). Cytolytic activity in the presence of Ly49A Ab (1/20 dilution of culture supernatant) is shown with triangles ({triangleup}). Representative data from three independent experiments are shown.

 
BALB/c mice rescued from MCMV infection by CD8 T cells expressing Ly49H chimeric receptor

Successful in vitro killing of m157-expressing cells by polyclonal CD8 T cells transduced with Ly49H-CD3{zeta} chimeric receptor prompted us to evaluate their ability to control MCMV infection in vivo. BALB/c mice lacking Ly49H expression are susceptible to MCMV infection and die within 2 wk of inoculation with 4 x 106 pfu of MCMV (Fig. 3A). We prepared CD8 T cells from BALB/c mice and transduced them with either Ly49HZ or Ly49Hcyto-del containing retroviruses. Mice received 5 x 107 CD8 T cells expressing the Ly49HZ or Ly49Hcyto-del receptor, and then received a lethal dose of MCMV infection within 1 h of the cell transfer. Mice receiving CD8 T cells expressing Ly49Hcyto-del or no cells died within 10 days, whereas mice receiving CD8 T cells expressing the Ly49HZ receptor survived for >1 mo after infection (Fig. 3A). We observed a positive correlation between the number of cells expressing Ly49HZ receptor transferred and survival in our preliminary studies (data not shown).


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 3. Protection of BALB/c mice from lethal MCMV infection by CD8 T cells expressing Ly49H-CD3{zeta} chimeric receptor and establishment of memory response. A, A total of 5 x 107 CD8 T cells expressing either Ly49HZ receptor ({blacktriangleup}), Ly49Hcyto-del ({triangleup}), or none ({blacksquare}) were injected into BALB/c mice (10 mice per group). Within 1 h of T cell transfer, the mice were infected with MCMV (4 x 106 pfu/mouse) and monitored for survival. B, Mice were infected with MCMV (4 x 106 pfu/mouse). Seventy-two hours after the infection, 5 x 107 CD8 T cells expressing either Ly49HZ receptor ({blacktriangledown}) or no cells ({blacksquare}) were injected into BALB/c mice (10 mice per group). C, Ten BALB/c mice that had been protected from initial MCMV infection by Ly49HZ-expressing CD8 T cells ({blacktriangleup}) were rechallenged with MCMV 40 days after initial infection without further CD8 T cell transfusion. As a control, naive uninfected BALB/c mice ({blacksquare}) (five mice per group) were infected with the same amount of virus. Representative data from two experiments are shown. D, Spleen cells from mice treated identically as in Fig. 3C ({triangleup}) were analyzed for the presence of GFP-positive CD8 T cells 10 days after rechallenge. Representative data from three mice are shown.

 
When the same number of CD8 cells transduced with Ly49HZ was transferred 3 days after the MCMV infection, all mice survived (Fig. 3B). This result indicates that delayed treatment after onset of infection is still capable of controlling replicating virus.

Forty days after the initial infection, surviving mice were tested for development of memory response to MCMV by rechallenging them with the same viral dose as in the initial challenge. Naive BALB/c mice died within 10 days, whereas all of the rechallenged mice survived, indicating the establishment of effective memory response in those mice (Fig. 3C). To evaluate the contribution of transfused CD8 T cells to memory response, we evaluated CD8 T cells in the spleens of the surviving mice 10 days after the rechallenge. In those mice, neither GFP-positive nor Ly49H-positive cells were found in the CD8 population (Fig. 3D).

Transient rescue of RAG-2-deficient B6 mice

Following the initial observation of BALB/c mice that do not have endogenous Ly49H-positive NK cells, we tested whether CD8 T cells expressing the Ly49HZ receptor can rescue mice lacking the adaptive immune system due to the defect in RAG-2 gene from lethal infection. This system is similar to the condition of human CMV infection in immunodeficient patients. It can also address the question whether endogenous or transferred T cells contribute to the anti-CMV memory response. B6-RAG-2-deficient (B6-RAG-2) mice lacking T and B lymphocytes but possessing Ly49H-positive NK cells received syngeneic CD8 T cells (5 x 107 cells, 50% positive for Ly49H surface expression) expressing either Ly49HZ or Ly49Hcyto-del and were challenged with a lethal dose of MCMV (1 x 109 pfu/mouse). All of the B6-RAG-2 mice and most of the B6-RAG2 mice receiving Ly49Hcyto-del-expressing CD8 T cells died within 10 days after infection, whereas B6-RAG-2 mice receiving Ly49HZ-expressing CD8 T cells survived (Fig. 4A). These data indicate that, with high-dose viral infection, endogenous NK cells are not sufficient to protect the host from acute viral expansion and death. Even in this condition, the acute-phase infection can be controlled by transferred CD8 T cells expressing virus-specific NK cell chimeric receptors. A second experiment was conducted under the same conditions, and the same short-term survival patterns as in the first experiment were obtained. Surviving mice monitored for an extended time started to die 29 days after the infection (Fig. 4B), suggesting that the transfusion of CD8 T cells, a mixture of Ly49HZ-positive and -negative cells, failed to develop long-term memory T cells. Various organs were harvested from a moribund B6-RAG-2 mouse on day 31, and the viral titer was determined for each organ. All organs contained very high viral titers (4.4 x 106, 2.0 x 107, 3.6 x 108, and 5.7 x 107 pfu in spleen, liver, salivary gland, and lung, respectively), indicating that late-phase death was due to reactivation of MCMV. From the salivary gland sample, m157 ORF gene was amplified by PCR and cloned PCR products were sequenced. All of the clones (14 of 14) contained various mutations that include deletion, point mutation, and premature termination in m157 (Table I). These data demonstrate that, similar to the study of low-dose MCMV infection of B6-RAG-2 mice (25), rescue of mice by Ly49HZ-expressing CD8 T cells from acute death due to high-dose viral infection does not prevent the appearance of mutant and delayed mortality. This also suggests that the transferred CD8 populations failed to generate effector/memory T cells in vivo.


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Protection of immunodeficient mice from lethal MCMV infection by CD8 T cells expressing Ly49HZ receptor. A, A total of 5 x 107 CD8 T cells expressing either Ly49HZ chimera ({blacktriangleup}), Ly49Hcyto-del ({triangleup}), or none ({blacksquare}) was injected into B6-RAG-2-deficient mice (five mice per group). Mice were then infected with MCMV (109 pfu/mouse) and monitored for survival. We used 109 pfu because B6-RAG-2-deficient mice are resistant at 4 x 106 pfu. B, In a separate experiment, mice (five mice per group) were treated in the same manner as in A and monitored for an extended period.

 

View this table:
[in this window]
[in a new window]

  		Table I. Mutation in m157 in a salivary gland from a B6-Rag2 mouse rescued by CD8 transfera

 
The fate of transferred CD8 T cells in vivo was monitored with or without MCMV infection in BALB/c mice. Even 24 h after transfer, there are very few, if any, GFP-positive cells in the spleen and lymph nodes, regardless of the MCMV infection (data not shown). Moreover, transferring CD8 T cells 5 days before infection failed to protect mice from MCMV infection (data not shown). These data indicate these transferred cells die shortly after the transfer regardless of the functional relevance.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we described the successful use of in vitro-modified CD8 T cells for protection of mice from lethal MCMV infection. Polyclonal CD8 T cells expressing virus-specific Ly49H-CD3{zeta} NK cell chimeric receptor can protect both virus-specific NK cell-deficient BALB/c mice and immunodeficient B6-RAG-2 mice from acute death caused by MCMV infection.

The effective protection of the host from pathogens requires both adaptive and innate immune systems. Innate immune cells use germline-encoded receptors for pathogen recognition and play an important role in early infection. However, it is difficult to manipulate cells of the innate immune system to generate specific immune responses to target pathogens. NK cells are unique among the cells in the innate immune system; they express certain virus-specific receptors rather than pattern recognition receptors expressed in DCs and macrophages. Thus, NK cells could be used to treat specific viral infections. However, it is difficult to manipulate and grow these cells both in vivo and in vitro. In contrast, adaptive immune responses mediated by T and B cells use clonally distributed receptors generated somatically. Following the innate response, these cells respond to pathogens and play an important role in the clearance of pathogens and the generation of memory immune responses. It has been shown that the transfer of in vitro-generated viral Ag-specific CD8 T cells efficiently restores viral immunity in immunodeficient humans (28). Although methods to proliferate activated T cells in vitro using mAb have been established (29), it is still difficult to generate pathogen-specific MHC-restricted effector T cells in vitro due to the low frequencies of such pathogen-specific T and B lymphocytes and the requirement of lengthy activation and differentiation of naive T lymphocytes. Furthermore, it is also difficult to establish Ag-specific T cells from individuals with impaired immune responses.

In this study, we successfully used two different immune cells to generate effective protection in mice lethally infected with MCMV. We used a chimeric receptor composed of germline-encoded NK receptor ectodomain and CD3{zeta} chain cytoplasmic domain to focus the cytolytic function of CD8 T cells to the target cells expressing viral Ags. These chimeric receptors, when expressed in T cell tumor or hybridoma cells, are capable of interacting with NK receptor-specific ligands and inducing a series of activation events similar to TCR-mediated stimulation in the cells (21). Our in vitro results clearly demonstrate that CD8 T cells expressing Ly49HZ, an MCMV m157 protein-specific receptor, can lyse target cells expressing the ligand. When these CD8 T cells were transferred into mice in vivo, the mice were protected from lethal MCMV infection. This protection was observed in both Ly49H-negative hosts and hosts lacking T and B lymphocytes, indicating that this method can be used to treat viral infection of hosts with different immune status. It should also be noted that use of the NK receptor/CD3{zeta} chain chimera allows us to use any pathogen-specific NK receptor for treatment of infection, regardless of the activating or inhibitory nature of the receptor (Fig. 2).

Although transfer of Ly49HZ-expressing CD8 T cells was effective even 3 days after infection (Fig. 3, A and B), the Ly49HZ-expressing CD8 T cells seem to be short-lived in vivo. In BALB/c mice, we did not find any transferred CD8 T cells 1 day after the cell transfer even without the MCMV infection, consistent with previous observation that majority of activated CD8 T cells transferred in vivo are trapped in the lung and liver (30). Moreover, in protected mice (Fig. 3C), no GFP-positive cells were found in the memory response to the secondary challenge. Thus, Ly49HZ-expressing T cells contributed to only the initial phase of host defense, similar to the role of NK cells as well as other innate immune cells under physiological conditions. Consistent with this finding, experiments with B6-RAG-2 mice also showed short-lived protection by Ly49HZ-expressing CD8 T cells. In this experiment, all mice survived the acute phase of MCMV infection but eventually succumbed due to subsequent expansion of the virus with mutations in the target molecule, m157. This observation is similar to that reported by French et al. (25), the only difference being that B6-RAG-2 mice receiving Ly49HZ-expressing CD8 T cells resisted 3 log order higher virus challenge. Because of the cytolytic effects of Ly49H-positive NK cells and Ly49HZ-expressing CD8 T cells, the MCMV with m157 mutation appears at the late stage of infection that can only be controlled by an adaptive immune system.

In BALB/c mice, a secondary response is mediated by endogenous T cells and in B6-RAG-2 mice, transferred CD8 T cells that contain both Ly49HZ-positive and -negative cells totally failed to mount long-term T cell-mediated control of MCMV. Thus, there is no evidence of the generation of memory CD8 T cells from transferred T cells. Numerous studies have demonstrated that the formation of memory T cells requires both TCR and costimulatory signaling at initial Ag encounter (31) and the generation and maintenance of memory CD8 T cells require CD4 T cell help (32, 33, 34). Furthermore, cytokines, particularly IL-7 and IL-15, are crucial for the maintenance of memory CD8 T cells (35, 36). Because transferred CD8 T cells were not observed 1 day after the transfer even without the infection, the methods used to generate effector CD8 T cells in this study, polyclonal activation and proliferation of CD8 T cells with IL-2, also account for the failure to generate memory CD8 T cells in vivo. It is also possible that signaling through the chimeric receptor does not support the generation of memory CD8 T cells, although both TCR and Ly49HZ receptor used the CD3{zeta} chain for T cell activation. Further study of the interaction of Ly49HZ-expressing CD8 T cells with authentic MHC/peptide Ags through TCR or with viral Ag through the NK receptor, may provide insights for the development of memory T cell and memory response to pathogens. Alternatively, transferred T cells may be actively recognized and rejected by NK cells in recipient mice in an NKG2D-dependent manner (37, 38). The NK cell depletion before the CD8 T cell transfer may prolong the survival of transferred cells.

Use of this technology in human viral infection requires identification of viral Ags by human NK receptors. Currently, no human NK receptors specific to human CMV molecules have been identified. However, numerous reports suggest interactions between viruses/viral Ags and human NK cells (39, 40, 41, 42). Human genes for inhibitory and activation receptors are found in two large genomic regions, the leukocyte receptor complex (LCR) and the NKC. The LCR loci contain many inhibitory and activation receptors with allelic polymorphism, similar to mouse NKC (43, 44). Recently, a role for the mouse NCR1 receptor, the homolog of human NKp46 located in the LCR, in the recognition of influenza-infected cells and in the protection of mice from influenza was demonstrated in vivo (45). Thus, a systematic search for both human genome and clinically relevant viral genomes for host-pathogen interaction should be conducted to identify virus-specific human receptors. For this purpose, the screening system using TCR assays previously used for the identification of ligands for orphan NK receptors would be ideal (21). When receptors specific to viral proteins, whether inhibitory or activation, are identified, our system would generate chimeric receptor-expressing CD8 T cell populations capable of attacking infected cells for treatment of human viral infection. We believe that the identification of virus-specific human receptors along with our approach will open up a new avenue to treat patients suffering from viral infection.


   Acknowledgments
 
We thank Dr. Wayne Yokoyama for reagents, Drs. Erik Peterson and Stephen Jameson for providing a critical review of the manuscript, and Diane Bjork and Michael Franklin for preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was partially supported by National Institutes of Health Grant R21AI064270 (to K.I.) and by American Society of Hematology Scholar Award (to K.I.). K.I.’s laboratory is located in a facility that was constructed with support from Research Facilities Improvement Program Grant Number CO6 CA062526-01 from the National Center for Research Resources, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Koho Iizuka, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, MN 55455. E-mail address: iizuk001{at}umn.edu; or Dr. Osami Kanagawa, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan. E-mail address: kanagawa{at}rcai.riken.jp Back

3 K.I. and C.N. contributed equally to this work. Back

4 Abbreviations used in this paper: DC, Dendritic cell; MCMV, murine CMV; ORF, open reading frame; NKC, NK gene complex; LCR, leukocyte receptor complex; h, human. Back

Received for publication October 26, 2006. Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Takeda, K., S. Akira. 2005. Toll-like receptors in innate immunity. Int. Immunol. 17: 1-14. [Abstract/Free Full Text]
  2. Scalzo, A. A., N. A. Fitzgerald, C. R. Wallace, A. E. Gibbons, Y. C. Smart, R. C. Burton, G. R. Shellam. 1992. The effect of the Cmv-1 resistance gene, which is linked to the natural killer cell gene complex, is mediated by natural killer cells. J. Immunol. 149: 581-589. [Abstract]
  3. Fishman, J. A., R. H. Rubin. 1998. Infection in organ-transplant recipients. N. Engl. J. Med. 338: 1741-1751. [Free Full Text]
  4. Tortorella, D., B. E. Gewurz, M. H. Furman, D. J. Schust, H. L. Ploegh. 2000. Viral subversion of the immune system. Annu. Rev. Immunol. 18: 861-926. [Medline]
  5. Webb, J. R., S. H. Lee, S. M. Vidal. 2002. Genetic control of innate immune responses against cytomegalovirus: MCMV meets its match. Genes Immun. 3: 250-262. [Medline]
  6. Beutler, B., K. Hoebe, X. Du, E. Janssen, P. Georgel, K. Tabeta. 2003. Lps2 and signal transduction in sepsis: at the intersection of host responses to bacteria and viruses. Scand. J. Infect. Dis. 35: 563-567. [Medline]
  7. Krug, A., A. R. French, W. Barchet, J. A. Fischer, A. Dzionek, J. T. Pingel, M. M. Orihuela, S. Akira, W. M. Yokoyama, M. Colonna. 2004. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21: 107-119. [Medline]
  8. Tabeta, K., P. Georgel, E. Janssen, X. Du, K. Hoebe, K. Crozat, S. Mudd, L. Shamel, S. Sovath, J. Goode, et al 2004. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc. Natl. Acad. Sci. USA 101: 3516-3521. [Abstract/Free Full Text]
  9. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, L. L. Lanier. 2002. Direct recognition of cytomegalovirus by activating and inhibitory NK cell receptors. Science 296: 1323-1326. [Abstract/Free Full Text]
  10. Smith, H. R., J. W. Heusel, I. K. Mehta, S. Kim, B. G. Dorner, O. V. Naidenko, K. Iizuka, H. Furukawa, D. L. Beckman, J. T. Pingel, et al 2002. Recognition of a virus-encoded ligand by a natural killer cell activation receptor. Proc. Natl. Acad. Sci. USA 99: 8826-8831. [Abstract/Free Full Text]
  11. Yokoyama, W. M., B. F. Plougastel. 2003. Immune functions encoded by the natural killer gene complex. Nat. Rev. Immunol. 3: 304-316. [Medline]
  12. Bubic, I., M. Wagner, A. Krmpotic, T. Saulig, S. Kim, W. M. Yokoyama, S. Jonjic, U. H. Koszinowski. 2004. Gain of virulence caused by loss of a gene in murine cytomegalovirus. J. Virol. 78: 7536-7544. [Abstract/Free Full Text]
  13. Lee, S. H., S. Girard, D. Macina, M. Busa, A. Zafer, A. Belouchi, P. Gros, S. M. Vidal. 2001. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat. Genet. 28: 42-45. [Medline]
  14. Brown, M. G., A. O. Dokun, J. W. Heusel, H. R. Smith, D. L. Beckman, E. A. Blattenberger, C. E. Dubbelde, L. R. Stone, A. A. Scalzo, W. M. Yokoyama. 2001. Vital involvement of a natural killer cell activation receptor in resistance to viral infection. Science 292: 934-937. [Abstract/Free Full Text]
  15. Lee, S. H., A. Zafer, Y. de Repentigny, R. Kothary, M. L. Tremblay, P. Gros, P. Duplay, J. R. Webb, S. M. Vidal. 2003. Transgenic expression of the activating natural killer receptor Ly49H confers resistance to cytomegalovirus in genetically susceptible mice. J. Exp. Med. 197: 515-526. [Abstract/Free Full Text]
  16. Loh, J., D. T. Chu, A. K. O’Guin, W. M. Yokoyama, H. W. Virgin, IV. 2005. Natural killer cells utilize both perforin and {gamma} interferon to regulate murine cytomegalovirus infection in the spleen and liver. J. Virol. 79: 661-667. [Abstract/Free Full Text]
  17. Lanier, L. L.. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225-274. [Medline]
  18. Lanier, L. L., B. C. Cortiss, J. Wu, C. Leong, J. H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391: 703-707. [Medline]
  19. Olcese, L., A. Cambiaggi, G. Semenzato, C. Bottino, A. Moretta, E. Vivier. 1997. Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells. J. Immunol. 158: 5083-5086. [Abstract]
  20. Furukawa, H., K. Iizuka, J. Poursine-Laurent, N. Shastri, W. M. Yokoyama. 2002. A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta2-microglobulin-deficient mice. J. Immunol. 169: 126-136. [Abstract/Free Full Text]
  21. Iizuka, K., O. V. Naidenko, B. F. Plougastel, D. H. Fremont, W. M. Yokoyama. 2003. Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nat. Immunol. 4: 801-807. [Medline]
  22. Fujio, K., Y. Misaki, K. Setoguchi, S. Morita, K. Kawahata, I. Kato, T. Nosaka, K. Yamamoto, T. Kitamura. 2000. Functional reconstitution of class II MHC-restricted T cell immunity mediated by retroviral transfer of the {alpha}beta TCR complex. J. Immunol. 165: 528-532. [Abstract/Free Full Text]
  23. Koga, Y., K. Tanaka, Y. Y. Lu, M. Oh-Tsu, M. Sasaki, G. Kimura, K. Nomoto. 1994. Priming of immature thymocytes to CD3-mediated apoptosis by infection with murine cytomegalovirus. J. Virol. 68: 4322-4328. [Abstract/Free Full Text]
  24. Jordan, M. C., J. L. Takagi. 1983. Virulence characteristics of murine cytomegalovirus in cell and organ cultures. Infect. Immun. 41: 841-843. [Abstract/Free Full Text]
  25. French, A. R., J. T. Pingel, M. Wagner, I. Bubic, L. Yang, S. Kim, U. Koszinowski, S. Jonjic, W. M. Yokoyama. 2004. Escape of mutant double-stranded DNA virus from innate immune control. Immunity 20: 747-756. [Medline]
  26. Karlhofer, F. M., R. K. Ribaudo, W. M. Yokoyama. 1992. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358: 66-70. [Medline]
  27. Tormo, J., K. Natarajan, D. H. Margulies, R. A. Mariuzza. 1999. Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand. Nature 402: 623-631. [Medline]
  28. Riddell, S. R., K. S. Watanabe, J. M. Goodrich, C. R. Li, M. E. Agha, P. D. Greenberg. 1992. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257: 238-241. [Abstract/Free Full Text]
  29. Riddell, S. R., P. D. Greenberg. 1990. The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J. Immunol. Methods 128: 189-201. [Medline]
  30. Engers, H. D., G. D. Sorenson, G. Terres, A. L. Glasebrook, C. Horvath, K. T. Brunner. 1981. Functional activity in vivo of cytolytic T lymphocytes generated in vitro. K. Resh, IV, and H. Kirchner, IV, eds. Mechanisms of Lymphocyte Activation 632 Elsevier/North-Holland, Amsterdam.
  31. Mueller, D. L., M. K. Jenkins, R. H. Schwartz. 1989. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7: 445-480. [Medline]
  32. Sun, J. C., M. A. Williams, M. J. Bevan. 2004. CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection. Nat. Immunol. 5: 927-933. [Medline]
  33. Sun, J. C., M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339-342. [Abstract/Free Full Text]
  34. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
  35. Marrack, P., J. Kappler. 2004. Control of T cell viability. Annu. Rev. Immunol. 22: 765-787. [Medline]
  36. Carrio, R., O. F. Bathe, T. R. Malek. 2004. Initial antigen encounter programs CD8+ T cells competent to develop into memory cells that are activated in an antigen-free, IL-7- and IL-15-rich environment. J. Immunol. 172: 7315-7323. [Abstract/Free Full Text]
  37. Rabinovich, B. A., J. Li, J. Shannon, R. Hurren, J. Chalupny, D. Cosman, R. G. Miller. 2003. Activated, but not resting, T cells can be recognized and killed by syngeneic NK cells. J. Immunol. 170: 3572-3576. [Abstract/Free Full Text]
  38. Cerboni, C., A. Zingoni, M. Cippitelli, M. Piccoli, L. Frati, and A. Santoni. Antigen-activated human T lymphocytes express cell surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK cell lysis. Blood. In Press.
  39. Biron, C. A., K. S. Byron, J. L. Sullivan. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N. Engl. J. Med. 320: 1731-1735. [Medline]
  40. Guma, M., A. Angulo, M. Lopez-Botet. 2006. NK cell receptors involved in the response to human cytomegalovirus infection. Curr. Top. Microbiol. Immunol. 298: 207-223. [Medline]
  41. Alter, G., M. Altfeld. 2006. NK cell function in HIV-1 infection. Curr. Mol. Med. 6: 621-629. [Medline]
  42. Khakoo, S. I., C. L. Thio, M. P. Martin, C. R. Brooks, X. Gao, J. Astemborski, J. Cheng, J. J. Goedert, D. Vlahov, M. Hilgartner, et al 2004. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305: 872-874. [Abstract/Free Full Text]
  43. McQueen, K. L., P. Parham. 2002. Variable receptors controlling activation and inhibition of NK cells. Curr. Opin. Immunol. 14: 615-621. [Medline]
  44. Trowsdale, J.. 2001. Genetic and functional relationships between MHC and NK receptor genes. Immunity 15: 363-374. [Medline]
  45. Gazit, R., R. Gruda, M. Elboim, T. I. Arnon, G. Katz, H. Achdout, J. Hanna, U. Qimron, G. Landau, E. Greenbaum, et al 2006. Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1. Nat. Immunol. 7: 517-523. [Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
I. A. Khan, R. Hakak, K. Eberle, P. Sayles, L. M. Weiss, and J. F. Urban Jr.
Coinfection with Heligmosomoides polygyrus Fails To Establish CD8+ T-Cell Immunity against Toxoplasma gondii
Infect. Immun., March 1, 2008; 76(3): 1305 - 1313.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iizuka, K.
Right arrow Articles by Kanagawa, O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iizuka, K.
Right arrow Articles by Kanagawa, O.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS