After completing this course, the reader will be able to:1. Describe the autoimmune pathogenesis of paraneoplastic neurological syndromes. Explain the clinical value of paraneoplastic antibody detection.3. Describe the general treatment approach to paraneoplastic neurological syndromes.

Objective:To determine the clinical features and presence in CSF of antineuronal antibodies in patients with pathologically proven autoimmune encephalitis derived from a cohort of patients with suspected Creutzfeldt-Jakob disease (CJD).Methods:The Dutch Surveillance Centre for Prion Diseases performed 384 autopsies on patients with suspected CJD over a 14-year period (1998–2011). Clinical information was collected from treating physicians. Antineuronal antibodies were tested in CSF obtained postmortem by immunohistochemistry on fresh frozen rat brain sections, by Luminex assay for the presence of well-characterized onconeural antibodies, and by cell-based assays for antibodies against NMDAR, GABABR1/2, GABAAR GLUR1/2, LGI1, Caspr2, and DPPX.Results:In 203 patients, a diagnosis of definite CJD was made, while in 181 a variety of other conditions were diagnosed, mainly neurodegenerative. In 22 of these 181, the neuropathologist diagnosed autoimmune encephalitis. One patient was excluded because of lack of clinical information. Inflammatory infiltrates were predominantly perivascular and consisted mainly of T cells. The predominant locations were basal ganglia and thalamus (90%) and temporal lobes and hippocampus (81%). In 6 patients (29%), antineuronal antibodies were detected in postmortem CSF, directed against Hu, NMDAR, GABABR1/2, Caspr2, and an unidentified synaptic antigen in 2. The most frequent symptoms were dementia (90%), gait disturbance (86%), cerebellar signs (67%), and neuropsychiatric symptoms (67%). Immunopathologic and clinical findings did not differ between autoantibody-negative patients and patients with antineuronal antibodies.Conclusions:It is important to consider immune-mediated disorders in the differential diagnosis of rapidly progressive neurologic deficits.

Comparison with previous studies suggests that hCG may have immunomodulatory activity and may modify the course of Hu-PNS, although well-established confounding factors may have contributed in this uncontrolled trial.

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did--and did not--elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays.

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AimIn paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies (Hu-PNS), Hu antigens expressed by the tumour hypothetically trigger an immune response that also reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized CD8+ T cell-mediated immune pathogenesis of these syndromes, we searched for circulating HuD-specific CD8+ T cells in a large cohort of Hu-PNS patients and controls.Patients and methodsBlood was tested from 43 Hu-PNS patients, 31 Hu antibody negative SCLC patients without PNS and 54 healthy controls. Peripheral blood mononuclear cells (PBMC) were stimulated with HuD protein-spanning peptide pools (15-mers) and individual HuD-derived peptides (9-mers) and analysed by cytokine flow cytometry and interferon-γ ELISPOT-assays. Additionally, HuD-based Class I HLA multimers were used to visualize HuD-specific CD8+ T cells.ResultsNo HuD-specific CD8+ T cells could be detected in the blood of Hu-PNS patients or controls.ConclusionsOur results do not support a role for HuD-specific CD8+ T cells in Hu-PNS. Further studies should focus on the detection of circulating HuD-specific CD4+ T cells and examine the antigen specificity of T cells in affected tissues.

To detect HuD-specific T cells in patients with Hu-antibody associated paraneoplastic neurological syndromes (Hu-PNS), we used short-term stimulation assays with HuD protein spanning peptide pools (PSPP) with purities of at least 70% and found reproducible false-positive CD81 T-cell responses in three of 127 individuals (two healthy controls and one Hu-PNS patient), which all shared HLA-A*2402 and HLA-B*1801. After testing the 15-mer peptides of the HuD antigen separately, we discovered that the same three 15-mers yielded the CD81 T cell response in those three individuals. This highly unusual result could not be reproduced when using new batches of peptides with a higher level of purity ([82% and [95%). Therefore, we assumed this response was not directed against the HuD peptides and analyzed the Key termsparaneoplastic neurological syndromes; Hu-antibodies; protein spanning peptide pools; T lymphocytes; mass spectrometry; CMV-encoded peptide; HLA-restriction PROTEIN spanning peptide pools (PSPP) are mixtures of overlapping (mostly 15-mer) peptides, that can efficiently stimulate both CD41 and CD81 T-cell responses and are valuable for the detection of T-cell responses against proteins (1,2). In most cases, pools consisting of peptides of 70% purity are used in screening studies for antigen-specific T cells (1), because the preparation of peptides of higher purity requires a significant investment. We used HuD PSPP of 15-mer peptides with purities of at least 70% in an earlier study to detect circulating HuD-specific T cells in patients with Hu-antibody associated paraneoplastic neurological syndromes (Hu-PNS) (3,4). HuD peptides were pooled and tested in short-term stimulation of peripheral blood mononuclear cells (PBMC) of Hu-PNS patients, small cell lung carcinoma (SCLC) patients and healthy controls (HC) followed by intracellular detection of interferon (IFN)-c (5). The results showed reproducible positive CD81 T-cell responses in only three of 127 individuals (two HC and one Hu-PNS patient), which all shared HLA-A*2402 and HLA-B*1801. Next, we tested the 15-mer peptides separately in the two responding HC (the Hu-PNS patient was no longer available) and showed that the same three 15-mers (peptide #38, #40, and #86) yielded the CD81 T-cell responses in both HC. Subsequently, two newly synthesized batches of those 15-mers with a higher purity ([82% and [93%, respectively) were tested and elicited no responses at all. Therefore, we concluded that the earlier, probably HLA-restricted,

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies (n = 18), SCLC without PNS (n = 19) and controls (n = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4+ and CD8+ T lymphocytes); (ii) increased proportions of activated CD4+ and CD8+ T cells; (iii) reduced numbers of terminally differentiated effector CD8+ T cells and cells with a cytotoxic T-cell phenotype (CD56+ and CD57+). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.