Evidence that endogenous ecotropic virus is not expressed in AKR thymic lymphoid cells of chimeric hosts.

AKR/J thymocytes derived from fetal liver cells do not produce virus when they differentiate in lethally irradiated B10.K mice, whereas spleen and bone marrow cells are virus producers. In contrast, B10.K thymocytes that differentiate in lethally irradiated AKR mice become virus producers. These results suggest that infection of the thymus in AKR mice is initiated in thymic stromal cells.

Identification of a variant of gross leukemia virus that induces disease in mice inoculated as adults.

Gross murine leukemia virus normally induces leukemia (thymic lymphoma) in mice inoculated as neonates, but not as adults. We have isolated an apparent variant of this virus which induces thymomas when inoculated i.p. into susceptible adult mice. Using H-2 congenic BALB and C57BL mice, susceptibility to virus-induced thymomagenesis was found to be linked to the H-2 complex. In addition, a radioresistant immune mechanism leading to inhibition of tumor growth was observed in mice with a C57BL but not a BALB background.

The genetic basis of susceptibility to leukemia induction in mice by 3-methylcholanthrene applied percutaneously.

Susceptibility to leukemia induction in mice by skin painting with 3-methylcholanthrene (MCA) is strain-specific, occurring only in strains relatively resistant to MCA-induced skin tumors. The Ah locus, which has a dominant allele (Ahb) for inducibility of the aryl hydrocarbon hydroxylase (AHH) enzyme system and a recessive allele (Ahd) for noninducibility, appears to be the major determinant of this trait. MCA-painted mice of strains and crosses carrying the Ahb allele usually show a high incidence of skin tumors (papillomas which may evolve into malignant tumors) and little or no leukemia, whereas in mice homozygous for the Ahd allele the treatment usually induces a high incidence of leukemia and few or no skin tumors. Among mice of a segregating backcross generation including both Ahb/Ahd heterozygotes and Ahd homozygotes, the occurrence of skin tumors was correlated directly with AHH inducibility and inversely with the leukemic response. Mice of Ahb strains with a high level of endogenous murine leukemia (MuLV) expression (C58, PL) show a much weaker skin tumor response than expected but no increase in leukemia incidence, and this observation tends to confirm the previous finding that MuLV infection of mice of low-MuLV strains results in reduced susceptibility to MCA tumorigenesis.

Neonatal exposure to thymotropic gross murine leukemia virus induces virus-specific immunologic nonresponsiveness.

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses.

A role for complement receptor-like molecules in iron acquisition by Candida albicans.

Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism.

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.

Rational design of cytotoxic T-cell inhibitors.

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 alpha-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analog's activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.

Construction of a DBA/2.Fv-2r congenic strain. Apparent lethality of the homozygous Fv-2r genotype: brief communication.

From the (C57BL/6 X DBA/2)F1 cross, 13 serial backcrosses to the DBA/2 parental mouse strain were bred with selection by progeny testing in each generation for the Fv-2s/Fv-2r heterozygous genotype. Intercrossing heterozygotes of the 13th backcross generation produced no Fv-2r/Fv-2r homozygotes. Homozygosity for the Fv-2r allele thus appeared to be lethal on a DBA/2 background and in the absence of protector gene(s) of the C57BL strain.

The Fv-2r resistance gene in mice: its effect on spleen colony formation by Friend virus-transformed cells.

To determine the mechanism by which the recessive Fv-2r gene confers complete resistance to spleen focus formation and to the induction of early splenomegaly characteristic of the Friend virus (FV) disease syndrome, we compared the parameters of FV infection in susceptible DBA/2 (D2) (Fv-1n, Fv-2s) and partially congenic D2.Fv-2r (Fv-1n, Fv-2r) mice. After infecting these mice with N-tropic FV complex, we followed the replication of spleen focus-forming virus (SFFV) and the generation of SFFV-transformed tumor colony-forming cells (CFC) in their spleens. By both parameters D2.Fv-2r mice were 20- to 100-fold less susceptible than DBA/2 controls, but the inhibition was only partial. Transplantation of washed spleen cells from SFFV-infected DBA/2 mice resulted in equal growth and recovery of tumor CFC from both D2.Fv-2r and Fv-2s mice. However, these tumor cells grew as colonies in Fv-2s mice, whereas they grew diffusely in the spleens of D2.Fv-2r hosts and did not develop into macroscopically or microscopically visible colonies. Thus the completeness of the resistance to spleen focus formation that defines the Fv-2r gene was reflected only in the complete suppression of the colonial growth of tumor cells, whereas the other parameters of infection showed no or only partial inhibition.

Signalling through sIgA on a novel murine B lymphoma.

A novel murine B lymphoma expressing membrane-associated IgA was isolated and used to compare mechanisms of signal transduction by sIgM and sIgA. Like other isotypes so far studied, crosslinking of sIgA by anti-immunoglobulin antibodies stimulates hydrolysis of inositol phospholipids and causes elevation of intracellular free calcium. Furthermore, signals generated through sIgA are coupled to elevation of c-fos proto-oncogene expression. Coupling appears to be through the protein kinase C rather than through the Ca2+ component of sIg signalling as phorbol diester, but the Ca2+ ionophore cannot mediate this effect. Thus these results, coupled with those from earlier studies, show that early signal transduction through surface immunoglobulin appears to be similar regardless of the particular isotype involved in binding ligand.

Differences between young and aged mice in susceptibility to Friend virus.

Friend virus (FV) is a murine leukemia virus that infects progenitor red blood cells and causes an erythroleukemia in susceptible mouse strains, resulting in splenomegaly. Several genetic loci of the host have been identified that affect erythroleukemia development, differentiation status of target cells and virus replication. Since age may change expression of these loci, age may affect FV disease. To explore this possibility, FV expression in four genetically diverse strains of mice of different ages was examined. Extent of viral replication and of disease were evaluated by measuring spleen focus forming units (SFFU), spleen weight and reverse transcriptase (RT) activity in target organs. Young DBA/2 and (C57BL/6 x DBA/2)F1 mice exhibited a greater level of virus expression than their aged counterparts in all parameters investigated. Young CBA/Ca mice had slightly higher spleen weights and SFFU values than aged CBA/Ca mice, but a definitive age-related change was not observed in the RT activity of the target organs. C57BL/6 mice, which are genetically resistant to the development of FV-induced erythroleukemia, exhibited a limited degree of virus replication that was not effected by the age of the animal. Our results indicate that the age of the mouse, as well as the genetic background, can contribute to the level of susceptibility to FV.

Altered macrophage antigen-presenting cell function following Friend leukemia virus infection.

To investigate the mechanism by which Friend leukemia virus (FV) causes immunosuppression, the ability of peritoneal macrophages to mediate antigen-specific T-cell activation following FV infection was examined. Decreased IL-2 production was observed when antigen-primed T cells were cultured with antigen-pulsed macrophages from mice infected with FV, compared to T cells cultured with macrophages from control mice. Macrophages from FV-infected mice demonstrated decreased phagocytic and pinocytic activity, suggesting that antigen uptake may be impaired in these cells. In addition, FV-infected mice had decreased numbers of MHC class II positive macrophages compared to uninfected controls, as measured by immunofluorescence. The alterations in antigen uptake and class II expression observed in macrophages from FV-infected mice may be the result of infection of these cells by FV, which was demonstrated by in situ hybridization using a FV-specific probe. The ability of FV to infect and modulate the functions of macrophages may account, at least in part, for the immunosuppression observed in FV-infected mice.

Environmental lighting alters the infection process in an animal model of AIDS.

In this study, we examined the effects of altered environmental lighting on the infection process of a murine leukemia virus, E-55(+), which induces a thymic lymphoma/leukemia in 100% of BALB.K mice inoculated as adults. One to two weeks after inoculation, high levels of proviral DNA are usually found. This is followed by an asymptomatic period of many weeks during which proviral DNA becomes essentially undetectable. Leukemia develops approximately 28 weeks postinoculation. In this experiment, one group of mice was exposed a consistent 10L: 14D cycle while a second was maintained in constant light (LL). A third group was exposed to a rotating cycle characterized by phase shifting a 10L: 14D cycle every three 24-h days (rLD). All cycles began 2 weeks prior to inoculation and were maintained thereafter. Animals were sacrificed at 1, 5, 10, and 15 weeks, and hematopoietic tissue was examined for proviral DNA content. At 1 week, LL- and rLD-exposed animals showed considerably less proviral DNA in bone marrow and spleen compared with controls. At 15 weeks, thymuses from controls were showing signs of infection whereas tissue from LL and rLD mice remained at background levels. We conclude that environmental lighting does alter the infective pattern displayed by this retrovirus, although whether this effect is mediated by changes in the target stem cells or through immunoenhancement has not yet been determined.

Induction of interferon in AKR mice by murine leukaemia viruses.

A high percentage of AKR mice develop spontaneous leukaemia which has shown to be associated with the early expression of ecotropic murine leukaemia virus (MuLV) and the subsequent expression of xenotropic as well as polytropic MuLVs. Generally, mice infected with any one of several groups of viruses, including MuLVs, have been shown to produce interferon (IF). However, we report here that AKR mice produce no IF, despite that fact that infectious, endogenous MuLV is expressed in these mice from birth.

Pentoxifylline- and caffeine-induced modulation of major histocompatibility complex class I expression on murine tumor cell lines.

The methylxanthines, pentoxifylline (PTX) and caffeine, modulated major histocompatibility complex class I expression on three constitutively class I-positive murine T cell lymphoma lines. On two cell lines, PTX or caffeine treatment enhanced H-2K and H-2D expression. Treatment with PTX and either interferon-gamma, interferon-alpha/beta, tumor necrosis factor, or lymphotoxin increased the levels of K and D expression above those observed following treatment with either PTX or cytokines alone. On the third cell line, PTX or caffeine treatment enhanced D expression and reduced K expression. Treatment with PTX and any of the cytokines resulted in a level of D expression greater than that seen following treatment with either PTX or cytokines alone. However, PTX inhibited the cytokine-induced enhancement of K expression. PTX and caffeine did not induce class I expression on three constitutively class I-negative murine T cell lymphoma lines. Dibutyryl cAMP modulated class I expression in the same manner as PTX and caffeine. The PTX- and caffeine-mediated enhancement of class I expression was at least partially blocked by an inhibitor of cAMP-dependent protein kinase A. These results demonstrate that PTX and caffeine are able to regulate class I expression and that this regulation involves a cAMP-dependent mechanism.

T cell receptor genes and T cell development in virus-transformed early T cell lines.

We have derived T cell lines from mice inoculated with Gross leukemia virus, which appear to represent early T cell developmental stages and to reflect normal T cell development. These cell lines may provide a breakthrough in the study of T cell development as Abelson transformants have done for the study of B cell development. Analysis of the TCR gene expression in these cell lines reveals that the sequence of rearrangement and expression of each TCR gene is not strictly ordered. Expression of RNA for the TCR alpha and -beta genes appears to be coordinated with rearrangement at the alpha and beta loci. This is not the case for gamma gene expression. Availability of the homogeneous populations of cells represented in these cells lines allows for a more detailed molecular analysis of T cell development than was previously possible.

Novel class I-like molecule expressed on a murine leukemia virus-transformed cell line.

A retrovirus-induced tumor cell line, which expresses no H-2K or H-2D class I molecules, appears to express a tumor-specific transplantation antigen which induces tumor rejection in vivo and cytotoxic T lymphocyte generation in vitro without prior immunization and thus resembles class I molecules. In addition, although these tumor cells express no detectable class I molecules, they do express beta 2 microglobulin and a 55- to 60-kDa beta 2 microglobulin-associated protein. Northern analysis demonstrated that these cells express no RNA hybridizing to class I probes, suggesting that neither the tumor-specific transplantation antigen nor the beta 2 microglobulin-associated protein, if these are different, are encoded by known class I genes.

Evidence for H-2-linked control of retrovirus production in Friend virus-induced tumor cell lines.

In Friend leukemia virus-induced tumor cell lines derived from mice congenic with respect to the H-2 complex, most cell lines expressing the H-2k haplotype continuously produced infectious exogenous virus in culture, whereas most cell lines expressing the H-2b or H-2d haplotype stopped producing virus during in vitro passage. This apparent H-2-linked control of virus production did not appear to be the result of alteration of the provirus or resistance to superinfection. The implications of this finding with respect to virus-induced leukemogenesis are discussed.

Use of H-2:H-7 congenic mice to study H-2-mediated resistance to Friend leukemia virus.

Congenic and double congenic mice expressing different H-7 alleles with varying H-2 haplotypes on the C57BL/10 (B10) background were tested for their susceptibility to Friend virus (FV) infection. Mice expressing the H-7b allele derived from BALB/c were susceptible to FV infection whereas mice expressing the H-7a allele of B10 were absolutely resistant. Coexpression of H-7b and the H-2a and H-2d haplotypes determined high susceptibility; mice expressing both H-7b and H-2b were relatively resistant compared to H-7b mice expressing H-2a or H-2d. Parallel experiments with BALB/c and BALB.B recipients did not demonstrate differences in susceptibility associated with H-2d and H-2b. Mapping experiments with the H-2h4 and H-2i5 recombinants indicated that the gene determining relative resistance to FV-induced spleen focus formation mapped to the K-end (KbAb) of H-2b. Female recipients expressing H-7b, regardless of their H-2 haplotype, were more susceptible to FV infection than their syngeneic male counterparts. These experiments demonstrate the utility of congenic and double congenic strains that define H-7 and Fv-2 on different H-2 haplotype backgrounds for the analysis of Fv-2:H-7:H-2 interactions in determining susceptibility to FV infection.