Robin Winkler-Pickett

Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells

We analyzed interleukin (IL) 12 and IL-23 production by monocyte-derived dendritic cells (mono-DCs). Mycobacterium tuberculosis H37Rv and zymosan preferentially induced IL-23. IL-23 but not IL-12 was efficiently induced by the combination of nucleotide-binding oligodimerization domain and Toll-like receptor (TLR) 2 ligands, which mimics activation by M. tuberculosis, or by the human dectin-1 ligand β-glucan alone or in combination with TLR2 ligands, mimicking induction by zymosan. TLR2 ligands inhibited IL-12 and increased IL-23 production. DC priming with interferon (IFN) γ strongly increased IL-12 production, but was not required for IL-23 production and inhibited IL-23 production induced by β-glucan. The pattern of IL-12 and IL-23 induction was reflected in accumulation of the IL-12p35 and IL-23p19 transcripts, respectively, but not IL-12/23p40. Although IL-23, transforming growth factor β, and IL-6 contained in the supernatants of activated mono-DCs played a role in the induction of IL-17 by human CD4+ T cells, IL-1β, in combination with one or more of those factors, was required for IL-17 production, and its production determined the differential ability of the stimuli used to elicit mono-DCs to produce soluble factors directing IL-17 production. Thus, the differential ability of pathogens to induce antigen-presenting cells to produce cytokines regulates the immune response to infection.

Glucocorticoid amplifies IL‐2‐dependent expansion of functional FoxP3+CD4+CD25+ T regulatory cells in vivo and enhances their capacity to suppress EAE

IL‐2 is crucial for the production of CD4+CD25+ T regulatory (Treg) cells while important for the generation of effective T cell‐mediated immunity. How to exploit the capacity of IL‐2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL‐2‐mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL‐2‐mediated selective expansion of Treg cells. We show in this study that short‐term simultaneous administration of Dex and IL‐2 markedly expanded functional suppressive Foxp3+CD4+CD25+ T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4+CD25+ T cells failed to suppress the proliferation of CD4+CD25– T cells. Pretreatment with Dex/IL‐2 remarkably increased the proportion of CD4+FoxP3+ cells and partially restored the function of splenic CD4+CD25+ T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL‐2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft‐vs.‐host disease.

Dissociation of NKT Stimulation, Cytokine Induction, and NK Activation In Vivo by the Use of Distinct TCR-Binding Ceramides

NKT and NK cells are important immune regulatory cells. The only efficient means to selectively stimulate NKT cells in vivo is α-galactosylceramide (αGalCer). However, αGalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, β-galactosylceramide (βGalCer) (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies using CD1d tetramers loaded with βGalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared with αGalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, βGalCer (C12), in contrast to αGalCer, failed to increase NK cell size, number, and cytolytic activity. Also in contrast to αGalCer, βGalCer (C12) is a poor inducer of IFN-γ, TNF-α, GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, αGalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while βGalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show βGalCer (C12) can effectively discriminate between NKT- and NK-mediated responses in vivo. These results indicate the use of different TCR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.

Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells

We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4+CD25+ T cells in C57BL/6 mice with MOG33–55‐induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down‐regulating splenic CD4+CD25+ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4+CD25+ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA‐4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4+CD25+ T cells. This property of PTx was attributable to its ADP‐ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4+CD25+ T regulatory (Treg) cells in vitro, but did so in vivo, presumably due to an indirect effect. Although the exact molecular target of PTx that reduces Treg activity remains to be defined, our data suggests that alteration of both distribution and function of splenic immunocytes should play a role. This study concludes that an underlying cause for the immunological adjuvanticity of PTx is down‐regulation of Treg cell number and function.

Cellular and molecular mechanisms of IFN-gamma production induced by IL-2 and IL-12 in a human NK cell line.

Interferon‐γ (IFN‐γ) is an important immunoregulatory protein produced predominantly by T cells and large granular lymphocytes (LGL) in response to different extracellular signals. In particular, two interleukins (ELs), IL‐2 and IL‐12, have been shown to be potent inducers of IFN‐γ gene expression in both T cells and LGL. Although it has been reported that there are some T cell lines that produce IFN‐γ in response to IL‐2 and IL‐12 stimulation, there has as yet been no report of a natural killer (NK) cell line that responds in a similar manner. In this report we present evidence that the cell line NK3.3 derived from human NK cells, responds to both IL‐2 and IL‐12, as measured by increases in IFN‐γ and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) cytoplasmic mRNA and protein expression. In addition, when used together EL‐2 and IL‐12 synergized in the induction of IFN‐γ and GM‐CSF and this synergy was attributed to an increased accumulation and stability of the IFN‐γ and GM‐CSF mRNAs. To investigate the signaling pathways involved in the gene induction, five inhibitors, cyclosporin A (CsA), transforming growth factor‐β, cydoheximide, genistein, and staurosporine A, were used in analyzing the effects of IL‐2 and IL‐12 on NK3.3 cells. The results suggest that activation of protein kinase C, but not new protein synthesis, is required for IL‐2 induction of IFN‐γ and GM‐CSF cytoplasmic mRNA. In contrast, IL‐12 induction of IFN‐γ cytoplasmic mRNA appears to only partially depend on activation of protein kinase C. Furthermore, both transforming growth factor‐β and genistein, a tyrosine kinase inhibitor, could suppress IL‐2 and IL‐12 signaling but CsA was generally inactive. It also was observed that suppression of cytokine gene expression by these agents was independent of the inhibition of proliferation. In addition, IL‐2 but not IL‐12 induced nudear factors NF‐κB and API, and regulation of the nudear levels of these two DNA binding protein complexes is correlated with IFN‐γ and GM‐CSF gene expression. These data indicate that IL‐2 and IL‐12 may have distinct signaling pathways leading to the induction of IFN‐γ and GM‐CSF gene expression, and that the NK3.3 cell line may serve as a novel model for dissecting the biochemical and molecular events involved in these pathways. J. Leukoc. Biol. 58: 225–233; 1995.

Class I MHC-Binding Characteristics of the 129/J Ly49 Repertoire

The Ly49 family of NK cell receptors and its MHC-binding characteristics have only been well characterized in C57BL/6 (B6) mice. Previous studies have shown that 129/J mice express unique Ly49 genes that are not found in the B6 strain. Screening of a 129/J cDNA library led to the discovery of 10 distinct full-length Ly49-related coding sequences (Ly49e, g, i, o, p, r, s, t, u, and v). Although 129/J mice share identical class I MHC (Kb and Db) transcripts with B6 mice, only one Ly49 is identical in the two strains (Ly49E). In addition to the previously characterized Ly49P, two new activating Ly49 proteins were discovered, Ly49R and U. The MHC specificity of the total 129/J Ly49 repertoire was evaluated with soluble class I MHC tetramers and found to be distinct compared with the B6 Ly49 repertoire. Ly49V bound to many types of class I MHC, suggesting that Ly49V+ NK cells may monitor host cells for a global down-regulation in MHC levels. An activating receptor, Ly49R, was shown to bind soluble class I molecules to a moderate degree, a result not previously observed for other activating Ly49 proteins. Furthermore, tetramer-binding results were confirmed functionally with cytotoxicity assays using sorted 129/J NK cells. This study shows that the Ly49 repertoire and its MHC-binding characteristics can be very different among inbred mouse strains. Ly49 divergence should be considered when using 129-derived embryonic stem cells for the production of gene-targeted mice, especially when an immune or NK-derived phenotype is under scrutiny.

Comparative studies of CD3- and CD3+ CD56+ cells: examination of morphology, functions, T cell receptor rearrangement, and pore-forming protein expression.

Both CD3- and CD3+ CD56+ effector cells can mediate non-MHC-restricted lysis in the absence of activation. Previous studies have shown that both of these subsets can be augmented with IL-2. In the present study, we have examined further the phenotypic markers expressed on these cells as well as the functional capacities of these subsets, including LAK activity, cytokine expression, and pore-forming protein (PFP) production. In addition, these populations were analyzed for clonality by Southern blot analysis of the T cell receptor beta chain gene constant region. The CD3-, CD56+ and CD3+, CD56+ lymphocytes were quite similar in their phenotypic markers, although the CD3+, CD56+ lymphocytes lacked high levels of IL-2 receptor beta chain and did not express CD16. The CD3+, CD56+ lymphocytes mediated non-MHC-restricted lysis, but failed to express LAK activity or be induced by IL-2 to secrete IFN gamma, a characteristic of the CD3-, CD56+ lymphocytes. The T cell receptor beta chain gene pattern of the CD3+, CD56+ lymphocytes was characteristic of a polyclonal cell population. Of interest, both populations of cells appeared morphologically to be large granular lymphocytes that contain PFP in their cytoplasmic granules. Therefore these CD56+ subsets provide a new model to study several questions related to non-MHC-restricted target cell lysis, including the identification of novel receptors involved in target cell recognition and/or triggering as well as the biochemical pathways implicated in cellular lysis.

In Vivo Regulation of Experimental Autoimmune Encephalomyelitis by NK Cells: Alteration of Primary Adaptive Responses

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vβ usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.

Ly-49 receptor expression and functional analysis in multiple mouse strains

We present data on the strain distribution and functional characteristics of the Ly‐49 receptors A, C/I, D, and G2 on DX5+ natural killer (NK) cells. We have examined tyrosine phosphorylation of the Ly‐49 molecules, regulation of NK cytotoxic functions, and in vivo marrow rejection capability. The flow cytometry results demonstrate a diverse and complex pattern of expression of the Ly‐49 receptors in the 11 strains examined. The vast majority of NK cells express Ly‐49s, although some NK1.1+ CD3+ cells also express these receptors. The results of our functional analysis indicate that H‐2Dd was able to inhibit the function of Ly‐49G2+ NK cells, not only in B6 mice, but also by NK cells derived from several haplotypes. The examination of Ly‐49 receptor tyrosine phosphorylation, which is a biochemical measure of inhibitory function, was consistently observed in the 11 mouse strains examined. In contrast, analysis of Ly‐49D function suggests its expression appears to be more restricted and that H‐2Dd is an activating ligand for this receptor. In addition, the in vivo examination of both inhibitory (Ly‐49G2) and activating (Ly‐49D) receptors demonstrated regulatory roles of these class I binding receptors in marrow transplantation. J. Leukoc. Biol. 66: 512–520; 1999.

Aberrant DAP12 Signaling in the 129 Strain of Mice: Implications for the Analysis of Gene-Targeted Mice

NK cells are implicated in antiviral responses, bone marrow transplantation and tumor immunosurveillance. Their function is controlled, in part, through the Ly49 family of class I binding receptors. Inhibitory Ly49s suppress signaling, while activating Ly49s (i.e., Ly49D) activate NK cells via the DAP12 signaling chain. Activating Ly49 signaling has been studied primarily in C57BL/6 mice, however, 129 substrains are commonly used in gene-targeting experiments. In this study, we show that in contrast to C57BL/6 NK cells, cross-linking of DAP12-coupled receptors in 129/J mice induces phosphorylation of DAP12 but not calcium mobilization or cytokine production. Consistent with poor-activating Ly49 function, 129/J mice reject bone marrow less efficiently than C57BL/6 mice. Sequence analysis of receptors and DAP12 suggests no structural basis for inactivity, and both the 129/J and C57BL/6 receptors demonstrate normal function in a reconstituted receptor system. Most importantly, reconstitution of Ly49D in 129/J NK cells demonstrated that the signaling deficit is within the NK cells themselves. These unexpected findings bring into question any NK analysis of 129/J, 129Sv, or gene-targeted mice derived from these strains before complete backcrossing, and provide a possible explanation for the differences observed in the immune response of 129 mice in a variety of models.