Rapid blood clearance of mouse IgG2a and human IgG1 in many nude and nu/+ mouse strains is due to low IgG2a serum concentrations

 We reported previously that the blood clearance of injected mouse IgG2a was extremely rapid in many strains of nude and nu/+ mice. In an attempt to determine the cause of this phenomenon, the levels of endogenous IgG2a in the blood of these mice was assayed. It was found that the serum level of IgG2a was extremely low in many of these mice, below 50 μg/ml, which is 20–100 times lower than the expected normal value. Great heterogeneity between individual mice was observed in their blood level of IgG2a, and there was an excellent correlation between low blood IgG2a levels and rapid clearance of injected IgG2a. Thus, the blood IgG2a levels are so low that a novel, previously undescribed effect occurs, namely the rapid clearance of small amounts of injected IgG2a. The clearance is due primarily to binding sites in the spleen and liver. The low level of endogenous IgG2a is not due to the lack of a thymus, since it occurs in nu/+ as well as nude mice, but can probably be attributed to the very clean environment in which these mice are raised. In assays of sera from approximately 50 mouse strains, low IgG2a levels were found in all nude colonies and also in some normal mouse strains. Some nude mice displayed relatively normal IgG2a clearance rates despite having low levels of endogenous IgG2a. In repeated bleedings of individual mice, IgG2a levels were found to fluctuate greatly. A similar clearance effect was observed with a human IgG1 Ab injected into mice. This rapid clearance of injected IgG, of certain subclasses, represents a practical problem for many experiments in which antibodies are used for diagnosis or therapy, and several methods of circumventing the problem are discussed. Received: 15 August 1977 / Accepted: 14 October 1997     

Tumor cells engineered with IL-12 and IL-15 genes induce protective antibody responses in nude mice

IL-12 and IL-15 stimulate T, B, and NK cell functions through independent mechanisms, and cooperative effects of these cytokines have been reported. The human MHC class I-negative small cell lung cancer cell line, N592, genetically engineered to secrete IL-15, N592/IL-15, showed a reduced tumor growth rate, while N592 cells engineered with IL-12, N592/IL-12, grew similarly to the wild-type N592, N592 parental cells (N592pc), in nude mice. However, N592 cells coexpressing both cytokines, N592/IL-12/IL-15 cells, were completely rejected by 100% of nude mice. Here we show that 60% of nude mice rejecting N592/IL-12/IL-15 cells were resistant to N592pc rechallenge. SCID mice rejected N592/IL-12/IL-15 cells, but did not develop resistance to N592pc rechallenge, suggesting a role of Ab responses. Among nude mice rejecting N592/IL-12/IL-15 cells, those developing resistance to N592pc rechallenge had significantly higher titers of anti-N592 IgG2b Abs than nonresistant nude mice. Induction of an Ig class switch in nude mice was related to the expression of IFN-gamma and CD40 ligand in the draining lymph nodes. An IgG2b, anti-N592 mAb, derived from N592/IL-12/IL-15-immunized nude mice splenocytes induced significant protection against N592pc, while an IgM mAb was ineffective. The protective IgG2b mAb, but not the IgM mAb, triggered Ab-dependent cell-mediated cytotoxicity by nude mouse splenocytes against N592pc. These data indicate that IL-12 and IL-15 synergistically trigger innate, immunity-mediated, anti-tumor effects, resulting in cytotoxic IgG Ab responses in T cell-deficient mice. Protective Ab responses may relate to both direct actions of IL-12 and IL-15 on B cells and to the activation of an innate immunity-B cell cross-talk.     

Rapid elimination of mouse/human chimeric monoclonal antibodies in nude mice

 At our laboratory we are currently evaluating the suitability of mouse/human chimeric monoclonal antibodies (cmAb) for use in radioimmunotherapy of patients with head and neck squamous cell carcinoma (HNSCC). We have developed cmAb containing the human constant IgG1 domain and the variable domains of murine mAb (mmAb) E48 and U36 respectively. We considered the tumour-bearing nude mouse to be a well-validated model for a first testing of the targeting capabilities of these cmAb in comparison with the mmAb. Therefore, 3 μg cmAb E48 (labelled with ¹²⁵I) and 3 μg mmAb E48 (labelled with ¹³¹I) were simultaneously injected into HNSCC-bearing nude mice and, at various assay times, mAb uptake in blood and other tissues was assessed. Remarkably, while in roughly 50% of the animals the biodistribution of the conjugates was similar, in the other animals cmAb E48 showed a much higher blood clearance than mmAb E48. This resulted in a lower tumour uptake of cmAb E48 in comparison with mmAb E48. To determine whether this phenomenon was related to mAb E48 or to the animal model, other cmAb-mmAb combinations were evaluated in the same way: cmAbs SF-25, 17-1A and U36 (all IgG1) were tested and all showed a rapid elimination in about 50% of the animals. Besides a decrease in blood concentration, an increase of cmAb levels in liver and spleen was observed within 24 h after injection. Isotype-specific enzyme-linked immunosorbent assays showed that mice that demonstrated a rapid elimination of cmAb from the blood had much lower endogenous IgG1, IgG2b and IgG3 titres than mice showing normal clearance. IgG2a levels were low in all mice. Biodistribution experiments with 3 μg chimeric 17-1A isoforms showed high blood clearance in a proportion of the mice for IgG1, IgG3 and IgG4, but not for IgG2. Increase of the cmAb dose to 100 μg resulted in a similar cmAb and mmAb biodistribution in all mice. Moreover, the biodistribution of the F(ab′)₂ fragment of an IgG1 cmAb was similar for all mice in contrast to that of coinjected whole IgG. On the basis of these results it can be hypothesized that, in mice with low endogenous IgG titres, cmAb with specific isotypes are rapidly removed from the blood (and ultimately from the body) by mediation of Fc-binding receptors. Apparently, in mice with high endogenous IgG titres or in mice receiving a high cmAb dose, these receptors are saturated. Furthermore, the rapid elimination of cmAb from nude mice, which may occur after injection at a low dose, is a phenomenon related to the nude mouse model. Received: 26 July 1996 / Accepted: 16 January 1997     

Circulating immunoglobulin G can play a critical role in clearance of intestinal reovirus infection.

Reoviruses are encapsidated double-stranded RNA viruses that cause systemic disease in mice after peroral (p.o.) inoculation and primary replication in the intestine. In this study, we define components of the immune system involved in the clearing of reovirus from the proximal small intestine. The intestines of immunocompetent adult CB17, 129, and C57BL/6 mice were cleared of reovirus serotype 3 clone 9 (T3C9) within 7 days of p.o. inoculation. Antigen-specific lymphocytes were important for the clearance of intestinal infection, since severe combined immunodeficient (SCID) mice failed to clear T3C9 infection. To define specific immune components required for intestinal clearance, reovirus infection of mice with null mutations in the immunoglobulin M (IgM) transmembrane exon (MuMT; B cell and antibody deficient) or beta 2 microglobulin gene (beta 2-/-; CD8 deficient) was evaluated. beta 2-/- mice cleared reovirus infection with normal kinetics, while MuMT mice showed delayed clearance of T3C9 7 to 11 days after p.o. inoculation. Adoptive transfer of splenic lymphocytes from reovirus-immune CB17 mice inhibited growth of T3C9 in CB17 SCID mouse intestine 11 days after p.o. inoculation. The efficiency of viral clearance by adoptively transferred cells was significantly diminished by depletion of B cells prior to adoptive transfer. Results in SCID and MuMT mice demonstrate an important role for B cells or IgG in clearance of reovirus from the intestines. Polyclonal reovirus-immune rabbit serum, protein A-purified immune IgG, and murine monoclonal IgG2a antibody specific for reovirus outer capsid protein sigma 3 administered intraperitoneally all normalized clearance of reovirus from intestinal tissue in MuMT mice. This result demonstrates an IgA-independent role for IgG in the clearance of intestinal virus infection. Polyclonal reovirus-immune serum also significantly decreased reovirus titers in the intestines of SCID mice, demonstrating a T-cell-independent role for antibody in the clearance of intestinal reovirus infection. B cells and circulating IgG play an important role in the clearance of reovirus from intestines, suggesting that IgG may play a more prominent functional role at mucosal sites of primary viral replication than was previously supposed.     

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.     

Ig heavy chain complex-linked genes influence the immune response in a murine cryptococcal infection.

A murine pulmonary infection with Cryptococcus neoformans (Cne) has been used to determine mechanisms regulating effective T cell-mediated immunity in the lungs. In BALB/c and C.B-17 mice, following intratracheal deposition of Cne, the fungus initially grows rapidly and is then progressively cleared from the lungs. Cne clearance in C.B-17 mice requires CD4 and CD8 T cells, IFN-gamma, and NO. Clearance in congenic BALB/c mice proceeds more slowly than in C.B-17 mice, even though the only genetic difference between these strains is at the Ig H chain-containing region of chromosome 12. Examination of the pulmonary immune response in the two strains revealed that both cleared lung Cne by T cell-dependent mechanisms and generated equivalent levels of NO. Furthermore, both strains recruited equal numbers of macrophages, lymphocytes, and neutrophils to the lungs, although BALB/c mice recruited higher numbers of eosinophils. Notably, leukocytes isolated from BALB/c lungs during infection secreted lower levels of IFN-gamma and higher levels of the Th2 cytokines IL-4 and IL-5 as compared with lung leukocytes from C.B-17 mice. Furthermore, serum levels of IgM, IgG1, IgG2a, and IgG3 anti-Cne Abs generated during infection were significantly greater in BALB/c mice than C.B-17 mice. These data suggest that although both BALB/c and C.B-17 mice clear pulmonary cryptococcosis through T cell-mediated mechanisms, Ig H chain-linked genes in BALB/c mice are associated with a decreased effectiveness of the host response, which we suggest might influence the balance in Th1/Th2 T cell subset development or increase anti-Cne Abs, or both.     

Virus-neutralizing activity mediated by the Fab fragment of a hemagglutinin-specific antibody is sufficient for the resolution of influenza virus infection in SCID mice

Antibodies (Abs) contribute to the control of influenza virus infection in vivo by reducing progeny virus yield from infected cells (yield reduction [YR]) and by inhibiting progeny virus from spreading the infection to new host cells (virus neutralization [VN]). Previous studies showed that the infection could be resolved in severe combined immunodeficiency (SCID) mice by treatment with hemagglutinin (HA)-specific monoclonal antibodies (MAbs) that exhibit both VN and YR activities but not by MAbs that exhibited only YR activity. To determine whether virus clearance requires both activities, we measured the therapeutic activity of an HA-specific MAb (VN and YR) and its Fab fragment (VN) by intranasal (i.n.) administration to infected SCID mice. Immunoglobulin G (IgG) and Fab cleared the infection with i.n. 50% effective doses (ED(50)s) of 16 and 90 pmol, respectively. To resolve an established infection solely by VN activity, Fab must be present in the respiratory tract at an effective threshold concentration until all infected cells have died and production of virus has ceased. Because IgG and Fab had different half-lives in the respiratory tract (22 and 8 h, respectively) and assuming that both operated mainly or solely by VN, it could be estimated that clearance was achieved 24 h after Ab treatment when both reagents were present in the respiratory tract at approximately 10 pmol. This dose was approximately 200 times larger than the respiratory tract-associated Ab dose resulting from administration of the intraperitoneal ED(50) (270 pmol) of IgG. This indicated that our procedure of i.n. administration of Ab did not make optimal use of the Ab's therapeutic activity.     

Clearance of IgE from serum of normal and hybridoma-bearing mice

The half-life of IgE in the mouse was investigated by using radiolabeled and unlabeled monoclonal antibodies of the IgE class. Quantitative serologic assays were used for the unlabeled antibodies. IgE was cleared rapidly upon i.v. inoculation; after 48 hr, less than 0.2% of the initial concentration remained in the serum. The IgE was cleared initially with a half-life of 1 to 2 hr, attaining a relatively constant value of 5 to 8 hr. The corresponding values for IgG1, determined as a control, were 11 to 12 hr and 9 to 11 days, respectively. The initial stage probably reflects equilibration with extravascular spaces. This is supported by experiments with mice in which IgE-secreting tumors were implanted and then resected; IgE was cleared from such mice with an average initial half-life of about 5 hr. The rates of clearance of inoculated IgE were approximately the same in mice bearing an IgE-secreting tumor and in normal mice. This suggests that the initial rapid clearance of IgE from normal mice is not due to adherence of IgE to saturable sites; such sites might be expected to be occupied in mice expressing high serum concentrations of IgE. This conclusion was supported by experiments in which 1-mg quantities of IgE were inoculated i.v. into normal mice daily for 6 days. Additional IgE injected on day 7 was cleared normally. The results obtained with tumor-bearing mice indicate that the reported failure to elicit an IgE response to an antigen in mice bearing IgE-secreting hybridomas cannot be attributed to rapid clearance of newly synthesized IgE in such mice, as compared with normal mice.     

Neuritogenic effects of T cell-derived IL-3 on mouse splenic sympathetic neurons in vivo

To determine the role played by lymphocytes and cytokines in the growth of sympathetic neurons in vivo, the innervation and cytokine levels were examined in the spleens of SCID mice that lack T and B cells. Splenic noradrenaline, nerve growth factor (NGF), and IL-1beta levels were elevated in SCID mice. Immunohistochemical examination revealed that the density of tyrosine hydroxylase-positive (TH(+)) fibers of splenic central arteries in SCID mice was increased compared with wild-type C.B-17 mice, while SCID mice had significantly fewer TH(+) fibers in their periarteriolar lymphatic sheaths (PALS). Two weeks after SCID mice were injected with C.B-17 splenic T cells, their TH(+) fiber staining increased in the PALS. IL-3 levels increased significantly in SCID mice following T cell reconstitution, and the administration of anti-IL-3 Ab blocked the above T cell-induced increase in innervation in the PALS. Anti-IL-3 treatment also inhibited the regeneration of splenic sympathetic neurons in C.B-17 mice after they were chemically sympathetomized with 6-hydroxydopamine. Depletion of NK cells by anti-asialo GM1 promoted the splenic innervation in SCID mice, while there were no significant changes in the innervation between CD8(+) T cell-deficient beta(2)-microglobulin knockout mice and their wild type. Our results suggest that T cells (probably CD4(+) Th cells but not CD8(+) CTLs) play a role in regulating the sympathetic innervation of the spleen; this effect appeared to be mediated, at least in part, by IL-3. On the contrary, NK cells may exert an inhibitory effect on the sympathetic innervation.     

Glomerular deposition of immune complexes made with IgG2a monoclonal antibodies

The factors that determine whether immune complexes (IC) are cleared safely from the circulation or are deposited in vulnerable tissues such as glomeruli are not well defined. To better understand how IC are handled, the present study examined the fate in vivo of three model IC preparations with different immunochemical characteristics. Radiolabeled IC were constructed with murine IgG1, IgG2a, or IgG3 anti-DNP mAbs bound to DNP-BSA, designated IgG1 IC, IgG2a IC, and IgG3 IC, respectively. The IC were infused i.v. into BALB/c mice, and clearance and tissue localization of the three IC probes were compared. The results indicate that the major portion of each IC preparation was cleared from the circulation by the liver. However, compared with the other two probes, IgG2a IC were preferentially deposited in the kidney. Histologic examination revealed the presence of IgG2a IC in glomeruli. The enhanced renal uptake of IgG2a IC could not be attributed solely to such characteristics as IC size, Ag/Ab ratio, Ab charge, or affinity. However, the preferential renal deposition of IgG2a IC was abrogated by complement depletion. Thus, enhanced renal uptake in normal mice was complement dependent. These data suggest that interactions between IC and the complement system can influence the propensity of IC to deposit in tissues susceptible to IC-mediated injury.     

Outer membrane protein-specific monoclonal antibodies protect SCID mice from fatal infection by the obligate intracellular bacterial pathogen Ehrlichia chaffeensis

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.     

Rat MAbs to the product of the c-erbB-2 proto-oncogene for diagnosis and therapy in breast cancer

The product of the c-erbB-2 protooncogene (p185) is a member of the EGF receptor family of transmembrane tyrosine kinases. Amplification of this gene and overexpression of the product has been observed in adenocarcinomas and has been correlated with poor prognosis in patients with breast and ovarian cancer. The very low levels of expression of p185 by normal adult tissues makes the receptor an almost tumor-specific target. We have prepared rat monoclonal antibodies against five distinct epitopes on the external domain of the c-erbB-2 product overexpressed by the breast cancer line BT474. The antibodies bind to the protein core of p185 and stain specifically the membranes in frozen sections of tumors overexpressing the c-erbB-2 product. Three of the antibodies, ICR12 (epitope A), ICR54, and ICR55 (epitope E), also stain the cell membrane in formalin-fixed, paraffin-embedded sections and bind to p 185 in Western blots. An investigation of the stability of the antigen-antibody complexes indicates that the majority are not readily internalized by SKOV3 cells growing in vitro. Antibodies ICR12 (IgG2a) and ICR55 (IgG2a), which are directed against separate epitopes on the c-erbB-2 p185, are both of high affinity and immunoreactivity (>75%) and localize specifically and stably to xenografted breast and ovarian carcinomas growing in athymic mice when labeled with¹²⁵I (up to 13% injected dose/g, ICR12 and ICR55) or a range of other radionuclides (up to 20% id/g, ICR12). We conclude that these antibodies may be useful as therapeutic vehicles for targeting radionuclides (imaging and therapy) or enzymes for activating prodrugs (ADEPT).     

Resistance of Trypanosoma cruzi to blood clearance induced by acute-phase immune mouse serum.

To investigate functional changes in Trypanosoma cruzi parasites induced during their interaction with the vertebrate host, we compared the blood clearance profiles of blood forms isolated from infected normal mice (Reg-Tc) or from infected mice immunodepressed after treatment with cyclophosphamide (Cy-Tc). Parasite blood numbers were measured at various time intervals in animals injected intravenously (i.v.) with 1-2 x 10(6) T. cruzi of either isolate. In the absence of added immune sera (spontaneous clearance), Reg-Tc and Cy-Tc were cleared from blood at similar rates. However, when acute immune mouse serum (Ac-IMS) was injected i.v. 2 min after inoculation of parasites, a significant proportion of Cy-Tc only was cleared from the blood an hour later, whereas Reg-Tc were not, their clearance profile being identical to that observed in mice injected with normal mouse serum. Cy-Tc susceptibility to Ac-IMS was not the result of a toxic effect of cyclophosphamide over T. cruzi as parasites recovered from animals immunodepressed by irradiation before infection were cleared similarly by acute serum. Contrary to Ac-IMS, chronic immune mouse serum induced similar rates of disappearance of Reg-Tc and Cy-Tc from blood. Our results suggest the occurrence of T. cruzi selection or modification during the acute phase, which leads to an increased parasite resistance to the clearance properties of acute-phase antibodies.     

Synergistic roles of antibody and interferon in noncytolytic clearance of Sindbis virus from different regions of the central nervous system

Sindbis virus (SINV) is an alphavirus that causes infection of neurons and encephalomyelitis in adult immunocompetent mice. Recovery can occur without apparent neurological damage. To better define the factors facilitating noncytolytic clearance of SINV in different regions of the central nervous system (CNS) and the roles of innate and adaptive immune responses at different times during infection, we have characterized SINV infection and clearance in the brain, brain stem, and spinal cords of severe combined immunodeficiency (SCID) and C57BL/6 (wild-type [WT]) mice and mice deficient in beta interferon (IFN-beta) (BKO), antibody (muMT), IFN-gamma (GKO), IFN-gamma receptor (GRKO), and both antibody and IFN-gamma (muMT/GKO). WT mice cleared infectious virus by day 8, while SCID mice had persistent virus replication at all sites. For 3 days after infection, BKO mice had higher titers at all sites than WT mice, despite similar IFN-alpha production, but cleared virus similarly. GKO and GRKO mice cleared infectious virus from all sites by days 8 to 10 and, like WT mice, displayed transient reactivation at 12 to 22 days. muMT mice did not clear virus from the brain, and clearance from the brain stem and lumbar spinal cord was delayed, followed by reactivation. Eighty-one days after infection, muMT/GKO mice had not cleared virus from any site, but titers were lower than for SCID mice. These studies show that IFN-beta is independently important for early control of CNS virus replication, that antiviral antibody is critical for clearance from the brain, and that both antibody and IFN-gamma contribute to prevention of reactivation after initial clearance.     

Clearance and organ localization of small DNA anti-DNA immune complexes in mice

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.     

Mice can recover from pulmonary influenza virus infection in the absence of class I-restricted cytotoxic T cells.

Intranasal exposure of athymic (nu/nu) BALB/c mice to influenza virus leads to a persistent infection of the respiratory tract from which the mice die, usually within 3 to 4 wk with symptoms of general cachexia. However, if these nude mice were injected 1 day after infection, with approximately 10(6) cells from individual virus-specific MHC class II-restricted Th cell clones, they showed greatly reduced mortality and the titers of infectious virus in their lungs were reduced, often to undetectable levels. By coinfecting mice with pairs of antigenically distinct viruses and subsequently determining the extent of clearance of each type of virus, it could be shown first that the clearance mechanism was immunologically specific but did not display the typical crossreaction of class I-restricted cytotoxic T (Tc) cells. In addition, neither primary nor memory Tc responses could be detected in these mice. Second, Th cell clones promoted clearance solely of those viruses that contained the specific Th cell determinant, i.e., Th cell-nonreactive bystander viruses were not cleared. These findings were compatible with virus clearance being effected either directly after recognition of infected class II-positive cells by the transferred Th cells or indirectly via promotion of a glycoprotein-specific antibody response. The latter seems to be the case because transfer of Th cells into infected T and B cell-deficient SCID mice did not result in virus clearance, although transfer of an anti-hemagglutinin antibody cocktail did. Thus, a virus-specific Tc cell response is not a requirement for recovery from a pulmonary influenza virus infection.     

The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.