CD40 ligand is pivotal to efficient control of virus replication in mice infected with lymphocytic choriomeningitis virus

CD40 ligand (CD40L) is an important molecule that is known to be involved in T-B collaboration and certain aspects of cell-mediated immunity. However, its role in antiviral immunity has not been clearly defined as of yet. Therefore, mice with a targeted defect in the gene encoding this molecule were infected with one of two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in the host. Infection with lymphocytic choriomeningitis virus is initially controlled primarily by CD8+ effector cells, whereas long-term immune surveillance also depends upon CD4+ cells and B cells. Our results reveal that the primary activation, clonal expansion, and differentiation of CD8+ T cells does not require expression of CD40L. However, lack of expression results in rapid impairment of CTL responsiveness and failure to permanently control virus replication. This happens not only in mice infected with the rapidly spreading virus strain but also at a late stage in mice infected with the strain of more limited potential for spreading. In the latter mice, virus replication is initially controlled very efficiently, but high levels of virus can be detected in the blood and internal organs approximately 6 mo after virus inoculation. Since the impairment of immune function seems to be more pronounced in CD40L-deficient mice than in mice lacking either CD4+ cells or B cells, these results indicate that CD40L is pivotal to sustain efficient antiviral immune surveillance, including CD8+ T cells, and suggest that CD40L is critically involved in cellular interactions in addition to T-B cooperation.     

Analysis of baby hamster kidney cells persistently infected with lymphocytic choriomeningitis virus

The possibility has been demonstrated of using the method of aggregate-haemagglutination for the detection of B. cereus exo-enterotoxin in both food products and culture media. It has been established that 0.004 mug/ml of enterotoxin can be detected by this method. The applied antisera to B. cereus enterotoxin did not yield cross reactions with enterotoxins produced by E. coli, Cl. perfringens, St. aureus, V. cholerae or Sh. dysenteriae.     

Enhanced virus clearance by early inducible lymphocytic choriomeningitis virus-neutralizing antibodies in immunoglobulin-transgenic mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin mu heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host's capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin mu heavy and the kappa light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.     

Cytolytically active memory CTL present in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection

Generally, it has been assumed that memory T cells are dormant and inactive cells in the absence of their specific Ag. Recent work has challenged this assumption by showing that a portion of the CD8+ memory T cell pool is in cycle. In this study, we demonstrate that a significant number of blast-size memory CD8+ T cells in mice, long after lymphocytic choriomeningitis virus (LCMV) infection, mediate cytolysis against highly sensitive targets without any in vivo or in vitro restimulation and expansion with Ag. Peptide-coated RMA-S targets were sufficiently sensitive to detect low but significant cytolytic activity in bulk 51Cr release assays in nonstimulated LCMV-specific splenic memory CTL populations. Most of the directly cytotoxic activity was against the GP33 epitope, and this persisted throughout the lifetime of the mouse following infection. The cytotoxic activity was not inhibited by cyclosporin A, indicating that these cells were already in an active state and not dependent on further stimulation in vitro. It was formally shown that the cytotoxic activity was mediated by the CD8+ CTL by sorting for the blast-size CD8+ population and by blocking target cell lysis with anti-CD8 Ab. Thus, at any time after the original infection some portion of the memory CD8+ T cell pool is cycling, and it remains cytolytically active long after resolution of the original infection. These CTL may provide a rapidly acting defense mechanism against reinfection.     

Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus

A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.     

Uncovering subdominant cytotoxic T-lymphocyte responses in lymphocytic choriomeningitis virus-infected BALB/c mice

The cytotoxic T-lymphocyte response against lymphocytic choriomeningitis virus (LCMV) in BALB/c mice is predominantly directed against a single, Ld-restricted epitope in the viral nucleoprotein (residues 118 to 126). To investigate whether any Kd/Dd-restricted responses were activated but did not expand during the primary response, we used a BALB/c mutant, BALB/c-H-2dm2, which does not express the Ld molecule. Splenocytes from LCMV-infected BALB/c mice were transferred into irradiated BALB/c-H-2dm2 mice and rechallenged with LCMV. Thus, they were exposed to an antigenic stimulus without the involvement of the immunodominant Ld-restricted epitope. In this adoptive transfer model, the donor splenocytes protected the recipient mice against chronic LCMV infection by mounting a potent Kd- and/or Dd-restricted secondary antiviral response. Analysis of a panel of Kd binding LCMV peptides revealed that residues 283 to 291 from the viral glycoprotein (GP(283-291)) comprise a major new epitope in the adoptive transfer model. Because the donor splenocytes were first activated during the primary infection in BALB/c mice, the GP(283-291) epitope is a subdominant epitope in BALB/c mice that becomes dominant after rechallenge in BALB/c-H-2dm2 mice. This study makes two points. First, it shows that subdominant CTL responses can be protective, and second, it provides a general experimental approach for uncovering subdominant CTL responses in vivo. This strategy can be used to identify subdominant T-cell responses in other systems.     

Variants able to cause growth hormone deficiency syndrome are present within the disease-nil WE strain of lymphocytic choriomeningitis virus

Persistent infection of C3H/St mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong leads to disordered growth and hypoglycemia. Both host and viral determinants contribute to this growth hormone (GH) deficiency syndrome (GHDS). Development of the GHDS correlates with the virus's ability to replicate in the GH-producing cells and cause reduced levels of GH synthesis. LCMV strain WE infects few GH-producing cells and does not cause GHDS in C3H/St mice. We show here that clonal variants isolated from the GHDS-nil WE population are able to replicate at high levels in GH-producing cells and cause GHDS in C3H/St mice. These variants are stably maintained, but phenotypically silent, within the GHDS-nil WE population.     

Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice

Antiviral cytotoxic T-cells are critical for control of lymphocytic choriomeningitis virus (LCMV) infection in mice. In H-2b mice, the antiviral response is directed against three Db-restricted epitopes in the viral nucleoprotein (NP396-404) and glycoprotein (GP276-286 and GP33-41). Our present data revealed a clear hierarchy among these three epitopes, in which NP396-404 is immunodominant, followed by GP33-41 and GP276-286, respectively. In order to identify additional CTL epitopes in the LCMV nucleoprotein and glycoprotein, we used the motifs for Db2- and Kb-binding peptides, combined with MHC class I-binding assays. Out of 23 Db motif-fitting peptides, we identified 4 Db binders, one of which (GP92-101) turned out to be a new CTL epitope. Among 28 Kb motif-fitting peptides, 12 bound Kb, and one of these (NP205-212) was a CTL epitope. Both newly identified CTL peptides were recognized by LCMV-immune splenocytes after secondary in vitro stimulation. Both peptides bound their MHC class I molecules with intermediate affinity (470 and 170 nM for GP92-101 and NP205-212, respectively). Responses against these peptides were weaker than the responses against the three major epitopes. None of the high affinity binders were new epitopes, suggesting that high affinity binders are either immunodominant epitopes or no epitopes at all. Thus, analysis of 51 Kb and Db motif-fitting peptides yielded 2 new, subdominant epitopes. Immunization of C57BL/6 mice with these peptides, or vaccinia virus recombinants expressing these epitopes as minigenes, protected against chronic LCMV infection, demonstrating that immunization with subdominant epitopes can confer protection against chronic viral infection.     

Right ventricular size is acutely decreased by inhaled nitric oxide in newborns with pulmonary hypertension

The pressure and volume demands of the right and left ventricles may dramatically change following selective pulmonary vasodilation in newborns with pulmonary hypertension. Thus, ventricular planimetry was performed by two-dimensional echocardiography in 35 newborns with lung disease and increased pulmonary vascular resistance who were treated with inhaled nitric oxide to determine the influence of therapy on right and left ventricular size and function. The end-diastolic and end-systolic areas of each ventricle were measured from apical 4-chamber images before, and 30 to 60 minutes after, the onset of 20 parts per million inhaled nitric oxide. Estimates of ventricular function were determined by the systolic decrease in ventricular area, (diastolic area - systolic area) x 100/diastolic area. Heart rate, systemic blood pressure, and left ventricular areas did not change. However, the oxygenation index, the proportion of right-to-left ductal shunt (nonrestrictive ductus arteriosus, n = 22), the systolic pulmonary arterial pressure (closed or restrictive ductus arteriosus, n = 13), and the right ventricular diastolic and systolic areas were decreased after nitric oxide inhalation. The baseline systolic decrease in left ventricular area was lower in a subgroup of patients who developed an increase in left ventricular diastolic area following nitric oxide inhalation. Thus, nitric oxide improves pulmonary hemodynamics and decreases right ventricular size in newborns with lung disease and pulmonary hypertension. However, newborns may develop an increase in left ventricular size if left ventricular function is decreased prior to therapy.     

Recombination of nonreplicative RNA precursors of Sindbis virus in infected cells overexpressing murine-inducible nitric oxide synthase

The Sindbis virus-based SINrep5 expression system is one of the most efficient vectors for gene transfer leading to fast and high expression of the gene of interest. This system was used to transfect vascular endothelial and smooth muscle cells using murine inducible nitric oxide synthase (miNOS) as a reporter gene. Infection of both cell types leads to high expression levels of miNOS. In addition, the harvested supernatant of these infected cells was used for further rounds of infections, demonstrating that recombination of the parental RNA with the helper RNA takes place and results in the production of infectious particles. As shown by RT-PCR, after recombination the miNOS gene is located in between the nonstructural and structural viral genes. This study demonstrates that despite claims in other publications, the Sindbis virus-based SINrep5 expression system leads to recombination and is thus not a safe system for in vitro and in vivo applications.     

Cytokine regulation on the synthesis of nitric oxide in vivo by chronically infected human polymorphonuclear leucocytes

1 We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E-deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins. 2 Lumen diameter and wall thickness were measured in pressurized arteries (approximately 250 microm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague-Dawley female rats by use of a dimension analysing system. 3 Treatment with a cyclo-oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (NG-methyl-L-arginine) caused a significant increase in basal tone only in the vitamin E-deprived rats (% tone: 6.2+/-1.1 vs 1.2+/-0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E-deprived rats compared to the controls (26.5+/-2.7 vs 16.4+/-3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E-deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (-17.4+/-4.0%; P<0.05) only the arteries from the vitamin E-deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress-strain relationship were not different between the two groups of rats. 4 In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo-oxygenase pathways but did not alter passive characteristics of mesenteric arteries.     

Macrophages infected with bovine leukaemia virus (BLV) induce humoral response in rabbits

BLV is a lymphotropic retrovirus which infects mainly B-cells. However, the possible infection of cells of the monocyte/macrophage lineage (M/M) might explain some aspects of the disease such as latency or disease progression. We infected sheep M/M with BLV either by culturing M/M with supernatant containing virus, or coculturing M/M with persistently infected cell lines. These BLV-infected M/M were inoculated into rabbits and the serological response was followed for two years. ELISA results using adsorbed sera showed a persistent production of specific antibodies from as early as the first week post inoculation. Two tests were used to detect the response against envelope glycoprotein gp51: Agar gel immunodiffusion (AGID) and a virus neutralization test read as syncytia inhibition (SI). Sera were positive by AGID after the second or third inoculation. Neutralizing titres (SI) were higher than those seen in control rabbits inoculated with persistently infected cell lines, suggesting that the virus may be expressed better in M/M. Gag-related proteins were analyzed by Western Blot (WB). Sera from rabbits inoculated with BLV-infected M/M recognized as many viral proteins as sera from BLV immunized control rabbits or infected cows, and this profile did not change with repeated inoculations. All these results suggest that BLV may infect M/M, where viral proteins are actively expressed to the point that they induce a humoral immune response in animals, and that animals get persistently infected.     

Basal nitric oxide synthesis in essential hypertension

BACKGROUND: There is indirect evidence that nitric oxide (NO) synthesis in vascular endothelium of patients with hypertension is altered. The aim of this study was to estimate more directly NO production in patients with untreated essential hypertension by measurement of synthesis of inorganic nitrate, which is the end product of NO oxidation in humans. Two separate studies were undertaken in patients with hypertension and appropriate healthy controls. METHODS: In the first study, ten patients and 13 controls were given a diet containing 82 mumoles nitrate per day for 2 days, with urinary and plasma nitrate measurement and 24 h ambulatory blood pressure monitoring on the 2nd day. In the second study, 11 patients and 11 controls were studied in the postabsorptive state; a bolus of 200 mg L[15N]2 arginine was administered intravenously over 10 min. 24 h ambulatory blood pressure monitoring was done and complete urine collections were made for the next 36 h. FINDINGS: In the first study, 24 h urinary nitrate excretion was lower in the hypertensive patients than in the control group (mean 450 [SEM 37] vs 760 mumoles [77] per 24 h; p < 0.001). There was an inverse correlation between average mean daytime ambulatory blood pressure and nitrate excretion (p = 0.007; r2 = -0.73). In the second study, mean 36 h urinary 15N nitrate excretion was significantly lower in the hypertensive than in the control group (1313 [50] vs 2133 [142] pmoles; p < 0.001). There was an inverse correlation also between average mean daytime ambulatory blood pressure and 24 h urinary 15N nitrate excretion expressed per mmole of creatinine (p = 0.002, r2 = -0.59). In addition, total urinary 15N nitrate excretion in the hypertensive group was significantly higher in women than in men (285 [16] vs 198 [14] micrograms 15N nitrate per kg; p = 0.026). INTERPRETATION: These data suggest that whole-body NO production in patients with essential hypertension is diminished under basal conditions. The origin of the NO we measured is not known, and we cannot tell whether the impaired synthesis is primary or secondary to a rise in blood pressure.     

Role of nitric oxide in the inhibition of cytochrome P450 in the liver of mice infected with Chlamydia trachomatis

In this study, we attempted to determine the effect of a systemic infection with Chlamydia trachomatis on cytochrome P450(CYP)-dependent metabolism in mice. Furthermore, we wanted to assess if these effects were mediated through NO. BALB/c(H-2d) female mice were inoculated intraperitoneally with the C. trachomatis mouse pneumonitis (MoPn) biovar, and induction of NO synthase (NOS) was detected by measuring [NOx] levels and inducible NOS protein content in peritoneal macrophages by Western blotting. Recovery of C. trachomatis from liver, lung, and spleen peaked at 4 days postinfection. Following cotreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, there was a significant increase in the intensity and the length of the infection. Six days after inoculation with C. trachomatis, CYP1A- and CYP2B-mediated metabolism in the liver of the mice was diminished up to 49% of control levels. However, when animals were treated with N(G)-nitro-L-arginine methyl ester at days 4 and 6 postinfection, the decrease in the metabolism of CYP1A and CYP2B was largely blocked. These results suggest that C. trachomatis infection can depress cytochrome P450 in a manner similar to other types of infections and that NO is likely to be a mediator of this depression. This finding may be of significance to patients taking drugs that are metabolized by phase I enzymes during infections with some bacteria such as C. trachomatis.     

Evidence for altered platelet nitric oxide synthesis in essential hypertension

OBJECTIVES: To determine whether platelet aggregation to collagen was abnormal in patients with essential hypertension and whether nitric oxide donors and inhibitors of nitric oxide synthesis affect platelet aggregation differently in hypertensives compared with healthy controls. DESIGN: Platelet aggregation assays were conducted ex vivo from both hypertensive and normal subjects simultaneously. METHODS: Platelet aggregation in response to collagen was measured in platelet-rich plasma from 16 patients with untreated essential hypertension and 16 healthy volunteers matched for age, sex and smoking habits. The effect of sodium nitroprusside (a nitric oxide donor) and NG-monomethyl-L-arginine (L-NMMA), a specific nitric oxide synthase inhibitor, was studied. RESULTS: In healthy controls L-NMMA caused a marked increase in platelet aggregation, whereas in hypertensive patients a small inhibition of aggregation was seen. This was significantly different from the response seen in normal controls. No difference was seen in the aggregatory response to collagen between hypertensive patients and healthy controls. Sodium nitroprusside caused inhibition of aggregation in hypertensive patients and in controls, but there was no significant difference in the degree of inhibition between the two groups. CONCLUSIONS: We conclude that in platelets from hypertensive patients there is a markedly reduced sensitivity to L-NMMA, which could be explained by a reduction in nitric oxide synthesis.     

Transfer of lymphocytic choriomeningitis disease in beta 2-microglobulin-deficient mice by CD4+ T cells

In this study we have investigated the role of CD4+, MHC class II-restricted cytotoxic T lymphocytes (CTLs) in the disease caused by lymphocytic choriomeningitis virus (LCMV) in beta 2-microglobulin deficient (beta 2m-) mice. Intracranial (i.c.) infection with LCMV resulted in death of six out of 11 beta 2m- mice. Mice that survived showed a marked loss in body weight. Death and loss of body weight could be prevented by immunosuppressing the mice with irradiation or cyclosporine prior to i.c. injection of LCMV. This treatment also prevented induction of virus-specific, MHC class II-restricted CTL following peripheral inoculation with LCMV. In vivo depletion of CD4+ cells with antibody also prevented death following i.c. injection whereas in vivo depletion of CD8+ cells had no effect. Disease could be transferred to recipient beta 2m- mice by adoptive transfer of beta 2m- derived immune spleen cells. Transfer of non-immune spleen cells did not result in illness. In vitro treatment of immune spleen cells with anti-CD4 antibody and complement eliminated class II-restricted CTL activity and also prevented mortality of recipients after adoptive transfer. Treatment with anti-CD8 antibody had no effect. We were unable to transfer LCM disease to beta 2m- recipients by adoptive transfer of immune spleen cells from C57BL/6 mice. These results suggest that, unlike normal mice, the pathology of LCM disease in beta 2m- mice is dependent upon virus-specific, CD4+CD8-, MHC class II-restricted T cells.     

Cyclic GMP and cGMP-binding phosphodiesterase are required for interleukin-1-induced nitric oxide synthesis in human articular chondrocytes

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.     

A critical role for neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons in persistent and acute infections of mice with lymphocytic choriomeningitis virus: implications for adoptive immunotherapy of virus carriers

This study demonstrates that neutralizing-antibody-producing B cells, CD4(+) T cells, and interferons (IFNs) are of key importance in virus control both in adoptive immunotherapy of persistent infection and in the late phase of acute infection with the WE strain of lymphocytic choriomeningitis virus (LCMV). We report the following results. (i) Clearance of LCMV-WE from C57BL/6 carrier mice by adoptive transfer of memory spleen cells requires B cells and CD4(+) T cells but not necessarily CD8(+) T cells. (ii) At the doses examined, CD8(+) T cells contribute to the initial reduction of viral titers but are alone not sufficient to clear the virus because they are exhausted. (iii) In the presence of functional IFN-gamma, virus clearance correlates well with the generation of neutralizing antibodies in the treated carrier mice. (iv) In the absence of receptors for IFN-gamma, virus clearance is not achieved. (v) Adoptive immunotherapy of mice persistently infected with a distinct virus isolate, LCMV-Armstrong, revealed only low levels of neutralizing antibodies; in this case, CD8(+) T cells were needed for virus clearance in addition to B and CD4(+) T cells. (vi) After low dose infection of C57BL/6 mice with LCMV-WE, virus is eliminated below detectable levels by CD8(+) T cells, but long-term (>2 months) virus control is usually not achieved in the absence of B cells or CD4(+) T cells; reappearance of the virus is paralleled either by exhaustion of virus-specific cytotoxic T lymphocytes or lethal immunopathology. These findings are of importance for adoptive immunotherapy strategies against persistent virus infections in humans.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.