Autoreactivity to Sulfatide by Human Invariant NKT Cells.

Invariant NKT (iNKT) cells are innate-like lymphocytes that recognize lipid Ags presented by CD1d. The prototypical Ag, α-galactosylceramide, strongly activates human and mouse iNKT cells, leading to the assumption that iNKT cell physiology in human and mouse is similar. In this article, we report the surprising finding that human, but not mouse, iNKT cells directly recognize myelin-derived sulfatide presented by CD1d. We propose that sulfatide is recognized only by human iNKT cells because of the unique positioning of the 3-O-sulfated ß-galactose headgroup. Surface plasmon resonance shows that the affinity of human CD1d-sulfatide for the iNKT cell receptor is relatively low compared with CD1d-α-galactosylceramide (KD of 19-26 µM versus 1 µM). Apolipoprotein E isolated from human cerebrospinal fluid carries sulfatide that can be captured by APCs and presented by CD1d to iNKT cells. APCs from patients with metachromatic leukodystrophy, who accumulate sulfatides due to a deficiency in arylsulfatase-A, directly activate iNKT cells. Thus, we have identified sulfatide as a self-lipid recognized by human iNKT cells and propose that sulfatide recognition by innate T cells may be an important pathologic feature of neuroinflammatory disease and that sulfatide in APCs may contribute to the endogenous pathway of iNKT cell activation.

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling.

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

CD40L stimulation of rat dendritic cells specifically favors the IL-12/IL-10 ratio resulting in a strong T cell stimulatory capacity.

Dendritic cells (DC) play an important role in immune responses and have been studied extensively in human and mouse models. CD40 triggering of DC has a pivotal role in their maturation process, obtaining the unique capacity to induce strong CD4 and CD8 T cell activation. Although rat models are frequently used for the understanding of the underlying mechanism of human diseases, relatively little is known about rat DC. To investigate the effect of CD40 triggering on rat DC, we cloned the rat CD40L gene and generated murine fibroblasts with stable expression (L-rCD40L). DC stimulated by L-rCD40L cells exhibited a strong T cell stimulatory capacity, associated with higher amounts of IFN-gamma as compared to LPS-stimulated DC. Analysis of cytokine production showed that LPS induced both IL-12 and IL-10 production, whereas CD40L induced high amounts of IL-12, but little IL-10 production by rat DC. This implies that the difference found in T cell stimulatory capacity by the stimulated DC is due to the cytokine profile of the DC at the time of T cell activation.

Generation and characterization of a novel anti-rat CD40L antibody with inhibitory activities in vitro and in vivo.

The CD40-CD40L interaction plays a critical role in cell-mediated immune responses. Blocking this interaction has been shown to be beneficial in the treatment of various diseases studied in murine models. Although rats are widely used to test therapeutic strategies in several disease models, a monoclonal antibody (mAb) to block the CD40-CD40L interaction in rats is not broadly available. In the present study we generated Armenian hamster fibroblasts expressing rat CD40L and used these to generate a novel anti-rat CD40L mAb (AS1). In vitro studies showed that AS1 was able to block CD40L-induced DC maturation and B cell proliferation. Most importantly, in vivo, AS1 inhibited B cell responses in a dose-dependent fashion, as measured by the production of OVA specific antibodies after subcutaneous immunization with OVA. AS1 was shown to be a powerful tool to modulate Ag presentation in vitro and in vivo. Elucidating the effect of AS1 in various rat models for human diseases will provide more insight into blocking the CD40-CD40L interaction as a therapeutic strategy to prevent human diseases.

Accelerated antibody-mediated graft loss of rodent pancreatic islets after pretreatment with dexamethasone-treated immature donor dendritic cells.

BACKGROUND: Allogeneic islets of Langerhans transplantation is hampered in its success as a curative treatment of type 1 diabetes by the absence of potent, specific, and nontoxic immunosuppressive drugs. Here, we assessed whether donor bone marrow-derived dexamethasone-treated dendritic cells (dexDCs) could prolong islet allograft survival in a full major histocompatibility complex mismatch rat model. METHODS: Rodent allogeneic islet transplantation was performed from DA rats to Lewis rats and vice versa. Permanently immature dendritic cells were generated from the bone marrow of DA and Lewis rats by treatment with dexamethasone. Animals were either vehicle or donor dexDCs pretreated. Serum was used to monitor glucose, C-peptide, and alloreactive antibodies. RESULTS: The transplantation of DA islets into Lewis recipients showed direct graft failure with reduced numbers of ß-cells when rats were pretreated with donor dexDCs. In the reverse model (Lewis islets into DA recipients), dexDC-treated DA recipients even showed a significantly accelerated rejection of Lewis islets. Immunohistochemical analysis of allograft tissue of dexDC-treated recipients showed a predominant natural killer cell infiltration and a presence of antibody reactivity in the absence of complement deposition. Alloreactive antibodies were solely found in dexDC-treated recipients. CONCLUSION: Our study shows that pretreatment with donor-derived dexDCs induces an antibody-mediated rejection in this islet transplantation rodent model.

Induction of donor-specific T-cell hyporesponsiveness using dexamethasone-treated dendritic cells in two fully mismatched rat kidney transplantation models.

BACKGROUND: Dendritic cells (DC) can exert powerful immune stimulatory as well as regulatory functions and are therefore important tools for therapeutic strategies. Dexamethasone (Dex) was previously shown to inhibit DC maturation and to induce regulatory properties both in vitro and in vivo. Here, we investigated the immunoregulatory role of DexDC in two different rat acute rejection models of kidney transplantation. METHODS: Rat DC were generated from BN and DA bone marrow in the presence of the corticosteroid, Dex. The function of Dex-modulated DC was analyzed in vitro and in vivo, using a BN to LEW and a DA to LEW renal transplantation model in the absence of other forms of immunosuppression. T cells of transplanted rats were isolated and restimulated with donor mature DC (lipopolysaccharide [LPS] or CD40L activated). T-cell responsiveness was analyzed by proliferative capacity and IFN-gamma production. RESULTS: Stimulation of Dex-modulated rat DC with LPS resulted in normal IL-10 production, whereas synthesis of IL-12 was impaired. In accordance, the capacity of LPS-DexDC to stimulate T-cell activation was decreased. In both renal transplantation models, treatment with donor-derived LPS-DexDC induced a significant donor-specific T-cell hyporesponse. However, pretreatment did not result in a prolonged graft survival. CONCLUSIONS: In two fully mismatched kidney transplantation models, donor-derived LPS-DexDC induce a donor-specific T-cell hyporesponse. However, in this setting allograft survival was not improved, suggesting an important role for T cells with indirect alloreactivity. Understanding the underlying mechanism involved in the rejection process will improve the development of a cell-based immunotherapy.