Abstract. The status of the immune system of patients with B-cell chronic lymphocytic leukemia (B-CLL) is not yet sufficiently characterized. Clinically, B-CLL patients present immunodeficiency increasing along with disease progression and signs of autoimmunity. In the current study, we evaluated the expression of FOXP3 in CD4 + CD25 hi T regulatory lymphocytes (Treg) and their influence on immune response against tumor and viral antigens in the complex system of peripheral blood mononuclear cells. In 80 B-CLL patients, the frequency of Treg (CD4 + CD25 hi FOXP3 + ) cells was significantly higher in B-CLL patients when compared to healthy volunteers (HV) and increased with the progression of the disease (median: 8.24% in stage A, 11.24% in stage B and 12.57% in stage C according to the Binet classification). The frequency of Treg showed no correlation with prognostic markers such as ZAP-70, CD38 and HLA-G. Notably, Treg frequency correlated with serum levels of TNF (r 2 =0.45, p=0.001). T-cell immune responses against epitopes derived from the tumor-associated antigens survivin, fibromodulin and RHAMM as well as from the influenza matrix protein were evaluated. Functionally, higher frequencies of Treg correlated with decreased T-cell responses against viral and tumor antigens. In conclusion, we detected higher frequencies of Treg in B-CLL patients than in HV. Furthermore, Treg constitute the crucial mechanism of immunosuppression in B-CLL patients. IntroductionThe immune status of patients with B-cell chronic lymphocytic leukemia (B-CLL) is poorly characterized (1,2). Clinically, CLL patients present immunodeficiency, along with signs of autoimmunity (3). Several studies have aimed to characterize the immunity of B-CLL, however, results were ambiguous and difficult to interpret especially towards active immunotherapy for those patients (4). The graft vs. leukemia effect (5) as well as the 'spontaneous' remissions associated with an increased immunity against viral antigens (6) suggest that CLL cells may be targeted by a T cell-mediated immune response. Naturally occurring tumor-reactive T cells were found in some B-CLL patients (7). During disease progression, patients displayed signs of immunosuppression resulting in an impaired immune response against tumor-associated antigens (TAA) as well as against viral and bacterial antigens. Several immunosuppressive mechanisms allow CLL cells to escape from immunosurveillance. Recently, enhanced frequencies of CD4 + CD25 hi T regulatory cells (Treg) in B-CLL were reported by Beyer et al (8). Apart from CD4 + CD25 hi T cells, other immunomodulatory factors such as cytokines IL-10 and TGF-ß may suppress immune responses (9,10). Defects of immune response might be induced by a direct cell-cell contact suggesting the immunosuppressive role of the B-CLL cell itself that might be mediated by the expression of the non-classic human leukocyte antigen (HLA)-G protein (11). HLA-G released in its soluble (sHLA-G) form may also inhibit immune responses against TAA since sHLA-G in...

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Both ZAP-70 and CD38 expression were shown to predict the clinical course of the disease, while ZAP-70 expression appeared to be more predictive than CD38 expression and more relevant in defining the cases of B-CLL responsive or refractory to first line chemotherapy. A simultaneous evaluation of ZAP-70 and CD38 expression allowed distinguishing the patients groups with the most favourable prognosis as well as those with the worst. Taken together we recommend assessing both ZAP-70 and CD38 protein expression for the definition of prognostic subgroups in patients with B-CLL.

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Abstract. Antigen targeted immunotherapies might represent a novel treatment for B-cell chronic lymphocytic leukemia (B-CLL). We screened the mRNA expression of tumorassociated antigens (TAAs) from the literature (fibromodulin, survivin, OFA-iLRP, BAGE, G250, MAGE1, PRAME, proteinase, syntaxin, hTERT, WT-1) and TAAs defined previously by serological analysis of cDNA expression libraries from leukemic cells (PINCH, HSJ2, MAZ, MPP11, RHAMM/CD168, NY-Ren60). Peripheral blood mononuclear cells from 43 B-CLL patients and 20 healthy volunteers (HVs) were examined by conventional and quantitative RT-PCR. mRNA of RHAMM/CD168, fibromodulin, syntaxin and NYRen60 was expressed in 55-90%, and mRNA of HSJ2, MAZ and OFAiLRP was expressed in 90-100% of the patients. No expression of WT-1, hTERT, BAGE, G250, MAGE1 or survivin was observed. Low (2-20%) expression frequencies of MPP11, PINCH, PRAME and proteinase were detected. RHAMM/CD168, fibromodulin, PRAME and MPP11 showed expression in B-CLL patients, but not in HVs. Because of the exquisite tissue expression of RHAMM/CD168 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. In MLPC, RHAMM specific responses by CD8 + HLA-A2/R3tetramer + CCR7-CD45RA high effector T-cells were detected. RHAMM/CD168 might be a possible target for future immunotherapies in both ZAP-70(+) and ZAP-70(-) B-CLL patients.

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Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies.