Antigen targeted immunotherapies might represent a novel treatment for B-cell chronic lymphocytic leukemia (CLL).Differential expression of molecules in CLL might define both prognostic markers and suitable targets for immunotherapy.We assessed the expression of RHAMM as well as RHAMM splicing variants in series of 72 CLL patients.Quantitative RT-PCR revealed higher RHAMM expression in high-risk CLL patients as well as in advanced stages of the disease.CLL cases with a higher RHAMM expression showed a significantly shorter median treatment-free survival.Among patients with mutated IgVH genes, an analysis of RHAMMexpression enabled to distinguish subgroup of patients with favorable prognosis.In lymph nodes, RHAMM staining correlated with a higher Ki-67 index and CD40L expression.Functionally, stimulation with CD40L enhanced RHAMMexpression in CLL.Because of the exquisite tissue expression of RHAMM and its high expression frequency in CLL patients, mixed lymphocyte peptide culture (MLPC), enzyme-linked immunosorbent spot (ELISpot) and flow cytometry were performed for antigen-specific T cells.CD8+ T cells primed with the RHAMM-derived epitope R3, which is restricted by HLA-A2, effectively lyse RHAMM+ CLL cells.Therefore, we initiated a phase Ⅰ clinical trial of R3 peptide vaccination.Six HLA-A2+ CLL patients were vaccinated four times at biweekly intervals with the R3 peptide (ILSLELMKL; 300μg/dose) emulsified in incomplete Freunds adjuvant; GM-CSF (100μg/dose) was administered concomitantly.Detailed immunological analyses were conducted throughout the course of peptide vaccination.No severe adverse events greater than CTC 1° skin toxicity were observed.Four patients exhibited reduced white blood cell counts during vaccination.In 5/6 patients, R3-specific CD8+ T cells were detected with the corresponding peptide/HLA-A2 tetrameric complex; these populations were verified functionally in 4/5 patients using ELISpot assays.In patients with clinical responses, we found increased frequencies of R3-specific CD8+ T cells that expressed high levels of CD107a and produced both IFN-γ and granzyme B in response to antigen challenge.Interestingly, vaccination was also associated with the induction of regulatory T cells in four patients.In conclusion, RHAMM expression appears to be of prognostic value as well as might reflect the proliferative capacity of CLL cells and might therefore represent interesting target for immunotherapy.Peptide vaccination in CLL patients is safe and can elicit specific CD8+ T-cell responses against the tumor antigen RHAMM.