In vivo immunosuppression by targeting a novel protease receptor.

Membrane receptors for blood proteases govern the clotting and fibrinolytic cascades, regulate signal transduction and control the growth of mesenchymal cells. Despite their importance in the development of vascular injury, it is unclear whether these mechanisms participate in the generation of an immune response. Here we report that targeting a factor Xa receptor, designated effector cell protease receptor-1 (EPR-1), with antisense oligonucleotide or with a monoclonal antibody (mAB 2E1) inhibited CD3/T-cell receptor-dependent lymphocyte proliferation. Immunosuppression was mediated by abolishing cytokine production and down-modulating membrane expression of the interleukin (IL)-2 receptor. In vivo administration of mAb 2E1 to severe-combined-immunodeficient mice injected with human peripheral blood leukocytes suppressed production of human immunoglobulin, abolished graft-versus-host disease, and protected these xenochimaeric mice from Epstein-Barr-virus-induced human lymphoproliferative disease. These observations indicate a new role for protease receptors in the regulation of the immune response, and identify a potential target for therapeutic immunosuppression in humans.

Characterization of hu-PBL-SCID mice with high human immunoglobulin serum levels and graft-versus-host disease.

A chimeric model consisting of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL) has recently been described (bu-PBL-SCID mice). These reports indicated a limited reconstruction of the transferred human immune system and functionality of the human graft. Herein we described modifications of the PBL transfer method that minimize transfer time and cellular manipulations, leading to a more effective population of SCID mouse recipients. Severe combined immune deficiency mice given 15 x 10(6) PBL had human IgG serum levels reaching 2 to 5 g/l, and all mice had detectable human anti-tetanus toxoid antibody levels when they received cells from donors with such levels. These transfers were associated also with clinical and histologic evidence of graft-versus-host disease, suggesting responsiveness of the human graft in the recipients. When Epstein-Barr virus seropositive (EBV+) donors were used, the chimeric mice also showed a high incidence of fatal lymphoproliferative disease 1 to 3 months after transfer of 15 x 10(6) PBL. The high level of immunoglobulin synthesis and immunoresponsiveness of the human cells with this transfer procedure may expand the use of these chimeric mice for the manipulations of human immune cells in vivo.

The hu-PBL-SCID mouse model. Long-term human serologic evolution associated with the xenogeneic transfer of human peripheral blood leukocytes into SCID mice.

We present a 2-year serologic analysis of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL, hu-PBL-SCID mice). After 10-20 x 10(6) PBL transfer, human IgG serum levels generally increased in the SCID mouse recipient for 2 months, and thereafter decreased without returning to zero for at least 2 years. Great variability existed between different hu-PBL-SCID mice with regard to Ig serum levels even when derived from the same donor's PBL aliquot. The ratio of IgM to IgG serum levels was lower in hu-PBL-SCID mice than in the donors. The half-life of human IgG in the SCID mouse is shorter than in the human (8 days vs 23 days), suggesting a much higher production of IgG than expected from serum levels. The majority of hu-PBL-SCID mouse sera analyzed by high resolution electrophoresis had a smear appearance suggestive of diverse human Ig, generally with superimposed multiple faint mIg. Few mice developed strong human mIg, associated with lymphoproliferative diseases. In the hu-PBL-SCID mouse model, the transfer of cells from donors making antibody with defined specificity against TT and nuclear antigen resulted in the appearance of these antibodies in only a minority of the recipients.

Immunization of hu-PBL-SCID mice and the rescue of human monoclonal Fab fragments through combinatorial libraries.

Antibodies are usually prepared from recently boosted animals and reflect ongoing immune responses. In humans, this is restrictive as ethical constraints generally prevent antigen-boosting. Therefore the rich memory compartment of human antibody responses remains largely untapped. Severe combined immune deficiency (SCID) mice populated with human cells allow the stimulation of human antibody memory without the usual constraints. Here we show how peripheral blood lymphocytes can be stimulated by antigen to produce large secondary responses after transfer to SCID mice. Specific monoclonal human Fab fragments can then be isolated from the mice by repertoire cloning even when the human donor's last contact with antigen was more than 17 years ago.

An autosomal recessive gene that delays expression of lupus in BXSB mice.

We report the generation and serologic, cellular, histologic, and genetic characteristics of a BXSB/MpJScr substrain, termed BXSB/MpJScr-ll/ll, that has lost early-life male lupus disease. Classic genetic analysis suggested that delayed disease expression results from the action of a single autosomal recessive gene. This putative gene, referred to as ll (long-lived), causes a significant delay in expression of autoimmune serology (total serum IgG and anti-nuclear antibodies levels), monocytosis, and of immune complex-mediated histopathologic changes such as glomerulonephritis, arteritis, and myocardial infarction. Presumably as a consequence of the delayed immunopathology male BXSB/MpJScr-ll/ll mice live three to four times longer than regular BXSB/MpJScr. This strain might be useful for analysis of single genes responsible for severe autoimmune disease expression.

Transfer of human systemic lupus erythematosus in severe combined immunodeficient (SCID) mice.

To study the role of peripheral blood leukocytes (PBL) in the pathogenesis of human systemic lupus erythematosus (SLE), we transferred PBL from 5 SLE patients into 15 severe combined immunodeficiency (SCID) mice. Such reconstituted mice showed long-term presence of auto-antibodies characteristic of the donor in their sera, as well as human immunoglobulin deposition, and in some cases mouse C3, in the renal glomeruli. SCID mice repopulated with PBLs from normal donors do not develop serologic abnormalities or immunodeposits. It is concluded that human SLE serology and some associated renal changes can be reproduced solely by PBL transferred from afflicted patients, and that SCID-human-SLE mice may serve as an in vivo laboratory model for the study of human SLE.

Genetic diversity of murine rheumatoid factors.

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.

Autoimmune mice and stimulated normal mice make lambda 1 with increased V region diversity.

Previous studies of the genetic bases of murine SLE have defined gene segments that encode the H chain and the kappa L chain of anti-DNA, anti-Sm, and anti-IgG autoantibodies. As a result of these studies, the genetic origins of autoantibody H chains and kappa L chains are better understood, but little remains known about the genetic bases of autoantibody lambda-chains. Thus, we have analyzed serologically the germ-line and somatic origins of lambda 1 L chains in antibodies of normal mice and in both antibodies and autoantibodies of autoimmune mice. This study finds an increased lambda 1 diversity in both Ag-stimulated mice and autoimmune mice. This study also finds that the lambda 1 L chains in antibodies of unstimulated normal mice have the gene segment-encoded variable region, V lambda 1. In contrast, additional genetic processes appear to make the lambda 1 V regions of antibodies in Ag-stimulated normal mice and the lambda 1 V regions of both antibodies and autoantibodies in autoimmune mice. The increased lambda 1 diversity that we found in both Ag-stimulated mice and autoimmune mice might be caused by mutational processes creating antibody diversities. Therefore, the same somatic processes might be able to make both antibody and autoantibody lambda 1 diversities.

Complexity, polymorphism, and connectivity of mouse Vk gene families.

To define the polymorphism and extent of the mouse immunoglobulin kappa (Igk) gene complex, we have analyzed restriction-enzyme digested genomic DNA from 33 inbred strains of mice with labeled DNA probes corresponding to 16 Vk protein groups (1 of them previously undescribed) and the Jk/Ck region (V, variable; J, joining; C, constant). These probes detected between 1 and 25 distinct restriction enzyme fragments (REF) that appeared in up to eight polymorphic patterns, thus defining eight mouse Igk haplotypes. The investigated portion of the Vk repertoire was estimated to encompass between 60 and 120 discernable Vk gene-containing REFs. In contrast to mouse VH gene families, several Vk gene families defined by these probes appeared to overlap. This observation has implications for Vk gene analyses by nucleic acid hybridization and raises the possibility that the Vk gene complex is a continuum of related sequences.

T-cell receptor alpha-chain variable-region haplotypes of normal and autoimmune laboratory mouse strains.

We used Southern blotting and mRNA analysis to characterize allelic polymorphisms among genes of the T-cell antigen receptor (TCR) alpha-chain variable-region (V alpha) locus in a large panel of normal and autoimmune-susceptible or autoimmune-contributing strains of laboratory mice. Four major V alpha haplotypes were defined on the basis of multiple restriction fragment length polymorphisms for each of nine V alpha subfamily probes used. Southern blotting also revealed haplotype-specific loss of bands within some V alpha subfamilies, consistent with the deletion of particular V alpha genes or sets of genes from haplotype to haplotype. In contrast to the situation in the V beta locus, however, deletion of entire V alpha subfamilies was not observed. The nature of V alpha allelic variability was further explored by using an RNase protection assay to analyze expressed V alpha mRNA sequences in thymocyte RNA. Such analysis revealed both shared and unique patterns of V alpha mRNA expression among the different haplotypes and supported the conclusion that haplotype differences sometimes involve V alpha gene deletions. Interestingly, a disproportionate number of, but not all, autoimmune-susceptible strains, including NZB, SJL, SWR, PL/J, and NOD, share a common V alpha haplotype. The identification of murine TCR V alpha haplotypes should provide a basis for understanding the role of TCR diversity in normal immunoregulatory and immune-response phenomena, as well as autoimmune-disease predisposition.

Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice.

We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.

Genomic organization and expression of B and T cell antigen receptor genes in murine lupus.

Studies with genomic DNAs of lupus mice and monoclonal autoantibodies suggest that autoantibody expression does not result from defects in immunoglobulin germline genes nor in mechanisms generating antibody repertoires. Genomic and expression abnormalities of T cell antigen receptor genes have been identified in lupus mice, but their possible contribution to disease manifestations remains to be established.

Elevated titers of cell-free interleukin 2 receptor in serum of lupus mice.

Activation/proliferation of mouse and human T and B cells is associated with expression and subsequent release of interleukin 2 receptors (IL 2R) into the milieu. The soluble form of IL 2R, at least in part, retains its ability to bind to IL 2 and to anti-receptor antibodies, but its exact structure remains unknown. Because systemic lupus erythematosus (SLE) is associated with T and B cell activation, we have used monoclonal anti-IL 2R antibodies in an ELISA to measure levels of IL 2R in sera of various lupus strains. High levels of the released receptor were found at an active clinical stage in sera of four autoimmune strains of mice homozygous for the lpr (lymphoproliferation) gene that causes T cell expansion, massive lymphoid organ enlargement, and promotes the autoimmune process. High levels were also found in lupus mice characterized primarily by B cell proliferation (BXSB males) and in (NZB X W)F1 mice characterized by T and B cell activation. Similarly high IL 2R serum levels could be induced experimentally in normal mice injected with immunostimulants such as bacterial lipopolysaccharide or Freund's complete adjuvant. The results indicate that IL 2R serum levels may provide a good marker of ongoing lymphoid cell activation/proliferation, and thus might be useful in the follow-up of patients with systemic autoimmune or other lymphoproliferative disorders. The biologic roles, if any, of the soluble form of IL 2R and its effects in normal and abnormal conditions remain to be determined.