Dendritic cells from malaria-infected mice are fully functional APC

Malaria infection has long been associated with diminished T cell responses in vitro and more recently in experimental studies in vivo. Suppression of T cell-proliferative responses during malaria has been attributed to macrophages in a variety of murine and human systems. More recently, however, attention has been directed at the role of dendritic cells in this phenomenon, with several studies suggesting that maturation of dendritic cells is inhibited in vitro by the presence of malaria-infected E. In the studies reported here, we have examined the function of dendritic cells taken directly from infected mice. We found that they express high levels of costimulatory proteins and class II MHC, can activate naive T cells to produce IL-2 as efficiently as dendritic cells from uninfected mice, and support high levels of IFN-gamma production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-alpha production by naive T cells. These same dendritic cells present parasite Ag to a malaria-specific T cell hybridoma, a finding that demonstrates that dendritic cells participate in the generation of Ag-specific immunity during infection. Our findings challenge the contention that dendritic cell function is inhibited by malaria infection.     

Mesangial cells of lupus-prone mice are sensitive to chemokine production

Infectious antigens may be triggers for the exacerbation of systemic lupus erythematosus. The underlying mechanism causing acceleration and exacerbation of lupus nephritis (LN) is largely unknown. Bacterial lipopolysaccharide (LPS) is capable of inducing an accelerated model of LN in NZB/W mice, featuring diffuse proliferation of glomerular resident cells. We hypothesized that mesangial cells (MCs) from LN subjects are more responsive to LPS than normal subjects. Cultured primary NZB/W and DBA/W (nonautoimmune disease-prone strain with MHC class II molecules identical to those of NZB/W) MCs were used. Monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) expressions either in the baseline (normal culture) condition or in the presence of LPS were evaluated by real-time PCR, ELISA, or western blot analysis. NF-κB was detected by ELISA, electrophoresis mobility-shift assay, and immunofluorescence. First, either in the baseline condition or in the presence of LPS, NZB/W MCs produced significantly higher levels of MCP-1 and OPN than the DBA/W MC controls. Second, NZB/W MCs expressed significantly higher levels of Toll-like receptor 4, myeloid differentiation factor 88, and NF-κB than the DBA/W MC controls, both receiving exactly the same LPS treatment. In conclusion, NZB/W MCs are significantly more sensitive than their normal control DBA/W MCs in producing both MCP-1 and OPN. With LPS treatment, the significantly elevated levels of both chemokines produced by NZB/W MCs are more likely due to a significantly greater activation of the Toll-like receptor 4-myeloid differentiation factor 88-associated NF-κB pathway. The observed abnormal molecular events provide an intrarenal pathogenic pathway involved in an accelerated type of LN, which is potentially infection triggered.     

CD4+ T cells from lupus-prone mice avoid antigen-specific tolerance induction in vivo

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4(+) AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88-104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+(Fas-lpr) and from H-2(k)-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL x CBA)F(1) or (MRL x B10)F(1) recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4(+) T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.     

Dendritic cells are stressed out in tumor

A recently paper published in Cell reports that dendritic cells (DCs) are dysfunctional in the tumor environment. Tumor impairs DC function through induction of endoplasmic reticulum stress response and subsequent disruption of lipid metabolic homeostasis.Tumors develop diverse strategies to escape tumor-specific immunity. Tumor-infiltrating dendritic cells (tDCs) are dysfunctional and/or mediate immune suppression¹. Cubillos-Ruiz et al.² showed in their recent Cell paper that tDCs exhibit an activation of the unfolded protein response (UPR), as indicated by the presence of high levels of spliced XPB1, and this may be attributed to reactive oxygen species (ROS) in tumor, which induces lipid peroxidation, leading to the endoplasmic reticulum (ER) stress in tDCs. Furthermore, they demonstrated that UPR activation in tDCs results in poor DC function, which is accompanied by impaired lipid metabolism and subsequent reduction of T cell anti-tumor immunity. Thus, these observations present a novel mechanism for tDC malfunction.ER stress is evoked by the presence of unfolded or chemically modified proteins. In short, the presence of damaged proteins is sensed by the proteins in the ER membrane. Of these, IRE1α can remove a short nucleotide sequence from mRNA encoding XBP1 protein. This splicing event facilitates XBP1 translation. XBP1 protein binds to consensus sequences in target genes and activates their expression³. Many XBP1 target genes are fatty acid synthesis enzymes. Enhanced production of fatty acids leads to the formation of lipid droplets inside the cytoplasm and extension of the ER compartment due to efficient intracellular membrane formation³. Therefore, this mechanism is a form of adaptation of the cell to the harsh environment, which sustains the production of functional proteins. In such a context, the article by Cuillos-Ruiz et al.² shows intriguing data on the XBP1 pathway in silencing DC function in the tumor environment.First, Cubillos-Ruiz et al.² observed that tDCs express high levels of spliced XBP1, its direct target genes and other markers for the ER stress response. Targeted deletion of XBP1 in CD11c⁺ DCs reveals an association of the XBP1-dependent ER stress response with immunosuppressive properties of tDCs. DC-specific deletion of XBP1 not only inhibits tumor growth and prolongs animal survival, but also reduces tumor peritoneal metastasis, ascites accumulation, and splenomegaly in an ovarian cancer-bearing mouse model.Next, the authors elucidated why the XBP1 pathway in tDCs is highly activated. Unexpectedly, neither typical tumor-associated cytokines nor hypoxia can efficiently stimulate XBP1 activation. Interestingly, tDCs contain high levels of lipid peroxidation byproducts bound to the proteins, which is associated with the production of ROS. Microarray analysis revealed that DC-specific XBP1 deletion downregulates both UPR pathway-dependent genes and lipid metabolism, which results in lower total lipid production, loss of lipid droplets in the cytoplasm and decreased production of triacylglycerides. This phenotype can be recapitulated by chemical inhibition of ROS formation or IRE1α and XBP1 signaling. Furthermore, XBP1-deficient DCs are potent stimulators of OT-1 T cells. Adoptive transfer of T cells isolated from metastatic tumor-bearing mice with DC-specific XBP1 deletion also shows their superior ability to control tumor growth.On the basis of these observations, the authors tested the effects of therapeutic intervention with the usage of nanocomplexes containing XBP1 siRNA. The size of lipid particles or nanocomplexes determines the anatomical location of specific drug delivery⁴. Additionally, they previously optimized the nanocomplexes for selective engulfing by DCs⁵. They found that administration of the nanoparticles containing XBP1 siRNA causes potent T cell activation, which is accompanied by reduced cancer metastatic foci and improved animal survival.The paper by Cubillos-Ruiz et al.² provides an important insight into DC biology in general. Since the identification of Toll-like receptor (TLR) signaling, the main direction in DC biology has been associated with PAMP and DAMP recognition in various degrees (Figure 1). However, the link between cellular metabolism in specific microenvironment and DC biology is poorly explored (Figure 1). The ER stress response has previously been observed in DCs^6,7. It is thought that in response to ER stress, XBP1 is crucial for DC generation, survival, and function^6,7. The conceptual link between XBP1 signaling and DC biology is as follows⁸: (i) stimulation of DCs leads to ER stress; (ii) ER stress activates UPR/XBP1 pathway; (iii) XBP1 activates lipid synthesis genes; (iv) lipids are used for the extension of the ER and Golgi compartment, which are of importance for cytokine production and secretion^9,10. Now, the authors challenged this concept in the context of cancer. The authors demonstrated that XBP1 signaling is strongly associated with poor T cell activation and limitation of XBP1 signaling leads to improved T cell function in the tumor environment (Figure 1). Thus, the paper sheds a new light on DC biology.Open in a separate windowFigure 1Double-faced role of XBP1 signaling pathway in DCs. Left: under immunostimulatory conditions, DCs receive signals from Toll-like receptors. NF-κB signaling induces a XBP1-dependent ER stress response, which enhances lipid metabolism. Formation of new membranes expands ER and Golgi compartments, which enhances cytokine production and secretion⁷. Right: In the tumor microenvironment, DCs are exposed to ROS, which also results in ER stress. However, in this case DC lipid metabolism is impaired and DCs acquire an immunosuppressive phenotype².Of course, as with any interesting work, the article raises more questions than answers. For example, which immune-related properties of DCs are “selectively” affected by the XBP1 pathway? Is there an actual cause-and-effect relationship between aberrant lipid accumulation and the immunosuppressive phenotype of tDCs? The authors observed no obvious change in PD-L1 (B7-H1) expression in DCs, but noticed reduced surface levels of peptide-loaded MHC-I complexes in wild-type tDCs as compared to XBP-deficient tDCs. However, it remains unknown whether and how DC cross-presentation is involved in tDC function regulated by ER stress, XBP1 activation, and lipid metabolism. Human ovarian cancer-associated DCs express high levels of B7-H1 and limited IL-12¹¹. It would be interesting to thoroughly examine the cytokine profile, and the B7 and TNF family members, along with lipid pathway manipulation in XBP1^−/− DCs. It is well known that ER morphology and function is substantial for the synthesis of membrane proteins and cytokines, which would be affected by the XBP1 pathway. Paradoxically, a restricted ER stress response can help immune reaction, which requires the expansion of the ER compartment. Another question is how and which lipid synthesis and metabolism pathway is targeted by XBP1 in DCs (or/and tumor cells in the same environment). Obviously, future studies are warranted to address these important questions.In conclusion, the paper by Cubillos-Ruiz et al.² opens an interesting chapter for scientifically and therapeutically exploring DC biology in a specific metabolic environment. Given that silencing XBP1 signaling in DCs enhances tumor immunity, and XBP1 is an intrinsic pro-tumor factor, it is reasonable to assume that targeting this pathway may be beneficial in patients with cancer and could kill two birds with one stone.     

Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion

Abstract Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40°C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.     

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.     

Failure of CD25+ T cells from lupus-prone mice to suppress lupus glomerulonephritis and sialoadenitis

The development of organ-specific autoimmune diseases in mice thymectomized on day 3 of life (d3tx mice) can be prevented by transferring CD4(+)CD25(+) T cells from syngeneic, normal adult mice. Using a d3tx model, we asked whether CD4(+)CD25(+) T cell deficiency contributes to glomerulonephritis (GN) in lupus-prone mice. New Zealand Mixed 2328 (NZM2328) mice spontaneously develop autoantibodies to dsDNA and female-dominant, fatal GN. After d3tx, both male and female NZM2328 mice developed 1) accelerated dsDNA autoantibody response, 2) early onset and severe proliferative GN with massive mesangial immune complexes, and 3) autoimmune disease of the thyroid, lacrimal gland, and salivary gland. The d3tx male mice also developed autoimmune prostatitis. The transfer of CD25(+) cells from 6-wk-old asymptomatic NZM2328 donors effectively suppressed dsDNA autoantibody and the development of autoimmune diseases, with the exception of proliferative lupus GN and sialoadenitis. This finding indicates that NZM2328 lupus mice have a selective deficiency in T cells that regulates the development of lupus GN and sialoadenitis. After d3tx, the proliferative GN of female mice progressed to fatal GN, but largely regressed in the male, thereby revealing a checkpoint in lupus GN progression that depends on gender.     

Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic (mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that N(omega)-nitro- L-arginine methyl ester (L-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca(2+)-free solution. In colon from mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca(2+) through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.     

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

Biosynthesis, transport and secretion of immunoglobulin in plasma cells

Conclusions Evidence has been accumulated that immunoglobulin is transported from the site of synthesis through the rough membranes into the smooth membranes and out of the cell. Parallel to this migration stepwise addition of different sugar residues to immunoglobulins takes place at different subcellular sites. The immediate secretion of [³H]sugar-labelled immunoglobulins (Fig. 3), in contrast to the lag in the secretion of the newly synthesized [³H]leucine-labelled immunoglobulin (Fig. 2) suggests that the protein accepts carbohydrate long after the synthesis, and some of it, shortly before leaving the cell. The form of immunoglobulin complete in the carbohydrate component (with two fucose residues) found secreted from plasma cells cannot be found inside. These results, therefore, support the hypothesis that the attachment of carbohydrate may be requisite for the transport of the protein to the outside of the cell. It will be discussed in a forthcoming paper (in preparation) what experimental evidence can be marshalled against the role of carbohydrate attachment as the sole requisite for the secretion of immunoglobulin from plasma cells. The results obtained by us with the immunoglobulin-producing cells show striking similarities to those of the thyroglobulin-producing cellular system (Herscovics, 1969; Whuret al., 1969).Finally, it should be pointed out that only a very crude separation of subcellular components can be anticipated to occur on sucrose density gradients of the sort used in our studies. It is, therefore, all the more surprising that such a clear difference has been observed in the two separated main subcellular fractions, the smooth and the rough membranes. While the techniques for the preparation and separation of subcellular fractions of secretory cells clearly need to be improved, it may well prove useful to use the transport of immunoglobulin and the varying composition of its carbohydrate component as a marker in the identification of subcellular fractions.     

Ig VH gene family repertoire of plasma cells derived from lupus-prone MRL/lpr and MRL/++ mice

VH gene family usage was determined in both spontaneous, in vivo activated plasma cells and LPS-induced plasma cells from individual MRL/lpr mice by using in situ hybridization. It was found that VH gene family expression in spontaneous plasma cells varied from mouse to mouse. Some mice expressed VH families in an apparently random manner similar to that obtained with polyclonal activation. Other mice showed an exaggerated expression of particular VH gene families. VH J558 was overrepresented most frequently, but overrepresentation of VH 7183, Q52, and 36-60 was also observed. Importantly, LPS-induced VH gene family expression in these same mice displaying biased VH family usage in spontaneous plasma cells, appeared normal with no evidence for similar biases in the LPS-induced repertoire. Anti-DNA antibody concentrations and the degree of glomerulonephritis were determined for each mouse to measure the severity of disease. The level of expression of the J558 family was positively correlated with disease severity. The results suggest that the initial autoantibody response is highly diverse but becomes more restricted as the disease progresses.     

Dendritic cells and macrophages are productively infected by poliovirus

Expression of the poliovirus receptor (PVR) on cells is a major host determinant of infection by poliovirus. Previously, the only immune cell type known to express PVR was the blood-derived monocyte, which is susceptible to infection at very low frequency. We demonstrate that professional antigen-presenting cells-macrophages and dendritic cells, generated upon differentiation of monocytes-retain expression of PVR and are highly susceptible to infection by type 1 Mahoney strain of poliovirus. Maximal cell-associated titers of virus are obtained within 6 to 8 h postinfection, and cell death and lysis occurs within 24 h postinfection. Similar kinetics are observed in cells infected with the Sabin 1 vaccine strain. Although protein synthesis and receptor-mediated endocytosis are inhibited upon poliovirus infection of these critical antigen-presenting cells, we demonstrate for the first time that functional presentation of antigen occurs in these infected cells via the HLA class II pathway.     

Chromatin-associated DNA endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal DNA

The influence of nucleosome structure on the activity of 2 chromatin-associated DNA endonucleases, pIs 4.6 and 7.6, from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined on DNA containing either psoralen monoadducts or cross-links. As substrate a reconstituted nucleosomal system was utilized consisting of a plasmid DNA and either core (H2A, H2B, H3, H4), or total (core plus H1) histones from normal or XPA cells. Both non-nucleosomal and nucleosomal DNA were treated with 8-methoxypsoralen (8-MOP) plus long-wavelength ultraviolet radiation (UVA), which produces monoadducts and DNA interstrand cross-links, and angelicin plus UVA, which produces monoadducts. Both normal endonucleases were over 2-fold more active on both types of psoralen-plus-UVA-damaged core nucleosomal DNA than on damaged non-nucleosomal DNA. Addition of histone H1 to the system reduced but did not abolish this increase. By contrast, neither XPA endonuclease showed any increase on psoralen-treated nucleosomal DNA, with or without histone H1. Mixing the normal with the XPA endonucleases led to complementation of the XPA defect. These results indicate that interaction of these endonucleases with chromatin is of critical importance and that it is at this level that a defect exists in XPA endonucleases.     

Permeant anions are not required for norepinephrine secretion from pheochromocytoma cells

The ‘chemiosmotic’ model for secretion proposed by Pollard and his colleagues (Int. Rev. Cytol. 58, 159–197, 1979) was tested with pheochromocytoma cells. Contrary to the prediction of this model, norepinephrine secretion did not require the presence of a permeant anion in the medium. Secretion was not blocked by replacing much of the Cl^? of the medium with isethionate or by replacing all of the Cl^? salts of the medium with isotonic sucrose. Biochemical evidence is presented to indicate that the cells secreted by the normal exocytotic mechanism in the sucrose medium. Making the normal bathing medium hypertonic with 300 mM sucrose increased the basal level of norepinephrine release, but alos suppressed secretion in response to a strong secretagogue (1 mM Ba²⁺). The data indicate that the Pollard model does not apply to pheochromocytoma cells, but suggest the possible involvement of osmotic pressure in exocytosis.     

Naive CD4+ T cells from lupus-prone Fas-intact MRL mice display TCR-mediated hyperproliferation due to intrinsic threshold defects in activation

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.