Syamal K. Datta

Polyspecificity of Monoclonal Lupus Autoantibodies Produced by Human-Human Hybridomas

We studied the serologic properties of monoclonal autoantibodies that were produced by hybridomas derived from lymphocytes of patients with systemic lupus erythematosus. The hybridomas were made by fusion of a human lymphoblastoid cell line, GM 4672 (derived from a patient with multiple myeloma), with peripheral-blood or splenic lymphocytes from six patients with lupus. Thirty monoclonal autoantibodies, selected for their ability to react with denatured DNA, were analyzed. Eighteen of them reacted with three or more additional polynucleotides, including native DNA, left-handed double-helical DNA (Z-DNA), poly(l), and poly(dT). Ten reacted both with nucleic acids and the phospholipid cardiolipin. The multiple binding reactions of the monoclonal autoantibodies may be explained by the presence of appropriately spaced phosphodiester groups in both the polynucleotides and the phospholipid. The sharing of antigenic groups by polymers of different natures may contribute to the apparent diversity of serologic reactions in systemic lupus erythematosus. These findings suggest that DNA itself need not be the immunogenic stimulus for autoantibody formation in this disease.

Mendelian segregation of loci controlling xenotropic virus production in NZB crosses

Abstract Quantitative xenotropic virus assays were done in crosses made between NZB and SWR mice. Two independently segregating autosomal dominant loci (Nzv-1 and Nzv-2) determined expression of infectious xenotropic virus. Nzv-1 alone or in association with Nzv-2 was responsible for high-grade virus expression, and low titers of virus were expressed when Nzv-2 was present and Nzv-1 was absent. Virus assays on mice of the second backcross and intercross generations confirmed this finding. The virologic phenotype of individual animals was stable for up to 1 year.

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Abstract Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K k , and Ia k . In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2 k and Ia k . This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1 + 2 + thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.

Pathogenic autoantibody-inducing γ δ T helper cells from patients with lupus nephritis express unusual T cell receptors

In previous work, we found that only 59 (15%) of 396 “autoreactive” T cell clones derived from five patients with lupus nephritis had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies and the majority (4959) of those autoimmune T helper (Th) clones were CD4+. Surprisingly, 7 of those Th clones were CD4−CD8− and γδ TCR+, capable of augmenting the production of pathogenic anti-DNA autoantibodies up to 125-fold. The γδ The clones responded in a MHC-nonrestricted manner to some endogenous autoantigen associated with heat shock proteins (HSP60) on the lupus B cells. The γδ TCR genes expressed by 4 of these Th clones were amplified and sequenced here. Three of the 4 Th clones, each from a different lupus patient, expressed a gene from the Vγ1 subgroup. Moreover, 2 of the Th clones expressed Vδ5, and the others Vδ1 or Vδ3. These TCRs are rarely expressed by peripheral blood γδ T cells of normal adult humans. The predominant γδ T cells in human peripheral blood express Vγ2 (Vγ9) and Vδ2 TCR genes, including HSP-responsive T cells. None of the lupus Th clones expressed this combination of TCR genes. In addition, some of these pathogenic autoantibody-inducing Th clones from the lupus patients had limited diversity and few N-nucleotide additions in their γδ TCR junctional regions (CDR3), thus resembling fetal γδ thymocytes early in ontogeny.

Splenocyte plaque assay for the detection of murine leukemia virus.

Summary A modified XC assay for murine leukemia virus (MuLV) employing splenocytes taken directly from the animal is described. This modification can be more than 1000 times more sensitive than XC plaque assays employing tissue extracts. This technique should lend itself readily to the quantitation of infectious MuLV in defined populations of lymphoid cells. This research is supported by USPHS Grants CA 10018 and AM 07937, and a Grant from the Damon Runyon Memorial Fund for Cancer Research.

Recovery of xenotropic virus but not ecotropic virus during graft-versus-host reaction in mice

Abstract Graft-versus-host reaction (GVHR) enhanced the expression of endogenous xenotropic C-type RNA viruses and not of ecotropic C-type viruses in the spleens of CAF-1 hybrid mice. This observation suggests a specific xenotropic virus response during the GVHR in mice and indicates that ecotropic viruses are not necessarily expressed during GVHR. The possible role of xenotropic viruses in the GVHR-induced malignancy is considered.

Origins of pathogenic anti-DNA idiotypes in the NZB X SWR model of lupus nephritis.

The investigations with the NZB X SWR model show that the development of systemic autoimmune disease is a multistep, multigene process. Severe lupus nephritis in the NZB X SWR hybrids results from the interaction of genes inherited from both the autoimmune NZB and the normal SWR parents. A similar genetic interaction occurs in the NZB X NZW hybrids, but in this model, both the parental strains are abnormal and the nature of the gene products or their mechanism of action is unknown. In the NZB X SWR model, we have been able to identify a restricted subpopulation of nephritogenic anti-DNA antibody idiotypes that are encoded by genes of the normal SWR parents. Thus, these are one set of genes that determine the development of severe lupus nephritis in the F1 hybrids. In addition, another set of genes allows for the expansion of B cells that produce such pathogenic anti-DNA idiotypes in the F1 hybrids since such B-cell clones remain dormant in the normal SWR parents. The latter category of genes, presumably specifying defects in immunoregulation, are probably inherited from the NZB parents or may be the result of complementation of genes inherited from both parents. Further investigations with the NZB X SWR model will help us define the immunoregulatory defects in SLE that are specific for the T and B cells involved in pathogenic autoantibody production.

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.

Susceptibility to lymphomas and expression of C-type RNA viruses during the graft versus host reaction☆

Abstract The relationship between C-type RNA viruses and the development of lymphomas was examined in mice undergoing the chronic GVHR. The GVHR was induced in F 1 hybrid recipients by administration of parental spleen cells; donor strains were selected from lines that expressed abundant, few or no detectable ecotropic viruses. There was no correlation between titer of C-type RNA viruses (both ecotropic and xenotropic) and the occurrence of lymphomas. In one combination that failed to express any detectable ecotropic virus, SWR → (SWR × NZB)F 1 , lymphomas occurred in relatively high incidence. The results suggested that antigen-stimulated lymphocytes, which proliferate in abundance during the GVHR, may be preferentially susceptible to malignant transformation by viruses that, in other cells, are only weakly oncogenic or even non-oncogenic. In the course of these experiments we also found that NZB mice posses a dominant gene (or genes) that determines high-grade expression of xenotropic virus in crosses with other strains that express little or no xenotropic virus.

Characterization of a retrovirus that cross-reacts serologically with canine and human systemic lupus erythematosus (SLE)☆

Abstract This report characterizes the SP104 virus, which was previously shown to contain an antigen that cross-reacts with an antigen present on surfaces of blood lymphocytes of human and canine patients with systemic lupus erythematosus (SLE). Morphologically, the virus was a Type C particle. By physicochemical characterization it was a typical retrovirus with a bouyant density of 1.15–1.17 g/cm 3 , high molecular weight RNA and RNA-dependent DNA polymerase. The virus had antigens that cross-reacted with p30, gp71, p12, and p15 of other murine retroviruses. Biologically, SP104 was characterized as a murine B-tropic virus that was only weakly oncogenic but highly efficient in eliciting the production of antinuclear antibody in mice. Nucleic acid hybridization experiments indicated that the RNA of SP104 virus had only partial identity with the other murine leukemia viruses tested. There was no evidence that the genetic sequences found in the SP104 virus were present in tissues from canine or human patients with SLE.