The lpr gene causes an intrinsic T cell abnormality that is required for hyperproliferation.

The lpr gene induces marked lymphoproliferation characterized by the massive accumulation of T cells of an unusual phenotype and concomitant autoimmune disease. To clarify the mechanism of the lpr effect, bone marrow cells from B6-lpr/lpr (Ly-1.2) and B6-+/+ (Ly-1.1) mice were transferred into lethally irradiated B6-lpr/lpr mice. As has been previously reported, recipients of the B6-lpr/lpr bone marrow showed the typical lpr phenotype with marked lymphadenopathy, splenomegaly and increased levels of autoantibodies; while the recipients of B6-+/+ bone marrow had normal sized lymph nodes and spleen and no autoantibodies. A third group of mice received an equal mixture of bone marrow cells from the B6-lpr/lpr and B6-+/+ donors. These mice showed both lymphadenopathy and autoantibody production comparable to that of recipients of the B6-lpr/lpr marrow alone. Immunofluorocytometric analysis of the lymphoid populations in these mixed bone marrow recipients established that the T cells from the lpr/lpr and +/+ donors were equivalently represented in the peripheral blood and thymus. In striking contrast, the T cells that accumulated in abnormally large numbers in the lymph nodes were almost entirely from the lpr donor. Their surface phenotype was Thy-1+(dull), Ly-1.2+(dull), Lyt-2-, L3T4-, 9F3+, and 3A1+, which is consistent with that found in intact lpr mice. These results indicate that the lpr gene causes an intrinsic defect directly within the T cells that accumulate in large numbers in lpr mice. In addition, the presence of the +/+ T cells cannot prevent the expression of the lpr abnormalities.

Anti-idiotypic antibodies to the Coombs antibody in NZB F1 mice.

The F1 hybrids of NZB and several normal mouse strains are known to produce less anti-erythrocyte (Coombs) autoantibody and develop a milder hemolytic anemia than their NZB parents. We have found that serum from some (NZB x CBA)F1 mice agglutinated erythrocytes from certain Coombs-positive NZB mice, often in extremely high titer, whereas other (CBA x NZB)F1 sera agglutinated erythrocytes from different individual NZB mice. The agglutination was due to antibody, but was not due to rheumatoid factor activity. Because F(ab')2 fragments of the F1 sera agglutinated erythrocytes coated with F(ab')2 fragments of the appropriate NZB sera, the observed reactivity was probably caused by idiotype-anti-idiotype interactions. In addition, because F1 sera could not agglutinate mouse erythrocytes coated with monovalent NZB Fab' fragments, the recognized idiotype probably involved the antigen-binding site. Anti-idiotypic antibodies against anti-erythrocyte autoantibodies may play an important role in the regulation of autoantibody formation.

Hidden autoantibodies against common serum proteins in murine systemic lupus erythematosus. Detection by in vitro plaque-forming cell assay.

The autoantibodies found in human and murine systemic lupus erythematosus (SLE) are generally directed against cells or components of cells such as nuclear antigens. This predilection may be due to the unusual immunogenicity of certain autoantigens, or to unusual patterns of antibody crossreactivity. Alternatively, the observed spectrum of reactivities may reflect the in vivo absorption of those autoantibodies directed against soluble antigens. To test whether hitherto undetected autoantibodies against serum proteins might exist in murine SLE, we developed assays that were independent of the possibility of absorption of autoantibodies by serum autoantigens; large numbers of plaque-forming cells (PFC) directed against mouse albumin and mouse transferrin were easily detected in the spleens of MRL/Mp-lpr/lpr, BXSB, and NZB mice. The secreted antibodies were relatively specific for the mouse proteins, since only limited cross-reactivity was seen with albumin and transferrins of other species in inhibition experiments. The production of these hidden antibodies could not be the result of diffuse polyclonal B cell activation, since the PFC to mouse transferrins and albumin were not always accompanied by comparable numbers of PFC against related albumins and transferrins. The results indicate that autoantibody production in murine lupus is a generalized phenomenon, not limited to the production of autoantibodies to nuclear or other cell-bound antibodies. However, the relative specificity of the autoantibodies for self-antigens indicates that diffuse polyclonal B cell activation cannot be the mechanism responsible, and argues that a selective mechanism, probably driven by antigen, accounts for production of autoantibodies in SLE.

Defective B cell tolerance in adult (NZB X MZW)F1 mice.

Hapten-specific tolerance was induced in vitro by trinitrophenyl-human gamma globulin (TNP32HGG) to a comparable degree in B cells from adult autoimmune (NZB X NZW)F1 (B/W) mice and normal BDF1, CBA/J, and DBA/1J mice. When a lower epitope density tolerogen (TNP7HGG) was used, B/W mice were significantly less sensitive than normal mice to the induction of B cell tolerance. This finding of defective B cell tolerance in adult B/W mice is consistent with previous reports that document other B cell abnormalities that may relate to the expression of autoimmune disease.

Autoantibodies in chronic graft versus host result from cognate T-B interactions.

A chronic graft-versus-host reaction (GVH) induced in nonautoimmune mice causes a syndrome that closely resembles SLE. In this model, donor T cells react against incompatible host Ia structures and generate excessive help, which activates a subpopulation of self-reactive B cells. We have studied whether these self-reactive B cells are activated by direct interaction with alloreactive T cells or by nonspecific bystander effects. Two types of chimeras were made: double-parental chimeras, differing at both Ia and Igh allotype [B6.C20 + bm12----(B6.C20 x bm12)F1]; and control chimeras [(B6.C20 x bm12)F1----(B6.C20 x bm12)F1]. A chronic GVH syndrome was induced in the chimeras by infusion of B6 or bm12 spleen cells. Coombs and antichromatin autoantibodies were measured using Igh allotype-specific immunoassays. The double-parental chimeras that received bm12 cells made autoantibodies principally of the Igha allotype, indicating that the bm12 T cells interacted only with the Iab-bearing host B cells. Conversely, double-parental chimeras that received B6 cells made mostly Ighb autoantibodies, indicating direct cognate interaction with the Iabm12-bearing host B cells. The control chimeras made autoantibodies of both allotypes. These results indicate that autoantibodies in chronic GVH result from direct T-B interactions and not from nonspecific T cell-derived factors.

Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I.

Mice homozygous for the lpr gene have a defect in fas (CD95), a cell surface receptor that belongs to the tumor necrosis factor receptor family and that mediates apoptosis. This genetic abnormality results in lymphoproliferation characterized by the accumulation of CD4-CD8- (double negative [DN]) T cells, autoantibody production, and background strain-dependent, end-organ disease. Our previous results suggested that major histocompatibility complex (MHC) class I may be involved in the development of DN cells. To test this hypothesis, we derived C57BL/6-lpr/lpr (B6/lpr) mice that were deficient for the beta 2-microglobulin gene (beta 2m lpr) and had no detectable class I expression. At 6 mo of age, compared with B6/lpr littermates with normal class I genes, these mice showed greatly reduced lymphadenopathy, mostly due to a dramatic decrease in the number of DN cells. Significant changes in the percentage of other T cell subsets were noted, but only gamma/delta+ T cells showed a marked increase in both percentage and absolute numbers. Analysis of T cell receptor V beta expression of the remaining DN T cells in beta 2m -lpr mice showed a shift to a CD4-like repertoire from a CD8-like repertoire in control B6/lpr mice, indicating that a small MHC class II selected DN population was unmasked in lpr mice lacking class I. We also found that the production of immunoglobulin G (IgG) autoantibodies (antichromatin and anti-single stranded DNA), total IgG and IgG2a, but not total IgM or IgM rheumatoid factor, was significantly reduced in the beta 2m -lpr mice. This work suggests that >90% of DN T cells in lpr mice are derived from the CD8 lineage and are selected on class I. However, a T cell subset selected on class II and T cells expressing gamma/delta are also affected by the lpr defect and become minor components of the aberrant DN population.

Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.

The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase. The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350). In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death. Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive. The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350. In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.

Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice.

Mice homozygous for the lpr gene develop autoantibodies and polyclonal B cell activation similar to what is seen in human systemic lupus erythematosus patients. We have previously shown that an lpr-specific intrinsic B cell defect was necessary for autoantibody production in this model. In the current study, we have further defined these autoantibody-producing B cells. Two major subsets of B cells have been described. B-1 cells (CD5+ B cells) can be distinguished from conventional B cells on the basis of phenotype, cytokine secretion, gene expression, anatomical location, and function. In addition, B-1 cells have been implicated in autoimmunity in several murine and human studies. To address the question of which B cell subset produces autoantibodies in lpr mice, we used immunoglobulin heavy chain (Igh) allotype-marked peritoneal (B-1 cell source) and bone marrow (conventional B cell source) cells from lpr mice to establish B cell chimeras. We used two general approaches. In one, we reconstituted sublethally irradiated mice with B-1 cells of one allotype and bone marrow cells of another allotype. In the second method, we suppressed endogenous B cells in neonatal mice with allotype-specific anti-IgM antibody, and injected peritoneal cells of another allotype. After antibody treatment was stopped, the mouse's conventional B cells recovered, but the B-1 subset was only reconstituted by the donor. In both types of chimeras, antichromatin, rheumatoid factor, and anti-single stranded DNA (ssDNA) autoantibodies were produced by the conventional B cell bone marrow source. In addition, an age-related decrease in peritoneal B-1 cells was seen, even in unmanipulated lpr mice. These data show that lpr B-1 cells are not important producers of autoantibodies. Conventional B cells are the source of autoantibodies directed at chromatin, ssDNA, and IgG.

An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimmunity.

Mice homozygous for the gene lpr develop marked lymphadenopathy and a spectrum of autoantibodies closely resembling that of human systemic lupus erythematosus. The unusual T cell phenotype of the expanded lymphocyte population and the T-dependence of several antibodies in this strain have suggested that primary T cell abnormalities underlie the autoimmune syndrome. Using double chimeras, we now show that expression of the lpr gene in B cells is absolutely necessary for autoantibody production. Combinations of anti-Thy 1.2 + C' treated bone marrow from congenic strains of C57BL/6 mice, differing only at the immunoglobulin heavy chain (Igh) and lpr loci, were transferred into lethally irradiated B6/lpr mice. Double chimerism was documented by allotype-specific surface IgD and IgM immunofluorescence assay of peripheral blood and by allotype-specific enzyme-linked immunosorbent assay for total IgM in serum. Despite the presence of both +/+ and lpr B cells, IgM and IgG2a anti-chromatin as well as IgM anti-IgG were entirely the products of lpr B cells. Total serum IgG2a and IgG1 were also dominated by the lpr phenotype but not to the same extent. A similar experiment using B6/lpr-Igha recipients confirmed these findings. Additional experiments in which B6/lpr recipients were infused with ratios of donor bone marrow favoring B6.C20 +/+ over B6/lpr showed that even though +/+ B cells were overrepresented, autoantibodies were only of the lpr allotype. In addition, in the presence of lpr B cells, normal B cells showed little response to an exogenous, T cell-dependent antigen. The data thus indicate that lpr B cells manifest an intrinsic abnormality which is essential for autoantibody production in the lpr model.

Stochastic control of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr autoimmune mice consistently show an approximately 25% incidence of the systemic lupus erythematosus marker autoantibody anti-Sm. In the present report, we show that the failure to find anti-Sm antibodies in three-quarters of 5-mo-old MRL/lpr mice was not an artifact of an insensitive assay, but rather that the mice fell into two populations as regards their anti-Sm positivity. Based on an extensive analysis of the incidence of anti-Sm positivity in 5-mo-old mice according to their cage of residence, we found no evidence for genetic, environmental, or parental influences on the propensity of an individual animal to become anti-Sm positive. Also, the gender of the mouse, its Sm antigen level, or its length of survival were not related to anti-Sm antibody, nor was the anti-Sm antibody status of either parent. Some animals became anti-Sm positive after 5 mo of age, but this was less likely than becoming positive before 5 mo of age. Finally, a survey of 205 autoimmune C57BL/6-lpr/lpr mice confirmed the uniqueness of the MRL background for this autoantibody response. These results together indicate that the possibility of making anti-Sm antibodies is under genetic control, but that the expression of this capability in an individual animal is governed by stochastic events. We hypothesize further that such random processes may involve the expression of particular immunoglobulin variable-region genes combined with mechanisms of extensive somatic mutation or positive feedback amplification, which would transmute an initial monoclonal response into an eventual polyclonal one.

Regulation of the anti-Sm autoantibody response in systemic lupus erythematosus mice by monoclonal anti-Sm antibodies.

The administration of certain monoclonal anti-Sm antibodies (2G7, 7.13) induced most MRL/lpr mice to become anti-Sm positive by 5 mo of age, although other anti-Sm monoclonals (Y2, Y12) suppressed the spontaneous response. Positive anti-Sm antibody enhancement occurred efficiently only in MRL/lpr mice and not in other systemic lupus erythematosus mice that have little spontaneous anti-Sm production. The enhancement by anti-Sm antibodies was specific for the anti-Sm response. The mechanism of the passive antibody enhancement was apparently not isotype- or idiotype-related. The fine specificity of the anti-Sm monoclonal antibody may be essential to its enhancing or suppressing effects, since both enhancing monoclonals recognized only the D Sm polypeptide, whereas both suppressing monoclonals saw the D and the B polypeptides. Furthermore, analysis of serial bleeds from unmanipulated MRL mice that developed anti-Sm positivity showed that the D specificity almost always appeared first. We hypothesize, therefore, that those animals in which an anti-Sm response is initiated by D-specific B-cell clones can become serologically positive with the aid of a positive feedback loop. In contrast, animals in which the initial specificity is for both B and D peptides would be prevented from developing a full anti-Sm response.

Antigen nonspecific effect of major histocompatibility complex haplotype on autoantibody levels in systemic lupus erythematosus-prone lpr mice.

MHC-linked genes strongly influence susceptibility to autoimmune diseases and also regulate responses to exogenous antigens. To begin to understand the mechanism of this MHC effect on disease, we have investigated MHC-congenic mouse strains that develop spontaneous autoimmunity because of the lpr gene. C57BL6/lpr (B6/lpr) mice (H-2b) are known to have substantial levels of autoantibodies to chromatin, single stranded DNA (ssDNA3), and IgG of different murine subclasses (rheumatoid factor). We have crossed the H-2d and the H-2bm12 (la mutant) haplotypes onto the B6/lpr background. Surprisingly, levels of all the autoantibodies were markedly lower in B6/lpr.H-2d, but levels in B6/lpr.H-2bm12 were no different from those in B6/lpr mice. The downregulating influence of the H-2d allele was dominant, and there was no effect on autoantibody fine specificities. The genetics of the H-2d effect and its diffuse influence on multiple autoantibody specificities, in addition to the lack of effect of the bm12 mutation, which modifies the peptide-binding groove of I-A, together raise the question of whether MHC-linked genes other than classical (IR) genes may be responsible for MHC disease associations in this model.

Fas, ceramide and serum withdrawal induce apoptosis via a common pathway in a type II Jurkat cell line.

Ceramide is a key mediator of apoptosis, yet its role in Fas-mediated apoptosis is controversial. Some reports have indicated that ceramide is either a primary signaling molecule in Fas-induced cell death, or that it functions upstream of Fas by increasing FasL expression. Other studies have suggested that ceramide is not relevant to Fas-induced cell death. We have approached this problem by studying ceramide-induced apoptosis in unique Jurkat cell clones selected for resistance to membrane-bound FasL-induced death. Resistance of the mutant Jurkat cells was specific for FasL killing, since the mutant clones were sensitive to other apoptotic stimuli such as cycloheximide and staurosporine. We tested the effects of serum withdrawal, one of the strongest inducers of ceramide, and of exogenous ceramide on apoptosis of both wild-type and FasL-resistant clones. Wild-type Jurkat cells were remarkably sensitive to serum withdrawal and to exogenous ceramide. In contrast all FasL-resistant mutant clones were resistant to these apoptosis-inducing conditions. In contrast to previous work, we did not detect an increase in FasL in either wild-type or mutant clones. Moreover activation of stress-activated protein kinases (JNK/SAPKs) after serum withdrawal and exogenous ceramide treatment was detected only in the wild-type and not in the resistant clones. Because of the parallel resistance of the mutant clones to Fas and to ceramide-induced apoptosis, our data support the notion that ceramide is a second messenger for the Fas/FasL pathway and that serum withdrawal, through production of ceramide, shares a common step with the Fas-mediated apoptotic pathway. Finally, our data suggest that activation of JNK/SAPKs is a common mediator of the three pathways tested.

Fas ligand (gld)- and Fas (lpr)-deficient mice do not show alterations in the extravasation or apoptosis of inflammatory neutrophils.

Apoptosis of neutrophils plays a critical role in the resolution of acute inflammation. Neutrophils from human peripheral blood express Fas (CD95) and are sensitive to Fas ligand (FasL)/Fas-mediated apoptosis. Mice carrying spontaneous mutations in the genes for fas ligand (B6/gld) or fas (B6/lpr) were used to assess the role of FasL/Fas in the kinetics and magnitude of neutrophil extravasation to the thioglycolate (TG)-inflamed peritoneum and in the spontaneous apoptosis of TG-elicited neutrophils. The results showed that TG-elicited neutrophils (defined by flow cytometry as GR-1/Ly-6G(hi) cells) from normal (B6) and B6/gld mice, but not from the Fas-deficient B6/lpr mice, express high levels of Fas. The TG-elicited neutrophil response began at 2 h, peaked at 4 h, and subsided by 24-48 h after TG administration in all three strains. However, the response was more prolonged in B6 mice, such that B6/gld and B6/lpr mice had fewer neutrophils at 6 h after TG administration than did B6 mice. Further studies showed that 4 h TG-elicited neutrophils from B6, B6/gld and B6/lpr mice undergo apoptosis in vitro at similar rates (as assessed through flow cytometry by the decrease in forward angle light-scatter and externalization of phosphatidylserine (PS; as detected by Annexin V-FITC) that occur as neutrophils undergo apoptosis). Fas expression was down-regulated on apoptotic neutrophils in conjunction with maximal PS externalization and decreased forward angle light-scatter. Collectively, these findings suggest that FasL/Fas-mediated apoptosis is not essential in regulating the lifespan of neutrophils during an acute inflammatory response. The abbreviated inflammatory response observed in FasL/Fas-deficient mice is likely to be a secondary effect of the gld/lpr autoimmune/lymphoproliferative syndrome, and not a direct effect of FasL/Fas on the ability of inflammatory neutrophils to undergo apoptosis.

The polypeptide structure of the Sm and RNP nuclear antigens.

Sm and nuclear ribonucleoprotein are ubiquitous nuclear antigens towards which important autoantibodies are directed in systemic lupus erythematosus and other human autoimmune syndromes. Using physicochemical techniques and affinity adsorptions, we have purified the polypeptide components of these antigens. The Sm antigen contained polypeptide chains of 15,000 and 17,000 mol. wt. The RNP antigen, which is known by immunochemical techniques to contain the Sm antigen, had the same two polypeptides as well as a larger one of 85,000 mol. wt. This larger peptide was quite labile and apparently broke down into smaller components with manipulation. In addition, the process of affinity purification of the Sm polypeptides gave a product which had increased positive charge. Amino acid analysis of the Sm polypeptides confirmed the presence of relatively large numbers of basic residues. The purified Sm antigen provided an effective reagent for the investigation of autoreactivity to Sm. The differences in structure from our results and those published by others are probably accounted for by the lability of the constituent polypeptides.

bca: an activation-related B-cell gene.

We have identified a novel activation related B-cell gene (bca) through differential hybridization screening of a murine B cell cDNA library. The deduced amino acid sequence predicted a protein of 482 amino acids with strong sequence similarity to the SH2 and SH3 domains present within the non-catalytic regions of several protein tyrosine kinases. Northern analysis of RNA from several murine B-cell lines revealed a transcript of 1.8 kb, which was not detected in T-cell and non-lymphoid cell lines. bca was transcribed at low levels in resting spleen cells from a variety of normal mouse strains and was strongly expressed in kidney RNA. bca expression was markedly increased in RNA prepared from mitogen activated B cells, and in freshly isolated spleen and lymph node cells of MRL/lpr and NZB autoimmune strains. The unique sequence of bca, which bears no obvious similarity to any specific class of proteins containing SH2 and SH3 domains, suggests that this gene encodes a novel protein potentially involved in B-cell signal transduction.

T- and B-cell abnormalities in systemic lupus.

A wealth of T- and B-cell abnormalities has been described in systemic lupus erythematosus (SLE). In general, T and B cells manifest evidence of intense in vivo activation, yet perform poorly when tested for in vitro responsiveness to exogenous antigen. The immune system behaves as if its commitment to self-responsiveness overrides the normal capacity to respond to immunogenic challenges. Many immunoregulatory abnormalities have been characterized, but their relation to disease remains controversial. Although a widely accepted unifying hypothesis for the vigorous autoantibody production and associated immune deficiency of SLE is lacking, it seems certain that the profound abnormalities of T and B cells in SLE are intertwined with the fundamental cause of this illness.

Early carotid endarterectomy for critical carotid artery stenosis after thrombolysis therapy in acute ischemic stroke in the middle cerebral artery.

BACKGROUND AND PURPOSE: Tissue plasminogen activator (tPA) has been shown to be effective for acute ischemic stroke. However, if a high-grade cervical carotid stenosis remains despite tPA therapy, patients are at risk for recurrent stroke. Carotid endarterectomy (CEA) has been shown to be effective in symptomatic patients with high-grade cervical carotid stenosis in reducing the risk of stroke, but it is unknown whether CEA can be performed safely after tPA thrombolysis. We describe our experience with 5 patients who underwent early (<48 hours) CEA for residual high-grade cervical carotid stenosis after thrombolytic therapy for acute ischemic stroke in the middle cerebral artery territory. METHODS: All patients had a critical (>99%) carotid artery stenosis on the symptomatic side after tPA therapy. All patients received intravenous tPA; 3 patients also received intra-aortic tPA. Three patients received intravenous heparin infusion immediately after administration of tPA. All patients showed marked improvement in their National Institutes for Health Stroke Scale scores after treatment with tPA. CEA was then performed within 45 hours (6 hours in 1 patient, 23 hours in 2, 26 hours in 1, and 45 hours in 1). RESULTS: All 5 patients underwent successful CEA. There were no complications related to surgery. At discharge, 2 patients had a normal examination, and the remaining patients had mild deficits. In a long-term follow-up of 5 to 22 months, no patient had a recurrent cerebrovascular event. CONCLUSIONS: Early CEA can be performed safely and successfully in patients after tPA treatment for acute ischemic stroke in appropriately selected patients.

Clonal analysis of peripheral T cell precursors in lpr mice.

MRL/Mp-lpr/lpr mice develop massive lymphadenopathy characterized by expansion of an unusual population of T cells with the Thy 1+, CD3+, CD4-, CD8- (double negative) phenotype. The role these cells play in accelerating the autoimmune syndrome seen in these mice is unknown. In order to better understand the origin of the expanded population of T cells, we have derived a panel hybridomas from double negative lpr lymph node cells. Surprisingly, eleven of twelve hybridomas selected for the absence of surface CD4 and CD8 do not express CD3. Six of eleven confirmed to have inherited the MRL T cell receptor locus have rearrangement at that locus, suggesting commitment to a T cell lineage. Only hybridoma 2.4, which expresses CD3, responds to ConA, anti-CD3 monoclonal antibody, and induces antibody production. The presence of CD3-, CD4-, CD8- T cells in the periphery of lpr mice confirms aberrant T cell development in these mice and suggests an intrinsic cell defect which is expressed early in lymphopoiesis.

Diminished expression of the T cell receptor on the expanded lymphocyte population in MRL/Mp-lpr/lpr mice.

MRL mice homozygous for the recessive lpr gene develop an accelerated autoimmune syndrome and massive lymphadenopathy. Because the function of the expanded lymph node population is unclear, we have studied the subunits of the T cell receptor for antigen (TcR). DNA and RNA were prepared from MRL/Mp-lpr/lpr (lpr) and congenic MRL/Mp(-)+/+ (+/+) mice by standard techniques and studied by Southern blot, northern blot, and dot blot analysis using the cDNAs TT11, specific for the TcR alpha chain; 86T5, specific for the TcR beta chain; and T3 delta; specific for the subunit of the T3 molecule. Surface protein was immunoprecipitated with antisera 8177, which recognizes TcR framework determinants, and resolved by diagonal SDS-PAGE. FACS analysis was performed with a monoclonal antibody to murine T3, and with the KJ16-133 and F23.1 monoclonal antibodies, which recognize determinants encoded by the V beta 8 subfamily of beta chain variable region genes. When compared with +/+ controls, surface TcR density as detected by immunofluorescence using all three antibodies was significantly diminished on lpr spleen and lymph node cells, as well as on lpr lymph node cells which had been depleted of L3T4+ and Ly2+ cells by negative selection. There appeared, however, to be selective expression of the genes encoding the epitopes binding F23.1. Southern blot analysis of DNA showed polyclonal rearrangements of the TcR beta chain genes. There were increased alpha, beta, and T3 delta RNA transcripts in the double negative lymph node cells. The paradoxical decrease in TcR surface expression in the setting of large quantities of full length transcript is yet to be explained.