Introduction  6 9 7 15 17 3 22 5 + + 2 13 18 25 + 18 25 + 19 20 24 + 1 + + Materials and methods Patients 12 1 21 21 Table 1 Patient characteristics at the time of study entry  Hu-PNS  SCLC  N 43 31 Age (median, range) a 61 (40–83) Gender (M/F) 15/28 21/10 Hu-Ab titre (median, range) 12,800 (400–204,800) Negative CMV serostatus (pos/neg) 29/14 17/14 Paraneoplastic neurological syndrome  NA  PSN 27   PEM 5   PCD 4   PLE/ BE 3   Pseudo-obstruction 2   Motor neuron disease 2  Tumour  No tumour b 0  SCLC 31 31   Limited 28 17   Extended 3 14  NSCLC 2 0  Prostate 1 0  Neuroblastoma 1 0 Prior treatment  None 27 27  Chemotherapy ± immunosuppression 16 4 Neurological symptoms  NA  Interval onset symptoms (study entry) c   Interval onset symptoms (diagnosis) c  d 34 (79%)  Modified Rankin score  NA  MRS = 2 7   MRS = 3 22   MRS = 4 10   MRS = 5 4  Hu-Ab CMV pos neg NA PSN PEM PCD PLE BE SCLC NSCLC MRS a  b  c  d  Reagents 19 20 147 155 245 254 86 95 248 256 315 323 362 370 154 163 495 503 To measure general T-cell responsiveness, we used phorbol myristate acetate (PMA) plus ionomycin, or phytohemagglutinin (PHA). A peptide pool containing 15-mers spanning CMV-pp65 (JPT) was used as positive- and negative antigen-specific control in CMV seropositive and seronegative individuals, respectively. Cytokine flow cytometry 14 6 2 14 + + − + + + + Detection of secreted cytokines The secretion levels of IFN-γ, TNF-α, interleukin (IL)-2, IL-4, IL-5 and IL-10 were measured in supernatants using a cytometric bead array (BD Biosciences). Based on CMV data (not shown), a positive result was defined as cytokine concentration >2 times the background (no antigen) and a minimum level of 50 pg/ml. IFN-γ ELISPOT 5 2 10 10 + 11 5 + + + Results Cytokine flow cytometry of PBMC after stimulation with HuD-derived peptides + + 1 1 Fig. 1 + panel a + panel b panel c Horizontal lines panel d 5 panels a–d NEG HuDmix PP65 IL TNF IFN SFC PHA  ELE  RLD  SLG  NLL  QLF  RLG  QYG  IFNγ-ELISPOT assay on PBMC stimulated with HuD-derived 9-mers 20 1 + + +  + 2 Fig. 2 + panel a panel b panel c + 12 panels a–c horizontal axes + + + +  Discussion We set out to detect HuD-specific T cells in the blood of Hu-PNS patients as they are postulated to play a pivotal role in the immunopathology of this disease. Although we applied three different approaches we could not detect circulating HuD-specific T cells. + + 14 +  + + + + 1 1 1 20 20 20 + 20 +  18 + 23 4 16 + 1 13 +  + 26 8