Reconstitution of the complement function in C1q-deficient (C1qa-/-) mice with wild-type bone marrow cells.

Besides Ab-independent and Ab-dependent activation of the complement classical pathway in host defense, C1q plays a key role in the processing of immune complexes and in the clearance of apoptotic cells. In humans, C1q deficiency leads to systemic lupus erythematosus-like symptoms in over 90% of the cases, thus making this defect a strong disease susceptibility factor. Similarly, C1q-deficient mice (C1qa-/-) develop systemic lupus erythematosus-like symptoms, such as autoantibodies and glomerulonephritis. We have previously provided evidence that C1q is produced by cells of the monocyte-macrophage lineage. In this study, we have tested whether transplantation of bone marrow cells would be sufficient to reconstitute C1q levels in C1qa-/- mice. C1qa-/- mice received a single graft of 10(7) bone marrow cells from wild-type (wt) donors after irradiation doses of 6, 7, 8, or 9 Gy. Engraftment was monitored by a Y chromosome-specific PCR and a PCR that differentiated wt from C1qa-/- genotype. Serum levels of C1q Ag and C1 function increased rapidly in the recipient mice, and titers reached normal levels within 6 wk after bone marrow transplantation. In wt mice that received C1qa-/- bone marrow, serum levels of C1q decreased constantly over time and became C1q deficient within 55 wk. These data clearly demonstrate that bone marrow-derived cells are the source of serum C1q and are competent to reconstitute normal C1q serum levels in C1q-deficient mice. Therefore, stem cell transplantation could be a therapy for patients with hereditary C1q deficiency.

Dynamic antigen presentation patterns of Listeria monocytogenes-derived CD8 T cell epitopes in vivo.

Little information exists regarding the presentation of antigenic peptides in infected tissues. In this study the in vivo presentation of four different CD8 T cell epitopes of Listeria monocytogenes was monitored. Peptide presentation was measured by a new, highly sensitive, ex vivo Ag presentation assay that was based on the testing of freshly isolated cells from infected spleens with peptide-specific CD8 T cell lines in an IFN-gamma-specific ELISPOT assay. Remarkably, the peptide presentation pattern of splenocytes and that of macrophages purified from spleens of L. monocytogenes-infected mice were different from those of in vitro infected macrophage-like cell lines. The in vivo Ag presentation pattern of splenocytes also exhibited dynamic changes during the first 48 h of infection. In vivo peptide presentation at later time points postinfection was biased toward immunodominant CD8 T cell epitopes, while at an early time point, 6 h postinfection, subdominant and dominant CD8 T cell epitopes were presented with similar strength. In summary, our studies show that Ag presentation during an infection is a highly dynamic process that only can be fully appreciated by the study of cells infected in their physiological environment.

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.

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.

Macrophages escape inhibition of major histocompatibility complex class I-dependent antigen presentation by cytomegalovirus.

The mouse cytomegalovirus (MCMV) m152- and m06-encoded glycoproteins gp40 and gp48, respectively, independently downregulate major histocompatibility complex (MHC) class I surface expression during the course of productive MCMV infection in fibroblasts. As a result, presentation of an immediate-early protein pp89-derived nonapeptide to H-2L(d)-restricted CD8(+) cytotoxic T cells is completely prevented in fibroblasts. Here we demonstrate that MCMV-infected primary bone marrow macrophages and the macrophage cell line J774 constitutively present pp89 peptides during permissive MCMV infection to cytotoxic T lymphocytes (CTL). In contrast to fibroblasts, expression of the m152 and m06 genes in macrophages does not affect surface expression of MHC class I. Assessment of pp89 synthesis and quantification of extracted peptide revealed a significantly higher efficiency of macrophages than of fibroblasts to process pp89 into finally trimmed peptide. The yield of pp89 peptide determined in MCMV-infected tissues of bone marrow chimeras confirmed that bone marrow-derived cells represent a prime source of pp89 processing in parenchymal organs. The finding that macrophages resist the viral control of MHC I-dependent antigen presentation reconciles the paradox of efficient induction of CMV-specific CD8(+) CTL in vivo despite extensive potential of CMVs to subvert MHC class I.

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.

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.

In vivo replication of recombinant murine cytomegalovirus driven by the paralogous major immediate-early promoter-enhancer of human cytomegalovirus.

Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502-8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.

Proliferation and MHC-unrestricted bystander lysis by virus-specific cytotoxic T cells following antigen self-presentation.

Cytotoxic T cells (CTL) not only act as effector cells, but can also serve as antigen-presenting cells (APC) for other CTL due to their expression of major histocompatibility complex (MHC) class I molecules. In the present study we show that independently derived CTL lines (CTLL) with specificity for an L(d)-presented nonapeptide corresponding to amino acids 168-176 of the immediate-early 1 (IE1) protein of murine cytomegalovirus not only lyse syngeneic but also allogeneic target cells, if the peptide is present during the cytolytic assay. Whereas a short peptide pulse is sufficient to render syngeneic cells susceptible to lysis, continued presence of soluble peptide is mandatory for the lysis of allogeneic target cells. This indicates a difference in the mechanisms involved. Syngeneic BALB/c B cell blasts (K(d)D(d)L(d)) and mutant BALB/c-H-2dm2 B cell blasts lacking the restricting Ld molecules (K(d)D(d)0) were lysed to a similar extent in the absence of the IE1 nonapeptide, provided that the IE1-specific CTL had been pre-incubated with the peptide before the cytolytic assay. Since the mutant cells cannot present the IE1 peptide, their lysis indicates an MHC-unrestricted, peptide-independent mode of recognition by the CTLL. In addition, proliferation of the CTLL takes place after incubation with the cognate peptide, even in the absence of professional APC. These data indicate inter-CTL antigen self-presentation, resulting in activation of the lytic machinery leading to peptide-independent bystander lysis of allogeneic as well as syngeneic target cells.

Reconstitution of CD8 T cells is essential for the prevention of multiple-organ cytomegalovirus histopathology after bone marrow transplantation.

Cytomegalovirus (CMV) infection in the period of temporary immunodeficiency after haematoablative treatment and bone marrow transplantation (BMT) is associated with a risk of graft failure and multiple-organ CMV disease. The efficacy of immune system reconstitution is decisive for the prevention of CMV pathogenesis after BMT. Previous data in murine model systems have documented a redundancy in the immune effector mechanisms controlling CMV. CD8 T cells proved to be relevant but not irreplaceable as antiviral effectors. Specifically, in a state of long-term in vivo depletion of the CD8 T-cell subset, CD4 T cells were educed to become deputy effectors controlling CMV by a mechanism involving antiviral cytokines. It is of medical importance to know whether one can trust in this 'flexible defence' in all clinical settings. It is demonstrated here that reconstitution of CD8 T cells is crucial for the prevention of fatal multiple-organ CMV disease under the specific conditions of BMT.

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 Ld were transplanted into the Ld 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 Ld-positive donor-derived and Ld-negative recipient-derived cells. Pulmonary infiltrates did not include cytolytic T cells directed against Ld. 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 Ld 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.

Control of murine cytomegalovirus in the lungs: relative but not absolute immunodominance of the immediate-early 1 nonapeptide during the antiviral cytolytic T-lymphocyte response in pulmonary infiltrates.

The lungs are a major organ site of cytomegalovirus (CMV) infection, pathogenesis, and latency. Interstitial CMV pneumonia represents a critical manifestation of CMV disease, in particular in recipients of bone marrow transplantation (BMT). We have employed a murine model for studying the immune response to CMV in the lungs in the specific scenario of immune reconstitution after syngeneic BMT. Control of pulmonary infection was associated with a vigorous infiltration of the lungs, which was characterized by a preferential recruitment and massive expansion of the CD8 subset of alpha/beta T cells. The infiltrate provided a microenvironment in which the CD8 T cells differentiated into mature effector cells, that is, into functionally active cytolytic T lymphocytes (CTL). This gave us the opportunity for an ex vivo testing of the antigen specificities of CTL present at a relevant organ site of viral pathogenesis. The contribution of the previously identified immediate-early 1 (IE1) nonapeptide of murine CMV was evaluated by comparison with the CD3epsilon-redirected cytolytic activity used as a measure of the overall CTL response in the lungs. The IE1 peptide was detected by pulmonary CTL, but it accounted for a minor part of the response. Interestingly, no additional viral or virus-induced antigenic peptides were detectable among naturally processed peptides derived from infected lungs, even though infected fibroblasts were recognized in a major histocompatibility complex-restricted manner. We conclude that the antiviral pulmonary immune response is a collaborative function that involves many antigenic peptides, among which the IE1 peptide is immunodominant in a relative sense.

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.

IFN-gamma is a prerequisite for optimal antigen processing of viral peptides in vivo.

The immediate early protein pp89 of mouse CMV is processed into the nonapeptide YPHFMPTNL, which is presented to CD8+ T lymphocytes by the H-2 Ld molecule. The tissue distribution of this peptide was determined during the course of mouse CMV infection. In tissues, there was no general correlation between peptide processing and infectious virus productivity. Immunosuppression by sublethal irradiation resulted in enhanced MCMV replication but did not increase the peptide yield and drastically reduced the peptide to plaque-forming unit rate in infected organs. IFN-gamma administration restored efficient peptide processing in the immunocompromised host, and neutralization of IFN-gamma in the immunocompetent host decreased peptide processing. This suggests that the efficiency of peptide processing after CMV infection in vivo is governed by IFN-gamma rather than by the productivity of virus infection.