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.     

Liver autoimmunity triggered by microbial activation of natural killer T cells

Humans with primary biliary cirrhosis (PBC), a disease characterized by the destruction of small bile ducts, exhibit signature autoantibodies against mitochondrial Pyruvate Dehydrogenase Complex E2 (PDC-E2) that crossreact onto the homologous enzyme of Novosphingobium aromaticivorans, an ubiquitous alphaproteobacterium. Here, we show that infection of mice with N. aromaticivorans induced signature antibodies against microbial PDC-E2 and its mitochondrial counterpart but also triggered chronic T cell-mediated autoimmunity against small bile ducts. Disease induction required NKT cells, which specifically respond to N. aromaticivorans cell wall alpha-glycuronosylceramides presented by CD1d molecules. Combined with the natural liver tropism of NKT cells, the accumulation of N. aromaticivorans in the liver likely explains the liver specificity of destructive responses. Once established, liver disease could be adoptively transferred by T cells independently of NKT cells and microbes, illustrating the importance of early microbial activation of NKT cells in the initiation of autonomous, organ-specific autoimmunity.     

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

Background  

Invariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Anti-HIV activity mediated by natural killer and CD8+ cells after toll-like receptor 7/8 triggering

We previously found that triggering TLR7/8 either by single stranded HIV RNA or synthetic compounds induced changes in the lymphoid microenvironment unfavorable to HIV. In this study, we used selective TLR7 and 8 agonists to dissect their contribution to the anti-HIV effects. While triggering TLR7 inhibited efficiently HIV replication in lymphoid suspension cells from tonsillar origin, its effect was inconsistent in peripheral blood mononuclear cells (PBMC). In contrast, triggering TLR8 showed a very prominent and overall very consistent effect in PBMC and tonsillar lymphoid suspension cells. Depletion of dendritic cells (DC), Natural killer cells (NK) and CD8+ T-cells from PBMC resulted in the reversal of TLR8 induced anti-HIV effects. Especially noteworthy, depletion of either NK or CD8+ T-cells alone was only partially effective. We interpret these findings that DC are the initiator of complex changes in the microenvironment that culminates in the anti-HIV active NK and CD8+ effector cells. The near lack of NK and the low number of CD8+ T-cells in tonsillar lymphoid suspension cells may explain the lower TLR8 agonist's anti-HIV effects in that tissue. However, additional cell-type specific differences must exist since the TLR7 agonists had a very strong inhibitory effect in tonsillar lymphoid suspension cells. Separation of effector from the CD4+ target cells did not abolish the anti-HIV effects pointing to the critical role of soluble factors. Triggering TLR7 or 8 were accompanied by major changes in the cytokine milieu; however, it appeared that not a single soluble factor could be assigned for the potent effects. These results delineate the complex effects of triggering TLR7/8 for an efficient antiviral defense. While the ultimate mechanism(s) remains unknown, the potent effects described may have therapeutic value for treating chronic viral diseases. Notably, HIV replication is blocked by TLR triggering before HIV integrates into the host chromosome which would prevent the establishment or maintenance of the latent reservoir.     

Similarities and distinctions between murine natural killer cells and lymphokine-activated killer cells

Pretreatment of mice with rabbit anti-asialo GM1 removes both natural killer (NK) effector cells and NK cells responsive to interleukin 2 (IL-2). Spleen cells from these mice do possess normal lymphokine-activated killer (LAK) activity. Young mice (less than 3 weeks of age) do not have NK activity and do not possess IL-2-inducible NK effector cells. Similarly to anti-asialo GM1-treated mice, LAK cells can be generated from these mice. While these experiments indicate clear distinctions between a certain level of NK and LAK precursors, the distinctions are not as clear when analyzing mice congenitally deficient in NK cells. Beige mice which lack NK effector cells and IL-2-inducible NK cells also lack the ability to generate LAK cells. The relationships and differences between NK- and LAK-cell precursors and effectors are discussed.     

Deficient invariant natural killer T cells had impaired regulation on osteoclastogenesis in myeloma bone disease

Recent research showed that invariant natural killer T (iNKT) cells take part in the regulation of osteoclastogenesis. While the role of iNKT cells in myeloma bone disease (MBD) remains unclear. In our study, the quantity of iNKT cells and the levels of cytokines produced by them were measured by flow cytometry. iNKT cells and osteoclasts were induced from peripheral blood mononuclear cells after activation by α‐GalCer or RANKL in vitro. Then, gene expressions and the levels of cytokines were determined by RT‐PCR and ELISA, respectively. The results showed that the quantity of iNKT and production of IFN‐γ by iNKT cells were significantly decreased in newly diagnosed MM (NDMM), and both negatively related with severity of bone disease. Then, the osteoclasts from healthy controls were cultured in vitro and were found to be down‐regulated after α‐GalCer‐stimulated, while there was no significant change with or without α‐GalCer in NDMM patients, indicating that the regulation of osteoclastogenesis by iNKT cells was impaired. Furthermore, the inhibition of osteoclastogenesis by iNKT cells was regulated by IFN‐γ production, which down‐regulated osteoclast‐associated genes. In conclusion, the role of α‐GalCer‐stimulated iNKT cells in regulation of osteoclastogenesis was impaired in MBD, as a result of iNKT cell dysfunction.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Adhesion and activation molecules expressed by human natural killer cells

Our study concerns the expression and the regulation of adhesion and activation receptors on human NK cells. In particular we provide evidence on: a) the expression on fresh human NK cells of VLA-4, VLA-5 and VLA-6, extracellular matrix (ECM) receptors of integrin family capable of mediating their adhesion to FN and LM; b) the role of PKC on the regulation of CD16, a differentiation antigen associated with FcR type III expressed by all NK cells, which mediate ADCC activity and trigger lymphokine production.     

Non-reducing end alpha-mannosylated glycolipids as potent activators for invariant Valpha19 TCR-bearing natural killer T cells

A novel invariant Valpha19-Jalpha33 T cell receptor alpha chain, first found in mammalian blood cells, was primarily expressed by natural killer T cell repertoire (Valpha19 NKT cell). Attempts have been made to find specific antigens for Valpha19 NKT cells. A series of alpha- and beta-glycosyl ceramides were synthesized and tested whether they had potential to stimulate the cells isolated from invariant Valpha19-Jalpha33 TCR transgenic mice (where the development of Valpha19 NKT cells is facilitated). Comprehensive examinations revealed substantial antigenic activity in alpha-ManCer that was presented by MR1, one of the MHC class Ib molecules. Next, naturally occurring and synthetic alpha-mannosyl glycolipids were further analyzed to determine structural requirements for natural ligands for Valpha19 NKT cells. As a result, alpha-mannosyl phosphatidyl inositols (PI) such as (alpha-Man)(2)-PI and alpha-Man-alpha-GlcNH(2)-PI (a partial structure of mycobacterial lipoarabinomannan and GPI-anchors) as well as alpha-ManCer derivatives were found to activate Valpha19 NKT cells in vivo and in vitro. Thus, Valpha19 NKT cells are possibly responsive to certain alpha-mannosyl glycolipids and may have roles in the innate and adaptative immune systems to protect against various antigens expressing alpha-mannosyl glycolipids and to regulate the adaptive immune system responding to the intracellular ligands.     

Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection

The invariant (i) natural killer (NK)T cells consistently express the Valpha14 chain of the T cell receptor (TCR) and recognize alpha-galactosylceramide (alpha-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1(-) iNKT cell population contracted, whereas NK1.1(+) iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following alpha-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1(-) iNKT cells from NK1.1(+) cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.     

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.     

An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells

The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.     

Natural killer and natural killer T cells in liver fibrosis

The liver lymphocyte population is enriched with natural killer (NK) cells, which play a key role in host defense against viral infection and tumor transformation. Recent evidence from animal models suggests that NK cells also play an important role in inhibiting liver fibrosis by selectively killing early or senescence activated hepatic stellate cells (HSCs) and by producing the anti-fibrotic cytokine IFN-γ. Furthermore, clinical studies have revealed that human NK cells can kill primary human HSCs and that the ability of NK cells from HCV patients to kill HSCs is enhanced and correlates inversely with the stages of liver fibrosis. IFN-α treatment enhances, while other factors (e.g., alcohol, TGF-β) attenuate, the cytotoxicity of NK cells against HSCs, thereby differentially regulating liver fibrogenesis. In addition, the mouse liver lymphocyte population is also enriched for natural killer T (NKT) cells, whereas human liver lymphocytes have a much lower percentage of NKT cells. Many studies suggest that NKT cells promote liver fibrogenesis by producing pro-fibrotic cytokines such as IL-4, IL-13, hedgehog ligands, and osteopontin; however, NKT cells may also attenuate liver fibrosis under certain conditions by killing HSCs and by producing IFN-γ. Finally, the potential for NK and NKT cells to be used as therapeutic targets for anti-fibrotic therapy is discussed. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Low numbers and altered phenotype of invariant natural killer T cells in recurrent varicella zoster virus infection

Invariant natural killer T (iNKT) cells play an important role in the immune response against various infectious agents. In this study we investigated their role in human defense against the varicella zoster virus. We observed decreased numbers of iNKT cells in patients who failed to control latent varicella zoster virus infection, e.g. underwent several reactivations of the virus. The residual population of iNKT cells expressed significantly higher levels of inhibitory receptor CD158a that was further up-regulated in the course of acute viral infection. Both of these abnormalities might contribute to impaired control of varicella zoster virus in human.     

Induction of activation antigens on human natural killer cells mediated through the Fc-gamma receptor

NK cells, defined here as lymphocytes bearing the CD16 Ag found on the NK cell Fc-gamma receptor (FcR), are known to enter a proliferative and activated state in response to stimulation with IL-2 as assessed by clonal expansion, short-term DNA synthesis, and de novo expression of lymphocyte-associated activation Ag. We have found that the FcR of NK cells acts as a signaling pathway through which IL-2-dependent activation may be greatly enhanced, allowing for more rapid induction of activation Ag and recruitment of an increased percentage of cells expressing surface markers of cellular activation. FcR-interactive agents, such as solid phase immobilized immune complexes or cross-linked CD16-specific mAb, work synergistically with rIL-2 to elicit a rapid expression of IL2R and transferrin receptors on greater than 50% of purified NK cells as early as day 3 of culture. IL-2 or FcR-interactive stimuli alone were weak or ineffective stimulators by comparison. In contrast to the induction of de novo activation Ag, DNA synthesis was elicited by IL-2 alone, but was not substantially or consistently enhanced by the subsequent addition of FcR-interactive stimuli.