Viral induction of AID is independent of the interferon and the Toll-like receptor signaling pathways but requires NF-kappaB.

Activation-induced cytidine deaminase (AID) is expressed in germinal centers of lymphoid organs during immunoglobulin diversification, in bone marrow B cells after infection with Abelson murine leukemia retrovirus (Ab-MLV), and in human B cells after infection by hepatitis C virus. To understand how viruses signal AID induction in the host we asked whether the AID response was abrogated in cells deficient in the interferon pathway or in signaling via the Toll-like receptors. Here we show that AID is not an interferon responsive gene and abrogation of Toll-like receptor signaling does not diminish the AID response. However, we found that NF-kappaB was required for expression of virally induced AID. Since NF-kappaB binds and activates the AID promoter, these results mechanistically link viral infection with AID transcription. Thus, induction of AID by viruses could be the result of several signaling pathways that culminate in NF-kappaB activation, underscoring the versatility of this host defense program.

AID for innate immunity to retroviral transformation.

AID is a cytidine deaminase essential for class switch recombination and somatic hypermutation during the humoral immune response. In this issue of Immunity, Gourzi et al. (2006) show that AID also plays a critical role in innate immunity to virally induced acute pro-B cell leukemia.

A role for activation-induced cytidine deaminase in the host response against a transforming retrovirus.

Activation-induced cytidine deaminase (AID) is specifically expressed in the germinal centers of lymphoid organs, where it initiates targeted hypermutation of variable regions of immunoglobulin genes in response to stimulation by antigen. Ectopic expression of AID, however, mediates generalized hypermutation in eukaryotes and prokaryotes. Here, we present evidence that AID is induced outside the germinal center in response to infection by the Abelson murine leukemia virus. The genotoxic activity of virally induced AID resulted in checkpoint kinase-1 (chk1) phosphorylation and ultimately restricted the proliferation of the infected cell. At the same time, it induced NKG2D ligand upregulation, which alerts the immune system to the presence of virally transformed cells. Hence, in addition to its known function in immunoglobulin diversification, AID is active in innate defense against a transforming retrovirus.

L3T4+ T cells regulate Abelson virus-induced lymphomagenesis.

To evaluate the role of T cells in regulation of lymphomagenesis, experiments were performed using Abelson murine leukemia virus (AMuLV). In vitro transformation of bone marrow target cells by this B lymphotropic retrovirus was inhibited by peripheral lymph node cells from naive mice. The inhibitory activity depended on Thy-1+ L3T4+ cells but did not require Lyt-2+ cells. In vivo depletion of L3T4+ T cells with a mAb (GK1.5) altered the course of AMuLV-induced lymphoma. L3T4 depletion of naturally resistant C57BL/6 mice resulted in dramatic susceptibility to lymphoma induction. Lymphoma cells from anti-L3T4-treated C57BL/6 mice infected with AMuLV displayed the B lineage transformation marker P1606C3. These studies reveal an important immunologic component of Abelson disease resistance involving L3T4+ T cells.

Regulation of expression of T cell gamma chain, L3T4 and Ly-2 messages in Abelson/Moloney virus-transformed T cell lines.

Recent results have suggested that T cells may exist in two distinct pathways, one expressing alpha and beta chain of the T cell receptor genes with either or both of the cell surface markers CD4 and CD8, while the other is negative for these cell surface markers and expresses the T cell-specific gamma chain genes. The relationship between these two pathways is not known. In this study, we have examined a series of either Abelson virus or Moloney virus-derived T cell lines for their expression of these T cell receptor and cell surface marker genes. Results indicate that the Abelson T cell lines do not express the cell surface markers CD4 and CD8, but express relatively high levels of gamma chain transcripts. After culture of these cell lines with the phorbol ester phorbol myristate acetate and interleukin 2, a down-regulation of these gamma chain transcripts can be observed. More interestingly, we found that the Moloney virus-derived T cell lines, which express the cell surface markers CD4 and/CD8, contain high levels of alpha and beta chain T cell receptor transcripts but little or no gamma transcripts even though they have rearranged these latter genes. The gamma transcripts, however, can be induced to high levels after culture with phorbol myristate acetate and interleukin 2. In the process, the cell surface markers CD4 and CD8 and their transcripts were dramatically down-regulated resulting in cells with high levels of gamma chain transcripts and a CD4-CD8- phenotype. The regulation of expression of these genes is reversible. Taken together, these results indicate that the T cell receptor gamma chain genes and those of the cell surface markers CD4 CD8 can be regulated in vitro by external factors and it opens up the possibility of studying the regulatory sequences associated with these genes.

A temperature-sensitive mutant of Abelson murine leukemia virus confers inducibility of IgM expression to transformed lymphoid cells.

Lymphoid cell lines were isolated that were inducible for the expression of surface immunoglobulin by shift from 35.5 to 39.5 degrees C after infection of mouse bone marrow cells with a mutagen-treated Abelson murine leukemia virus. Virus produced by one of the cell lines (ts49) transmitted the temperature-sensitive phenotype to new lymphoid transformants as well as to NIH/3T3 cells. In addition, the tyrosine autophosphorylating activity of the p120gag-abl protein synthesized in ts49-transformed cells was found to be temperature-sensitive. Shift experiments using ts49-transformed lymphoid cells showed that at 39.5 degrees C they synthesize increased amounts of mu and kappa chain RNA and protein, and that they can be further induced to secrete IgM when treated with lipopolysaccharide.

Transformed lymphocytes from Abelson-diseased mice express levels of a B lineage transformation-associated antigen elevated from that found on normal lymphocytes.

Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen-expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease.

Phenotypic variation in clonal Abelson virus lymphoma cells.

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.

The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity.

Three antisera against the mouse v-abl gene product were used to identify two potential human c-abl gene products in the chronic myelogenous leukemia cell line K562. Two antipeptide sera were generated in rabbits using the predicted amino acid sequence of the mouse v-abl gene product. One antiserum was made against a polypeptide overlapping the in vivo tyrosine phosphorylation site of murine P120gag-abl and what is believed to be a homologous tyrosine phosphorylation site of the predicted normal human c-abl gene product (v-abl 263-280). The second antipeptide serum, abl 389-403, was generated against a predicted hydrophilic peptide of the v-abl gene product. Immunoprecipitation from K562 cells metabolically labeled with [32P]orthophosphate by a mouse tumor regressor and abl 389-403 antipeptide sera detected two proteins of 190,000 and 240,000 Da. Both proteins were labeled primarily at serine and, to a much lesser extent, at tyrosine residues. Immune complex kinase assays using conditions that allow the tyrosine phosphorylation of P120gag-abl showed that in vitro phosphorylation of P190 and P240 occurs primarily at tyrosine residues. The detection of these enzymatically active human c-abl gene products is a rare observation which may be in part attributed to the c-abl gene translocation from chromosomes 9 to 22 occurring in the vast majority of chronic myelogenous leukemia patients.

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures.

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.

Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix: architecture of the protein.

We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule.

Cytotoxic T cell response against lymphoblasts infected with Moloney (Abelson) murine leukemia virus. Methodological aspects and H-2 requirements.

A method for infection of lymphocytes with Moloney(Abelson) murine leukemia virus [M(A)-MuLV] is described. Only lymphoblasts obtained after stimulation of normal spleen cells by the B cell mitogen lipopolysaccharide (LPS) were satisfactory targets for virus-specific, secondary cytotoxic T lymphocytes (CTL), whereas spleen cells stimulated by the T cell mitogen concanavalin A were not. The secondary CTL response against M(A)-MuLV could be efficiently measured using M(A)-MulV-infected LPS blasts as stimulating cells for secondary in vitro restimulation and as target cells for virus-specific destruction. Cold target inhibition demonstrated virus specificity of CTL. The T cell character of the cytotoxic cells was demonstrated by their sensitivity to anti-Thy-1.2 treatment. Using syngeneic virus-infected LPS blasts as target and stimulator, CTL responses were measured with effector cells from C57BL mice of the H-2b haplotype and of recombinant haplotypes sharing either K or D alleles with H-2b. In analogy with previous studies on Moloney virus-specific CTL, it was observed that C57BL/6 (H-2b) effector cells predominantly lysed Db-compatible, virus-infected target cells; B10.A(5R), (KbDd) effector cells showed a poor CTL response against syngeneic, virus-infected target cells. The combined findings indicate the existence of an Ir gene in the H-2D region regulating the CTL response against Moloney leukemia virus.

Partial maturation and light chain restriction of Abelson virus-transformed B cell precursors.

The induction of partial maturation in an in vitro derived Abelson virus-transformed murine lymphoid cell subline (ABC-1/AT1) is described. Pre-B (cytoplasmic, mu chain-positive) lymphocytes were induced from presumptive B cell precursors by prostaglandin E1, butyric acid, lipopolysaccharide and interferon. Maturation was independent of alterations in cellular growth rate and could be achieved in the absence of cell division. The AT1 subline was found to be restricted to the expression of a single light chain type (lambda) indicating a possible B cell lineage-committed precursor as the target for viral transformation.

Production of anti-self H-2 antibodies by hybrid mice immune to a viral tumour.

During the course of immunization of hybrid mice (genotype H-2b/k) with a parental Abelson virus-induced lymphoma (genotype H-2b/b), antibodies were produced to the H-2Kb or H-2Db antigens of the immunizing cells. Such 'anti-self H-2' antibodies demonstrate the existence of autoreactive B-cell clones in hybrid mice, and pose intriguing questions for the nature of self-tolerance.

Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1- Abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas.

Hybrids between pseudodiploid Thy-1.1+ lymphomas and Thy 1.2- pseudodiploid Abelson-leukemia-virus-induced-(ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1+ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2+ or Thy 1.2-. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1+, Thy 1.2- hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1+ lymphomas and pseudotetraploid Thy 1.2- Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1+ revertants were isolated from one of the Thy-1- hybrids by cell sorting. - These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparently cis-active, regulatory mechanisms which determine the ability of the Thy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.

Transformation by Abelson murine leukemia virus: properties of the transformed cells.

Ab-MuLV transforms mouse 3T3 fibroblasts, macrophages, and lymphocytes, which have properties of immature cells in the B-lymphocyte differentiation pathway. Differences in expression of intracellular immunoglobulin and surface antigens the lymphoma lines suggest that more than one B-lymphocyte differentiation stage may be transformed, each of which must be before the B lymphocyte acquires cell-surface immunoglobulin receptors for antigen and antigen sensitivity. The plasmacytomas induced by Ab-MuLV plus pristane tested thus far show no evidence of infection by Ab-MuLV. The transformation of these immunoglobulin-secreting cells may be due to causes other than the transforming elements of the Ab-MuLV genome.