Susanna Cardell

Murine CD1d-Restricted T Cell Recognition of Cellular Lipids

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.

Prevention of diabetes in nonobese diabetic mice mediated by CD1d-restricted nonclassical NKT cells

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Vα14-Jα18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Vα3.2+Vβ9+ NKT cells producing high levels of IFN-γ but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.

CD1high B cells: a population of mixed origin

A specialized subpopulation of T lymphocytes is reactive to the MHC class I‐like molecule CD1d. It is not clear which cells are the major antigen‐presenting cells in vivo in the activation of CD1‐restricted immune responses. We have characterized a subset of B lymphocytes expressing six‐ to eightfold higher levels of CD1 than the bulk of B cells. The cells have a surface phenotype (CD21high, CD23low, IgMhigh, IgDlow) found previously to characterize B cells residing in the splenic marginal zones. CD1high B cells localize preferentially to the spleen, and appear late in ontogeny, at 3 – 4 weeks of age. The CD1high B cells were present in mice lacking conventional helper T cells, ruling out an exclusive origin from T cell‐dependent immune responses. Still, some CD1high B cells had been involved in T cell‐dependent immune responses as suggested by mutations in their rearranged immunoglobulin gene regions. The population could still be found in mice with severely reduced B cell reactivity to bacterial lipopplysaccharides (C3H / HeJ mice) and in mice unable to respond to thymus‐independent type 2 antigens (NFR.Xid mice), as well as in germ‐free mice, indicating that bacterial antigens are not major stimuli for the induction of CD1high B cells. In contrast, the CD1high B cell population was severely reduced in CD19‐deficient mice. Taken together, the results imply that the CD1high population is heterogenous and of mixed origin, dependent for its development or maintenance on signaling through the CD19 molecule.

CD1d-Specific NK1.1+ T Cells with a Transgenic Variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Vα14-Jα281 rearrangement and a limited set of TCR Vβ segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Vα3.2/Vβ9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-γ, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the “non-Vα 14” diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.

The role of CD1d-restricted NK T lymphocytes in the immune response to oral infection with Salmonella typhimurium

CD1d‐restricted natural killer T (NKT) cells belong to the innate‐like lymphocytes which respond rapidly to stress and infectious challenge. We have studied murine CD1d‐restricted NKT cells in the early immune response to virulent Salmonella enterica serovar Typhimurium after oral infection. In the liver and spleen, neutrophil and macrophage numbers had increased several‐fold by day 5 post‐infection, while the frequency of B and T lymphocytes decreased. These cellular changes occurred independently of CD1d‐restricted NKT cells, and further, CD1d‐restricted T cells did not influence the bacterial load. However, in CD1d+ mice NK1.1+ T cells and invariant CD1d‐restricted T cells were activated by the infection, as demonstrated by an increase in size, up‐regulation of CD69 and production of IFN‐γ. The NK1.1 antigen was down‐modulated on these cells during the course of infection, while TCR levels were unaffected. While dendritic cells (DC) up‐regulated CD1d‐levels upon 24 h of in vitro exposure to the bacteria, increased CD1d expression was not evident on DC in vivo during infection. Furthermore, in vitro re‐stimulation of CD1d‐restricted T cells isolated from infected mice demonstrated a significant skewing of the cytokine profile, with suppressed IL‐4 and increased IFN‐γ production.

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11.

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 × NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.

Surface receptors identify mouse NK1.1+ T cell subsets distinguished by function and T cell receptor type.

Natural killer T (NKT) lymphocytes rapidly produce several cytokines, including IL‐4 and IFN‐γ, upon activation, and act as regulatory cells at an early interphase of innate and adaptive immune responses. They have been implicated as important elements in diverse immune responses including the regulation of autoimmune disease, the immune response to infections, and the prevention of tumor metastasis. The broad spectrum of their activities suggested that functionally different subsets of NKT cells may exist. We demonstrate two functionally distinct splenic NKT populations identified by the expression of CD49b and CD69, respectively. Each NKT subset was represented by the amplified transgenic NKT cell population in a distinct transgenic mouse line expressing a CD1d‐restricted TCR. CD49bhigh CD69– NKT cells, termed NKT1 cells by us, were high producers of IFN‐γ after stimulation, but essentially devoid of IL‐4‐synthesizing cells. Most NKT1 cells used diverse (non‐Vα14‐canonical) TCR. The CD69+ CD49–/low NKT cell population, which we term NKT2, produced large quantities of IL‐4 and substantial amounts of IFN‐γ upon activation and were dominated by cells using the canonical Vα14‐Jα18 T cell receptor. Knowledge of the unique roles of the different NKT cell subsets in specific situations will be essential for our understanding of NKT cell biology.

Differential regulation of Ly49 expression on CD4+ and CD4-CD8- (double negative) NK1.1+ T cells.

The pan‐NK cell marker NK1.1, present in some mouse strains, is also found on a subset of TCRα β + lymphocytes termed NKT cells. These cells are primarily CD4+ or CD4–CD8– (double negative, DN), and both NKT subpopulations contain cells reactive with the MHC class I‐like molecule CD1d. Murine NK cells express clonally distributed inhibitory receptors of the Ly49 family that bind to different alleles of MHC class I molecules and transmit negative signals regulating NK cell function. Ly49 receptors are also found on TCRα β + NK1.1+ T cells. To investigate the potential role of inhibitory Ly49 markers in the regulation of NKT cells, we have done a thorough analysis of their expression on different NKT populations. The CD4+ and DN NK1.1+ T cell subsets have traditionally been dealt with as one NK1.1+ T cell population, but we found dramatic differences between these two NKT cell subsets. We demonstrate here expression of Ly49 receptors on DN, but not on CD4+, NK1.1+ T cells in spleen and liver. Absence of the specific MHC class I ligand in the host was associated with elevated levels of expression and, to a greater extent than has been found for NK cells, increased the frequencies of Ly49‐positive cells within the DN subset, while CD4+ NK1.1+ cells remained negative. In the thymus and bone marrow both NK1.1+ T cell subsets contained high frequencies of Ly49‐positive cells. Results from in vitro stimulation of DN NKT cells further suggest that activation and expansion of NKT cell subsets are regulated by the Ly49 receptors.

The complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant α-chain paired with β-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like α-galactosylceramide (α-GalCer). iNKT cells can be visualized with α-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-γ secretion after α-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes α-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to α-GalCer presented by rat or mouse CD1d and efficiently bound α-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to α-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent α-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Vα14) α-chains and wild-type or mutated BV8S2 (Vβ8.2) β-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.

MHC‐dependent and ‐independent modulation of endogenous Ly49 receptors on NK1.1+ T lymphocytes directed by T‐cell receptor type

Natural killer (NK) T lymphocytes are thought to act as regulatory cells directing early events during immune responses. Murine NKT cells express inhibitory receptors of the Ly49 family. These receptors have a well‐established and crucial role in modulating NK cell activities, but their physiological role in regulating NKT cells is not well understood, nor is the influence of major histocompatibility (MHC) ligands on endogenous Ly49 expression. We have further investigated how the expression of inhibitory NK receptors is regulated on NKT cells, and demonstrate a non‐random expression of ligated Ly49 molecules on CD1d‐restricted NKT cells. The nature of the T‐cell receptor on the NKT cell crucially determines the profile of expressed Ly49 isoforms. Further, we show that MHC class I ligands efficiently modulate the expression levels of the inhibitory receptors, and the frequencies of cells positive for the Ly49 members. In addition, we find a several‐fold increase in Ly49C/I‐expressing NKT cells in adult thymus, apparently independent of MHC class I molecules. Abundant expression of Ly49 receptors on NKT cells, and the striking differences found in Ly49 isoform patterns on NKT‐cell subsets differing in T‐cell receptor expression, suggest that the pattern of Ly49 expression is tuned to fit the T‐cell receptor and to emphasize further a role for these receptors in NKT immunity.