Persistent infection of mice by lactate dehydrogenase-elevating virus: effects of immunosuppression on virus replication and antiviral immune responses

Maximum plasma titers (10(9)-10(10) ID50/ml) of lactate dehydrogenase-elevating virus (LDV) in mice are observed one day after infection, but then decrease 4-5 log during the next 5 weeks to attain a persistent steady-state level for the remainder of the life of the animal. The decrease in plasma LDV level during the first 5 weeks after infection and long-term viremia were not affected by lethal X-irradiation of the mice, daily injections of cyclosporin A or depletion of the mice of T cells by treatment with anti-CD4, anti-CD8, or anti-Thy1.2 monoclonal antibodies, although these treatments inhibited the formation of anti-LDV antibodies. LDV viremia was also the same in nu/nu and nu/+ Swiss mice, though the former did not mount an anti-LDV immune response, while the latter did. The appearance of anti-LDV neutralizing antibodies in infected mice 1-2 months after infection or the injection of infected mice with high doses of anti-LDV neutralizing monoclonal antibodies also did not affect the level of LDV viremia. Repeated treatments of infected mice with either cyclophosphamide or dexamethasone caused 1-2 log increases in plasma LDV titers. Although cyclophosphamide treatment prevented the formation of anti-LDV antibodies, dexamethasone caused an increase in plasma LDV levels without affecting anti-LDV antibody formation. We conclude that an anti-LDV immune response does not play a significant role in controlling LDV replication in mice. The data support the view that within 1 day after infection of a mouse, all LDV-permissive macrophages, which appear to be the only cells supporting LDV replication in the mouse, are destroyed as a result of a cytocidal infection by LDV. Subsequently, LDV replication is limited by the rate of generation of new permissive macrophages. The steady-state viremia attained about 5 weeks after infection reflects a balance between LDV replication in permissive macrophages as they arise and LDV inactivation and clearance.     

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.     

Immune response to lactate dehydrogenase-elevating virus: serologically specific rabbit neutralizing antibody to the virus.

A rabbit was immunized with large amounts of the lactate dehydrogenase-elevating virus (LDV) over a 9-month period. The plasma from this rabbit possessed an anti-LDV IgG titer of 1:80,000 as measured by the enzyme-linked immunosorbent assay (ELISA) method and a neutralizing titer of 1:1,000 for the homologous strain of LDV. LDV neutralization at 4 degrees C followed single-hit kinetics. In contrast, mouse anti-LDV IgG in plasma of chronically LDV-infected mice failed to neutralize LDV at 4 degrees C and neutralization at 37 degrees C was slow, biphasic, and inefficient compared with the neutralization caused by rabbit anti-LDV IgG, even though high levels of anti-LDV IgG were detectable in mouse plasma by the ELISA method. Rabbit anti-LDV IgG neutralized one heterologous strain of LDV as rapidly as it did the homologous strain, but failed to significantly neutralize five other strains of LDV, all of which were originally isolated from different mouse strains bearing transplantable tumors. The results indicate clear serological differences between LDV strains. Cross-reactions between the strains, however, were observed by ELISA, using the antibody induced during persistent infection of mice with each LDV strain. Immunoglobulin M (IgM) from mice infected for 15 days with the various strains bound equally to our LDV strain. IgG obtained from 2-month-infected mice also cross-reacted, but to a varying extent which partly correlated with the specificity detected by neutralization. Both rabbit and mouse anti-LDV IgG enhanced the infectivity of LDV at a low multiplicity of infection for primary cultures of peritoneal mouse macrophages.     

Immune complexes that bind to ELISA plates not coated with antigen in mice infected with lactate dehydrogenase-elevating virus: relationship to IgG2a- and IgG2b-specific polyclonal activation of B cells.

We have further investigated the nature of IgG-containing complexes of 150-300 kD that rapidly appear in the circulation of mice of various strains after infection with lactate dehydrogenase-elevating virus (LDV) and are recognized and quantitated by their binding in the presence of 0.05% Tween 20 to certain enzyme-linked immunosorbent assay (ELISA) plates with high protein affinity that have not been coated with protein antigen (5). These binding complexes have been found to contain primarily IgG2a or, in some mice, IgG2b. Their isotype specificity and time course of formation correlated with those of the polyclonal production of immunoglobulins in these mice, as measured by increases in total IgG2a or IgG2b in the circulation. In contrast, anti-LDV antibodies exhibited much broader isotype specificities in all mouse strains investigated. Depletion of BALB/c mice of CD4+T cells or lack of T cells in nude Swiss mice only partly reduced the polyclonal activation of B cells and the formation of ELISA plate-binding complexes, whereas anti-LDV antibody formation was completely blocked. Only a small proportion of the total IgG2a or IgG2b formed as a result of the LDV-induced polyclonal activation of B cells was recovered in plate-binding complexes, which sedimented in sucrose density gradients between 150 and 300 kD. Diverse monoclonal antibodies of different IgG isotopes did not bind to the plates at concentrations at which LDV-induced immune complexes exhibited binding activity. We suggest that the LDV-induced immune complexes do not contain anti-LDV antibodies, but are complexes of auto-antibodies and self-antigen(s). However, additional features must be responsible for the high affinity of these complexes for ELISA plates since various immune complexes formed in vitro failed to bind to the plates, and binding activity of the immune complexes formed in LDV-infected mice could not be regenerated in vitro once the complexes had been dissociated by a low pH treatment.     

Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus

BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody-LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti-LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus.     

Immune response to lactate dehydrogenase-elevating virus: isolation of infectious virus-immunoglobulin G complexes and quantitation of specific antiviral immunoglobulin G response in wild-type and nude mice.

Lactate dehydrogenase-elevating virus (LDV) causes a normally benign persistent infection of mice, resulting in a life-long viremia characterized by the presence of circulating infectious immune complexes, impaired clearance of certain enzymes from the blood, and modification of the host immune response to various heterologous antigens. In this study, we isolated infectious immunoglobulin G (IgG)-LDV complexes in the plasma of persistently infected mice by adsorption to and elution from protein A-Sepharose CL-4B. We found that practically all infectious LDV in the plasma of persistently infected mice is complexed to IgG. LDV infectivity in these complexes was partially neutralized, but could be reactivated by treatment with 2-mercaptoethanol. We also quantitated total plasma IgG and anti-LDV IgG in wild-type and nude Swiss and BALB/c mice as a function of the time after infection with LDV by radial immunodiffusion and an enzyme-linked immunosorbent assay, respectively. Total plasma IgG levels nearly doubled in BALB/c mice during 150 days of infection. IgG levels in uninfected nude mice were only 20% of those in uninfected BALB/c mice, but during infection with LDV increased to approximately those found in uninfected BALB/c mice. Anti-LDV IgG levels were almost as high in nude mice as in normal BALB/c mice. Isoelectric focusing of purified IgG from BALB/c mice showed that LDV infection resulted in the enhanced synthesis of all 16 normal IgG fractions that we could separate by this method, which suggests that LDV infection results in polyclonal activation of IgG-producing lymphocytes.     

Regulation of maternal-fetal virus transmission in immunologically reconstituted SCID mice infected with lactate dehydrogenase-elevating virus.

Immunodeficient SCID (C.B-17 scid/scid) mice with persistent lactate dehydrogenase-elevating virus (LDV) infection failed to produce IgG anti-LDV antibodies, and during chronic infection transmitted virus infection to 95% of their offspring. In contrast, normal mice infected 15 or more days prior to giving birth produced IgG anti-LDV antibodies and transmitted LDV infection to only 0-46% of their fetuses. Transplacental transmission of LDV infection was dependent on the timing of maternal infection. Adoptive transfer of immune competence to LDV-infected SCID mice resulted in fetal protection from maternally transmitted virus infection. Fetal protection correlated with the presence of maternal IgG anti-LDV but not with fetal levels of IgG anti-LDV, and the levels of viremia in nonimmune SCID mice did not affect transplacental virus transmission. These results demonstrate the importance of maternal immunity in protecting the fetus from infection, and validate the use of this mouse model for investigation of immune mechanisms of transplacental virus transmission.     

Infection of SCID mice with lactate dehydrogenase-elevating virus stimulates B-cell activation

Mice of the C.B-17 strain homozygous for the scid mutation (SCID mice) were infected with lactate dehydrogenase-elevating virus (LDV), and plasma samples obtained at intervals up to 42 days postinfection were analyzed for total immunoglobulins, anti-LDV antibodies, virus-specific immune complexes, and viremia levels. The mice responded to LDV infection with transient increases in total blood IgM, production of IgM-antigen complexes and IgM anti-LDV, as well as increased blood IgG2a. However, SCID mice failed to make a specific IgG2a anti-LDV immune response, and their blood LDV levels were elevated about 100-fold relative to those of control mice. The results suggest a role for IgG antibodies in the regulation of viremia and demonstrate a viral pathway of B-cell differentiation in SCID mice.     

Immunoglobulins that bind to uncoated ELISA plate surfaces: appearance in mice during infection with lactate-dehydrogenase-elevating virus and in human anti-nuclear antibody-positive sera

Immunoglobulins present in the blood plasma of mice infected with lactate-dehydrogenase-elevating virus (LDV) were found to bind strongly in the presence of 0.05% Tween 20 to the uncoated surfaces of wells of certain ELISA plates with previously recognized high protein-binding capacity. The binding was readily distinguishable from non-specific background binding of immunoglobulins present in normal mouse plasma. The binding components absorbed to protein A and had molecular weights in the 150-300 kDa range. Binding of the purified IgG fraction was progressively inhibited by increasing the concentration of Tween 20 in the diluent and by preincubation of the fraction at pH 3-4 for 10 min. The appearance of plate-binding IgM and IgG during LDV infection corresponded approximately with previously reported time courses of appearance of IgM- and IgG-containing circulating immune complexes and of specific IgM and IgG anti-LDV antibodies in LDV-infected mice. We conclude that complexes of IgG and IgM with LDV antigens have a much higher affinity for ELISA plates with high protein-binding capacity than uncomplexed immunoglobulins. Immune complexes did not significantly bind to ELISA plates with low protein-binding capacity, which, therefore, are suitable for measuring specific antiviral antibodies. Preliminary experiments with human anti-nuclear antibody-positive serum samples demonstrated markedly elevated non-specific binding of immunoglobulins to high-binding-capacity ELISA plates.     

Detection of the binding of IgG2a and IgG2b on the surface of macrophages from mice chronically infected with lactic dehydrogenase virus.

This study showed that sera from mice chronically infected with lactic dehydrogenase virus (LDV) contained virus-antibody complexes (IC). IgG2a and IgG2b, but not IgG1, IgG3, IgM or IgA, were demonstrated on the surface of macrophages from chronically infected mice. These results suggest that IC in the circulation may bind to Fc receptors for IgG2a and IgG2b on the surface of macrophages and lead to the modulation of macrophage function seen in chronically LDV-infected mice.     

Clearance of lactate dehydrogenase by SJL/J mice infected with lactate dehydrogenase-elevating virus

The plasma level of lactate dehydrogenase (LDH) activity rises to about ten times the normal level by 4 days after infection of mice with lactate-dehydrogenase elevating virus (LDV). The levels of seven other enzymes are also increased, but to a lesser degree. SJL/J mice demonstrate a unique, genetically controlled 20-fold increase in the plasma level of LDH enzyme after LDV infection, as well as enhanced levels of the other plasma enzymes elevated by LDV infection. Comparison of virus infection in SJL/J and Swiss mice as well as in cultures of peritoneal exudate cells made from them indicated that the time course and extent of virus replication was similar for the two strains of mice. The rate of clearance of intravenously injected rabbit or mouse LDH was found to be impaired to a similar extent in LDV-infected SJL/J and Swiss mice. The effect of LDV infection on the levels of endogenous LDH released as a result of injection of carbon tetrachloride or tumor growth was also similar in the two strains of mice. These results suggest that LDV infection may specifically induce a greater influx of LDH into the plasma of SJL/J mice from an as-yet-unknown source than in other strains of mice.     

Effect of prostaglandin E2 on plasma lactic dehydrogenase activity in (NZB x NZW)F1 mice with a chronic infection of lactic dehydrogenase virus.

The effect of prostaglandin E2(PGE2) on blood LDH values was investigated in (NZB x NZW)F1 mice with or without an established infection with lactic dehydrogenase virus (LDV). Plasma LDH decreased in infected mice treated with PGE2, but increased in infected mice treated with indomethacin, an inhibitor of PGE2. However no significant effect on LDH occurred in uninfected mice treated with PGE2 or indomethacin. To investigate the mechanisms of decreased LDH activities resulting from treatment with PGE2, clearance tests were performed. Clearance of LDH-5, but not LDH-1, was faster in PGE2-treated mice than in non-treated mice, whether or not they were infected with LDV. The results suggest that enhanced clearance of LDH-5 in mice treated with PGE2 may account for the fall in plasma LDH in LDV-infected mice.     

CD4+ T cells play a major role for IgM and IgG anti-DNA production in mice infected with Plasmodium yoelii

The infection by a non-lethal strain of Plasmodium yoelii induces the formation of autoantibodies such as anti-DNA and anti-Sm antibodies in mice. The extent of the relative increase in serum levels of IgM and IgG anti-DNA and anti-Sm antibodies and their kinetics were found to be similar to those of anti-hapten antibodies and of total IgM and IgG levels. This strongly suggested that anti-DNA and anti-Sm autoantibody responses observed in malaria-infected mice are a result of polyclonal activation of B cells. The analysis of the IgG subclasses reacting with DNA antigen showed significant levels of the T cell-dependent isotypes, IgG1 and IgG2. The role of T cells in the activation of autoreactive B cells was confirmed by using athymic nude mice. Indeed, BALB/c-nu/nu and C57BL/6-nu/nu mice failed to produce IgG anti-DNA antibodies after infection with P. yoelii. Moreover, the reconstitution of BALB/c nude mice with lymph node cells from congenic euthymic BALB-Igb mice showed the activation of autoreactive B cells in nude mice by T cells from euthymic mice. Studies in mice depleted of CD4+ T cells strongly suggested that malaria-induced anti-DNA antibodies were almost entirely dependent on the presence of CD4+ T cells, as this depletion significantly decreased IgM anti-DNA antibodies and completely abolished the IgG anti-DNA production, including the IgG3 subclass in infected mice. In contrast, depletion of the CD8+ T cell subset had no effect on the production of autoantibody in malaria-infected mice. Our results indicate that CD4+ T cells play a major role for both IgM and IgG anti-DNA production during the course of malaria infection.     

Isotypically restricted activation of B lymphocytes by lactic dehydrogenase virus

Lactic dehydrogenase elevating virus (LDV) was found to selectively stimulate IgG2a synthesis in infected mice. Within one week after infection, the production of IgG2a increased nearly 50-fold whereas that of IgM, IgA, IgG1 and IgG3 remained virtually unchanged. IgG2b synthesis was also enhanced but to a lesser extent. Several observations suggested that this stimulation of IgG2 production resulted from a polyclonal B cell activation: (a) the isoelectric focusing patterns of IgG2a before and after LDV infection were exactly the same, (b) the frequency of clones with anti-LDV activity in hybridoma collections derived from infected mice was extremely low (less than 4/1000) and (c) the proliferative response elicited by LDV in unsensitized animals was comparable with that induced by lipopolysaccharide. The effect of LDV on immunoglobulin synthesis was drastically reduced in nude mice but was not affected by the X-linked B lymphocyte defect of animals carrying the xid mutation.     

Lactic dehydrogenase virus infection inhibits allergic eosinophil reaction and IL-5 gene expression in vivo.

The effects of lactic dehydrogenase virus (LDV) infection on allergic eosinophil reaction and IL-5 gene expression were studied. LDV infection suppressed antigen-induced eosinophil recruitment into the peritoneal cavity in sensitized mice. The elevation of IL-5 gene expression in the spleen and mesenteric lymph nodes 6 h after ovalbumin challenge was significantly suppressed in LDV-infected mice compared with uninfected (control) mice. The expression of the interferon-gamma and IL-2 genes in the spleen, but not in mesenteric lymph nodes, was significantly suppressed in LDV-infected mice compared with control mice. The present results suggest, that suppression of IL-5 gene expression by LDV infection may not be mediated by a mutual inhibitory mechanism between Th1 and Th2 cells.     

Lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of macrophages yet fails to affect their phagocytic activity

The effect of acute infection of mice with lactic dehydrogenase virus (LDV) on two major functions of peritoneal macrophages was tested. Using a macrophage-dependent T cell proliferative assay to test the antigen-presenting capacity of LDV-infected macrophages we found that LDV impairs the capacity of antigen-presenting cells to trigger memory T lymphocytes. Endocytosis of antigen by LDV-infected macrophages was similar to that of uninfected cells. In addition, the proportion of intracellular antigen versus membrane-bound antigen in LDV-infected cells were similar to that observed in uninfected mice. It appears therefore, that the impaired immunogenic effect of LDV-infected macrophages results from reduced immunogenicity of the membrane-bound antigen.Testing the phagocytic activity of peritoneal macrophages we found that the uptake of radiolabeled antibody-coated sheep erythrocytes or bacteria (E. coli) by infected cells was similar to that by uninfected macrophages. In addition, LDV failed to affect the ability of peritoneal macrophages in a nitroblue tetrazolium reduction reaction which serves as an alternative parameter for measuring phagocytic activity. Our results support the assumption that LDV, which probably propagates in the cells of the reticuloendothelial system, impairs some of the immunogenic functions of macrophages and thereby affects macrophage-dependent immune responses.     

Immune responses in mice infected with lactic dehydrogenase virus. IV. Functional status of the macrophage during acute LDV infection.

Macrophages from uninfected and lactic dehydrogenase virus (LDV)-infected mice were compared with respect to the affinity and number of their Fc receptors for IgG2a; no differences were found regarding these parameters. When the uptake of DNP-BGG by macrophages from uninfected and acutely LDV-infected mice was compared, again no differences were observed. However, when the per cent membrane-bound DNP-BGG was determined as a function of time after antigen uptake in these two groups, more DNP-BGG was found membrane-bound on the macrophages from the LDV-infected mice, than on uninfected macrophages. In view of the fact that humoral immunity is enhanced during acute LDV infection, these data provide a positive correlation between increased retention of membrane-bound antigen and enhanced humoral immune responses.     

Non-tolerant B cells cause autoimmunity in anti-CD8 IgG2a-transgenic mice

Using a pair of γ2a/x immunoglobulin genes, transgenic mice were generated to study tolerance induction in B cells that express IgG2a autoantibodies. The transgenic IgG2a specifically binds CD8 α chains of the CD8.2 allotype expressed on the surface of CD8⁺ T cells, but not CD8 molecules expressed by the CD8.1 allele. Thus, IgG2a transgenic mice expressing the CD8.1 allele were used as controls to monitor B cell development and mice expressing CD8.2 were used to study B cell tolerance. Both types of mice showed transgenic γ2a expression on the surface of B cells. Expression of endogenous heavy chain alleles was strongly inhibited in immature B cell subsets, whereas mature B cells co-expressed transgenic γ2a and endogenous IgM/D. The transgenic x chain expression leads only to partial allelic exclusion of endogenous light chains. B cells that express high levels of transgenic CD8.2-specific IgG2a were identified using soluble CD8-Ig. In CD8.1⁺ and in CD8.2⁺ mice, we found no differences in expression and maturation of transgenic anti-CD8.2 IgG2a⁺ B cells. High levels of serum anti-CD8.2 IgG2a antibodies led to the elimination of CD8⁺ T cells, causing a severe defect in cytotoxic immune responses. These results show that tolerance induction is incomplete in the CD8.2⁺ mice, either because IgG2a⁺ B cells are resistant to censoring mechanisms or because the secreted CD8-specific IgG2a antibodies render the CD8 autoantigen inaccessible to the B cells. This contrasts strongly with the efficient induction of B cell tolerance in mice expressing anti-CD8.2 IgM autoantibodies.     

Extensive CD4 cross-linking inhibits T cell activation by anti-receptor antibody but not by antigen.

Anti-CD4 mAbs have been shown to inhibit T cell activation in a variety of ways. We have tested a panel of IgG and IgM anti-CD4 mAbs for their effects on the activation of a cloned T cell line by antigen presented by syngeneic accessory cells, by soluble anti-T cell receptor antibodies, and by mitogenic lectins. Both IgM and IgG mAbs to CD4 inhibit responses to mitogenic lectins. However, IgM, but not IgG, anti-CD4 antibodies inhibit T cell activation by mAbs specific for the TCR. This inhibitory activity appears to be due to the signaling effects of IgM mAbs, as cross-linked IgG antibodies mimic the behavior of the IgM anti-CD4 antibodies. Inhibition of T cell activation correlates with the ability of IgM and of cross-linked IgG anti-CD4 antibodies to induce tyrosine phosphorylation of the CD4-associated tyrosine kinase p56lck and an unknown substrate, pp32. Surprisingly, we find that the IgM anti-CD4 mAbs tested had no effect on the specific antigen recognition, despite their potent inhibitory effects on the other responses of the same cloned T cell line. These results suggest that multivalent CD4 interactions with ligands such as MHC class II molecules are inhibitory of T cell activation, but that this inhibition can be reversed when CD4 and the TCR bind the same ligand. We discuss the possible implications for positive intrathymic selection of these findings on signaling through CD4.     

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.