Two antigenic peptides from genes m123 and m164 of murine cytomegalovirus quantitatively dominate CD8 T-cell memory in the H-2d haplotype.

The importance of CD8 T cells for the control of cytomegalovirus (CMV) infection has raised interest in the identification of immunogenic viral proteins as candidates for vaccination and cytoimmunotherapy. The final aim is to determine the viral "immunome" for any major histocompatibility complex class I molecule by antigenicity screening of proteome-derived peptides. For human CMV, there is a limitation to this approach: the T cells used as responder cells for peptide screening are usually memory cells that have undergone in vivo selection. On this basis, pUL83 (pp65) and pUL123 (IE1 or pp68 to -72) were classified as immunodominant proteins. It is an open question whether this limited "memory immunome" really reflects the immunogenic potential of the human CMV proteome. Here we document an analogous focus of the memory repertoire on two proteins of murine CMV. Specifically, ca. 80% of all memory CD8 T cells in the spleen as well as in persisting pulmonary infiltrates were found to be specific for the known IE1 peptide 168YPHFMPTNL176 and for the peptide 257AGPPRYSRI265, newly defined here, derived from open reading frame m164. Notably, CD8 T-cell lines of both specificities protected against acute infection upon adoptive transfer. In contrast, the natural immune response to acute infection in draining lymph nodes and in the lungs indicated a somewhat broader specificity repertoire. We conclude that the low number of antigenic peptides identified so far for CMVs reflects a focused memory repertoire, and we predict that more antigenic peptides will be disclosed by analysis of the acute immune response.     

Enrichment of immediate-early 1 (m123/pp89) peptide-specific CD8 T cells in a pulmonary CD62L(lo) memory-effector cell pool during latent murine cytomegalovirus infection of the lungs

Interstitial cytomegalovirus (CMV) pneumonia is a clinically relevant complication in recipients of bone marrow transplantation (BMT). Recent data for a model of experimental syngeneic BMT and concomitant infection of BALB/c mice with murine CMV (mCMV) have documented the persistence of tissue-resident CD8 T cells after clearance of productive infection of the lungs (J. Podlech, R. Holtappels, M.-F. Pahl-Seibert, H.-P. Steffens, and M. J. Reddehase, J. Virol. 74:7496-7507, 2000). It was proposed that these cells represent antiviral "standby" memory cells whose functional role might be to help prevent reactivation of latent virus. The pool of pulmonary CD8 T cells was composed of two subsets defined by the T-cell activation marker L-selectin (CD62L): a CD62L(hi) subset of quiescent memory cells, and a CD62L(lo) subset of recently resensitized memory-effector cells. In this study, we have continued this line of investigation by quantitating CD8 T cells specific for the three currently published antigenic peptides of mCMV: peptide YPHFMPTNL processed from the immediate-early protein IE1 (pp89), and peptides YGPSLYRRF and AYAGLFTPL, derived from the early proteins m04 (gp34) and M84 (p65), respectively. IE1-specific CD8 T cells dominated in acute-phase pulmonary infiltrates and were selectively enriched in latently infected lungs. Notably, most IE1-specific CD8 T cells were found to belong to the CD62L(lo) subset representing memory-effector cells. This finding is in accordance with the interpretation that IE1-specific CD8 T cells are frequently resensitized during latent infection of the lungs and may thus be involved in the maintenance of mCMV latency.     

The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.     

Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope.

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.     

Murine model of interstitial cytomegalovirus pneumonia in syngeneic bone marrow transplantation: persistence of protective pulmonary CD8-T-cell infiltrates after clearance of acute infection

Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62L(lo) and CD62L(hi) subsets of CD44(+) memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral "stand-by" memory-effector T cells persist in the lungs to prevent virus recurrence from latency.     

Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.     

Cytolytic CD4 cells: Direct mediators in infectious disease and malignancy

CD4 T cells have traditionally been regarded as helpers and regulators of adaptive immune responses; however, a novel role for CD4 T cells as direct mediators of protection against viral infections has emerged. CD4 T cells with cytolytic potential have been described for almost 40 years, but their role in host protection against infectious disease is only beginning to be realized. In this review, we describe the current literature identifying these cells in patients with various infections, mouse models of viral infection and our own work investigating the development of cytolytic CD4 cells in vivo and in vitro. CD4 CTL are no longer considered an artefact of cell culture and may play a physiological role in viral infections such as EBV, CMV, HIV and influenza. Therefore, vaccine strategies aimed at targeting CD4 CTL should be developed in conjunction with vaccines incorporating B cell and CD8 CTL epitopes.     

Experimental Preemptive Immunotherapy of Murine Cytomegalovirus Disease with CD8 T-Cell Lines Specific for ppM83 and pM84, the Two Homologs of Human Cytomegalovirus Tegument Protein ppUL83 (pp65)

CD8 T cells are the principal antiviral effectors controlling cytomegalovirus (CMV) infection. For human CMV, the virion tegument protein ppUL83 (pp65) has been identified as a source of immunodominant peptides and is regarded as a candidate for cytoimmunotherapy and vaccination. Two sequence homologs of ppUL83 are known for murine CMV, namely the virion protein ppM83 (pp105) expressed late in the viral replication cycle and the nonstructural protein pM84 (p65) expressed in the early phase. Here we show that ppM83, unlike ppUL83, is not delivered into the antigen presentation pathway after virus penetration before or in absence of viral gene expression, while other virion proteins of murine CMV are processed along this route. In cytokine secretion-based assays, ppM83 and pM84 appeared to barely contribute to the acute immune response and to immunological memory. Specifically, the frequencies of M83 and M84 peptide-specific CD8 T cells were low and undetectable, respectively. Nonetheless, in a murine model of cytoimmunotherapy of lethal CMV disease, M83 and M84 peptide-specific cytolytic T-cell lines proved to be highly efficient in resolving productive infection in multiple organs of cell transfer recipients. These findings demonstrate that proteins which fail to prime a quantitatively dominant immune response can nevertheless represent relevant antigens in the effector phase. We conclude that quantitative and qualitative immunodominance are not necessarily correlated. As a consequence of these findings, there is no longer a rationale for considering T-cell abundance as the key criterion for choosing specificities to be included in immunotherapy and immunoprophylaxis of CMV disease and of viral infections in general.     

Control of Cytomegalovirus in Bone Marrow Transplantation Chimeras Lacking the Prevailing Antigen-Presenting Molecule in Recipient Tissues Rests Primarily on Recipient-Derived CD8 T Cells

Cytomegalovirus (CMV) infection during the transient immunodeficiency after bone marrow transplantation (BMT) develops into disease unless antiviral CD8 T cells are restored in due course. Histoincompatibility between donor and recipient is associated with increased risk. Complications may include a rejection response against the foreign major histocompatibility complex (MHC) antigens and a lack of antiviral control resulting from a misfit between donor-derived T cells and the antigenic viral peptides presented in recipient tissues. Here we have established a murine model of CMV disease after experimental BMT performed across a single MHC class I disparity. Specifically, BALB/c bone marrow cells expressing the prevailing antigen-presenting molecule L^d were transplanted into the L^d gene deletion mutant BALB/c-H-2^dm2, an experimental setting that entails a selective risk of host-versus-graft but not graft-versus-host response. The reconstituted T-cell population proved to be chimeric in that it consisted of L^d-positive donor-derived and L^d-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against L^d. This finding implies that the infection did not trigger a host-versus-graft response. Notably, upon adoptive transfer, donor-derived CD8 T cells preferentially protected tissues of donor genotype, whereas recipient-derived CD8 T cells protected tissues of either genotype. We infer from these data that the focus on immunodominant antigens presented by L^d within the donor cell population distracted the donor T cells from protecting recipient tissues and that protection in the chimeras was therefore primarily based on recipient T cells. As a consequence, T-cell chimerism after BMT should give a positive prognosis with respect to control of CMV.Cytomegaloviruses (CMV) are kept under tight immune control (for reviews, see references 22 and 23). As a consequence, acute CMV infection is resolved rapidly and does not result in disease unless the host is immunologically immature or immunocompromised. Bone marrow (BM) transplantation (BMT) as a therapy of hematological malignancies is associated with a transient immunodeficiency. Accordingly, during the period of immunocompromise, transmission of donor-type CMV with the transplant as well as recurrence of CMV from latency established within the organs of the transplantation recipient both entail a risk for destructive virus replication in tissues resulting in multiple-organ CMV disease (16). In BMT recipients, CMV-induced interstitial pneumonia is a frequent and endangering manifestation of CMV disease (11, 27). However, CMV infection does not inevitably result in fatal disease. It appears that CD8 T-cell reconstitution is the decisive parameter in the control of CMV after BMT. Clinical data have shown that both efficient reconstitution of CD8 T cells (41) and supplementation of antiviral CD8 T cells by preemptive cytoimmunotherapy with T-cell lines (42, 50) correlate with a reduced risk of human CMV disease, whereas combined in vivo-ex vivo T-cell depletion, intended as a prophylaxis against graft-versus-host (GvH) disease, accidentally resulted in an increased incidence of CMV infections in BMT patients (14). Aspects of these clinical problems can be approached experimentally in a murine model of BMT and concurrent infection with murine CMV (for an overview, see reference 35). Specifically, depletion of CD8 T cells, but not of CD4 T cells, performed in vivo during the phase of reconstitution after BMT abolished the development of protective antiviral immunity, with an inevitably lethal outcome (34, 47) resulting from multiple-organ pathology (34), including BM aplasia (29, 30). Likewise, an insufficient endogenous reconstitution was successfully supplemented by experimental adoptive cytoimmunotherapy with antiviral CD8 T cells. Again, CD4 T cells were not effective (36, 37, 39, 47). Altogether, clinical data on human CMV infection and experimental data from the murine model have so far been concordant and have identified CD8 T cells as the principal effectors controlling CMV infections after BMT.These findings imply that all conditions which lower the efficacy of CD8 T-cell reconstitution will increase the risk for progression of asymptomatic CMV infection to fatal CMV disease. Histoincompatibility between graft and recipient is a factor likely to negatively influence the restoration of antiviral immunity. Accordingly, even though cases of severe human CMV disease have been reported also after autologous BMT (27, 40), the incidence of CMV-related complications is generally higher after histoincompatible BMT (51). In clinical BMT, donor and recipient are usually matched in major histocompatibility complex (MHC) class II molecules, whereas differences in minor histocompatibility loci and in MHC class I loci are tolerated if unavoidable. Complications caused in the CMV-infected recipient by histoincompatibility may include (i) an impaired engraftment of transplanted cells in the recipient BM stroma, (ii) an immunological GvH response as well as a host-versus-graft (HvG) response directed against the foreign minor or major histocompatibility molecules, and (iii) a lack of antiviral T-cell control resulting from an inappropriate repertoire of viral antigenic peptides presented by infected tissue cells of the transplantation recipient.In a first attempt to dissect these possibilities, we have established a murine model of experimental BMT performed across a single MHC class I disparity, namely, the presence and absence of the L^d molecule in BALB/c mice (MHC class I molecules K^d, D^d, and L^d) and the L^d gene deletion mutant BALB/c-H-2^dm2 (44), respectively. Depending on the choice of donor and recipient for the BMT, immunogenetical GvH and HvG conditions can be studied separately (35). Work presented herein focuses on the HvG setting with BALB/c as the donor strain and the mutant as the recipient. Hence, after incomplete depletion of hematopoietic cells of the recipients, this model entails a risk for graft rejection caused by a recipient response directed against the donor MHC class I molecule L^d. In addition, presentation of viral peptides by L^d, including the immunodominant IE1 nonapeptide of murine CMV (18, 38), is confined to donor-derived hematopoietic cells and their progeny, whereas the parenchymal and stromal sites of cytocidal infection (34) lack L^d as the prevailing peptide presenter. The aim of the study was to investigate the influence of this particular MHC class I disparity on the control of murine CMV after BMT.     

Use of transgenic HLA A*0201/Kb and HHD II mice to evaluate frequency of cytomegalovirus IE1-derived peptide usage in eliciting human CD8 cytokine response

Unlike the pp65 protein of human cytomegalovirus (CMV), which has an immunodominant peptide, pp65(495-503), recognized by human CD8(+) cells in the context of HLA A*0201, the fine peptide specificity for CMV IE1 has shown no such immunodominance. With the use of transgenic HLA A*0201/Kb and HHD II mice, a selected pool of IE1 peptides, including IE1(p256-264), IE1(p297-304), and IE1(p316-324), were shown to stimulate cytolytic T-lymphocyte lysis in the context of HLA A*0201. Based on an intracellular gamma interferon response, IE1(p297-304), a previously unrecognized CD8 epitope, triggered a prominent response to CMV IE1 in HLA A*0201 subjects.     

Prostaglandin E2 Production and T Cell Function in Mouse Adenovirus Type 1 Infection following Allogeneic Bone Marrow Transplantation

Adenovirus infections are important complications of bone marrow transplantation (BMT). We demonstrate delayed clearance of mouse adenovirus type 1 (MAV-1) from lungs of mice following allogeneic BMT. Virus-induced prostaglandin E₂ (PGE₂) production was greater in BMT mice than in untransplanted controls, but BMT using PGE₂-deficient donors or recipients failed to improve viral clearance, and treatment of untransplanted mice with the PGE₂ analog misoprostol did not affect virus clearance. Lymphocyte recruitment to the lungs was not significantly affected by BMT. Intracellular cytokine staining of lung lymphocytes demonstrated impaired production of INF-γ and granzyme B by cells from BMT mice, and production of IFN-γ, IL-2, IL-4, and IL-17 following ex vivo stimulation was impaired in lymphocytes obtained from lungs of BMT mice. Viral clearance was not delayed in untransplanted INF-γ-deficient mice, suggesting that delayed viral clearance in BMT mice was not a direct consequence of impaired IFN-γ production. However, lung viral loads were higher in untransplanted CD8-deficient mice than in controls, suggesting that delayed MAV-1 clearance in BMT mice is due to defective CD8 T cell function. We did not detect significant induction of IFN-β expression in lungs of BMT mice or untransplanted controls, and viral clearance was not delayed in untransplanted type I IFN-unresponsive mice. We conclude that PGE₂ overproduction in BMT mice is not directly responsible for delayed viral clearance. PGE₂-independent effects on CD8 T cell function likely contribute to the inability of BMT mice to clear MAV-1 from the lungs.     

Induction of long-term memory CD8(+) T cells for recall of viral clearing responses against influenza virus

Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.     

IL-4 bypasses the immune suppressive effect of cyclosporin A during the in vitro induction of murine cytotoxic T lymphocytes

We have analyzed at the clonal level the effect of IL-4 on the immune suppressive action of cyclosporin A (CsA) during the in vitro primary activation of anti-MHC alloantigen-reactive murine CD8+ CTL. Although neither IL-4 nor IL-2 alone were able to overcome the CsA-mediated suppression, the addition of IL-4 in the presence of IL-2 restored in a dose-dependent manner the induction of cytolytic activity. On the other hand, CsA greatly impaired proliferative responses of alloantigen-reactive CD8 T cells, thereby operationally dissecting proliferative responsiveness from acquisition of cytolytic activity during primary activation of alloantigen-reactive CD8+ T cells. The existence of a CsA-resistant induction pathway for Ag-specific CD8+ T cell-mediated cytolytic activity may be of relevance for experimental and clinical organ transplantation.     

Modulation of HLA-A*0201-restricted T cell responses by natural polymorphism in the IE1(315-324) epitope of human cytomegalovirus

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8(+) CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV(+) donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1(315-324), an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1(315-324), recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1(315-324) epitope variants.     

Preemptive CD8 T-Cell Immunotherapy of Acute Cytomegalovirus Infection Prevents Lethal Disease,Limits the Burden of Latent Viral Genomes,and Reduces the Risk of Virus Recurrence

In the immunocompetent host, primary cytomegalovirus (CMV) infection is resolved by the immune response without causing overt disease. The viral genome, however, is not cleared but is maintained in a latent state that entails a risk of virus recurrence and consequent organ disease. By using murine CMV as a model, we have shown previously that multiple organs harbor latent CMV and that reactivation occurs with an incidence that is determined by the viral DNA load in the respective organ (M. J. Reddehase, M. Balthesen, M. Rapp, S. Jonjic, I. Pavic, and U. H. Koszinowski. J. Exp. Med. 179:185–193, 1994). This predicts that a therapeutic intervention capable of limiting the load of latent viral genome should also reduce the risk of virus recurrence. Here we demonstrate the benefits and the limits of a preemptive CD8 T-cell immunotherapy of CMV infection in the immunocompromised bone marrow transplantation recipient. Antiviral CD8 T cells prevented CMV disease and accelerated the resolution of productive infection. The therapy also resulted in a lower load of latent CMV DNA in organs and consequently reduced the incidence of recurrence. The data thus provide a further supporting argument for clinical trials of preemptive cytoimmunotherapy of human CMV disease with CD8 T cells. However, CD8 T cells failed to clear the viral DNA. The therapy-susceptible portion of the DNA load differed between organs and was highest in the lungs. The existence of an invariant, therapy-resistant load suggests a role for immune system evasion mechanisms in the establishment of CMV latency.Recurrence of productive infection by reactivation of latent viral genome in the immunocompromised host is a feature common to the members of the herpesvirus family (39; reviewed in reference 38). Specifically, in the case of human cytomegalovirus (CMV), the human herpesvirus type 5, primary as well as recurrent infection during the temporal immunodeficiency early after bone marrow (BM) transplantation (BMT) entails a risk of graft failure and severe organ manifestations of CMV disease (8, 44). Early findings by Quinnan et al. (24) have suggested a correlation between efficient reconstitution of the cellular immune response and the control of post-BMT CMV infection, and more recent clinical data have attributed this control to the reconstituted CD8 T cells (35). Accordingly, restoration of antiviral immunity in the critical phase before the reconstitution by BMT becomes effective should diminish the risk of CMV disease. Experimental research with the model of murine CMV infection has positively demonstrated the antiviral and protective efficacy of adoptively transferred acutely sensitized (31, 34) or memory (28) CD8 T cells recovered from immune donors as well as of short-term CD8 T-cell lines propagated in culture (32). These studies have been pivotal for clinical trials of a preemptive CD8 T-cell immunotherapy of post-BMT human CMV infection in patients (37, 43).Infection of the BMT recipient can accidentally result from the transmission of infectious virus, however, productive infection is more commonly initiated by reactivation of latent CMV in either the transplant or the recipient’s own organs or, occasionally, both (11). For the murine model system, we have previously demonstrated the existence of multiple organ sites of CMV latency at which the latent viral DNA is retained after the resolution of productive primary infection and after clearance of the viral genome from hematopoietic leukocytic cells in BM and blood (27). In accordance with the wide distribution of the latent viral DNA, recurrence was found to occur focally in any of the organs, which led us to propose the concept of multifocal CMV latency and recurrence (27). Most importantly, the incidence of recurrence was found to correlate with the load of latent viral DNA in the respective tissue. Specifically, low virus dissemination and rapid control of infection in immunocompetent adult mice resulted in a low load and was associated with a low risk of recurrence, whereas the delayed control of infection in neonatal mice resulted in a high load and was associated with a high risk. Furthermore, there were also organ-specific differences. In accordance with the high incidence of interstitial CMV pneumonia after BMT, the lungs were identified as having a high load of latent CMV (2, 17).It is apparent that antiviral CD8 T cells generated during primary infection as well as memory cells present during latency do not eradicate latently infected cells under physiological conditions, since latency would not exist if they did. However, it has been open to question whether adoptive transfer of antiviral CD8 effector cells could prevent the escape of virus into latency. We will show here that modulation of primary infection by experimental CD8 T-cell immunotherapy has indeed had an effect on the load of latent viral DNA in tissues. The effect of the therapy is of relevance, since the load of latent viral DNA can be kept below the threshold required for effective recurrence. Our data thus provide a further supporting argument for clinical trials of cytoimmunotherapy. Interestingly, however, the data also predict that no dosage of CD8 T cells will prevent the establishment of latency.     

Visualization and characterization of respiratory syncytial virus F-specific CD8(+) T cells during experimental virus infection

CTL play a major role in the clearance of respiratory syncytial virus (RSV) during experimental pulmonary infection. The fusion (F) glycoprotein of RSV is a protective Ag that elicits CTL and Ab response against RSV infection in BALB/c mice. We used the strategy of screening a panel of overlapping synthetic peptides corresponding to the RSV F protein and identified an immunodominant H-2K(d)-restricted epitope (F(85-93); KYKNAVTEL) recognized by CD8(+) T cells from BALB/c mice. We enumerated the F-specific CD8(+) T cell response in the lungs of infected mice by flow cytometry using tetramer staining and intracellular cytokine synthesis. During primary infection, F(85-93)-specific effector CD8(+) T cells constitute approximately 4.8% of pulmonary CD8(+) T cells at the peak of the primary response (day 8), whereas matrix 2-specific CD8(+) T cells constituted approximately 50% of the responding CD8(+) T cell population in the lungs. When RSV F-immune mice undergo a challenge RSV infection, the F-specific CD8(+) T cell response is accelerated and dominates, whereas the primary response to the matrix 2 epitope in the lungs is reduced by approximately 20-fold. In addition, we found that activated F-specific effector CD8(+) T cells isolated from the lungs of RSV-infected mice exhibited a lower than expected frequency of IFN-gamma-producing CD8(+) T cells and were significantly impaired in ex vivo cytolytic activity compared with competent F-specific effector CD8(+) T cells generated in vitro. The significance of these results for the regulation of the CD8(+) T cell response to RSV is discussed.     

Fewer CTL, not enhanced NK cells, are sufficient for viral clearance from the lungs of immunocompromised mice

Activation of the aryl hydrocarbon receptor (AhR) causes numerous defects in anti-viral immunity, including suppressed CTL generation and impaired host resistance. However, despite a reduced CTL response, mice that survive infection clear the virus. Therefore, we examined the contribution of NK cells and pro-inflammatory cytokines to viral clearance in influenza virus-infected mice exposed to TCDD, the most potent AhR agonist. Infection caused transient increases in pulmonary TNFalpha, IL-1, and IFNalpha/beta levels, but neither the kinetics nor magnitude of this response was affected by AhR activation. No IL-18 was detected at any time point examined. Exposure to TCDD enhanced NK cell numbers in the lung but did not affect their IFNgamma production. Furthermore, depletion of NK cells did not alter anti-viral cytolytic activity. In contrast, removal of CD8+ T cells ablated virus-specific cytolytic activity. These results demonstrate that the pulmonary CTL response to influenza virus is robust and few CTL are necessary for viral clearance.     

Dense bodies of human cytomegalovirus induce both humoral and cellular immune responses in the absence of viral gene expression

Infection of fibroblast cell cultures with human cytomegalovirus (HCMV) leads to the production of significant amounts of defective enveloped particles, termed dense bodies (DB). These noninfectious structures contain major antigenic determinants which are responsible for induction of both the humoral and the cellular immune response against HCMV. We tested the hypothesis that, by virtue of their unique antigenic and structural properties, DB could induce a significant immune response in the absence of infectious virus. Mice were immunized with gradient-purified DB, which were either left untreated or subjected to sequential rounds of sonication and freeze-thawing to prevent cellular entry. Titers of neutralizing antibodies induced by DB were in a range comparable to levels present in convalescent human sera. The virus-neutralizing antibody response was surprisingly durable, with neutralizing antibodies detected 12 months following primary immunization. The HCMV-specific major histocompatibility complex class I-restricted cytolytic T-cell (CTL) response was assayed using mice transgenic for the human HLA-A2 molecule. Immunization with DB led to high levels of HCMV-specific CTL in the absence of de novo viral protein synthesis. Maximal total cytolytic activity in mice immunized with DB was nearly as efficient as the cytolytic activity induced by a standard immunization with murine cytomegalovirus. Furthermore, DB induced a typical T-helper 1 (Th1)-dominated immune response in mice, as determined by cytokine and immunoglobulin G isotype analysis. Induction of humoral and cellular immune responses was achieved without the concomitant use of adjuvant. We thus propose that DB can serve as a basis for the future development of a recombinant nonreplicating vaccine against HCMV. Finally, such particles could be engineered for efficient delivery of antigens from other pathogens to the immune system.