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A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acid–sensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members. Trafficking to endolysosomes and function of SLC15A4 were dependent on the Adaptor protein 3 (AP-3) complex. Importantly, SLC15A4 was required for trafficking and colocalization of nucleic acid–sensing TLRs and their ligands to endolysosomes and the formation of the LAMP2+VAMP3+ hybrid compartment in which IFN-I production is initiated. Collectively, these findings define mechanistic processes by which SLC15A4 controls endosomal TLR function and suggest that pharmacologic intervention to curtail the function of this transporter may be a means to treat lupus and other endosomal TLR-dependent diseases.

Considerable evidence indicates that autoimmune disease expression depends on both genetic and environmental factors. Among potential environmental triggers, occupational airway exposure to crystalline silica and virus infections have been linked to lupus and other autoimmune diseases in both humans and mouse models. Here, we hypothesized that combined silica and virus exposures synergize and induce autoimmune manifestations more effectively than single exposures to either of these factors, particularly in individuals with low genetic predisposition. Accordingly, infection with the model murine pathogen lymphocytic choriomenigitis virus (LCMV) in early life, followed by airway exposure to crystalline silica in adult life, induced lupus-like autoantibodies to several nuclear self-antigens including chromatin, RNP and Sm, concurrent with kidney lesions, in non-autoimmune C57BL/6 (B6) mice. In contrast, given individually, LCMV or silica were largely ineffectual in this strain. These results support a multihit model of autoimmunity, where exposure to different environmental factors acting on distinct immunostimulatory pathways complements limited genetic predisposition and increases the risk of autoimmunity above a critical threshold.

IL-7 is known to be vital for T cell homeostasis but has previously been presumed to be dispensable for TCR-induced activation. Here, we show that IL-7 is critical for the initial activation of CD4(+) T cells in that it provides some of the necessary early signaling components, such as activated STAT5 and Akt. Accordingly, short-term in vivo IL-7Rα blockade inhibited the activation and expansion of autoantigen-specific CD4(+) T cells and, when used to treat experimental autoimmune encephalomyelitis (EAE), prevented and ameliorated disease. Our studies demonstrate that IL-7 signaling is a prerequisite for optimal CD4(+) T cell activation and that IL-7R antagonism may be effective in treating CD4(+) T cell-mediated neuroinflammation and other autoimmune inflammatory conditions.

Hemorrhagic fever (HF) viruses, such as Lassa, Ebola, and dengue viruses, represent major human health risks due to their highly contagious nature, the severity of the clinical manifestations induced, the lack of vaccines, and the very limited therapeutic options currently available. Appropriate animal models are obviously critical to study disease pathogenesis and develop efficient therapies. We recently reported that the clone 13 (Cl13) variant of the lymphocytic choriomeningitis virus (LCMV-Cl13), a prototype arenavirus closely related to Lassa virus, causes in some mouse strains endothelial damage, vascular leakage, platelet loss, and death, mimicking pathological aspects typically observed in Lassa and other HF syndromes. This model has the advantage that the mice used are fully immunocompetent, allowing studies on the contribution of the immune response to disease progression. Moreover, LCMV is very well characterized and exhibits limited pathogenicity in humans, allowing handling in convenient BSL-2 facilities. In this chapter we outline protocols for the induction and analysis of arenavirus-mediated pathogenesis in the NZB/LCMV model, including mouse infection, virus titer determination, platelet counting, phenotypic analysis of virus-specific T cells, and assessment of vascular permeability.

Hemorrhagic fever (HF) viruses, such as Lassa, Ebola, and dengue viruses, represent major human health risks due to their highly contagious nature, the severity of the clinical manifestations induced, the lack of vaccines, and the very limited therapeutic options currently available. Appropriate animal models are obviously critical to study disease pathogenesis and develop efficient therapies. We recently reported that the clone 13 (Cl13) variant of the lymphocytic choriomeningitis virus (LCMV-Cl13), a prototype arenavirus closely related to Lassa virus, causes in some mouse strains endothelial damage, vascular leakage, platelet loss, and death, mimicking pathological aspects typically observed in Lassa and other HF syndromes. This model has the advantage that the mice used are fully immunocompetent, allowing studies on the contribution of the immune response to disease progression. Moreover, LCMV is very well characterized and exhibits limited pathogenicity in humans, allowing handling in convenient BSL-2 facilities. In this chapter we outline protocols for the induction and analysis of arenavirus-mediated pathogenesis in the NZB/LCMV model, including mouse infection, virus titer determination, platelet counting, phenotypic analysis of virus-specific T cells, and assessment of vascular permeability.

Type I IFN and nucleic acid–sensing TLRs are both strongly implicated in the pathogenesis of lupus, with most patients expressing IFN-induced genes in peripheral blood cells and with TLRs promoting type I IFNs and autoreactive B cells. About a third of systemic lupus erythematosus patients, however, lack the IFN signature, suggesting the possibility of type I IFN–independent mechanisms. In this study, we examined the role of type I IFN and TLR trafficking and signaling in xenobiotic systemic mercury-induced autoimmunity (HgIA). Strikingly, autoantibody production in HgIA was not dependent on the type I IFN receptor even in NZB mice that require type I IFN signaling for spontaneous disease, but was dependent on the endosomal TLR transporter UNC93B1 and the endosomal proton transporter, solute carrier family 15, member 4. HgIA also required the adaptor protein-3 complex, which transports TLRs from the early endosome to the late endolysosomal compartments. Examination of TLR signaling pathways implicated the canonical NF-κB pathway and the proinflammatory cytokine IL-6 in autoantibody production, but not IFN regulatory factor 7. These findings identify HgIA as a novel type I IFN–independent model of systemic autoimmunity and implicate TLR-mediated NF-κB proinflammatory signaling from the late endocytic pathway compartments in autoantibody generation.

The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus--a prototype of Old World arenaviruses closely related to Lassa fever virus--elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell-mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6-8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.

In vitro evidence suggests that plasmacytoid dendritic cells (pDCs) are intimately involved in the pathogenesis of lupus. However, it remains to be determined whether these cells are required in vivo for disease development, and whether their contribution is restricted to hyperproduction of type I IFNs. To address these issues, we created lupus-predisposed mice lacking the IFN regulatory factor 8 (IRF8) or carrying a mutation that impairs the peptide/histidine transporter solute carrier family 15, member 4 (SLC15A4). IRF8-deficient NZB mice, lacking pDCs, showed almost complete absence of anti-nuclear, anti-chromatin, and anti-erythrocyte autoantibodies, along with reduced kidney disease. These effects were observed despite normal B-cell responses to Toll-like receptor (TLR) 7 and TLR9 stimuli and intact humoral responses to conventional T-dependent and -independent antigens. Moreover, Slc15a4 mutant C57BL/6- Fas lpr mice, in which pDCs are present but unable to produce type I IFNs in response to endosomal TLR ligands, also showed an absence of autoantibodies, reduced lymphadenopathy and splenomegaly, and extended survival. Taken together, our results demonstrate that pDCs and the production of type I IFNs by these cells are critical contributors to the pathogenesis of lupus-like autoimmunity in these models. Thus, IRF8 and SLC15A4 may provide important targets for therapeutic intervention in human lupus.

The demonstration in humans and mice that nucleic acid-sensing TLRs and type I IFNs are essential disease mediators is a milestone in delineating the mechanisms of lupus pathogenesis. In this study, we show that Ifnb gene deletion does not modify disease progression in NZB mice, thereby strongly implicating IFN-α subtypes as the principal pathogenic effectors. We further document that long-term treatment of male BXSB mice with an anti–IFN-α/β receptor Ab of mouse origin reduced serologic, cellular, and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment in BXSB mice was clearly evident when applied early in the disease process, but only partial reductions in some disease characteristics were observed when treatment was initiated at later stages. A transient therapeutic effect was also noted in the MRL-Faslpr model, although overall mortality was unaffected. The combined findings suggest that IFN-α/β receptor blockade, particularly when started at early disease stages, may be a useful treatment approach for human systemic lupus erythematosus and other autoimmune syndromes.

The activation of immune cells is mediated by a network of signaling proteins that can undergo post-translational modifications critical for their activity. Methylation of nucleic acids or proteins can have major effects on gene expression as well as protein repertoire diversity and function. Emerging data indicate that indeed many immunologic functions, particularly those of T cells, including thymic education, differentiation and effector function are highly dependent on methylation events. The critical role of methylation in immunocyte biology is further documented by evidence that autoimmune phenomena may be curtailed by methylation inhibitors. Additionally, epigenetic alterations imprinted by methylation can also exert effects on normal and abnormal immune responses. Further work in defining methylation effects in the immune system is likely to lead to a more detailed understanding of the immune system and may point to the development of novel therapeutic approaches.

Evidence strongly suggests that excessive or protracted signaling, or both, by cell-surface or intracellular innate immune receptors is central to the pathogenesis of most autoimmune and autoinflammatory rheumatic diseases. The initiation of aberrant innate and adaptive immune responses in autoimmune diseases can be triggered by microbes and, at times, by endogenous molecules--particularly nucleic acids and related immune complexes--under sterile conditions. By contrast, most autoinflammatory syndromes are generally dependent on germline or de novo gene mutations that cause or facilitate inflammasome assembly. The consequent production of proinflammatory cytokines, principally interferon-alpha/beta and tumor necrosis factor in autoimmune diseases, and interleukin-1beta in autoinflammatory diseases, leads to the creation of autoamplification feedback loops and chronicity of these syndromes. These findings have resulted in a critical reappraisal of pathogenetic mechanisms, and provide a basis for the development of novel diagnostic and therapeutic modalities for these diseases.

The discovery of molecular sensors that enable eukaryotes to recognize microbial pathogens and their products has been a key advance in our understanding of innate immunity. A tripartite sensing apparatus has developed to detect danger signals from infectious agents and damaged tissues, resulting in an immediate but short-lived defense response. This apparatus includes Toll-like receptors, retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, and nucleotide-binding and oligomerization domain-like receptors; adaptors, kinases and other signaling molecules are required to elicit effective responses. Although this sensing is beneficial to the host, excessive activation and/or engagement by self molecules might induce autoimmune and other inflammatory disorders.

We have devised a method of using intracellular combinatorial libraries to select antibodies that control cell fates. Many agonist antibodies have been selected with this method, and the process appears to be limited only by the availability of a phenotypic selection system. We demonstrate the utility of this approach to discover agonist antibodies that engage an unanticipated target and regulate macrophage polarization by selective induction of anti-inflammatory M2 macrophages. This antibody was used therapeutically to block autoimmunity in a classic mouse model of spontaneous systemic lupus erythematosus.—Han, K. H., Gonzalez-Quintial, R., Peng, Y., Baccala R., Theofilopoulos, A. N., Lerner, R. A. An agonist antibody that blocks autoimmunity by inducing anti-inflammatory macrophages.

Among T cell subsets, gamma delta T cells uniquely display an Ag receptor-based tissue distribution, but what defines their preferential homing and homeostasis is unknown. To address this question, we studied the resources that control gamma delta T cell homeostasis in secondary lymphoid organs. We found that gamma delta and alpha beta T cells are controlled by partially overlapping resources, because acute homeostatic proliferation of gamma delta T cells was inhibited by an intact alpha beta T cell compartment, and both populations were dependent on IL-7 and IL-15. Significantly, to undergo acute homeostatic proliferation, gamma delta T cells also required their own depletion. Thus, gamma delta T cell homeostasis is maintained by trophic cytokines commonly used by other types of lymphoid cells, as well as by additional, as yet unidentified, gamma delta-specific factors.

Efforts to develop effective anti-tumor immunotherapies are hampered by the difficulty of overcoming tolerance against tumor antigens, which in most instances are normal gene products that are over-expressed, preferentially expressed or re-expressed in cancer cells. Considering that lymphopenia-induced homeostatic T cell proliferation is mediated by self-peptide/MHC recognition and that the expanded cells acquire some effector functions, we hypothesized that this process could be used to break tolerance against tumor antigens. Studies by us and others in several mouse models demonstrated that availability of tumor antigens during homeostatic T cell proliferation indeed leads to effective anti-tumor autoimmunity with specificity and memory. This effect appears to be mediated by reduction in the activation threshold of low-affinity tumor-specific T cells, leading to their preferential engagement and expansion. In its simplicity, this approach is likely to have application in humans, since it relies on conventional lymphopenia-inducing cancer therapies, infusion of autologous lymphocytes and, optimally, tumor-specific vaccination.

Objective To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery. Materials and methods Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens. Results Human tonsil pieces survived a long period of time in SCID mice, while polyclonal human T- and B-lymphocytes persisted in close vicinity within the implantation area; however, little or no graft-versus-host disease was detectable. Not surprisingly, local development of lymphoproliferative disease was often observed in animals receiving lymphoid implants from donors previously infected by the Epstein-Barr virus. One month after surgery, higher serum levels of human IgG were found in SCID mice transplanted with tonsil pieces (2 × 10 7 cells/animal) than in animals injected with 5 × 10 7 tonsil cells in suspension (1.9 vs. 0.3 mg/mL, p 0.002). Importantly, the production of human IgG in hu-ton-SCID mice remained polyclonal for at least 6 months and was linked to the presence of cells within the implants. Immunization of hu-ton-SCID mice with hepatitis B core, a de novo antigen, did not produce a significant IgG immune response; however immunization with tetanus toxoid (TT), a thymus-dependent recall antigen, yielded high ( > 700-fold increase in anti-TT IgG levels) and long-lasting ( > 6 months) secondary immune responses. Conclusion The hu-ton-SCID mouse xenochimeric model described in this report may improve our understanding of human lymphoid cell interactions, secondary immune responses, and lymphomagenesis.

Following exposure to some types of antigen (superantigens), responsive T cells expand and then decline in numbers, a phenomenon that has been called 'peripheral deletion'. This process may play a role in limiting autoimmune reactions and in the maintenance of immune homeostasis. Here we describe experiments on peripheral deletion in mice carrying the lpr/lpr defect, which has been shown to be due to defective production of the CD95/Fas molecule. Young lpr/lpr mice with no apparent immunologic abnormalities display a defect in bacterial superantigen-induced peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion. Apoptotic death of the expanded T cell population associated with such peripheral deletion in normal animals is dramatically reduced in the mutant mice. Further, the levels of Fas on responding cells in normal mice increases and decreases together with increases and decreases in cell numbers, suggesting that cells with the highest levels of Fas are preferentially deleted. These observations are consistent with the known ability of CD95 to transduce a signal leading to apoptosis, and they implicate this signal transduction pathway in peripheral deletion. In contrast, bacterial superantigen-induced deletion of thymocytes appears to be fully functional in these mice, and thus Fas/APO-1 does not appear to be required for this process. Further, antibody ligation of the TCR on activated T cells from normal or young lpr/lpr mice can induce apoptosis and therefore under some circumstances this phenomenon is not dependent upon CD95/Fas. Thus, to avoid autoreactivity and ensure immune homeostasis, several different apoptotic mechanisms exist in peripheral T lymphocytes, only some of which involve Fas.(ABSTRACT TRUNCATED AT 250 WORDS)

We continue our efforts to define the pathogenesis of systemic lupus-like autoimmunity in predisposed mouse models by focusing on the role of endosomal nucleic acid-sensing TLRs. We reported that NZB mice deficient for the common receptor for type I IFNs showed significant reductions in all disease parameters. To differentiate whether the pathogenic effects were mediated by the multiple IFNα subtypes and/or the single IFNβ, we created congenic NZB mice lacking Ifnb and found that disease severity was unaltered, strongly implicating IFNα subtypes as the principal effectors. We then documented that long-term treatment of male BXSB mice with an anti-IFNAR antibody of mouse origin reduced serologic, cellular and histologic disease manifestations and extended survival, suggesting that disease acceleration by the Tlr7 gene duplication in this model is mediated by type I IFN signaling. The efficacy of this treatment was greater when applied relatively early in the disease process, but reductions in some disease characteristics, especially kidney pathology, were evident even when treatment was initiated at later stages, and a transient therapeutic effect was also noted in the MRL-Faslpr model. The combined findings suggest that antibody-mediated IFNAR blockade may be a useful treatment approach in human SLE and probably other autoimmune syndromes in which these cytokines appear to play a pathogenic role. We have also hypothesized that engagement of nucleic acid-sensing TLRs may be responsible for spreading the aberrant response beyond ANAs to encompass the broader spectrum of disease-associated autoantibody specificities. Using MRL-Faslpr mice congenic for the 3d mutation of the Unc93b1 gene, in which signaling by all endosomal TLRs (TLR3, TLR7, TLR9) is extinguished, we indeed found reductions not only in autoantibodies against nucleic acids and associated proteins (anti-chromatin, anti-RNP, anti-Sm), but also in a broad panel of autoantibody specificities, particularly against antigens known to contain or bind to nucleic acids, such as cardiolipin, β2-glycoprotein 1 and myeloperoxidase. Surprisingly, even anti-erythrocyte autoantibodies and hemolytic anemia were significantly reduced in NZB mice congenic for the 3d mutation compared with wild-type controls. Thus, almost the entire gamut of autoantibodies in lupus can be traced to the initial engagement of nucleic acid-sensing TLRs. To examine whether engagement of B-cell intrinsic nucleic acid-sensing TLRs is required for autoantibody production in vivo, we generated mixed bone marrow chimeras of B6-Faslpr mice that were either 3d/IgHb or WT/IgHa and measured allotype-specific IgM RF and IgG2a anti-chromatin responses. Strikingly, both autoantibodies were derived almost exclusively from the wild-type donor B cells. This finding underscores the essential significance of B-cell nucleic acid sensors in loss of tolerance and production of autoantibodies in lupus and provides a specific target for intervention. Evidence suggests that plasmacyotoid dendritic cells (pDC), the major producers of type I IFNs, are likely to be involved in the pathogenesis of lupus. One ENU phenovariant identified by Beutler and colleagues, named feeble, showed abrogation of both TLR7-induces and TLR9-induced type I IFN and proinflammatory cytokine production by pDCs, while leaving intact pDC development and TLR responses by other cells. The feeble phenotype was mapped to a mutation in Slc15a4, which encodes the peptide/histidine transporter 1 (PHT1), one of the four members of the solute carrier 15 (Slc15) family of proteins. In preliminary collaborative studies with Beutler and colleagues, lupus-prone B6Faslpr mice congenic for the feeble mutation showed significant reduction in hypergammaglobulinemia, lymphadenopathy and mortality. These findings provide direct evidence for the role of pDC in the pathogenesis of systemic autoimmunity and suggests that pharmacologic agents that interfere with Sle15a4 function may be useful in treating lupus and other diseases in which these cells appear to be involved.

Lupus is characterized by disturbances in lymphocyte homeostasis, as demonstrated by the marked accumulation of activated/memory T cells. Here, we provide evidence that proliferation of the CD8+ precursors for the accumulating CD4⁻CD8⁻ T cells in MRL-Fas(lpr) lupus-predisposed mice is, in part, driven by commensal antigens. The ensuing lymphadenopathy is associated with increased production of IL-7 due to expansion of fibroblastic reticular cells, the primary source of this cytokine. The excess IL-7 is not, however, consumed by CD4⁻CD8⁻ T cells due to permanent down-regulation of IL-7Rα (CD127), but instead supports proliferation of autoreactive T cells and progression of autoimmunity. Accordingly, IL-7R blockade reduced T cell activation and autoimmune manifestations even when applied at advanced disease stage. These findings indicate that an imbalance favoring production over consumption of IL-7 may contribute to systemic autoimmunity, and correction of this imbalance may be a novel therapeutic approach in lymphoproliferative and autoimmune syndromes.

During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered γ2a-reactive superantigen (γ2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Faslpr lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.

Acute lymphopenia-induced homeostatic proliferation (HP) of T cells promotes antitumor immunity, but the mechanism is unclear. We hypothesized that this is due to a lack of inhibitory signals that allows activation of T cells with low affinity for self-antigens. Tumors resist immunity in part by expressing inhibitory molecules such as PD-1 ligand 1 (PD-L1), B7-H4, and TGF-β. In irradiated mice undergoing HP, we found that T cells displayed a severe deficit in the activation-induced expression of inhibitory molecules PD-1 and CTLA-4, and TGF-β1–induced expression of Foxp3. HP T cells were also less suppressed by B7-H4/Ig and, unlike control T cells, failed to produce IL-10 in response to this molecule. This deficiency in regulation was reversed as normal T-cell numbers were restored. We conclude that T cells are weakly regulated by inhibitory molecules during the acute phase of HP, which could explain their increased effectiveness in cancer immunotherapy.

International audience; Previous studies have shown that the antibodies of the preimmune repertoire are able to bind to various auto- and xenoantigens including chemical haptens. Sequence analysis of two such murine monoclonal IgM natural autoantibodies showed that they are encoded by unmutated germ-line variable regions of the light and heavy chain (V alpha and VH) genes which were also found in various murine immune responses, like phenyl-oxazolone, dinitrophenyl, arsonate, phosphorylcholine and influenza virus hemagglutinin. These data raised the question as to whether induced antibodies possessing germ-line sequence are also able to react with autoantigens. To study this problem, anti-poly(Glu60Ala30Tyr10) (GAT) and anti-alprenolol (Alp) monoclonal antibodies, carrying similar VH and V alpha genes and the same IgG1 isotype, were examined for their capacity to react with several self and non-self antigens. The results showed that: (a) the anti-GAT antibodies tested reacted with different autoantigens, such as murine tubulin, actin and myosin as well as trinitrophenyl (TNP) and bovine serum albumin. Similarly, one of the anti-Alp showed weak reactivities for myosin, DNA, actin and TNP; (b) in contrast two other anti-Alp antibodies did not react with any of the tested antigens. Since the major differences between the oligoreactive anti-GAT and the monoreactive anti-Alp antibodies are in the complementarity determining regions (CDR) our results suggest that the observed cross-reactions are mediated by hypervariable loops. Sequence comparison of these antibodies indicate a possible correlation between cross-reactivity and the presence of aromatic and charged amino acids in the CDR.

Identification of expanded clones engaged in immune and autoimmune responses is still imperfect, since they are often diluted by irrelevant cells expressing diverse specificities. To efficiently characterize T cell receptors expressed by clonally expanded lymphocytes in rheumatoid arthritis (RA) and other inflammatory conditions, we developed an assay system, termed sequence enrichment nuclease assay (SENA). Key elements of SENA are the efficiency of heat-denatured DNA strand reassociation, which increases exponentially with concentration, and the elimination of unhybridized sequences by single-strand-specific DNase. T cell clonal expansions were identified primarily in synovial fluids, but also in peripheral blood of RA patients. Synovial fluids had more prominent expansions in the CD8 than the CD4 subset, whereas clonal expansions in the CD4 subset predominated among peripheral blood lymphocytes. Dominant clones exhibited diverse sequences with no clear conservation of junctional motifs, although the same amino acid sequence was identified in two patients. In most instances, dominant clones in the blood were discordant to those in the corresponding synovial fluid, suggesting local stimulation or preferential sequestration of T cells displaying particular specifities.

To define age-associated alterations in the immune system at the molecular level, we have analyzed TCR V beta gene expression patterns at the fetal, neonatal, adult, and advanced ages of mice. In contrast to V gamma and VH genes, V beta genes rearranged without any preference related to their chromosomal organization. Endogenous superantigen-mediated clonal deletions were registered for the first time at the neonatal stage, presumably reflecting the late developmental appearance of these molecules. Such deletions, once established, were maintained throughout life with little, if any, leakage in this process. Furthermore, bone marrow transplantation and other studies indicated that an involuted thymus maintained its capacity to perform both its functions, i.e. positive and negative selection. Although overall V beta repertoires showed remarkable stability with advanced age, modifications in expression levels for some V beta, particularly those associated with the CD8 subset and presumably reflecting antigenic stimulation, were recorded. Mice with lupus and early-life thymic involution were fully capable of deleting endogenous superantigen-reactive V beta clones, and even lupus mice with a genetic defect in the apoptosis-promoting Fas gene were normal in this regard. The results indicate that, aside from some anticipated clonal expansions induced by antigenic stimulation, age-associated alterations in immune functions are not caused by any profound changes in the overall TCR repertoire.