Chromosome 17/17q gain and unaltered profiles in high resolution array-CGH are prognostically informative in neuroblastoma.

The prognostic relevance of chromosome 17 gain in neuroblastoma is still discussed. This investigation specifies the frequency, type, size, and transcriptional relevance in a large patient cohort. Primary tumor material of 202 patients was analyzed using high-resolution oligonucleotide array-based comparative genomic hybridization (aCGH) and correlated with clinical and survival data. A subset (n = 145) was correlated for differentially expressed genes (DEG) by microarray analysis. Chromosome 17 aCGH analysis showed numerical gain in 94/202 patients (47%), partial gain in 93/202 patients (46%), and no gain in 15/202 patients (7%). The frequency of partial gain was higher in stage 4 neuroblastoma (stage 1 15%; stage 2 12%; stage 3 16%; stage 4S 7%; and stage 4 50%). Overall survival (OS) was superior in patients with numerical gain compared with patients with partial gain or no gain (5-y-OS: 0.95 ± 0.02 vs. 0.63 ± 0.05 vs. 0.60 ± 0.13; P < 0.001). Gene expression analysis demonstrated 95/130 DEGs between tumors with numerical or partial chromosome/no gain. Only one DEG (CCKBR) was detected comparing tumors with partial gain and those with no gain. In patients with partial gain, the distribution of breakpoints did not correlate with stage and 11q status, but with MYCN amplification and 1p status. The "best" breakpoints in cases with partial 17q gain were at 42.5 Mb for event-free and 26.6 Mb for OS. Numerical gain of chromosome 17 is associated with a better prognosis than partial and no gain. The group of tumors with partial gain was similar to the group without gain with respect to stage distribution, outcome, and gene expression profile.

Nitric oxide stress in sporadic inclusion body myositis muscle fibres: inhibition of inducible nitric oxide synthase prevents interleukin-1ß-induced accumulation of ß-amyloid and cell death.

Sporadic inclusion body myositis is a severely disabling myopathy. The design of effective treatment strategies is hampered by insufficient understanding of the complex disease pathology. Particularly, the nature of interrelationships between inflammatory and degenerative pathomechanisms in sporadic inclusion body myositis has remained elusive. In Alzheimer's dementia, accumulation of ß-amyloid has been shown to be associated with upregulation of nitric oxide. Using quantitative polymerase chain reaction, an overexpression of inducible nitric oxide synthase was observed in five out of ten patients with sporadic inclusion body myositis, two of eleven with dermatomyositis, three of eight with polymyositis, two of nine with muscular dystrophy and two of ten non-myopathic controls. Immunohistochemistry confirmed protein expression of inducible nitric oxide synthase and demonstrated intracellular nitration of tyrosine, an indicator for intra-fibre production of nitric oxide, in sporadic inclusion body myositis muscle samples, but much less in dermatomyositis or polymyositis, hardly in dystrophic muscle and not in non-myopathic controls. Using fluorescent double-labelling immunohistochemistry, a significant co-localization was observed in sporadic inclusion body myositis muscle between ß-amyloid, thioflavine-S and nitrotyrosine. In primary cultures of human myotubes and in myoblasts, exposure to interleukin-1ß in combination with interferon-γ induced a robust upregulation of inducible nitric oxide synthase messenger RNA. Using fluorescent detectors of reactive oxygen species and nitric oxide, dichlorofluorescein and diaminofluorescein, respectively, flow cytometry revealed that interleukin-1ß combined with interferon-γ induced intracellular production of nitric oxide, which was associated with necrotic cell death in muscle cells. Intracellular nitration of tyrosine was noted, which partly co-localized with amyloid precursor protein, but not with desmin. Pharmacological inhibition of inducible nitric oxide synthase by 1400W reduced intracellular production of nitric oxide and prevented accumulation of ß-amyloid, nitration of tyrosine as well as cell death inflicted by interleukin-1ß combined with interferon-γ. Collectively, these data suggest that, in skeletal muscle, inducible nitric oxide synthase is a central component of interactions between interleukin-1ß and ß-amyloid, two of the most relevant molecules in sporadic inclusion body myositis. The data further our understanding of the pathology of sporadic inclusion body myositis and may point to novel treatment strategies.

CD28 costimulation Impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation.

Adoptive T-cell transfer showed promising efficacy in recent trials raising interest in T cells with redirected specificity against tumors. T cells were engineered with a chimeric antigen receptor (CAR) with predefined binding and CD3ζ signaling to initiate T-cell activation. CD28 costimulation provided by a CD28-CD3ζ signaling CAR moreover improved T cell activation and persistence; however, it failed to meet the expectations with respect to mounting attacks against solid tumors infiltrated with regulatory T (Treg) cells. We revealed that a CD28 CAR-redirected T-cell attack is accompanied by higher numbers of Treg cells infiltrating the tumor and is less efficient against cancer cells in presence of Treg cells than a CD3ζ CAR T-cell attack. Deletion of the lck binding moiety in the CD28 CAR endodomain, however, improved redirected anti-tumor activity in presence of Treg cells without impairing interferon-γ (IFN-γ) secretion, proliferation, and cytolysis. CD28 modification abrogated interleukin-2 (IL-2) induction upon CAR engagement which in turn is no longer available to sustain Treg cell persistence. CARs with the modified CD28 endodomain thereby expedite the implementation of adoptive T-cell therapy in patients with a variety of cancer types that are heavily infiltrated by Treg cells.

Rational development of high-affinity T-cell receptor-like antibodies.

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Leukemia inhibitory factor deficiency modulates the immune response and limits autoimmune demyelination: a new role for neurotrophic cytokines in neuroinflammation.

The neurotrophic cytokines ciliary neurotrophic factor and leukemia inhibitory factor (LIF) play a key role in neuronal and oligodendrocyte survival and as protective factors in neuroinflammation. To further elucidate the potential of endogenous LIF in modulating neuroinflammation, we studied myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LIF knockout mice (LIF(-/-) mice). In the late phase of active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, LIF(-/-) mice exhibited a markedly milder disease course. The inflammatory infiltrate in LIF(-/-) mice was characterized by an increase in neutrophilic granulocytes early and fewer infiltrating macrophages associated with less demyelination later in the disease. In good correlation with an effect of endogenous LIF on the immune response, we found an Ag-specific T cell-priming defect with impaired IFN-gamma production in LIF(-/-) mice. On the molecular level, the altered recruitment of inflammatory cells is associated with distinct patterns of chemokine production in LIF(-/-) mice with an increase of CXCL1 early and a decrease of CCL2, CCL3, and CXCL10 later in the disease. These data reveal that endogenous LIF is an immunologically active molecule in neuroinflammation. This establishes a link between LIF and the immune system which was not observed in the ciliary neurotrophic factor knockout mouse.

Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2.

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4(+)C25(+) Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4(+)CD25(-) T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4(+)CD25(-) T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.