The role of NK cells in the development of autoantibodies

The systemic lupus erythematosus (Sle1) interval from the NZM2410 mouse strain has been shown to be responsible for high levels of autoantibody production against antinuclear antibodies (ANA) when transferred into C57BL/6 mice. B cells derived from the B6.Sle1 strain are required for the production but help from both T-dependent and independent sources have been documented. Using radiation chimeras constructed in a strain of mice that is chronically depleted of Natural killer (NK) cells, but not NKT cells, we have examined the role of NK cells in the development of ANA in this context. Our results show that in the presence of intact T cell help depletion of NK cells does not affect ANA production. However, when T cell help is compromised, the prevalence of animals producing ANA is significantly decreased suggesting that NK cells can provide help for the T-independent production of ANA. Further experiments provide a possible mechanism for the NK-cell dependence.     

Antigen-specific responses and ANA production in B6.Sle1b mice: a role for SAP

B6.Sle1b mice, which contain the Sle1b gene interval derived from lupus prone NZM2410 mice on a C57BL/6 background, present with gender-biased, highly penetrant anti-nuclear antibody (ANA) production. To obtain some insight into the possible induction mechanism of autoantibodies in these mice we compared antigen-specific T dependent (TD) and T independent (TI-II) responses between B6.Sle1b and B6 mice before the development of high ANA titers. Our results show that B6.Sle1b mice mount enhanced responses to a TI-II antigen. Additionally, the memory T cell response generated by a TD antigen also increased. This enhancement correlates with the greater ability of B cells from B6.Sle1b mice to present antigen to T cells. The SLAM Associated Protein (SAP) is critical for signaling of many of the molecules encoded by the SLAM/CD2 gene cluster, candidates for mediating the Sle1b phenotype; therefore, we also investigated the effect of sap deletion in these strains on the TD and TI-II responses as well as on ANA production. The results of these studies of responses to non-self-antigens provide further insight into the mechanism by which responses to self-antigens might be initiated in the context of specific genetic alterations.     

Antigen-specific responses and ANA production in B6.Sle1b mice: A role for SAP

B6.Sle1b mice, which contain the Sle1b gene interval derived from lupus prone NZM2410 mice on a C57BL/6 background, present with gender-biased, highly penetrant anti-nuclear antibody (ANA) production. To obtain some insight into the possible induction mechanism of autoantibodies in these mice we compared antigen-specific T dependent (TD) and T independent (TI-II) responses between B6.Sle1b and B6 mice before the development of high ANA titers. Our results show that B6.Sle1b mice mount enhanced responses to a TI-II antigen. Additionally, the memory T cell response generated by a TD antigen also increased. This enhancement correlates with the greater ability of B cells from B6.Sle1b mice to present antigen to T cells. The SLAM Associated Protein (SAP) is critical for signaling of many of the molecules encoded by the SLAM/CD2 gene cluster, candidates for mediating the Sle1b phenotype; therefore, we also investigated the effect of sap deletion in these strains on the TD and TI-II responses as well as on ANA production. The results of these studies of responses to non-self-antigens provide further insight into the mechanism by which responses to self-antigens might be initiated in the context of specific genetic alterations.     

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4+ T‐cell compartment

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8–12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4⁺ T‐cell dependent, since old (40–50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age‐related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4⁺ T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T‐cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T‐cell homeostasis may drive the onset of some autoimmune features.     

Natural killer T-cell populations in C57BL/6 and NK1.1 congenic BALB.NK mice-a novel thymic subset defined in BALB.NK mice

Natural killer (NK) T lymphocytes are a subpopulation of T lymphocytes regarded as early regulators of immune responses. The majority of NKT cells are restricted by the CD1d molecule. NKT cells have mostly been studied in one single mouse strain, C57BL/6 (B6), because of the absence of NK1.1 in other common mouse strains, and the lack of other reliable surface markers for CD1d-restricted cells. To investigate NKT cell subsets in a mouse strain of a genetic background different from B6, we have back-crossed the NKT cell marker NK1.1 from the B6 mouse to the BALB/c mouse strain. We show that NKT cells in the congenic BALB.B6-NK1.1(b) mouse share many characteristics with their B6 counterparts, but seem to be deficient in the functional NKT cell subtype characterized by low interleukin-4 and high interferon-gamma production, and surface expression of CD49b but not CD69. Moreover, in the thymus but not the spleen of BALB.B6-NK1.1(b) mice we find a novel Valpha14-Jalpha18 invariant NKT cell subset which is devoid of a set of NK markers, suggesting that these cells represent a less differentiated NKT cell stage, and carries high levels of the T-cell receptor and uses a skewed T-cell receptor Vbeta-repertoire.     

In Vivo Generation of Mouse Natural Killer Cells: Role of the Spleen and Thymus

The role of the spleen and thymus was investigated in the natural killer (NK) cell system. These NK cells have the ability to kill a variety of tumour cells as demonstrated in vitro in short-term ⁵¹Cr release assays. The work presented deals with three observations: (1) the effect of splenectomy on the levels of NK activity in the blood and lymph nodes. (2) the effect of splenectomy on the reconstitution of irradiated animals with bone marrow cells, and (3) the level of NK activity in adult thymectomized, irradiated animals which were reconstituted with bone marrow or thymus cells from either high or low NK activity animals. Thus, for the third point, chimaeras were established between histocompatible strains of mice A. BY (a low NK strain), and C57B1/6 (a high NK strain). The ability of T cells from one strain could then be observed to either help or suppress the NK activity of the bone-marrow-derived cells. The data presented show that the absence of a spleen does not affect MK activity or reconstitution of NK cells in irradiated animals. Further, T cells from one strain do not affect NK activity of animals reconstituted with bone marrow cells from a histocompatible strain. Thus T cells from a low NK stain (A. BY) did not suppress the high activity of C57B1/6 cells, and, conversely, the C57B1/6 T cells did not compensate for the low NK activity of A. BY cells.     

Lupus resistance is associated with marginal zone abnormalities in an NZM murine model

The NZM2410 and NZM TAN (TAN) are two of 27 inbred strains derived from an intercross between the NZW and NZB strains. NZM2410 mice develop a highly penetrant lupus nephritis mediated by three susceptibility loci, Sle1, Sle2 and Sle3. These three loci have been combined on a C57BL/6 background in a triple congenic strain that reconstitutes the NZM2410 autoimmune phenotype. Remarkably, inspite of the presence of Sle1, Sle2 and Sle3, TAN mice display a mild autoimmune phenotype reminiscent of NZW. Contrary to the lupus-prone strains, the majority of TAN CD4(+) T cells are in a na?ve-inactivated stage. TAN mice show B-cell developmental abnormalities similar to lupus-prone mice, such an accumulation of transitional T1 cells and peritoneal B-1a cells. TAN mice show, however, a unique expansion of the splenic marginal zone, in which B cells express high levels of CD5 and CD9, fail to migrate to the follicles in response to LPS, and show sub-optimal binding of T-independent type 2 antigens. Therefore, TAN mice present a functional silencing of marginal zone B cells, which have been previously implicated with autoimmune process. The TAN strain thus provides a novel model for the analysis of the genetic determinants of B-cell autoreactivity.     

The in vitro production of anti-nuclear antibodies by human peripheral blood mononuclear cells. Demonstration of T cell requirement and soluble inducing factor(s) for anti-nuclear antibodies triggering in patients with systemic lupus erythematosus.

Peripheral blood mononuclear cells (PBMC) of 29 patients with systemic lupus erythematosus (SLE) and 14 normal individuals were investigated for the in vitro production of anti-nuclear antibodies (ANA). Twenty-eight of 29 SLE patients but only one control spontaneously produced ANA in unstimulated PBMC. Pokeweed mitogen induced ANA synthesis in six controls. No detectable ANA was observed in B cell enriched fraction except in two cases of SLE. Recombination of B + T cell enriched fractions and PBMC supernatants from SLE patients could induce B cells to synthesize ANA. These results indicate that: (1) SLE patients spontaneously produced ANA in vitro whereas controls rarely did; (2) autoreactive clones exist in normal individuals but are kept under control and (3) T cell help is required for ANA triggering.     

Systemic IFN-alpha drives kidney nephritis in B6.Sle123 mice

The impact of IFN-alpha secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental C57BL/6 (B6) congenic partner using an adenovirus (ADV) expression vector containing a recombinant IFN-alpha gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFN-alpha. Most IFN-alpha-induced phenotypes were similar in B6 and B6.Sle123 mice; however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFN-alpha exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFN-alpha were identical in both strains, severe glomerulonephritis occurred only in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum anti-nuclear antibody levels. In summary, the predominant impact of systemic IFN-alpha in this murine model is an exacerbation of mechanisms mediating end organ damage.     

Diseases caused by reactions of T lymphocytes to incompatible structures of the major histocompatibility complex. IV. Autoantibodies to nuclear antigens.

We studied the requirements for induction of ANA formation in non-irradiated F1 hybrid mice undergoing a chronic graft-versus-host reaction (GVHR) after the injection of parental-strain lymphocytes. T lymphocytes in the donor cell inoculum were both needed and sufficient for the induction of ANA formation. For optimal ANA formation, the F1 recipient mice had to differ at H-2 from the parental donor strain. ANA belonged to the IgG1, IgG2, IgM and IgA (sub)classes of immunoglobulin. IgG ANA occurred in maximal serum titres of 1 in 5,120. ANA were not donor anti-host alloantibodies. At least some ANA were true autoantibodies, i.e. of F1 origin, because they carried the Ig allotypic markers characteristic of the F1 hybrid recipients. These findings are consistent with the concept that the pathogenic mechanism underlying autoantibody formation during the GVHR is an abnormal T-B-cell co-operation. In this process, donor T cells react against foreign histocompatibility antigens of the F1 recipient and generate non-specific help for B cells, including the autoreactive B cells.     

Ly108 expression distinguishes subsets of invariant NKT cells that help autoantibody production and secrete IL-21 from those that secrete IL-17 in lupus prone NZB/W mice

Lupus is a systemic autoimmune disease characterized by anti-nuclear antibodies in humans and genetically susceptible NZB/W mice that can cause immune complex glomerulonephritis. T cells contribute to lupus pathogenesis by secreting pro-inflammatory cytokines such as IL-17, and by interacting with B cells and secreting helper factors such as IL-21 that promote production of IgG autoantibodies. In the current study, we determined whether purified NKT cells or far more numerous conventional non-NKT cells in the spleen of NZB/W female mice secrete IL-17 and/or IL-21 after TCR activation in vitro, and provide help for spontaneous IgG autoantibody production by purified splenic CD19⁺ B cells. Whereas invariant NKT cells secreted large amounts of IL-17 and IL-21, and helped B cells, non-NKT cells did not. The subset of IL-17 secreting NZB/W NKT cells expressed the Ly108^loCD4⁻NK1.1⁻ phenotype, whereas the IL-21 secreting subset expressed the Ly108^hiCD4⁺NK1.1⁻ phenotype and helped B cells secrete a variety of IgG anti-nuclear antibodies. α-galactocylceramide enhanced the helper activity of NZB/W and B6.Sle1b NKT cells for IgG autoantibody secretion by syngeneic B cells. In conclusion, different subsets of iNKT cells from mice with genetic susceptibility to lupus can contribute to pathogenesis by secreting pro-inflammatory cytokines and helping autoantibody production.     

Cytokines production of U5A2-13-positive T cells by stimulation with glycolipid alpha-galactosylceramide

We have previously established and reported a novel monoclonal antibody (mAb), U5A2-13, which recognizes a phenotypically similar population of natural killer (NK)-like T cells. Using U5A2-13 mAb, we now describe the functional properties of U5A2-13(+) T cells in both NK1.1-positive or -negative mouse strains. Similar to NK1.1(+) T cells, hepatic U5A2-13(+) T cells of C57BL/6 (NK1.1(+) strain) mice, but not U5A2-13(-) T cells, could be induced to produce large amounts of IL-4 and IFN-gamma by stimulation with glycolipid alpha-galactosylceramide (alpha-GalCer) present on dendritic cells (DC) in a dose-dependent manner. The abundant production of these cytokines from U5A2-13(+) T cells of BALB/c (NK1.1(-) strain) mice is similar to that noted in C57BL/6 mice. Cytokine production by cultures stimulated with DC of beta2-microglobulin-deficient mice was significantly less than that of cultures stimulated with DC of intact mice. Overall, U5A2-13(+) T cells recognize alpha-GalCer presented by CD1d, indicating that U5A2-13(+) T cells can be used to analyze NK-like T cell function in various strains of mice.     

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

Act1 is a negative regulator of B‐cell activation factor of the TNF family (BAFF) and CD40L‐induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti‐nuclear IgG autoantibody (ANA‐IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T‐cell dependent or T‐cell independent manner, we investigated the role of T‐cell help during Act1‐mediated autoimmunity. Act1‐deficiency was bred onto C57Bl/6 (B6.Act1^?/?) mice and B6.TCRβ^?/?TCRδ^?/?Act1^?/? (TKO) mice were generated. While TCRβ/δ‐sufficient B6.Act1^?/? mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA‐IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1^?/? nor TKO mice developed SjS‐like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1^?/? mice. Interestingly, BAFF‐driven transitional B‐cell abnormalities, previously reported in BALB/C.Act1^?/? mice, were intact in B6.Act1^?/? mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE‐like disease in B6.Act1^?/? mice, but not BAFF‐driven transitional B‐cell differentiation.     

Suppression of natural killer cell activity in young and old mice

Spleen cells from female mice of the recombinant inbred strain BPS lack natural killer (NK) cytolytic activity, and can suppress the cytolytic activity of normal NK effector cells. The suppressor cells are not typical B cells, T cells, macrophages, or NK cells; they lack the characteristics and surface markers of each of these cell types. In BPS mice, suppressor cell activity is a dominant and significant characteristic of spleen cells at every age tested (2 weeks to 18 months). In other strains of mice which are normally classified as high-responders in NK assays, such as the C57BL strain, these suppressor cells are less prominent but can be detected when separated (on the basis of their higher density) from other spleen cell populations. As mice of the high-responder strains age, however, the suppressor cells become a significant part of the spleen cell population.     

Natural killer T cells and innate immune B cells from lupus-prone NZB/W mice interact to generate IgM and IgG autoantibodies

Lupus-prone NZB/W F1 mice develop glomerulonephritis after T helper cell-dependent isotype switching of autoantibody secretion from IgM to IgG at about 6 months of age. We compared innate immune natural killer (NK) T cells and conventional T cells for their capacity to help spontaneous in vitro immunoglobulin and autoantibody secretion of innate immune (B-1 and marginal zone) and conventional (follicular) B cell subsets from NZB/W F1 mice. We found that purified NKT cells not only increased spontaneous secretion of IgM and IgM anti-double-stranded (ds)DNA antibodies by B-1 and marginal zone B cells, but also facilitated secretion of IgG anti-dsDNA antibodies predominantly by B-1 B cells. Few IgM or IgG anti-dsDNA antibodies were secreted by follicular B cells, and conventional T cells failed to provide potent helper activity to any B cell subset. All combinations of T and B cell subsets from normal C57BL/6 mice failed to generate vigorous IgM and IgG secretion. NZB/W NKT cell helper activity was blocked by anti-CD1 and anti-CD40L mAb. In conclusion, direct interactions between innate immune T and B cells form a pathway for the development of IgM and IgG lupus autoantibody secretion in NZB/W mice.     

Murine lupus susceptibility locus Sle2 activates DNA-reactive B cells through two sub-loci with distinct phenotypes

The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B-cell differentiation, which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b and Sle2c. Sle2 also enhances the breach of B-cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a-expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that have a critical role in B-cell tolerance.     

Association of extensive polymorphisms in the SLAM/CD2 gene cluster with murine lupus

Susceptibility to autoimmunity in B6.Sle1b mice is associated with extensive polymorphisms between two divergent haplotypes of the SLAM/CD2 family of genes. The B6.Sle1b-derived SLAM/CD2 family haplotype is found in many other laboratory mouse strains but only causes autoimmunity in the context of the C57Bl/6 (B6) genome. Phenotypic analyses have revealed variations in the structure and expression of several members of the SLAM/CD2 family in T and B lymphocytes from B6.Sle1b mice. T lymphocytes from B6.Sle1b mice have modified signaling responses to stimulation at 4-6 weeks of age. While autoimmunity may be mediated by a combination of genes in the SLAM/CD2 family cluster, the strongest candidate is Ly108, a specific isoform of which is constitutively upregulated in B6.Sle1b lymphocytes.     

Enhanced expression of stem cell antigen-1 (Ly-6A/E) in lymphocytes from lupus prone mice correlates with disease severity

B6.Sle1 mice, congenic for the NZM2410-derived lupus susceptibility locus, Sle1 on chromosome 1 exhibit many of the features seen in human lupus including activated lymphocytes and high titers of antinuclear autoantibodies. Among the different surface molecules that were aberrantly expressed on the B6.Sle1 lymphocytes was Ly-6A/E. Splenic B- and T-lymphocytes but not myeloid cells from B6.Sle1 mice exhibited enhanced levels of Ly-6A/E compared to B6 controls. In particular, MZ B cells, GC B cells and B-cell blasts expressed the highest levels of Ly-6A/E in both strains, with the levels being even higher on B6.Sle1 derived cells. Following stimulation with LPS or anti-IgM, there was a profound up-regulation in Ly-6A/E, particularly on MZ B cells and B-cell blasts. CD4 and CD8 T cells also up-regulated Ly-6A/E after stimulation with anti-CD3 and anti-CD28. These studies were extended to additional autoimmune strains including B6.Sle3, B6.Sle1.lpr and BXSB. Importantly, Ly-6A/E levels on lymphocytes were commensurate with the degree of disease exhibited by these lupus strains. Finally, it appears that increased interferon levels, in addition to antigen receptor stimulation, may also be a factor accounting for elevated Ly-6A/E in lupus. Given these observations it is important to elucidate the functional role of Ly-6A/E in lupus in future studies.     

NK cell-mediated immunopathology during an acute viral infection of the CNS

Natural killer (NK) and cytotoxic T (Tc) cells are prime effector populations in the antiviral response of the host. Tc cells are essential for recovery from many viral diseases but may also be responsible for immunopathology. The role of NK cells in recovery from viral infections is less well established. We have studied acute virulent Semliki Forest virus (vSFV) infection of the central nervous system in C57BL/6J mice, which was mainly controlled by NK cells without marked Tc cell involvement. We show that mice with defects in the Fas and/or granule exocytosis pathways of cytotoxicity are more resistant to lethal vSFV infection than wild-type mice. On the other hand, mice defective in the IFN-gamma response are more sensitive than wild-type mice, whereas mice lacking the Tc cell compartment (beta-2 microglobulin-deficient mice) exhibit susceptibility similar to wild-type mice. The additional finding that depletion of NK cells significantly delayed the mean time to death but did not prevent mortality in SFV-infected B6 mice suggests that cytolytic activity of NK cells is detrimental, while IFN-gamma production is beneficial for recovery from SFV infection. This is the first study illustrating an NK cell-mediated immunopathological outcome to an acute viral infection.     

A lupus-susceptibility C57BL/6 locus on chromosome 3 (Sle18) contributes to autoantibody production in 129 mice

Epistatic interactions between the non-autoimmune strains 129 and C57BL/6 (B6), used for generating gene-targeted animals, can induce a lupus-like disease. Genome-wide scan analyses of testcross progeny between these two strains have identified several lupus susceptibility loci, with the strongest linkage to the production of autoantibodies (auto-Abs) displayed by an interval on chromosome 1 of 129 origin (Sle16). However, the contribution of B6 loci to the lupus phenotype remained unknown. We used a congenic approach to deduce the contribution to the autoimmune traits of the B6 genomic interval on chromosome 3 (Sle18), previously shown to be linked to antinuclear Ab production. This interval, when transferred on a 129 background (a strain termed 129.B6-Sle18), promoted auto-Ab production targeting a broad spectrum of autoantigens, expansion of activated CD4(+)T and B cells and mild glomerulonephritis. Surprisingly, these immunological and serological defects were accompanied by a significant increase in the percentage of regulatory T cells (Tregs; CD4(+) Foxp3(+)). However, these cells, that expressed lower levels of Foxp3, had no impaired regulatory function when tested in vitro. These findings illustrate further the efficacy of congenic dissection for functional characterisation of individual lupus susceptibility loci and highlight the contribution of loci derived from non-autoimmune strains to the disease pathogenesis.