Mark L. Lang

Glycolipid-activated NKT cells support the induction of persistent plasma cell responses and antibody titers

NKT cell activation with CD1d‐binding glycolipid α‐galactosylceramide (α‐GC) enhances antibody responses to co‐administered T‐dependent antigen. The efficacy of α‐GC relative to other CD1d‐binding glycolipids and adjuvants is not known. There is little information on how NKT cells affect antibody production beyond initial booster‐stimulated recall responses. We therefore tested the hypothesis that α‐GC stimulates induction of plasma cells and antibody responses as effectively as Th1‐ and Th2‐skewing variants of α‐GC and several other adjuvants. C57BL/6 and CD1d–/– mice were immunized with nitrophenol‐conjugated keyhole limpet hemocyanin (NP‐KLH) plus α‐GC or NP‐KLH plus adjuvants before administration of an NP‐KLH booster and assessing antibody responses and plasma cell frequency. α‐GC boosted long‐term antibody responses as efficiently as all other agents tested and induced plasma cells that were detected in bone marrow 13u2004weeks after immunization. We then determined whether NKT cells were required in the presence of other adjuvants. CD1d–/– mice had a reduced induction of plasma cells in response to NP‐KLH/Alum as compared to C57BL/6 mice. However, NKT cells were not required for the continued presence of those cells that were induced. Although NKT cells are capable of inducing persistent plasma cell responses, they may not play a major role in supporting longevity post‐induction.

Bacillus anthracis lethal toxin disrupts TCR signaling in CD1d-restricted NKT cells leading to functional anergy.

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen.

Type II NKT cells facilitate Alum-sensing and humoral immunity

Alum‐based adjuvants facilitate vaccine‐driven humoral immunity, but their mechanism of action remains poorly understood. Herein, we report that lack of type II NKT cells is associated with intact, mature B cells but dampened humoral immunity following immunization with Alum‐adsorbed T‐dependent antigen. Type II NKT cells facilitated production of IL‐4, IL‐5, IL‐10, IL‐13, and antibody by LN and splenocyte cultures following Alum/antigen administration in vivo and antigen restimulation in vitro. Addition of IL‐4 and IL‐5 to type II NKT‐deficient cultures restored in vitro antibody production. Intracellular staining revealed that Alum‐primed type II NKT cells coordinated IL‐4 secretion by T cells. Alum did not significantly affect CD1d expression in vivo, but addition of CD1d‐blocking mAb diminished cytokine production and in vitro antibody production. Type II NKT cells therefore function as part of the Alum‐sensing apparatus and in a CD1d‐dependent manner, facilitate TH2‐driven humoral immunity. This may have important consequences for understanding the mechanism of action of Alum‐containing vaccines.

How do natural killer T cells help B cells

CD1d-restricted natural killer (NKT) cells are important contributors to antigen-specific antibody responses. There is, therefore, considerable interest in the design and implementation of strategies to appropriately activate NKT cells and boost vaccine-induced protective antibody responses. In order to achieve these goals, investigators are examining the mechanisms by which NKT cells enhance antibody responses. Although information is limited, it is now appreciated that both cognate and noncognate interactions between CD1d-expressing B cells and NKT cells drive enhanced antibody responses. NKT cells may provide B-cell help in the form of direct receptor-mediated interactions as well as by secretion of soluble effectors, including cytokines. In this article, we review the evidence in support of these mechanisms and discuss how they likely take place in the context of interactions of NKT cells with other cell types, such as dendritic cells and helper T cells. We also discuss the evidence that NKT cells affect discrete differentiation events in the multistep process by which a naive B cell experiences antigen and develops into a memory B cell or an antibody-secreting plasma cell. Since most information on NKT cells and humoral immunity has been derived from murine studies, we discuss what is known about human NKT cells and humoral immunity. We offer thoughts on whether the findings in murine systems will translate to humans.

BAFF- and APRIL-Dependent Maintenance of Antibody Titers after Immunization with T-Dependent Antigen and CD1d-Binding Ligand

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide–containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell–derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell–derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.

Regulation of Anthrax Toxin-Specific Antibody Titers by Natural Killer T Cell-Derived IL-4 and IFNγ

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4−/− mice and IFNγ−/− mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4−/− mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.

Reduction of CD1d expression in vivo minimally affects NKT-enhanced antibody production but boosts B-cell memory

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

Differential contribution of dendritic cell CD1d to NKT cell-enhanced humoral immunity and CD8+ T cell activation

CD1d‐restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T‐dependent, antigen‐derived peptide on MHC class II and CD1d‐binding glycolipid α‐GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d‐expressing, DTR‐transgenic DCs and CD1d+ or CD1d− nontransgenic DCs. Following DT‐mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d+ and CD1d− DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d+ DCs blocked in vivo expansion of antigen‐specific cytotoxic (CD8+) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT‐enhanced CD8+ T cell expansion, it is dispensable for specific antibody production.

Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL‐22 binding protein

High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patients possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag‐Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6‐weeks old Rag‐Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (Pu2009<u20090.0005) and increased colon tumors multiplicities by 84% (Pu2009<u20090.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (Pu2009<u20090.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)‐22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)‐γ systemically. Notably, IL‐22 binding protein (IL‐22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL‐22 BP.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids.