Peter J. Kim

Invariant NKT Cells Inhibit Autoreactive B Cells in a Contact- and CD1d-Dependent Manner

Autoantibody production is a hallmark of autoimmune diseases, such as lupus and rheumatoid arthritis. Accumulating evidence suggests a role of invariant NKT (iNKT) cells in their pathogenesis. Mechanisms underlying the role of iNKT cells in these diseases, however, remain unclear. In this study, we show that iNKT cells suppress IgG anti-DNA Ab and rheumatoid factor production and reduce IL-10–secreting B cells in a contact-dependent manner, but increase total IgG production and enhance activation markers on B cells via soluble factors. In vivo reconstitution with iNKT cells also reduces autoantibody production in iNKT-deficient mice and in SCID mice implanted with B cells. Using an anti-DNA transgenic model, we found that autoreactive B cells spontaneously produce IL-10 and are activated in vivo. In the presence of activated iNKT cells, these autoreactive B cells are selectively reduced, whereas nonautoreactive B cells are markedly activated. Because iNKTs recognize CD1d, we reasoned that CD1d might play a role in the differential regulation of autoreactive versus nonautoreactive B cells by iNKT cells. Indeed, autoreactive B cells express more CD1d than nonautoreactive B cells, and CD1d deficiency in lupus mice exacerbates autoantibody production and enhances Ab response to a self-peptide but not to a foreign peptide. Importantly, iNKT cells fail to inhibit autoantibody production by CD1d-deficient B cells. Thus, iNKT cells inhibit autoreactive B cells in a contact- and CD1d-dependent manner but activate nonautoreactive B cells via cytokines. Such ability of iNKTs to suppress autoantibody production, without causing global suppression of B cells, has important implications for the development of iNKT-based therapy for autoimmune diseases.

Brief Treatment with iNKT Cell Ligand α-Galactosylceramide Confers a Long-term Protection Against Lupus

CD1d presents glycolipid antigens such as α-galactosylceramide (αGalCer) to invariant natural killer T cells (iNKT). We have reported that activated iNKTs inhibit IL-10-producing autoreactive B cells, while promoting or leaving intact the normal B cell responses, making iNKT modulation an attractive therapeutic modality. Here, we report that a brief treatment of young lupus-prone (NZB/NZW)F1 (BWF1) mice with two injections of αGalCer conferred a long-term protection against lupus. Long-term repeated administrations of αGalCer, however, afforded no clinical benefit. These disparate clinical effects correlated with iNKT responsiveness. While a brief treatment with αGalCer enhanced iNKT responses upon in vitro recall, the long-term αGalCer treatment resulted in reduced iNKT responses in BWF1 mice. The improvement in disease with αGalCer treatment was associated with the reduced IL-10 production. Furthermore, iNKTs directly inhibited IL-10-secreting cells in vivo in reconstituted SCID mice and inhibited IL-10-secreting B cells in vitro in co-cultures. Thus, a brief treatment with a CD1d-binding glycolipid enhances iNKT responses, reduces IL-10 production, and delays the onset of lupus, whereas long-term repeated treatments induce marked iNKT hyporesponsiveness and do not affect disease outcome in BWF1 mice. Identifying glycolipid regimens that can modulate iNKT responsiveness will have important implications for developing iNKT-based therapies for autoimmune diseases.

Homeostatic Regulation of Marginal Zone B Cells by Invariant Natural Killer T Cells

Marginal zone B cells (MZB) mount a rapid antibody response, potently activate naïve T cells, and are enriched in autoreactive B cells. MZBs express high levels of CD1d, the restriction element for invariant natural killer T cells (iNKT). Here, we examined the effect of iNKT cells on MZB cell activation and numbers in vitro and in vivo in normal and autoimmune mice. Results show that iNKT cells activate MZBs, but restrict their numbers in vitro and in vivo in normal BALB/c and C57/BL6 mice. iNKT cells do so by increasing the activation-induced cell death and curtailing proliferation of MZB cells, whereas they promote the proliferation of follicular B cells. Sorted iNKT cells can directly execute this function, without help from other immune cells. Such MZB regulation by iNKTs is mediated, at least in part, via CD1d on B cells in a contact-dependent manner, whereas iNKT-induced proliferation of follicular B cells occurs in a contact- and CD1d-independent manner. Finally, we show that iNKT cells reduce ‘autoreactive’ MZB cells in an anti-DNA transgenic model, and limit MZB cell numbers in autoimmune-prone (NZB×NZW)F1 and non-obese diabetic mice, suggesting a potentially new mechanism whereby iNKT cells might regulate pathologic autoimmunity. Differential regulation of follicular B cells versus potentially autoreactive MZBs by iNKT cells has important implications for autoimmune diseases as well as for conditions that require a rapid innate B cell response.

Langerhans Cells Maintain Local Tissue Tolerance in a Model of Systemic Autoimmune Disease

Systemic autoimmune diseases such as lupus affect multiple organs, usually in a diverse fashion where only certain organs are affected in individual patients. It is unclear whether the “local” immune cells play a role in regulating tissue specificity in relation to disease heterogeneity in systemic autoimmune diseases. In this study, we used skin as a model to determine the role of tissue-resident dendritic cells (DCs) in local and systemic involvement within a systemic lupus disease model. Skin-resident DCs, namely, Langerhans cells (LCs), have been implicated in regulating tolerance or autoimmunity using elegant transgenic models, however, their role in local versus systemic immune regulation is unknown. We demonstrate that although lymphocytes from skin-draining lymph nodes of autoimmune-prone MRL/MpJ-Faslpr/lpr (MRL-lpr) mice react spontaneously to a physiological skin self-Ag desmoglein-3, epicutaneous applications of desmoglein-3 induced tolerance that is dependent on LCs. Inducible ablation of LCs in adult preclinical MRL-lpr and MRL/MpJ-Fas+/+ mice resulted in increased autoantibodies against skin Ags and markedly accelerated lupus dermatitis with increased local macrophage infiltration, but had no effect on systemic autoantibodies such as anti-dsDNA Abs or disease in other organs such as kidneys, lung, and liver. Furthermore, skin-draining lymph nodes of LC-ablated MRL-lpr mice had significantly fewer CD4+ T cells producing anti-inflammatory cytokine IL-10 than LC-intact controls. These results indicate that a skin-resident DC population regulates local tolerance in systemic lupus and emphasize the importance of the local immune milieu in preventing tissue-specific autoimmunity, yet have no effect on systemic autoimmunity.

Disparate effects of depletion of CD1d-reactive T cells during early versus late stages of disease in a genetically susceptible model of lupus.

Some T cells react with lipid antigens bound to antigen-presenting molecule CD1d. Numbers and functions of a subset of such lipid-reactive T cells are reduced in patients with systemic lupus erythematosus (SLE) and their relatives, as well as in genetically susceptible and chemically induced animal models of lupus-like disease. We have reported that the germline deletion of CD1d exacerbates lupus, suggesting a protective role of these cells in the development of lupus. The use of a knockout mouse model in this study, however, did not allow examination of the role of these cells at different stages of disease. Here, we describe an approach to deplete CD1d-dependent T cells, which allowed us to investigate the role of these cells at different stages of disease in genetically lupus-prone NZB/NZW F1 (BWF1) mice. Repeated intravenous injections of large numbers of CD1d-transfected cells resulted in ∼50–75% reduction in these cells, as defined by the expression of CD4, NK1.1 and CD122, and lack of expression of CD62 ligand. TCR γδ +NK1.1+ cells were also reduced in the recipients of CD1d-transfected cells as compared with control recipients. Such depletion of CD1d-reactive T cells in preclinical BWF1 mice resulted in disease acceleration with a significant increase in proteinuria and mortality. In older BWF1 mice having advanced nephritis, however, such depletion of CD1d-reactive T cells resulted in some disease improvement. Taken together, these data as well as our published studies suggest that CD1d-reactive T cells protect against the development of lupus in animal models. However, these cells appear to be unable to suppress established lupus nephritis in these animals, and might even play a disease aggravating role in late stages of disease.

Intrinsic hyporesponsiveness of invariant natural killer T cells precedes the onset of lupus

Patients with systemic lupus erythematosus (SLE) display reduced numbers and functions of invariant natural killer T (iNK T) cells, which are restored upon treatment with corticosteroids and rituximab. It is unclear whether the iNK T cell insufficiency is a consequence of disease or is a primary abnormality that precedes the onset of disease. To address this, we analysed iNK T cell function at different stages of disease development using the genetically lupus‐susceptible NZB × NZW F1 (BWF1) model. We found that iNK T cell in‐vivo cytokine responses to an iNK T cell ligand α‐galactosylceramide (α‐GalCer) were lower in BWF1 mice than in non‐autoimmune BALB/c and major histocompatibility complex (MHC)‐matched NZB × N/B10.PL F1 mice, although iNK T cell numbers in the periphery were unchanged in BWF1 mice compared to control mice. Such iNK T cell hyporesponsiveness in BWF1 mice was detected at a young age long before the animals exhibited any sign of autoimmunity. In‐vivo activation of iNK T cells is known to transactivate other immune cells. Such transactivated T and B cell activation markers and/or cytokine responses were also lower in BWF1 mice than in BALB/c controls. Finally, we show that iNK T cell responses were markedly deficient in the NZB parent but not in NZW parent of BWF1 mice, suggesting that BWF1 might inherit the iNK T cell defect from NZB mice. Thus, iNK T cells are functionally insufficient in lupus‐prone BWF1 mice. Such iNK T cell insufficiency precedes the onset of disease and may play a pathogenic role during early stages of disease development in SLE.

Germline deletion of β2 microglobulin or CD1d reduces anti-phospholipid antibody, but increases autoantibodies against non-phospholipid antigens in the NZB/W F1 model of lupus

Introductionβ2-microglobulin (β2m) is required for the surface expression of MHC class I and class I-like proteins such as CD1d, Qa1 and neonatal Fc receptor (FcRn), all of which may impact the development of autoimmunity. Since CD1d is known to bind and present phospholipid antigens to T cells, we asked if the deficiency of β2m or CD1d will impact the development of anti-phospholipid antibodies as compared to other aspects of lupus autoimmunity.MethodsWe introgressed the β2m-null genotype onto the NZB and NZW backgrounds for 12 to 14 generations to generate genetically lupus-susceptible (NZB/NZW)F1 (BWF1) mice that are β2m-deficient (β2m°). Circulating immunoglobulins (Ig), rheumatoid factor (RF), anti-DNA and anti-cardiolipin (anti-CL) antibodies, and renal disease were analyzed in these and CD1d-deficient (CD1d°) BWF1 mice that we had previously generated.ResultsWhereas β2m° BWF1 mice had reduced serum IgG, they had increased mortality, nephritis, serum IgG anti-DNA antibody and RF as compared to heterozygous and wild-type littermates. These effects were recapitulated in CD1d° BWF1 mice, except that they also had increased serum IgG as compared to control littermates. Intriguingly, both β2m° and CD1d° mice had lower serum anti-CL antibody levels than in control littermates. Such CD1d dependence of anti-CL antibody production is not mediated by CD1d/glycolipid-reactive iNKT cells, as these cells reduced the production of RF and anti-DNA antibodies but had no effect on anti-CL antibodies.ConclusionsWe report a novel dichotomous role of β2m and CD1d, whereby these molecules differently regulate autoimmunity against phospholipid versus non-phospholipid autoantigens.

Abstract 3979: In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC A previous murine study (B. Farsaci et al, Int. J. Cancer, 2010) showed that mice administered an anti-cancer vaccine prior to treatment with the pan-BCL-2 inhibitor obatoclax (GX15-070) showed a substantial reduction of metastatic mouse lung tumors due to increased apoptotic resistance of mature CD8T cells and decreased Treg function. The purpose of this study was to evaluate activity of obatotclax on human lymphocyte populations. In vitro effects of obatoclax on human T cell subsets (naive, central memory, effector-memory and terminal effector T cells) at different maturation states (CD69+ early-activated T cells and CD69- mature T cells) and regulatory T cells (Tregs) were evaluated at multiple concentrations (0.1 - 10 uM). Purified CD8 T cells or whole PBMCs from normal donors were activated for 3-4 days with anti-CD3 and anti-CD28 monoclonal antibodies (short-term culture) or maintained in an IL7/IL15-enriched medium for 7 additional days (long-term culture). After short- or long-term cultures, isolated CD8Ts and whole PBMCs were treated with obatoclax or control (DMSO) for 24 hours, and their viability and differentiation status were analyzed by flow cytometry. The results indicate that (1) long-term cultured CD8 and CD4T cells were more resistant to obatoclax induced cell death than those in the short-term culture condition, (2) obatoclax increased the percentage of total memory CD8 and CD4T cells (central memory + effector memory) in the long-term culture condition, and (3) obatoclax increased the apoptosis of regulatory T cells (Tregs) and induced a profound down-regulation of FOXP3 in a dose dependent manner. It is postulated that first, for an optimum combination effect, an immune-stimulating agent directed at T cells needs to precede obatoclax treatment long enough to allow T cells to acquire resistance to obatoclax. Then secondly, with proper treatment scheduling, memory T cells can be viably maintained, thereby establishing a sustainable source of cytotoxic T cells to kill tumors. Finally, because human regulatory T cells are sensitive to obatoclax, there is a potential for obatoclax to tip the numerical and functional balance of effector T cells and Tregs towards effector T cells in human tumor microenvironment. These conclusions support the preclinical murine in vivo study, and provide the rationale for combining obatoclax with an immunotherapeutic regimen in clinical studies. Citation Format: Peter Kim, James Hodge, Italia Grenga, Renee Donahue, Jeffrey Schlom, Benedetto Farsaci. In vitro analysis of pan-BCL-2 inhibitor GX15-070 (obatoclax) on human lymphocytes for the feasibility of combination immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2013-3979