Activation of autoreactive T cells that help nucleobindin-injected mice produce anti-DNA antibodies

Nucleobindin (Nuc) is a DNA- and calcium-binding protein that was originally identified as an anti-DNA antibody-enhancing factor in MRL/MpJ-lpr/lpr (MRL/lpr) mice. In MRL/lpr mice, both expression of Nuc mRNA in enlarged lymph nodes and serum concentration of Nuc protein are shown to increase as disease progresses. Administration of recombinant (r) Nuc to young MRL/MpJ-+/+ and normal BALB/c mice leads to augmentation or induction of IgG anti-double-stranded (ds) DNA autoantibodies. In this study, spleen cells prepared from MRL/lpr mice were found to show a proliferative response to rNuc, indicating existence of T cells that are specific to this autoantigen in peripheral lymphoid tissues. Furthermore, CD4+ T cell lines were generated from a BALB/c mouse that had been producing anti-dsDNA antibodies as a result of repeated injections of rNuc. These T cell lines were confirmed to be autoreactive, because they proliferated in culture with syngeneic but not with allogeneic spleen cells without addition of any exogenous antigens. Their proliferation was enhanced by rNuc, and inhibited by an anti-MHC class II monoclonal antibody. Upon in vivo inoculation, these T cells provided help for rNuc-injected BALB/c mice to produce anti-DNA antibodies. These results suggest that Nuc is able to activate autoreactive peripheral T cells through an MHC-class II pathway leading to acceleration or induction of anti-DNA antibody production when it is excessively produced in lupus-prone mice or experimentally administered into normal mice.     

Involvement in lupus disease of idiotypes Id.F-423 and Id.IV-228 defined, respectively, upon foetal and adult MRL/Mp-lpr/lpr DNA-binding monoclonal autoantibodies.

The derivation of a monoclonal IgG3K autoantibody, designated F-423, from a foetal MRL/Mp-lpr/lpr mouse is described. It has immunochemical properties similar to DNA-binding monoclonal antibodies derived from adult mice with lupus disease in that it reacts with single-stranded DNA and, to a lesser extent, with double-stranded DNA and some forms of RNA. Its similarities to antibodies from adults extend further: it carries a public idiotype, Id.F-423, that can also be detected on antibodies from adult MRL and (NZB x NZW)F1 mice, and F-423 itself expresses other idiotypes defined originally on antibodies from adult lupus mice of both strains. Its potential involvement in pathological processes is demonstrated by two observations: (i) immunization of young MRL/Mp-+/+ mice with antibody F-423 induced the nephritic and immunological changes associated with systemic lupus erythematosus; and (ii) heterologous rabbit anti-Id.F-423 anti-idiotypic antibodies suppressed the progression of lupus disease in adult MRL/Mp-lpr/lpr mice. Similar effects were found with monoclonal antibody IV-228, an antibody derived from an adult MRL mouse and previously known to be directly nephrotoxic, and with anti-Id.IV-228 antibodies. It is concluded that even during foetal life mice of lupus-prone strains have lymphocytes capable of making pathogenic autoantibodies long before symptoms of lupus disease appear.     

IL-33 Neutralization Suppresses Lupus Disease in Lupus-Prone Mice

IL-33 is a new member of the IL-1 family that plays a role in inflammation. In this study, we evaluated the potential of IL-33 inhibition as a treatment for systemic lupus erythematosus (SLE) using the lupus-prone model MRL/lpr mice and the underlying mechanisms of action. We treated mice with anti-mouse IL-33 antibody (anti-IL-33Ab) via intraperitoneal injection every other day from week 14 until week 20 for 6 weeks. A control group received the same amount of IgG control. Renal damage and mouse survival were compared. Cytokines, antibodies, immune complex, Tregs, myeloid-derived suppressor cells (MDSCs), and Th17 cells were also analyzed. Correlations between serum IL-33 and SLE disease activity index in human SLE were also investigated. MRL/lpr mice treated with anti-IL-33Ab showed reduced proteinuria and reduced serum anti-dsDNA levels. Nephritis, immune complex deposits, and the circulating antibodies and immune complex besides the mortality were significantly reduced by anti-IL-33Ab. Anti-IL-33Ab remarkably increased Tregs and MDSCs and reduced the Th17 cells and IL-1β, IL-6, and IL-17 levels in MRL/lpr mice. These results suggest that IL-33 inhibition may inhibit SLE via expansion of Tregs and MDSCs and inhibition of Th17 cells and proinflammatory responses, indicating that blockade of IL-33 has a protective effect on SLE.     

Functional deficiencies of spleen dendritic cells in autoimmune MRL/lpr mice

We investigated functional aspects of splenic dendritic cells (DC) obtained from MRL/MpJ-lpr/lpr (MRL/l) mice, and their normal counterparts, MRL/MpJ-+/+ (MRL/+) mice. In vitro antigen-presenting activity of DC obtained from MRL/l mice was impaired compared with that of DC from MRL/+ mice. Another major functional aspect of DC, i.e., stimulator cell activity in the allogeneic mixed lymphocyte reaction (MLR), was also found to be impaired in MRL/l mice. MRL/l mice and MRL/+ mice were also examined for co-operation between macrophages (M luminal diameter) and DC. We found peritoneal resident M luminal diameter obtained not only from MRL/+ mice but also from MRL/l mice released a soluble factor which enhanced the activity of DC from the respective mice. Furthermore, DC from both MRL/l and MRL/+ mice responded to the culture supernatant of a mouse macrophage hybridoma clone, S44, which releases a factor that enhances the antigen-presenting activity of DC, and their activity as stimulator cells in the allogeneic MLR was enhanced. However, the degree of enhancement was less in MRL/l mice than in MRL/+ mice.     

A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-lpr/lpr mouse: specificity and idiotype in serum immunoglobulins

Anti-double stranded (ds) DNA antibodies in the sera of lupus-prone MRL/Mp-lpr/lpr (MRL/l) mice were determined by an enzyme-linked immunosorbent assay in parallel with anti-single stranded (ss) DNA, anti-left-handed Z-DNA and anti-poly(ADP-ribose) antibodies. The serum levels of these antibodies in these mice increased with age, and in particular anti-dsDNA antibodies appeared in mice more than 14 weeks old, along with progressive lymphadenopathy. We therefore established a hybridoma producing monoclonal anti-dsDNA antibody (2C10) from an 8-month-old MRL/l mouse. Monoclonal antibody 2C10 did not react with either poly(dT) or poly(I), which are major cross-reactants with previously reported monoclonal MRL mouse autoantibodies. Antibody 2C10 showed preference for phi X-174 replicative form DNA and calf thymus dsDNA over ssDNA. 2C10 idiotype (Id) was present in the sera of MRL/l mice, but only occasionally at high levels even in the aged mice tested. This result suggested that many Ids with anti-dsDNA antibody activity may contribute to lupus pathogenesis in this strain of mouse.     

Anti-nRNP anti-nuclear antibody-secreting cells are represented in the B-lymphocyte repertoire of normal and MRL/MP-lpr/lpr lupus mice.

Spleen cells from MRL-lpr/lpr, CBA and BALB/c mice were cultured in vitro and assayed for production of anti-nuclear antibodies. Spleen cells from all species produced IgM antibodies to a nRNP (U1-RNP)-specific antigen and to double-stranded DNA (dsDNA) after stimulation with LPS. The specificity of the anti-nRNP antibodies was shown, by immunoblotting, to be directed against the 33,000 MW polypeptide of nRNP/Sm. CBA mice produced more IgM autoantibody in vitro than MRL/lpr or BALB/c mice. In contrast, IgG anti-nRNP and anti-dsDNA antibody were not produced by any of the strains. Our data show that anti-nRNP and anti-dsDNA precursor B cells are part of the normal murine immune repertoire and are not confined to the MRL/lpr strain. This suggests that the spontaneous development of anti-nRNP and anti-dsDNA antibodies associated with systemic lupus erythematosis (SLE) is dependent on clonal stimulation and removal of suppressive influences.     

Plasma cells in systemic lupus erythematosus: the long and short of it all

Plasma cells can be classified as long- or short-lived. The lifespan of a plasma cell largely depends on whether it arises from a germinal center or an extrafollicular locus and most importantly whether it can find a survival niche in the spleen or BM. In systemic lupus erythematosus (SLE) patients, long-lived plasma cells are believed to be responsible for the production of anti-RNA and anti-cardiolipin antibodies, whereas short-lived plasma cells, which are more susceptible to anti-proliferation therapies, are the main producers of anti-DNA antibodies. A previous study showed that transient overexpression of interferon-α (IFN-α), a cytokine that plays a pathogenic role in SLE, accelerates disease onset in lupus-prone NZB/W mice. In this issue of the European Journal of Immunology, the same group report that IFN-α induces large numbers of short-lived plasma cells, accompanied by high titers of anti-dsDNA antibodies in NZB/W, but not BALB/c, mice. Our commentary discusses this interesting observation in the context of the previous data regarding plasma cell differentiation and conveys our view about the clinical implications with respect to therapies that target plasma cells in SLE patients.     

Effect of genetic deficiency of terminal deoxynucleotidyl transferase on autoantibody production and renal disease in MRL/lpr mice

Terminal deoxynucleotidyl transferase (TdT) places non-template-coded nucleotides (N additions) in the VH CDR3 of T cell receptors and immunoglobulins. Amino acids coded for by N additions are important in autoantibody binding of dsDNA in lupus. We hypothesized that a genetic lack of TdT would modulate disease in lupus-prone mice. To test this hypothesis, we derived TdT-deficient MRL/lpr mice. Serum levels of anti-dsDNA antibodies and anti-dsDNA producing splenocytes were significantly lower in the TdT(-) versus TdT(+) littermates. Albuminuria, glomerular IgG deposition, and pathologic renal disease were significantly reduced in the TdT(-) mice. Sequence analysis of anti-dsDNA hybridomas derived from TdT(-) mice revealed a lack of N additions, short VH CDR3 segments, yet the presence of VH CDR3 arginines. Thus, the genetic absence of TdT reduces autoantibody production and clinical disease in MRL/lpr mice, confirming the importance of N additions in the autoimmune response in these mice.     

Spontaneous B cell hyperactivity in autoimmune-prone MRL mice

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.     

Monoclonal antibody therapy that targets phospholipid-binding protein delays lupus activity in MRL/lpr mice

Systemic lupus erythematosus is an autoimmune syndrome characterized by the development of autoantibodies to a wide range of antigens. Together with B cells, respective self-reactive T cells have an important contribution in disease progression as being responsible for inflammatory cytokines secretion, B cell activation and promoting amplification of the autoimmune response. Annexin A1 is expressed by many cell types and binds to phospholipids in a Ca²⁺-dependent manner. Abnormal expression of annexin A1 was found on activated B and T cells in both murine and human autoimmunity suggesting its potential role as a therapeutic target. In the present study, we have investigated the possibility to suppress autoimmune manifestation in spontaneous mouse model of lupus using anti-annexin A1 antibody. Groups of lupus-prone MRL/lpr mice were treated with the anti-annexin A1 monoclonal antibody, and the disease activity and survival of the animals were following up. Flow cytometry, ELISA assays, and histological and immunofluorescence kidney analyses were used to determine the levels of Annexin A1 expression, cytokines, anti-dsDNA antibodies and kidney injuries. The administration of this monoclonal antibody to MRL/lpr mice resulted in suppression of IgG anti-dsDNA antibody production, modulated IL-10 secretion, decreased disease activity and prolonged survival compared with the control group.     

Concomitant early appearance of anti-ribonucleoprotein and anti-nucleosome antibodies in lupus prone mice

OBJECTIVE: To gain insights on initial stages of the autoimmune response in lupus prone mice taking advantage of new sensitive and quantitative techniques for the detection of autoantibodies specific for RNA- (ribonucleoproteins) and DNA-protein (chromatin) complexes. METHODS: DNA and nucleosome antibodies were detected by ELISA, antibodies to SmB, U1A-RNP, Ro52, Ro60 and La by a new radioligand assay, using de novo synthesized radio-labeled antigens. RESULTS: Analysis of anti-chromatin (including anti-nucleosome, anti-dsDNA and anti-histone antibodies) and of anti-snRNP antibodies (including anti-U1A-RNP, anti-SmB, anti-Ro52, anti-Ro60, anti-La antibodies) was performed in sequential sera from B/W, MRL+/+, MRL Yaa and MRL lpr/lpr mice. In a cohort of 105 MRL+/+ mice of different ages, 59, 51, and 57 mice were positive for anti-nucleosome, anti-SmB and anti-U1A-RNP, respectively. None of them was positive for anti-dsDNA. Importantly, antibody positivities were not randomly distributed but were significantly clustered in individual mice. Appearance of DNA- and RNA-protein complex antibodies started at approximately 18-20 weeks of age, preceding that of the anti-dsDNA (or anti-histone) antibodies that only started at 30-32 weeks. Anti-nucleosome, anti-SmB and anti-U1A-RNP antibody responses did not display any cross-reactivity as demonstrated by inhibition and adsorption experiments. CONCLUSION: These data indicate that anti-nucleosome and anti-snRNP antibodies appear early and concomitantly in lupus prone mice even though they do not share any cross-reactivity. These results fit with the assumption that their production is triggered by tightly physically associated nucleosomes and snRNP autoantigens contained in the same apoptotic bodies.     

M-CSF transgenic mice: role of M-CSF in infection and autoimmunity.

In this study, transgenic CD2F1 mouse lines (C-1.1-C-1.11) bearing a transgene encoding the murine growth factor M-CSF under the control of the liver specific alpha-1-antitrypsin gene promoter were generated. Transgenic C-1.4 mice showed elevated expression of transgene-encoded M-CSF in the liver and displayed a 2-3-fold increase of M-CSF plasma levels and of macrophage numbers in the liver as compared with non-transgenic littermates. M-CSF transgenic mice showed increased resistance against sublethal i.v. infections with Listeria monocytogenes as compared with infected non-transgenic mice. To investigate the influence of M-CSF in murine systemic lupus erythematosus (SLE), the M-CSF transgenic mouse line C-1.4 was bred into the genetic background of SLE-prone MRL+/+ mice. The resulting C-1.4/MRL transgenic mice bearing increased endogenous M-CSF levels showed consistently lower levels of anti-ss-DNA autoantibodies as compared with non-transgenic MRL+/+ mice. The life span of the C- 1.4/MRL transgenic mice and the severity of the disease in these mice remained unchanged as compared with their non-transgenic littermates. It is concluded that in addition to M-CSF further factors must be involved in the acceleration of the autoimmune disease in SLE prone MRL/lpr mice.     

Enhancement of the activities of glycosyltransferases involved in the biosynthesis of mucin-type sugar chains in autoimmune MRL lpr/lpr mouse T cells

Lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-+/+ (+/+) mice were compared as to their cell surface lectin-binding sites and glycosyltransferase activities. T cells from enlarged LN of lpr mice expressed a higher amount of binding sites for lectins reactive to mucin-type sugar chains than normal +/+ mouse T cells. Correspondingly, glycosyltransferase activities involved in the biosynthesis of mucin-type sugar chains were higher in lpr mouse T cells than in +/+ T cells. The activities of UDP-N-acetylgalactosamine (GalNAc):polypeptide GalNAc transferase and UDP-galactose (Gal):asialo bovine submaxillary mucin (BSM) Gal transferase were found to be elevated. The activity of UDP-Gal:asialo-agalacto transferrin Gal transferase, which is involved in the biosynthesis of complex type sugar chains, was also increased in lpr mice but to a smaller extent than the mucin-type Gal transferase activities. An abnormality in sialyltransferase activity was also found in lpr T cells.     

A novel pseudovirus encoding Diphtheriatoxin A fragment eliminates autoantibody-producing B cells and inhibits lupus in the BWF1 mouse model

Lupus-associated anti-dsDNA Abs plays an important role in the triggering and development of human systemic lupus erythematosus (SLE). The method of targeting B cells that produce anti-dsDNA Abs has become a promising way to treat autoantibody-mediated SLE in the immunotherapy field. In this study, we reported a novel pseudovirus, which consisted of the peptide DWEYSVWLSN and a plasmid encoding the Diphtheriatoxin A fragment (DTA). Our data demonstrated that vaccination of lupus-prone (BWF1) mice with the pseudovirus could eliminate anti-dsDNA antibody-producing B cells, reduce serum anti-dsDNA antibody levels, impede the development of proteinuria and improve survival. These findings suggested that this novel pseudovirus ablated autoreactive B cells and might represent a promising therapeutic strategy for autoantibody-mediated diseases.     

Methyl- rich diet ameliorates lupus-like disease in MRL/lpr mice

Genetic susceptibility is necessary but not sufficient for systemic lupus erythematosus (SLE) to appear indicating that environmental factors are also key components in the disease onset. Aberrant DNA methylation profile positively correlates with the development of lupus-like disease in MRL/lpr mice. In the present study, we evaluate the effect of long term administration of methyl-rich diet in MRL mice. The results showed that supplemented diet decreased the levels of proteinuria and of anti-dsDNA antibodies and modulated cytokine profiles. Limited kidney failure and prevented development of skin lesions in MRL/lpr mice were another positive effects of the high-dose methyl diet. These data suggest that it is possible to modulate the disease course by altering the amount of particular dietary micronutrients and that nutrition-mediated changes in DNA methylation may have potential clinical relevance.     

The central and multiple roles of B cells in lupus pathogenesis

Summary: A standard view of B cells in systemic autoimmunity is that they promote lupus by producing autoantibodies (autoAb). However, this view is incomplete because recent studies have revealed that autoimmune disease can be dissociated from autoAb deposition. Furthermore, the spontaneous T-cell activation and organ infiltration in systemic lupus erythematosus patients and animal models are difficult to explain entirely via a direct autoAb-mediated mechanism. In this review, we describe work addressing the B-cell functions of autoantigen presentation and autoAb production in lupus pathogenesis. In the J_HD-MRL-Fas^lpr strain (JHD/lpr), a B-cell-deficient version of the lupus-prone MRL-Fas^lpr (MRL/lpr) mouse, spontaneous nephritis and dermatitis is abrogated, demonstrating that B cells have a primary role in disease. B cells play a similar role in Fas-intact, lupus-prone MRL mice. To address the role of autoantigen presentation, we analyzed transgenic mice which have B cells that cannot secrete immunoglobulin (mIgM transgenic mice). The restoration of B cells without antibody caused substantial interstitial nephritis and vasculitis although less marked than the intact MRL/lpr controls. To address the role of autoAb, we infused serum from aged MRL/lpr mice into J_HD/lpr mice. At most, mild to no nephritis was observed in the infused mice. These results indicate that B cells are promoting autoimmunity in mechanisms other than autoAb secretion, and we describe a model depicting these B-cell roles in the context of other inflammatory events in lupus.     

Synthesized OVA323-339MAP octamers mitigate OVA-induced airway inflammation by regulating Foxp3 T regulatory cells

Background

Prolactin is secreted from the pituitary gland and other organs, as well as by cells such as lymphocytes. Prolactin has an immunostimulatory effect and is associated with autoimmune diseases that are characterised by abnormal B cell activation, such as systemic lupus erythematosus (SLE). Our aim was to determine if different splenic B cell subsets express the prolactin receptor and if the presence of prolactin influences these B cell subsets and correlates with development of lupus.

Results

Using real-time PCR and flow cytometry, we found that different subsets of immature (transitional) and mature (follicular, marginal zone) B cells express different levels of the prolactin receptor and are differentially affected by hyperprolactinaemia. We found that transitional B cells express the prolactin receptor at higher levels compared to mature B cells in C57BL/6 mice and the lupus-prone MRL/lpr and MRL mouse strains. Transitional-1 (T1) B cells showed a higher level of prolactin receptor expression in both MRL/lpr and MRL mice compared to C57BL/6 mice. Hyperprolactinaemia was induced using metoclopramide, which resulted in the development of early symptoms of SLE. We found that T1 B cells are the main targets of prolactin and that prolactin augments the absolute number of T1 B cells, which reflects the finding that this B cell subpopulation expresses the highest level of the prolactin receptor.

Conclusions

We found that all B cell subsets express the prolactin receptor but that transitional B cells showed the highest prolactin receptor expression levels. Hyperprolactinaemia in mice susceptible to lupus accelerated the disease and increased the absolute numbers of T1 and T3 B cells but not of mature B cells, suggesting a primary effect of prolactin on the early stages of B cell maturation in the spleen and a role of prolactin in B cell differentiation, contributing to SLE onset.     

Murine lupus strains differentially model unique facets of human lupus serology

Systemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by the production of anti-nuclear autoantibodies that lead to subsequent end organ damage. Previous array-based studies in patients with SLE have shown that high immunoglobulin (Ig)G anti-nuclear autoantibody reactivity was associated with severe renal lupus, whereas IgM polyreactivity was associated with less severe disease. To ascertain how different murine lupus strains recapitulate these different autoantibody profiles seen in patients, serum from New Zealand black (NZB)/NZ white (W) F(1), Murphy Roths large (MRL)/lpr, NZ mixed (M)2410 and BXSB strains were compared using a comprehensive array-based screen. The array results were verified using enzyme-linked immunosorbent assays (ELISAs). Serum from MRL/lpr mice exhibited high levels of IgG anti-nuclear antibodies as well as anti-glomerular antibodies and variable levels of antibodies to myosin, Matrigel and thyroglobulin. Elevated anti-nuclear IgG antibodies were associated with severe nephritis in this strain. In contrast, NZM2410 mice exhibited lower IgG autoantibody levels with less severe nephritis but a significantly higher polyreactive IgM autoantibody profile. ELISA analysis confirmed these results. The NZB/NZW F(1) and BXSB strains exhibited an intermediate serological profile. Hence, just as in patients with SLE, whereas strong IgG reactivity to nuclear antigens is associated with severe renal disease, a polyreactive IgM seroprofile is also less ominous in murine lupus.     

Anti-alpha-actinin antibodies: a new marker of lupus nephritis

The exact role of anti-ds (double stranded) DNA antibodies in the pathogenesis of kidney injury in lupus nephritis remains a focus of continuing investigation. One theory explaining the pathogenicity of anti-dsDNA antibodies in lupus nephritis is direct cross-reactivity with renal antigens. Several years ago, alpha-actinin was identified as a major cross-reactive target for pathogenic anti-dsDNA antibodies in murine SLE. Indeed, binding of a nephritogenic murine anti-dsDNA antibody was stronger to the alpha-actinin derived from a lupus prone mouse mesangial cell line as compared to alpha-actinin in a non-autoimmune mouse mesangial cell line. Furthermore, we recently showed that immunization of non-autoimmune mice with alpha-actinin induces anti-chromatin antibodies, glomerular IgG deposition and proteinuria. In humans, anti-alpha-actinin autoantibodies (Ab) were associated with anti-dsDNA Ab in SLE. In those patients, anti-alpha-actinin rather than anti-dsDNA Ab were significantly associated with glomerulonephritis and disease activity. The anti-alpha-actinin reactivity was associated with high avidity anti-dsDNA Ab. Moreover, the anti-alpha-actinin response was related to the actin-binding site of alpha-actinin. Taken together, these studies indicate that detection of anti-alpha-actinin Ab, in association with anti-dsDNA Ab, may constitute a new marker in lupus nephritis.     

Expression of Heterozygous lpr Gene in MRL Mice

The presence of the homozygous lpr gene (lpr/lpr) in MRL mice has been regarded as mandatory for the development of the early onset of lupus disease, T-cell dysfunction, and polyclonal B-cell activation. Congeneic MRL mice lacking the lpr gene (MRL +/+) display neither the lupus disease nor the immunological abnormalities within the first year of life. In this study we examined the cellular functions of MRL mice heterozygous at the lpr locus. The results indicate that MRL mice heterozygous at the lpr locus display intermediate delayed-type hypersensitivity compared with homozygous lpr/lpr mice on the one hand and congeneic +/+ mice on the other. Furthermore, proliferative responses to concanavalin A, measured by uptake of [3H]thymidine, were significantly lower in MRL mice heterozygous at the lpr locus than in +/+ mice, but significantly higher than in homozygous MRL lpr/lpr mice. Polyclonal B-cell activation, assessed by measurement of frequencies of IgG-secreting spleen cells, a prominent feature in MRL lpr/lpr mice, was significantly lower in lpr/+ mice and totally absent in +/+ mice. Furthermore, spleen cells spontaneously secreting auto-antibodies were found in large numbers in MRL lpr/lpr mice and in considerably lower but still significant numbers in heterozygous MRL +/lpr mice. In contrast, spleen cells from matched MRL +/+ mice did not display any spontaneous autoantibody production. Taken together, these data provide evidence for immunomodulatory properties of the heterozygous lpr gene.