Markus Sköld

Role of CD1d-Restricted NKT Cells in Microbial Immunity

The discovery that T cells recognize lipid and glycolipid molecules presented by CD1 proteins has greatly expanded the number of potential microbial antigens targeted by the immune system following infection. The ability of CD1d-restricted NKT cells to activate innate and adaptive immune responses has led to the idea that these cells can modulate immunity to infectious agents. In addition, CD1d-restricted NKT cells may directly contribute to host resistance as they express a variety of effector molecules that could mediate an antimicrobial effect. Although much has been learned about CD1d-restricted NKT cells through the use of the synthetic antigen α-galactosylceramide (αGalCer), the field has been hampered by the paucity of information about the physiological self and microbial lipid antigens that can be presented by CD1d. Here we review the literature stating that CD1d-restricted NKT cells contribute to host defense against microbial pathogens.

In Vivo Depletion of CD11c+ Cells Delays the CD4+ T Cell Response to Mycobacterium tuberculosis and Exacerbates the Outcome of Infection

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.

Structural Features of the Acyl Chain Determine Self-phospholipid Antigen Recognition by a CD1d-restricted Invariant NKT (iNKT) Cell

Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0–18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.

Interplay of Cytokines and Microbial Signals in Regulation of CD1d Expression and NKT Cell Activation

In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-γ alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-γ and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-γ-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.

Tuberculosis Triggers a Tissue-Dependent Program of Differentiation and Acquisition of Effector Functions by Circulating Monocytes

The origin and function of the different myeloid cell subsets that appear in the lung during pulmonary tuberculosis are unknown. Herein we show that adoptively transferred monocytes give rise to many of the macrophage and dendritic cell (DC) subsets that appear following aerosol infection with virulent Mycobacterium tuberculosis. Monocyte differentiation in infected peripheral tissue is surprisingly heterogeneous and results in the formation of five distinct myeloid subsets, including both classically activated macrophages, that produce inducible NO synthase via an IFN-γ-dependent mechanism, and DC. In contrast, monocytes recruited to draining pulmonary lymph nodes are functionally different and acquire a mature DC phenotype. Thus, while monocytes are recruited to the lungs of uninfected mice, their differentiation and acquisition of myeloid effector functions are dramatically altered in the presence of inflammation and bacteria and are dependent on tissue localization. Therefore, our results support a model in which recruited monocytes are well poised to influence multiple aspects of host immunity to infections in the lungs. This report provides the first direct evidence for monocyte differentiation into both the macrophage and DC lineages in vivo following infection with a live human pathogen.

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.

Synovial fibroblasts self‐direct multicellular lining architecture and synthetic function in three‐dimensional organ culture

OBJECTIVE To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. METHODS FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. RESULTS FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo-lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. CONCLUSION This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.

α-Galactosylceramide as a Therapeutic Agent for Pulmonary Mycobacterium tuberculosis Infection

RATIONALE Invariant natural killer T (iNKT) cells are a unique subset of T cells that recognize lipid antigens presented by CD1d molecules. Recent studies have shown that iNKT cells can protect mice against Mycobacterium tuberculosis (Mtb) infection. We sought to determine whether pharmacological activation of iNKT cells by α-galactosylceramide (α-GalCer) could be used to treat tuberculosis (TB). OBJECTIVES We hypothesized that α-GalCer, either alone or in combination with isoniazid, could be used to treat pulmonary TB. METHODS The ability of α-GalCer-activated iNKT cells to suppress Mtb replication was evaluated using an in vitro coculture system. To test its potency in vivo, mice infected with virulent Mtb were treated with α-GalCer alone or in combination with isoniazid. MEASUREMENTS AND MAIN RESULTS Quantitative colony-forming unit counts were compared for both experimental systems. Our results show that α-GalCer plus isoniazid controls bacterial growth better than α-GalCer or INH alone, and single or multiple α-GalCer administrations prolong the survival of the mice infected via the aerosol route. CONCLUSIONS Our results demonstrate that α-GalCer administration can improve the outcome of Mtb infection, even when transmitted by the aerosol route. However, a combination of isoniazid and α-GalCer treatment has a synergistic effect on infection control. We conclude that more efficient treatment of TB will be achieved through a combination of classic chemotherapy and modulation of the host immune response.

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‐NK1.1b 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‐γ production, and surface expression of CD49b but not CD69. Moreover, in the thymus but not the spleen of BALB.B6‐NK1.1b mice we find a novel Vα14‐Jα18 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 Vβ‐repertoire.