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The Journal of Immunology, 2004, 172: 6239-6250.
Copyright © 2004 by The American Association of Immunologists

Visualizing the Viral Burden: Phenotypic and Functional Alterations of T Cells and APCs during Persistent Infection 1

Dirk Homann2, Dorian B. McGavern and Michael B. A. Oldstone

Department of Neuropharmacology, Division of Virology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Persistent viral infections continue to present major public health problems. Failure to achieve virus control confronts the immune system with a chronic viral burden that may involve immune cells themselves and directly compromise the functionality of effector lymphocytes and APCs. In this study we use the lymphocytic choriomeningitis virus system for persistent viral infection of its natural murine host and use analytical techniques for direct ex vivo visualization of virus-infected immune cells. We report that virtually all cells of the immune system can be infected, but the distribution of the viral burden is differentially allocated to lymphocyte and APC subsets of defined phenotypes. Importantly, the profile of immune cell infection found in the blood is broadly representative for the pattern of cellular infection in most organs and is independent of the presence of Abs or complement. By direct comparison of virus-infected and uninfected cell subsets, we demonstrate that lymphocytic choriomeningitis virus-infected T cells show preferential activation, skewed cytokine profiles, and increased apoptosis. In contrast, increased activation of APCs is generalized and independent of the presence of viral Ag. Our data indicate that specific patterns of immune cell infection are associated with distinct forms of immunostimulatory and immunosuppressive alterations that may provide insights into autoimmune processes associated with infectious disease and offer clues for therapeutic interventions aimed at restoration of complete immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Persistent viral infections pose a particular challenge to the immune system. Failure to effectively control a viral infection results in the persistence of virus, viral proteins, or genes that may coexist indefinitely in the presence of an immune response that can be impaired to varying degrees. The precise balance between persisting virus and immune response depends on multiple factors, such as the nature of the infecting virus, the route of infection, and the initial viral burden, as well as the immune status of the infected host. The interplay among these parameters ultimately determines the spectrum of possible clinical symptoms associated with viral disease (1). The mechanisms by which viruses subvert the immune system and potentially establish persistence are manifold (2), and recent work suggests that persistent virus infections are associated with functional impairment and physical deletion of specific CD8+ T cells normally required for effective virus control (3, 4). Perhaps the most simple strategy for immune evasion is the capacity of some viruses, notably HIV, hepatitis B virus, HSV, EBV, human T cell leukemia virus, polio, and measles, to infect lymphocytes and promote their direct destruction, functional impairment, or sensitization to immunopathological attack (5). A recent report, for example, has demonstrated that HIV preferentially infects HIV-specific CD4+ T cells, which may contribute to the eventual loss of specific CD4+ T cell immunity (6).

The lymphocytic choriomeningitis virus (LCMV) 3 system has been especially useful in modeling persistent viral infections in vivo, as many of the viral and host determinants can be deliberately controlled (7). In particular, two models of LCMV persistence have been extensively studied that differ with regard to two crucial parameters: the presence or the absence of an LCMV-specific T cell response and generalized or specific immunosuppression (8). In the first model, infection of immunocompetent adult mice with certain LCMV isolates results in an abortive CTL response, progressive functional impairment, and eventual physical deletion of LCMV-specific CD8+ T cells in an epitope-dependent fashion (9, 10, 11, 12). The hallmark of this model is a generalized immunosuppression that has been proposed to arise as a consequence of high affinity virus-receptor interactions, infection, and destruction of APCs (13, 14, 15, 16). More recent work from our laboratory has documented impaired dendritic cell (DC) maturation as well as phenotypic and functional DC alterations that can persist even beyond T cell-mediated virus control in many (but not all) tissues (17).

In the second model, the subject of the present investigations, LCMV infection in utero or at birth leads to a life-long virus carrier state by deletion of LCMV-specific T cells (18, 19, 20, 21, 22). However, no generalized immune suppression is observed (23, 24, 25, 26), and specific T and B cell responses to many Ags are apparently unimpaired (13, 26, 27, 28). Moreover, persistently LCMV-infected mice superinfected with heterologous viruses, such as influenza, vaccinia, herpes simplex, or Pichinde virus (the latter also an arenavirus), exhibited relatively normal CTL responses (8, 27) and demonstrated normal or even enhanced resistance to ectromelia or eastern equine encephalomyelitis virus, respectively (29, 30). Although both models of LCMV persistence are characterized by high viral loads in lymphatic and nonlymphatic tissues, the divergent impact on heterologous immunity remains poorly defined.

In the present study we have provided a detailed and comprehensive analysis of persistent infection initiated in utero or at birth. Our results demonstrate that under these conditions, LCMV can infect all subclasses of immune cells in lymphatic and nonlymphatic tissues and is detectable in defined APC subsets at levels that are, to our knowledge, unprecedented. In contrast to infection of adult mice with immunosuppressive LCMV isolates, infection in utero is associated with a generalized activation of DCs and is accompanied by specific activation of T cells that are infected with the virus. Nevertheless, virus-infected immune cells appear to be more prone to apoptosis, and specific T cell responses to heterologous viruses are numerically (but not functionally) reduced. These findings suggest a model in which the balance between specific immunostimulatory and immunosuppressive events determines the functionality of heterologous immunity under conditions of viral persistence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and viruses

C57BL/6J (B6) mice (KbIAbDb) were obtained from the rodent breeding colony at The Scripps Research Institute (La Jolla, CA). B cell-deficient µMT/µMT mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient for the complement component C3 were a gift from Dr. M. Carrol (Harvard Medical School, Boston, MA). Persistently LCMV-infected mice were the offspring of dams infected with LCMV Armstrong clone 53b at birth (31). In some experiments mice infected at birth were also used for experiments; no differences were noted compared with offspring from persistently infected dams. Mice were bred and maintained under specific pathogen-free conditions at The Scripps Research Institute. All persistently infected and uninfected control mice were evaluated at 4–6 mo of age, no differences were observed between male and female mice. For vesicular stomatitis virus (VSV) infection, mice were challenged with 2 x 106 PFU of VSV Indiana i.v. Pichinde virus infection was performed by i.p. inoculation of 2 x 107 PFU of Pichinde strain AN3739.

Tissue preparation

Cells were obtained from spleen, lymph nodes, blood, Peyer’s patches (PP), and bone marrow according to standard procedures. For isolation of cells from liver and kidney, mice were anesthetized, then sacrificed by total body perfusion through the left ventricle with PBS. Organs were cut into small pieces, resuspended in HBSS/2% FCS, forced through a 100-µm pore size mesh, and centrifuged. The pellet was then resuspended in 35% Percoll (Sigma-Aldrich, St. Louis, MO) and centrifuged at 600 x g, and resulting pellet was treated with 0.83% Tris-buffered ammonium chloride to lyse residual RBCs.

Abs and peptides

The Abs, fluorochromes, and peptides used have been described previously (32, 33). In addition, the following Abs and reagents were used: CD11b (M1/70), CD11c (HL3), CD25 (7D4), CD43 (1B11), CD80/B7.1 (16-10A1), CD86/B7.2 (GL1), CD127 (SB/14), CD178/Fas ligand (FasL; MFL3), Kb (AF6-88.5), I-Ab (AF6-120.1), IL-4 (11B11), IL-10 (JES5-16E3), active caspase-3 (C92-605), and isotype controls (R3-34 and G235-2356; BD PharMingen, La Jolla, CA) as well as CD38 (33), CD40 (1C10), and TRAIL (N2B2; eBioscience, San Diego, CA). The 113 mAb raised in mice (IgG2a) and specific for LCMV-nucleoprotein (NP) was produced in our laboratory. The Ab was purified from ascites using a protein A column (Pharmacia, Peapack, NJ) and directly conjugated to Alexa 488 (Molecular Probes, Eugene, OR) or Cy5 (Amersham Pharmacia Biotech, Piscataway, NJ) using reagents and protocols provided by the manufacturers. Peptides were obtained form Peptidogenic (Livermore, CA); their MHC restriction and amino acid sequences are indicated: LCMV: gp33–41 (Db) KAVYNFATC; VSV (34, 35): N52–59 (Kb) RGYVYQGL, gp415–433 (IAb) SSKAQVFEHPHIQDAASQL; Pichinde virus (36): NP16–25 (Db) RGLSNWTHPV; NP38–45 (Kb) SALDFHKV; NP122–132 (Db) VYEGNLTNTQL; NP205 (Kb) YTVKFPNM; OVA257–264 (Kb) SIINFEKL.

Flow cytometry

Reagents and procedures for 5-h polyclonal (PMA/ionomycin) and specific (virus peptides) in vitro restimulation and for surface and intracellular Ab staining have been described previously (32, 33). Functional avidities were determined by calculating the peptide concentration required to elicit IFN-{gamma} production in 50% of epitope-specific T cells (32, 33). Direct ex vivo stains for detection of intracellular LCMV-NP were performed using reagents and protocols identical with those used for intracellular cytokine staining (32).

In vitro T cell priming

TCR transgenic CD8+ T cells from naive p14 mice (specific for LCMV-gp33 epitope) and OT-I mice (specific for OVA257 epitope) were isolated by negative selection (37). In parallel, spleen cells obtained from persistently infected and uninfected mice were depleted of T cells (37). Purified CD8+ T cells were labeled with CFSE (32), and 5 x 105 CD8+ T cells were cultured with 5 x 105 T-depleted APCs (96-well, U-bottom plates, complete RPMI 1640) in the presence or the absence of 10–7 M gp33 or OVA257 peptides. Seventy-two hours later, cultures were harvested, and progressive CFSE dilution of CD8+ T cells was determined by flow cytometry.

In situ analysis of apoptotic cells

Spleens were harvested from persistently infected mice, embedded in OCT (VWR International, West Chester, PA), and frozen on dry ice. Six-micron frozen sections were cut, stained using the ApopTag Red apoptosis detection kit (Chemicon International, Temecula, CA), washed, stained with guinea pig anti-LCMV/biotinylated rat anti-CD4 Abs (BD PharMingen; 1 h at room temperature), washed, and stained with Cy5 anti-guinea pig/streptavidin fluorescein dichlorotriazine (Jackson ImmunoResearch Laboratories, West Grove, PA) (1 h at room temperature). To quantify the frequencies of apoptotic cells that contained LCMV Ag, three-color (fluorescein diclorotriazine, rhodamine, and Cy5) analyses were performed using a MRC1024 confocal microscope (Bio-Rad, Richmond, CA) fitted with a krypton-argon mixed gas laser (excitation, 488, 568, and 647 nm) and a x40 oil objective. Twenty random fields (covering 1.68 mm2 of tissue) were captured from the splenic white pulp of three independent mice. Fields were analyzed to determine the number of apoptotic cells, then apoptotic cells were counted to establish the percentage carrying LCMV Ags.

Statistical analyses

Data handling, analysis, and graphic representation were performed using PRISM 3 (GraphPad, San Diego, CA). Statistical significance was calculated by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Visualizing viral Ag in lymphocytes and APCs

To combine phenotypic characterization of various immune cell subsets with the detection of viral Ag, we established a flow cytometry-based assay using the LCMV NP-specific 113 Ab. The LCMV-NP was chosen as a suitable target Ag because expression of the gp is down-regulated under conditions of persistent infection (38). Spleen cells from 4- to 6-mo-old mice infected with LCMV in utero were stained for surface markers and intracellular LCMV-NP as described in Materials and Methods. Fig. 1A demonstrates that viral NP is readily detectable in all major lymphocyte populations. The preferential infection of CD4+ T cells at frequencies of ~5% is consistent with an earlier report using immunofluorescence staining with polyclonal anti-LCMV sera (39). Lower fractions of LCMV-NP+ cells were found in other major lymphocyte populations, including CD8+ T cells (1.3%), B cells (1.5%), and NK cells (2.3%), whereas NK T cells, identified by coexpression of CD3 and DX5 surface markers, demonstrated viral NP at frequencies of up to 10% (Fig. 1, A and D, and data not shown). As APCs are a phenotypic and functionally heterogeneous population (40), we distinguished five different populations based on the expression profiles of CD11c and CD11b (Fig. 1B). CD11c–/CD11b+ cells, comprising predominantly macrophages, showed a moderate degree of infection of ~5%. Surprisingly, CD11b-expressing CD11c+ cells exhibited a profound extent of viral Ag burden. Almost one-third of "myeloid" DCs, conforming to a CD11c+/CD11b+ phenotype, carried detectable viral NP, whereas infection of the CD11c+/CD11b++ population, characterized by a lower level of CD11c, but enhanced CD11b expression (and potentially related to a subset described as containing immature myeloblasts and DC precursors (41, 42)), could exceed 50% of cells in individual mice (Fig. 1, C and D). In contrast, "lymphoid" DCs, identified by lack of CD11b expression, contained viral Ag at lower frequencies of ~10%.



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  		FIGURE 1. Visualizing virus-infected cells of the immune system. A, Spleen cells obtained from uninfected and persistently LCMV-infected mice, aged 4–6 mo, were stained for surface Ags and intracellular LCMV-NP as detailed in Materials and Methods. LCMV-NP in B220+ B cells, as well as CD4+ and CD8+ T cells (black tracings), was detected in cells from persistently infected mice at frequencies indicated by the values on the right. Control experiments with spleen cells from uninfected mice (gray-shaded histograms) demonstrated background LCMV-NP staining of <0.1% (values on the left). B, Delineation of five distinct APC subpopulations according to CD11b/CD11c expression profiles. C, Detection of LCMV-NP in five distinct APC populations of persistently infected (black tracings, values on the right) and uninfected (gray-shaded histograms, values on the left) control mice. Boxed numbers in lower right corners are provided to facilitate identification of the corresponding APC subset as displayed in B. D, Left panels, Frequencies of LCMV-NP+ lymphocyte and APC populations. Right panels, Absolute numbers of phenotypically defined immune cell populations () and corresponding LCMV-NP+ subpopulations ({blacksquare}) in spleens of persistently infected mice. Values represent the SEM of 8–12 mice tested in multiple independent experiments.

 
Comprehensive analysis of viral burden in immune cells throughout the body

We next assessed whether the differential patterns of immune cell infection were also observed in other lymphatic and nonlymphatic tissues. The data obtained for LCMV infection of lymphocyte and APC subsets in spleen (Fig. 1D) were compared with corresponding cell populations in blood, mesenteric lymph nodes (MLN), Peyer’s patches (PP), thymus, bone marrow, liver, and kidney. Although APC populations in organs such as lymph node, liver, and thymus exhibit tissue-specific phenotypic and functional characteristics (40, 43, 44, 45, 46), we have used the simple distinction according to CD11b/CD11c expression to facilitate direct comparison of APC infection in different organs. We have also included an evaluation of two minor cell subsets: CD4+CD8+ T cells that carry viral Ag at frequencies of ~20%, and B220+CD4+ cells (Fig. 1D). Interestingly, approximately two-thirds of B220+CD4+ cells expressed TCR and showed a degree of viral infection comparable to CD4+ T cells, whereas the TCR-negative population demonstrated elevated levels of viral Ag reminiscent of DC infection (~25%). In support of this observation, B220 has been described as a marker for murine plasmacytoid DCs (47).

Results from our comprehensive overview are displayed according to three different parameters: 1) as a percentage of viral NP+ cells within a given cell population (Fig. 2, A–C), 2) as absolute numbers of total and NP+ cells in defined subsets (Fig. 3), and 3) as the relative fraction individual cell subsets contribute to the entire pool of infected cells (Fig. 2D). Approximately 60–95% of NP+ cells in all tissues were characterized by the expression of CD4, CD11c, and/or B220, indicating that the differential contributions of infected immune cell populations to the overall pattern of infection (while a function of the tissue-specific cell subset composition) were similar. Nevertheless, several organs revealed unique signatures of immune cell infection compared with secondary lymphatic tissues (spleen, MLN, and PP). For example, the viral burden in the kidney was particularly high and was not completely accounted for by our analyses of various immune cells, a likely consequence of immune complex deposition in this organ (48). The unique distribution of lymphocyte populations in the thymus was associated with a diverging pattern of NP+ cells: the relative abundance of CD4+CD8+ thymocytes made this subset, although only ~0.3% contained viral Ag, the largest fraction of infected thymocytes. B cells, when analyzed in terms of absolute numbers, were also shown to be an important reservoir for viral Ag due to their relative preponderance in tissues such as spleen and PPs. As for the different APC populations, CD11c+ cells expressing CD11b++ or CD11b+ constituted the subset with the highest viral burden in all tissues and dominated infected cell populations in the blood. It should also be noted that enumeration of all cell subsets in persistently infected mice did not reveal significant differences compared with uninfected, LCMV-immune, or persistently infected mice successfully treated by adoptive immunocytotherapy (data not shown). Thus, despite tissue-specific differences in immune cell subset composition and degree of infection, the patterns of infection were quite similar in most organs, and the distribution of cellular infection in the blood was broadly representative for the differential infection of lymphocyte and APC populations throughout the entire body.



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  		FIGURE 2. Viral burden in lymphatic and nonlymphatic organs. A, Viral burden in lymphatic and nonlymphatic tissues as quantified by intracellular LCMV-NP stains and displayed as the percentage of cells recovered from indicated organs. B and C, Frequencies of LCMV-NP+ subpopulations in defined immune cell subsets of persistently infected mice were determined by a combination of cell surface and intracellular LCMV-NP staining. Note that the frequencies of NP+ B220+CD4+ and CD4+CD8+ cells in kidneys could not be determined due to insufficient cell numbers. Values in A–C represent the SEM of 4–12 mice analyzed in multiple independent experiments. D, Relative contributions of infected immune cell subsets to overall infection. Note that the contribution of defined cell subsets to infection in MLN and thymus is >100%, indicating that some cell subsets, distinguished by the indicated markers, are also represented in other subsets. In the kidney, cells of unidentified phenotype apparently contribute to the overall relatively high infection. Error bars have been omitted in D to improve the clarity of data representation.

 


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  		FIGURE 3. Infected cells of the immune system in lymphatic and nonlymphatic tissues. Absolute numbers of defined cell immune cell populations () and corresponding LCMV-NP+ subpopulations ({blacksquare}) were determined in various lymphatic and nonlymphatic organs. Values represent the SEM of at least four mice tested in multiple experiments. Note that the maximal x-axis values for blood, MLN, PP, liver, and kidneys differ from the corresponding spleen cell figures (Fig. 1D), as fewer cells were recovered from these organs. Absolute cell numbers in blood were estimated by multiplying white blood counts (WBC; per cubic millimeter) by a coefficient of 5850/100 g of body weight. The median cell count of one larger and one smaller PP was multiplied by a coefficient of 7.4 for an approximation of absolute cell numbers in PP (72 ). Cell counts in kidneys refer to combined absolute numbers in both kidneys. For an estimate of total bone marrow (BM) cells, cells retrieved from two femurs were multiplied by a coefficient of 7.9, as previously described (73 ). ND, not detected.

 
Patterns of immune cell infection are independent of Abs and complement

The profound degree of DC infection found under conditions of persistent infection is in agreement with our recent report that CD11c+ DCs express high levels of {alpha}-dystroglycan ({alpha}DG), a receptor for LCMV (14). In contrast, macrophages or lymphocytes express significantly less {alpha}DG, and T cells are refractory to LCMV infection in vitro. A possible explanation for the high degree of infection observed in immune cells that do not express detectable levels of {alpha}DG is Ag capture via Abs or complement. In viral infections, Abs or the soluble complement components, C3 and C4, may directly bind the virus and promote cell infection via binding to the cellular Fc or complement receptors (49). To test these hypotheses, persistently infected B cell-deficient and C3-deficient mice were evaluated for the extent of immune cell infection. As shown in Fig. 4, the pattern of differential APC infection was completely independent of the presence of Abs or complement C3. Similarly, infection of lymphocytes was not altered by B cell/Ab or C3 deficiency (data not shown).



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  		FIGURE 4. APC infection in the absence of Abs or complement. Infection of splenic APC subsets in persistently LCMV-infected mice deficient for B cells and Abs (µMT/µMT mice) or the soluble complement component C3 (C3–/–) is shown. Values indicate the percentages of APC subsets that stained positively for intracellular LCMV-NP. Representative data of three mice analyzed are presented. Concurrent evaluation of persistently infected B6 control mice gave results comparable to those displayed in Fig. 1, C and D.

 
Phenotypic and functional alterations of APCs in persistent viral infection

The principal advantage of our flow cytometry-based assay for identification of virus-infected cells lies in the possibility to compare phenotype and function of infected and noninfected cellular subpopulations within the same sample. When DCs (identified by expression of CD11c and absence of CD3{epsilon}) from uninfected and persistently infected mice were analyzed for the expression of costimulatory markers, we found larger fractions of B7.1-, B7.2-, and CD40-expressing DCs in both NP– and NP+ populations of persistently infected mice (Fig. 5, A and B). Similarly, persistent infection was associated with an overall increase in MHC-I expression, whereas levels of MHC-II expression remained unaffected (Fig. 5C). In agreement with our finding that persistent infection does not delete particular DC subsets, the fractions of CD8-, CD4-, or B220-expressing DCs were comparable in uninfected mice and in NP+ and NP– compartments of persistently infected mice (not shown). It appears from these phenotypic analyses that persistently infected mice harbor more activated DCs independent of viral Ag presence in individual cells, a finding that may impact on the capacity of these mice to mount immune responses to heterologous Ags. To test this idea, we compared the capacity of APCs from uninfected and persistently infected mice to prime a CD8+ T cell response to the model Ag OVA in vitro. As shown in Fig. 5D, proliferation of primary OVA-specific CD8+ T cells induced by persistently LCMV-infected compared with uninfected APCs was indeed more pronounced. Moreover, activation of CD8+ T cells by persistently infected APCs was specific as it required the presence of cognate Ag (OVA257 peptide; Fig. 5D). Control experiments using LCMV gp33-specific CD8+ T cells revealed, in addition, that APCs from persistently infected donors could induce proliferation of gp33-specific CD8+ T cells without addition of exogenous peptide Ag (Fig. 5D). These data demonstrate that in persistent neonatal LCMV infection, viral gp can be effectively processed and presented to stimulate naive LCMV-specific T cells.



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  		FIGURE 5. Phenotypic and functional alterations of virus-infected APCs. A, Spleen cells from uninfected (upper dot plots) and persistently infected mice (lower dot plots) were stained for the indicated costimulatory markers and gated on CD11c+/CD3{epsilon}– DCs. B, Bar diagrams indicating the fraction of B7.1-, B7.2-,or CD40-expressing CD11c+/CD3{epsilon}– DCs from uninfected mice ({square}) as well as the LCMV-NP– () and LCMV-NP+ ({blacksquare}) subpopulations of persistently infected mice. C, Expression levels of MHC class I (Kb) and class II (I-Ab) by CD11c+/CD3{epsilon}– DCs from uninfected mice ({square}) as well as the LCMV-NP– () and LCMV-NP+ ({blacksquare}) subpopulations of persistently infected mice. GMFI, geometric mean of fluorescence intensity. Values indicate the SEM of three or four mice tested. Statistical significance was calculated between NP+ or NP– and uninfected populations. D, In vitro priming of naive TCR transgenic CD8+ T cells by uninfected or persistently infected APCs. Purified, CFSE-labeled OT-I (specific for OVA257) or p14 (specific for LCMV-gp33) CD8+ T cells were cultured with T-depleted APCs from uninfected or persistently LCMV-infected donors in the absence or the presence of cognate Ag (SIINFEKL or KAVYNFATC peptides). CD8+ T cell proliferation was determined by progressive CFSE dilution. Note that persistently infected APC can effectively prime gp33-specific CD8+ T cell responses in the absence of exogenously added gp33 peptide. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant.

 
Phenotypic and functional alterations of T cells in persistent viral infection

In contrast to the generalized APC activation, phenotyping of CD8+ and CD4+ T cells from persistently and uninfected mice demonstrated that the majority of activation/memory markers tested was preferentially increased in NP+ T cell subsets. In this study virus-infected T cells contained larger fractions of CD44high cells (and CD62Llow cells; not shown), and activation markers such as CD69, CD25, CD38, CD11b, and CD43 were also found on larger subsets of NP+ CD8+ and CD4+ T cells compared with NP– T cells and T cells from uninfected mice (Fig. 6). The presence of increased CD25-expressing CD4+ T cells prompted us to enumerate these cells in persistently and uninfected mice. Although it is well possible from the preceding data that virus infection activates T cells, the CD4+CD25+ T cell phenotype is also associated with regulatory function, and increased numbers of CD4+CD25+ regulatory T cells have been found in another model of persistent viral infection (50). Indeed, persistently infected mice had up to ~4-fold increased numbers of CD4+CD25+ T cells, only ~10% of which were infected with LCMV (not shown). However, despite the substantial expansion of uninfected T cells that conform to the regulatory phenotype (CD4+CD25+), the activation-associated changes observed under conditions of persistent LCMV infection represent a confounding parameter, and further studies will be required to determine whether this T cell subset is indeed involved in immunoregulatory functions. Lastly, comparable total T cell numbers were found in persistently infected and uninfected mice (not shown), but persistent infection was associated with a relative depletion of naive phenotype (CD44low) T cells (CD44lowCD8+, 52.4 ± 2.6 vs 64.4 ± 0.5%; CD44lowCD4+, 66.7 ± 4.9 vs 76.9 ± 0.5%).



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  		FIGURE 6. Phenotypic alterations of virus-infected T cells. A, Spleen cells from uninfected (upper dot plots) and persistently infected mice (lower dot plots) were stained for CD8, CD4, LCMV-NP, and the indicated activation markers. Dot plots are gated on CD8+ or CD4+ T cells. B, Bar diagrams indicate the fraction of memory/activation marker-expressing CD8+ or CD4+ T cells from uninfected mice ({square}) as well as the LCMV-NP– () and LCMV-NP+ ({blacksquare}) subpopulations of persistently infected mice. Values indicate the SEM of three or four mice tested in two or three independent experiments. Statistical significance was calculated between LCMV-NP– and -NP+ populations. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (significance between NP+ and uninfected populations in B, upper left plot is indicated by (*)).

 
T cell functionality under conditions of persistent infection was first evaluated by determining cytokine profiles after polyclonal stimulation. Compared with T cells from uninfected mice, both CD8+ and CD4+ T cells from persistently infected mice had a greater capacity to produce IFN-{gamma}, but impaired potential to produce IL-2 (Fig. 7A). In addition, TNF-{alpha} production was diminished, particularly for CD4+ T cells. When the potentials for IFN-{gamma} production by CD8+ and CD4+ T cells were compared in NP+ and NP– T cells, both populations contained more IFN-{gamma}+ cells than uninfected control mice, yet the small NP+ subpopulations consistently contained greater fractions of IFN-{gamma} producers (Fig. 7, B and C). Thus, enhanced IFN-{gamma} production by T cells correlates with the generalized activation status in persistently infected mice and is most pronounced in virus-infected T cells.



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  		FIGURE 7. Functional alterations of virus-infected T cells. A, Spleen cells from uninfected (left dot plots) or persistently infected (right dot plots) mice were stimulated for 5 h with PMA/ionomycin and stained for CD8 or CD4 as well as intracellular IFN-{gamma}, TNF-{alpha}, and IL-2. IL-2-expressing cells are color-coded in red; their overall frequencies are indicated by the boxed values in red. B, Polyclonally stimulated CD4+ T cells from uninfected (left plot) and persistently infected (right plot) mice stained for intracellular IFN-{gamma} and LCMV-NP. C, Summary of IFN-{gamma}-producing CD8+ and CD4+ T cell populations from uninfected mice ({square}) as well as the LCMV-NP– () and LCMV-NP+ ({blacksquare}) subpopulations of persistently infected mice. Representative data from two or three independent experiments are presented; values represented by bar diagrams are the SEM (three mice per group). *, p < 0.05; ***, p < 0.001 (statistical significance was calculated between LCMV-NP– and -NP+ populations).

 
We next evaluated the impact of the persistently LCMV-infected microenvironment on the capacity to generate a primary T cell response against heterologous viruses. In B6 mice, the dominant T cell responses against VSV are directed against the MHC class I-restricted N52 as well as the class II-restricted gp415 epitope (34, 35). Fig. 8A illustrates that in addition to the N52-specific CD8+ T cell response, gp415-specific CD4+ T cell responses in VSV-infected B6 can be readily visualized by intracellular IFN-{gamma} staining. Both responses were significantly reduced when tested in persistently LCMV-infected mice superinfected with VSV (Fig. 8, A and B). Interestingly, a very small subset of VSV-specific T cells stained positively for LCMV-NP, although it remains unclear at what stage of the VSV-specific T cell response these cells became infected with LCMV. Further functional analyses demonstrated that despite its numerical reduction, the VSV-specific T cell response was characterized by enhanced IFN-{gamma} production at the single-cell level (Fig. 8B), and functional avidities of VSV-specific CD8+ T cells generated in persistently LCMV-infected mice were not compromised, but were actually slightly higher than those in VSV-infected control mice (Fig. 8C). Similar results were obtained in superinfection experiments using the arenavirus Pichinde. Pichinde virus-specific T cell epitopes have recently been mapped (36) and, with the exception of the NP205 epitope, are not cross-reactive with LCMV epitopes. As anticipated, Pichinde infection of persistently LCMV-infected mice failed to generate NP205-specific T cell responses (due to deletion of specific precursor T cells in the LCMV-infected thymus) and the CD8+ T cell responses to NP16, NP38 and NP122 were reduced compared with those in Pichinde-infected control mice (Fig. 8D). In addition, no immune deviation toward increased IL-4 or IL-10 production by VSV- or Pichinde virus-superinfected mice was observed (not shown). We conclude that T cell responses specific for heterologous viruses are quantitatively reduced, possibly due to fewer naive precursor T cells, but are functionally slightly enhanced by the presence of an underlying persistent viral infection.



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  		FIGURE 8. Heterologous T cell immunity in persistently LCMV-infected hosts. A, Uninfected and persistently LCMV-infected mice were challenged with VSV, and N52-specific CD8+ (upper plots) and gp415-specific CD4+ (lower plots) T cell responses were enumerated by intracellular IFN-{gamma} staining 7 days later. B, Left panel, Frequencies of N52-specific CD8+ and gp415-specific CD4+ T cell populations of VSV-challenged control ({blacksquare}) or persistently LCMV-infected mice (). Right panel, IFN-{gamma} expression levels (geometric mean of fluorescence intensity (GMFI)) by VSV N52-specific CD8+ T cells from control ({blacksquare}) or persistently LCMV-infected mice (). C, Functional avidities of VSV N52-specific CD8+ T cells were determined by stimulating spleen cells 7 days after VSV infection with graded doses of N52 peptide. Values listed are the peptide concentrations required to elicit a detectable IFN-{gamma} response in 50% of specific T cell populations. The lower these values, the higher the functional avidity. D, Frequencies of specific CD8+ T cells in Pichinde virus-infected mice (legend as in B). Data are from three or four mice tested in one representative of two or three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (statistical significance for heterologous T cell responses was calculated between persistently LCMV-infected and uninfected animals).

 
Survival of LCMV-infected immune cells

In the final set of experiments, we questioned whether the persisting LCMV infection would affect the survival of immune cells. Although LCMV is considered to be a noncytopathic virus, persistent LCMV infection may be associated with reduced survival of infected cells. However, given an average frequency of ~3% infected cells, we expected no discernible differences in global analyses. We therefore used our flow cytometry-based assay to evaluate survival and apoptosis simultaneously in LCMV-NP– and -NP+ cells. As detection of intracellular LCMV-NP requires fixation and permeabilization of the cells, we could not use some of the commonly used apoptosis assays. Instead, we assessed the potential for survival indirectly by determining expression levels of IL-7R{alpha} and Bcl-2. IL-7 is essential for the survival of developing and mature T cells, and IL-7R{alpha}-deficient mice show a pronounced loss of T cells that can be averted by transgenic overexpression of Bcl-2 (51, 52). Bcl-2 is a critical survival factor for lymphocytes and has recently been shown to also control the longevity of DCs (53). When tested in persistently LCMV-infected mice, we observed lower IL-7R{alpha} expression by CD8+ and CD4+ T cells, with NP+ cells showing the greatest reduction (Fig. 9A). Analysis of Bcl-2 levels was less conclusive, but lower expression levels specifically in NP+ CD4+ T cells (as well as B220+ cells) were noted. For a more direct assessment of apoptosis, we analyzed caspase-3 activation, which precedes chromatin condensation and degradation. Ex vivo detectable active caspase-3 expression was found in <1% of lymphocytes in uninfected mice or NP– populations of persistently infected mice, but was found in up to 10% of NP+ B220+ and CD8+ T cells as well as ~4% of NP+ CD4+ T cells and CD11c+ DCs (Fig. 9B). We subsequently visualized DNA fragmentation directly by in situ TUNEL assays. Fig. 9C shows that viral infection of CD4+ T cells is associated with altered cell morphology, loss of cell surface-restricted CD4 expression, and fragmentation of DNA. The correlation between virus infection and cell death was confirmed by an extended quantitation of apoptotic cells that showed that 72% of dying (TUNEL+) CD4+ T cells were also infected with LCMV. These findings demonstrate that long term persistence of LCMV may lead to reduced longevity and increased death, primarily among cells directly infected with the virus, potentially by cell-intrinsic alterations in response to virus infection or activation of innate defense mechanisms.



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  		FIGURE 9. Survival and death of virus-infected immune cells. A, Expression of IL-7R{alpha} by splenic CD4+ and CD8+ T cells from uninfected ({square}) and persistently infected mice (, NP– subset; {blacksquare}, NP+ subset). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (statistical significance was calculated between LCMV-NP– and -NP+ populations; in addition, IL-7R{alpha} expression was significantly different between NP– and uninfected populations). B, Percentages of active caspase 3-expressing cells in uninfected and persistently infected mice. Data were obtained from three or four experiments, with three mice per group. Statistical significance was calculated between LCMV-NP– and -NP+ populations. C, Spleen sections prepared from persistently infected mice were stained for CD4, LCMV, and DNA fragmentation by TUNEL assay and were analyzed by three-color confocal microscopy, as detailed in Materials and Methods. Arrows in merged image point at LCMV-infected, apoptotic CD4+ T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have provided a comprehensive quantitation of the viral burden associated with neonatal persistent LCMV infection. Overall, cells of the immune system are infected at a fraction of 2–4% in most tissues. The extent and pattern of infected immune cells are comparable in many lymphatic and nonlymphatic tissues and are largely independent of the host age (data not shown), suggesting that the combination of systemic infection and cellular trafficking in the absence of specific T cell responses leads to an organism-wide equilibration of virus infection. As a consequence, the distribution of viral Ags in blood-borne cells is broadly representative for the infection of immune cells in other tissues of the host. The most important questions arising form these observations relate to the survival, phenotype, and function of virus-infected immune cells as well as an integration of these findings into the host’s altered capacity to mount effective immune responses.

However, we need to address first whether the cells positive for viral Ag are productively infected with LCMV. Our flow cytometry-based assay allows for rapid detection of viral Ag in defined immune cell subsets, yet it does not measure the levels of infectious virus that may be present, as shown in previous studies on CD4+ T cell infection, at ~10-fold lower frequencies (39, 54). Our results cannot exclude the uptake of viral Ags in the absence of actual infection, but this possibility seems unlikely for several reasons. 1) The frequencies of cells carrying LCMV nucleic acid sequences in both unfractionated and purified CD4+ T cell populations are comparable to those obtained by NP staining in the current study (54, 55). 2) During persistent LCMV infection, both viral gp and NP RNA are present at low levels (J. C. de la Torre, unpublished observations), but the synthesis, stability, or both of NP and gp proteins are differentially regulated, resulting in robust NP, but restricted gp expression levels (38), which compromises the generation of infectious virus. 3) According to the defective ribosomal product hypothesis (56), presentation of antigenic epitopes preferentially originates from newly synthesized polypeptides that are degraded shortly after translation. In fact, display of an LCMV-NP epitope was recently shown to rely on NP neosynthesis rather than a long-lived pool of cellular NP (57). 4) NP does not have budding properties, and thereby its release from infected cells appears to be highly unlikely (58). 5) Finally, experiments to visualize the in vivo uptake of soluble protein Ag require the administration of very large Ag quantities, its uptake is limited to APCs, and Ag becomes undetectable within 18 h due to rapid degradation and processing by APCs (59). Together, these considerations suggest that the presence of viral NP as well as the presentation of LCMV epitopes by persistently infected APCs are the consequences of continuous transcription, translation, and post-translational processing of viral proteins, rather than the acquisition of isolated NP in the absence of infection.

How, then, are immune cells infected in our persistent infection model? The preferential targeting of defined APC subsets may be due in part to the LCMV Armstrong->clone 13 genotype mutations observed in persistent neonatal infection (60) and use of {alpha}DG as a cellular receptor by the mutant virus (61). We have shown that {alpha}DG is highly expressed by CD11c+ cells, but is not detectable on lymphocytes and macrophages with the assays used (14). Consistent with this finding, we observed that CD11c– macrophages, previously shown to be a target for LCMV infection (29), exhibited only a moderate degree of infection. However, preliminary experiments failed to demonstrate a direct correlation between the extent of {alpha}DG and NP expression in different CD11c-bearing APC subsets (not shown). As we have ruled out an acquisition of viral Ags via Ab or complement binding, other mechanisms must be operative in the preferential infection of individual APC populations. Using fluorescent beads with the approximate size of the LCMV virion, we found a partial correlation between bead uptake and the presence of LCMV-NP (i.e., the extent of bead uptake and viral NP expression were most pronounced in CD11b-expressing CD11c+ APCs; not shown). In this context it is also of note that CD11b+CD11c+ DCs were recently identified as a population with particular capacity for uptake and processing of virus-like particles (62). Therefore, the possibility arises that receptor-independent mechanisms (63) play a role in the acquisition of viral particles, which could contribute to the pattern of APC infection seen in our experimental system.

The extraordinarily high degree of infection in defined APC subsets makes these cells significant contributors, despite their limited numbers, to the overall viral burden. This finding is particularly surprising considering the reported absence of generalized immunosuppression (8, 13, 26, 27). An explanation for this phenomenon may come from our observation that persistent viral infection can create a relatively stable microenvironment that favors a widespread up-regulation of costimulatory APC marker and MHC-I expression in the absence of significantly reduced MHC-II levels. Although our findings are in contrast with those obtained in some models of acute DC infection, it is important to note that DCs infected de novo are subject to dynamic regulation by cell intrinsic as well as extrinsic parameters. Infection of DCs in vitro or in vivo with MCMV resulted in initial up-regulation of B7.2 and CD40, but subsequent down-regulation of B7.1, B7.2, CD40, and MHC classes I and II and impaired capacity for T cell priming (64). Similarly, CMV infection of human DC resulted in enhanced expression of B7.1, B7.2, and CD40, whereas MHC class I and II expression was reduced (65). Interestingly, up-regulation of Fas ligand and TRAIL was proposed to delete activated T cells in this in vitro model system. Although we observed some Fas ligand up-regulation (but concurrent TRAIL down-regulation) by virus-infected DCs evaluated directly ex vivo, the biological significance is unclear given that FasL expression was increased from ~1.0% among uninfected to ~2.5% of infected DCs (not shown).

Ultimately, we would like to argue that it is the immunological visibility of APCs, i.e., the presentation of immunogenic viral epitopes and the expression of costimulatory molecules in conjunction with the presence or the absence of specific T cells, that shapes the specific phenotype of persistent viral disease. Although LCMV-infected DCs appear to be more prone to apoptosis in our model of persistent neonatal virus infection, restricted viral gp expression (38) does not impair the effective processing and presentation of gp-derived epitopes that allow for the induction of primary LCMV-specific CD8+ T cell responses, and the globally enhanced activation status of DCs may, in fact, facilitate heterologous T cell responses. Therefore, further therapeutic manipulations to increase DC functionality are probably not of benefit for virus control. This is in striking contrast to persistent infection of adult mice with immunosuppressive LCMV strains, which leads to high levels of DC infection associated with compromised maturation, functional impairment, phenotypic deactivation, and epitope-dependent alterations of effective LCMV Ag presentation (12, 14, 15, 17). In this model, it is the interplay between DCs and LCMV-specific T cell immunity that apparently results in mutual immunosuppression by progressive exhaustion of T cell responses and stunted DC functionality. Thus, therapeutic interventions at the levels of both DCs and specific T cells are warranted in the latter model.

Although the use of immunosuppressive LCMV isolates in immunocompetent mice results in very limited infection of T cells (14), our study of neonatal persistent LCMV infection also provides important insights into the biology of virus-infected T cells. In agreement with previous studies (39, 54), we found CD4+ T cells to be the major T cell compartment infected with LCMV. Yet all other T cell populations, including CD8+, CD4+CD8+, {gamma}{delta}TCR, and NK T cells, demonstrated readily detectable infectability. The emerging picture for virus-infected T cells suggests two opposing trends that shape their biological activity. Even a "noncytopathic" virus such as LCMV can cause apoptosis of virus-infected T cells in vivo, and analysis of specific T cell responses to heterologous viral challenges has revealed a previously unappreciated numerical reduction of these responses under conditions of persistent viral infection. Yet, virus-infected CD8+ and CD4+ T cells, as opposed to the corresponding LCMV-NP– subsets, clearly demonstrated enhanced expression of activation/memory markers and cytokine profiles commonly associated with effector T cells (enhanced IFN-{gamma} and reduced IL-2/TNF-{alpha} expression) with the capacity for rapid IFN-{gamma} production preferentially residing in virus-infected T cells. Furthermore, effector T cells specific for VSV or Pichinde virus produced larger quantities of IFN-{gamma} and exhibited slightly enhanced functional avidities.

In summary, our analyses have revealed two diverging trends in a persistent virus infection: 1) immunostimulatory processes that may enhance adaptive immunity (generalized activation of APCs, specific activation of virus-infected T cells, and increased functionality of heterologous T cell responses), as well as 2) immunosuppressive events that may impair effective immune responses (increased apoptosis of virus-infected T cells and APCs, numerical reduction of heterologous T cell responses, and increased numbers of CD4+CD25+ cells with potential regulatory function). In this respect the model of neonatal persistent LCMV infection reproduces the pathology of HIV infection characterized by virus-mediated killing of CD4+ T cells and generalized activation of the immune system (reviewed Ref.66). The recently reported enhanced turnover of effector-memory T cells in HIV-infected individuals (67) may be a consequence of such chronic immune activation and carries several important implications for the pathogenesis of HIV disease: enhanced proliferation of memory T cells may drain the naive T cell pool, resulting in reduced capacity to generate primary and memory responses to heterologous Ags, and expansions of T cells capable of producing proinflammatory cytokines may increase bystander pathology associated with HIV infection (66). Some of these hypotheses can, in fact, be validated by our study of persistent LCMV infection, as we found reduced fractions of naive T cells, diminished primary T cell responses, and enhanced IFN-{gamma} production. Furthermore, preliminary gene array experiments indicate that persistent infection is associated with profound activation of the IFN system (not shown), and cytokines such as IFN-{gamma}, IL-12, and IL-18 can promote accelerated turnover of memory T cells in an Ag-nonspecific fashion (68).

Our observations also provide insights into the paradoxical relation between infectious and autoimmune diseases. Although infectious pathogens, in particular viruses, can cause or precipitate autoimmunity (69, 70), they may also prevent the onset of autoimmune diseases (71). Neonatal persistent LCMV infection exemplifies this paradox because it can exacerbate or manifest lupus-like disease (in NZWxNZB F1 or NZW mice, respectively) and produce symptoms of type 1 diabetes (in BALB/c mice), yet it prevents diabetes in nonobese diabetic mice (7). Our findings suggest that the particular balance between immunostimulatory and immunosuppressive processes acquire differential significance in the context of distinct autoimmune diseases. Thus, enhanced DC function may contribute to more effective priming of autoimmune responses in the lupus model, whereas increased death and altered immune regulation of T cells in nonobese diabetic mice may curtail the development of autoimmune diabetes.


   Acknowledgments
 
We thank Juan Carlos de la Torre for his insights and comments during the preparation of this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Training Grant AG00080, Juvenile Diabetes Foundation International Fellowship 3-1999-629, and a grant from Studienstiftung des Deutschen Volkes (to D.H.) as well as National Institutes of Health Grants AI09484 and AI45927 (to M.B.A.O.). Back

2 Address correspondence and reprint requests to Dr. Dirk Homann, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box 140, Denver, CO 80262. E-mail address: dirk.homann{at}uchsc.edu Back

3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; DC, dendritic cell; {alpha}DG, {alpha}-dystroglycan; FasL, Fas ligand; MLN, mesenteric lymph node; NP, nucleoprotein; PP, Peyer’s patch; VSV, vesicular stomatitis virus. Back

Received for publication December 8, 2003. Accepted for publication March 10, 2004.


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