Mark A. Exley

Extreme Th1 bias of invariant Vα24JαQ T cells in type 1 diabetes

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic β-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result,. We therefore investigated a series of at-risk non-progressors and type1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4−CD8− Vα24JαQ+ T cells compared with their non-diabetic sibling. All 56 Vα24JαQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-γ upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-γ. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-γ. These results support a model for IDDM in which Th1-cell-mediated tissue damage is initially regulated by Vα24JαQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis

While Crohn disease (CD) has been clearly identified as a Th1 inflammation, the immunopathogenesis of its counterpart inflammatory bowel disease, ulcerative colitis (UC), remains enigmatic. Here we show that lamina propria T (LPT) cells from UC patients produce significantly greater amounts of IL-13 (and IL-5) than control cells and little IFN-gamma, whereas comparable cells from CD patients produce large amounts of IFN-gamma and small amounts of IL-13. We then show that stimulation of UC LPT cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing transfected CD1d induces substantial IL-13 production. While this provided firm evidence that the IL-13-producing cell is an NK T (NKT) cell, it became clear that this cell does not express invariant NKT cell receptors characteristic of most NKT cells since there was no increase in cells binding alpha-galactosylceramide-loaded tetramers, and alpha-galactosylceramide did not induce IL-13 secretion. Finally, we show that both human NKT cell lines as well as UC CD161(+) LPT cells are cytotoxic for HT-29 epithelial cells and that this cytotoxicity is augmented by IL-13. These studies show that UC is associated with an atypical Th2 response mediated by nonclassical NKT cells producing IL-13 and having cytotoxic potential for epithelial cells.

Loss of IFN-gamma production by invariant NK T cells in advanced cancer.

Invariant NK T cells express certain NK cell receptors and an invariant TCRα chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-γ, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (α-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-γ and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-γ production and a striking decrease in their IFN-γ:IL-4 ratio. The IFN-γ deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-γ and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.

Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of α-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.

CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung.

CD1d-restricted T cells are implicated as key players in host defense against various microbial infections. However, the mechanisms involved and the role they play, if any, at the mucosal surfaces where pathogenic infections are initiated is unknown. In a murine pneumonia model established by intranasal application of Pseudomonas aeruginosa, CD1d−/− mice showed markedly reduced pulmonary eradication of P. aeruginosa compared with wild-type mice; this was associated with significantly lower amounts of macrophage inflammatory protein-2 and reduced numbers of neutrophils within the bronchoalveolar lavage fluid. Corollarily, treatment of mice with α-galactosylceramide—a lipid that activates CD1d-restricted T cells—increased the amount of interferon-γ; this was associated with rapid pulmonary clearance through enhanced phagocytosis of P. aeruginosa by alveolar macrophages. These results reveal a crucial role played by CD1d-restricted T cells in regulating the antimicrobial immune functions of macrophages at the lung mucosal surface.

NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance

In a model of systemic tolerance called Anterior Chamber-Associated Immune Deviation (ACAID), the differentiation of the T regulatory (Tr) cells depends on NK T cells and occurs in the spleen. We now show that the CD1d-reactive NK T cell subpopulation, required for development of systemic tolerance, expresses the invariant Vα14Jα281 TCR because Jα281 knockout (KO) mice were unable to generate Ag-specific Tr cells and ACAID. The mechanism for NK T cell-dependent differentiation of Ag-specific Tr cells mediating systemic tolerance was studied by defining the cytokine profiles in heterogeneous and enriched NK T spleen cells. In contrast to there being no differences in most regulatory cytokine mRNAs, both mRNA and protein for IL-10 were increased in splenic NK T cells of anterior chamber (a.c.)-inoculated mice. However, IL-10 mRNA was not increased in spleens after i.v. inoculation. Finally, NK T cells from wild-type (WT) mice, but not from IL-10 KO mice, reconstituted the ACAID inducing ability in Jα281 KO mice. Thus, NK T cell-derived IL-10 is critical for the generation of the Ag-specific Tr cells and systemic tolerance induced to eye-inoculated Ags.

CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes

Human T cells expressing CD161 and an invariant T‐cell receptor (TCR) α‐chain (Vα24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T‐cell population, and whether alterations in CD1d expression contribute to the regulation of Vα24invt T‐cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000‐MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d‐reactive Vα24invt T‐cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d–, but low‐level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d–. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up‐regulated by granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) activation. These results demonstrate constitutive CD1d expression by human professional antigen‐presenting cells and that post‐translational processing of CD1d may contribute to regulation of the activity of CD1d‐specific T cells.

CD1d function is regulated by microsomal triglyceride transfer protein.

CD1d is a major histocompatibility complex (MHC) class I–related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-α chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell–mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.

Disseminated Varicella Infection Due to the Vaccine Strain of Varicella-Zoster Virus, in a Patient with a Novel Deficiency in Natural Killer T Cells

An 11-year-old girl presented with a papulovesicular rash and severe respiratory distress 5 weeks after receiving varicella vaccine. Restriction fragment length-polymorphism analysis of virus isolated from an endotracheal-tube aspirate and from bronchoalveolar lavage revealed that this patients illness was due to the Oka vaccine strain of varicella. An extensive immunologic analysis failed to identify a known diagnostic entity to explain her susceptibility to this attenuated vaccine strain. Analysis of her lymphocytes on separate occasions, months after recovery from her illness, revealed a profound deficiency of natural killer T (NKT) cells and of NKT-cell activity, suggesting that NKT cells contribute to host defense against varicella virus.

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.