Irradiation of Nf1 mutant mouse models of spinal plexiform neurofibromas drives pathologic progression and decreases survival

Background: Genetically susceptible individuals can develop malignancies after irradiation of normal tissues. In the context of therapeutic irradiation, it is not known whether irradiating benign neoplasms in susceptible individuals promotes neoplastic transformation and worse clinical outcomes. Individuals with Neurofibromatosis 1 (NF1) are susceptible to both radiation-induced second malignancies and spontaneous progression of plexiform neurofibromas (PNs) to malignant peripheral nerve sheath tumors (MPNSTs). The role of radiotherapy in the treatment of benign neoplasms such as PNs is unclear. Methods: To test whether radiotherapy promotes neoplastic progression of PNs and reduces overall survival, we administered spinal irradiation (SI) to conditional knockout mouse models of NF1-associated PNs in 2 germline contexts: Nf1 fllfl ; PostnCre + and Nf1 fl/- ; PostnCre + . Both genotypes develop extensive Nf1 null spinal PNs, modeling PNs in NF1 patients. A total of 101 mice were randomized to 0 Gy, 15 Gy (3 Gy × 5), or 30 Gy (3 Gy × 10) of spine-focused, fractionated SI and aged until signs of illness. Results: SI decreased survival in both Nf1 fllfl mice and Nf1 fl/- mice, with the worst overall survival occurring in Nf1 fl/- mice receiving 30 Gy. SI was also associated with increasing worrisome histologic features along the PN-MPNST continuum in PNs irradiated to higher radiation doses. Conclusions: This preclinical study provides experimental evidence that irradiation of pre-existing PNs reduces survival and may shift PNs to higher grade neoplasms

Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system

The Histone Deacetylase Inhibitor LBH589 (Panobinostat) Modulates the Crosstalk of Lymphocytes with Hodgkin Lymphoma Cell Lines

Epigenetic changes have been implicated in the malignant phenotype of Hodgkin Reed Sternberg (HRS) cells in Hodgkin lymphoma (HL), where HRS survival and proliferation depends on the microenvironment. The histone-deacetylase (HDAC) inhibitor LBH589 (panobinostat) showed clinical efficacy but its impact on the HRS microenvironment is unclear. Hence, we analysed the effects of LBH589 on lymphocytes and also potential combination therapies. In lymphocyte-target cell killing assays, LBH589-treatment triggered an enhanced lymphocyte-dependent lysis of HL cells despite of mild lymphocytopenic effects. In co-culture experiments of lymphocytes with HL cells, LBH589 suppressed the IFNgamma-release but increased the TNFalpha secretion. Recombinant TNFalpha boosted the lymphocyte-dependent lysis of HL target cells. In HL cell lines, LBH589 induced cell death, autophagy, and an increase of MICA/B that are ligands to natural killer cell receptors. The combination of LBH589 with Brentuximab Vedotin was inefficient due to down-regulation of CD30 as a target. Combination with gemcitabine revealed highly significant effects, suggesting a potential combination for future therapy. Based on these data we suggest that LBH589 favourably modulates the cytokine network and lymphocyte activity in the HL microenvironment

Interleukin-6 is essential for zwitterionic polysaccharide-mediated abscess formation

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4(+) T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4(+) T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4(+) T-cells and causes IL-17 expression by CD4(+) T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6- specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation

Oligoclonal CD4+ T Cells Promote Host Memory Immune Responses to Zwitterionic Polysaccharide of Streptococcus pneumoniae▿

Zwitterionic polysaccharides of the normal flora bacteria represent a novel class of antigens in that they correct systemic CD4+ T-cell deficiencies and direct lymphoid organogenesis during colonization of the host. Presentation of these polysaccharides to CD4+ T cells depends on major histocompatibility complex class II- and DM-dependent retrograde transport from lysosomes to the cell surface. Yet the phenotype and clonality of the immune response to the polysaccharide in the mature host immune system have not been studied. Using the zwitterionic capsular polysaccharide Sp1 of Streptococcus pneumoniae, a transient member of the bacterial flora, in an experimental mouse model of cellular immunity, we demonstrated the accumulation of TH1- and TH17-polarized CD4+ CD44high CD62low CD25− memory T cells. Subcutaneous immunization with Sp1 resulted in an increase of serum immunoglobulin G (IgG), predominantly of the IgG1 subclass, and suggested the presence of a humoral memory response to the polysaccharide. CD4+ T cells stimulated with polysaccharide in vitro and in vivo showed a nonrestricted pattern for the T-cell receptor (TCR) β-chain variable region, as demonstrated by semiquantitative reverse transcription-PCR and flow cytometry. Clonotype mapping of in vivo and in vitro polysaccharide-activated CD4+ T cells revealed clonotypic TCR transcripts. Taken together, the data show the induction of clonal expansion of CD4+ T cells by polysaccharides of commensal bacteria. Cellular and humoral memory host responses imply the ability of these polysaccharides to mediate the expansion of T cells via recognition within the CDR3 region of the TCR

LBH589 modulated lymphocyte secretion of cytokines.

<p>(<b>A</b>) ELISA for IFNgamma from supernatants of cultured cells. Left panel: PBMCs and L428 cells were cultured separately. L428 cells were co-cultured with PBMCs (middle panel) and CD3+ cells (right panel; t = 36 hours). In indirect co-cultures, cells were separated by the membrane. The ratios of effector to tumour cells were 1∶1 with PBMCs and 1∶2 with CD3+ cells. PBMC/L428 co-culture did not show significant results due to high intra-group variance. For CD3+ cells, a significant IFNgamma increase in direct co-culture compared with indirect co-culture (p = 0.0198) as well as a significant downregulation upon LBH589-treatment (p = 0.0192) was observed (two-way ANOVA analysis; solid lines indicate the comparison of indirect versus direct co-culture; dashed lines the effect of LBH589 [also in B) and C]). (<b>B</b>) ELISA for TNFalpha and settings like in A). For both, PBMC/L428 and CD3+/L428 co-cultures, the effect of indirect vs. direct co-culture was not significant (p = 0.6637 and p = 0.3187, respectively), however the LBH589 treatment increased the TNFalpha secretion significantly (middle and right subpanels; p = 0.0059 and 0.0006, respectively; two-way ANOVA analysis). (<b>C</b>) ELISA for TNFalpha release in lymphocyte/L428 co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each; and 1∶10 in NK cell co-cultures. LBH589 increased significantly the TNFalpha secretion in co-cultures of L428 with CD4+, CD8+ and NK cells (p = 0.0001, p = 0.0017 and p = 0.0253, respectively). The direct versus indirect co-culture method showed a significant impact on CD4+ (p = 0.0006) and CD8+ lymphocytes (p = 0.0328; two-way ANOVA analysis). The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures.</p

LBH589 enhanced cytotoxicity and worked synergistically with gemcitabine.

<p>(<b>A/B</b>) Killing assays in which target cells and PBMCs were pre-treated for 4 h with (<b>A</b>) 10 nM LBH or (<b>B</b>) 200 ng/ml TNFalpha. The panels show one representative experiment of three. The ratios of effector to target cells are indicated below the axis. In addition, the summary bar graph for the effector to target cell ratio 50∶1 of three independent experiments is given for both experimental settings (paired, two-sided t-test). (<b>C/D</b>) Combination regimens were tested with sublethal doses adjusted to each compound and cell line. L428 cells and L540 cells were treated with 10 nM LBH589 (LBH) and (<b>C</b>) Gemcitabine (GMZ; 500 ng/ml for L428 and 0.5 ng/ml for L540; n = 3) or (<b>D</b>) Everolismus (RAD; 5 µM for L428; 0.5 µM for L540; n = 3 each; paired, two-sided t-test).</p