Involvement Of Autoreactive T Lymphocytes In Pathogenesis Of Chronic Lymphocytic Leukemia (CLL): Specific T-Cell Immune Responses Against Autoantigens Recognized By CLL Cells

The etiology of chronic lymphocytic leukemia (CLL) has remained unknown but recently it has been speculated that antigens stimulation could be involved in the pathogenesis of CLL. Cytoskeletal proteins exposed on the surface of apoptotic cells could represent autoantigens involved in the development of CLL since monoclonal antibodies derived from CLL-specific B-cell receptors (BCR) recognized MYHIIA (non-muscle myosin heavy chain IIA), vimentin, and cofilin-1. Since the humoral immune response against these autoantigens could play a crucial role in pathogenesis of CLL we suspect that it might be accompanied by T-cell immune responses. We defined HLA-A2-restricted epitopes in silico using two independent computer prediction algorithms. Next 36 most immunogenic peptides derived from MYHIIA, cofilin-1 and vimentin were selected and synthesized. To determine whether peptides could bind to HLA-A2 also in vivo we performed cellular-peptide binding affinity assay utilizing T2 cell line. The expression of HLA-A2 was measured using flow cytometry with FITC-conjugated anti-HLA-A2 monoclonal antibodies. Fluorescence index (FI) for particular peptides was calculated as mean fluorescence intensity of HLA-A2 on T2 cells with peptide versus without peptide. We selected peptides with the highest FI M6 (KLKDVLLQV, FI=0.43), M1 (GLLRVISGV, FI=0.20), M2 (YLLEKSRAI, FI=0.16), M3 (VLMIKALEL, FI=0.15) derived from MYHIIA, V1 (SLQEEIAFL, FI=4.93) and V4 (ILLAELEQL, FI=1.49) derived from vimentin and C1 (ALYDATYET, FI=0.60) C7 (LQANCYEEV, FI=0.68), C11 (ILEEGKEIL FI=0.88) derived from cofilin-1. Mixed lymphocyte/peptide cultures (MLPC) were performed to define immunogenicity of synthesized peptides in vitro. CD8⁺ cells from HLA-A2 positive 15 CLL patients and 8 healthy volunteers were selected by magnetic-activated cell sorting. Afterwards, CD8^- antigen presenting cells were irradiated and pulsed for 2 hours with respective peptides derived from MYHIIA, vimentin and cofilin-1 or control peptide at a concentration of 10 μg/ml and without peptide for negative control. Cells were supplemented with IL-2 and IL-7 on day +1 and restimulated every week. At day +21 of MLPC, cells were harvested and evaluated for their specific cytotoxicity in ELISpot assays with peptides, with the unspecific positive control Pokeweed Mitogen (PWM), or without peptide, as negative control. The ELISpot assay was performed under the conditions recommended by the Cancer Immunotherapy Monitoring Panel. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 71%, vimentin in 83% and cofilin-1 in 50% CLL patients. Additionally, we found positive responses in 37.5% healthy volunteers. Finally, we assessed the frequency of the specific T cells in 14 CLL patients along with detailed phenotypical characteristics using dextrameric peptide-specific complexes in 7-colour panel using FACSCantoII Flow Cytometer ex vivo. To characterize naïve/effector/effector-memory and memory cells were stained for CD8, CD3, CCR7, CD45RA with exclusion of CD19+, CD56+ and CD4+ cells and 7AAD+ dead cells. We detected low frequency of peptide-specific T cells, with the highest level of 3.6% CD8⁺ cells specific for M3 peptide. Most cells (83%) represented effector-memory phenotype. Subsequently, we isolated RNA from 100 CLL patients to assess MYHIIA, VIM, and CFL-1 expression using qRT-PCR. Quantity mean values for gene expression were calculated according to the Standard Curve method. GAPDH was used as a constitutive gene. All three genes were overexpressed with no difference between males and females, Binet stages A, B and C nor different cytogenetic and molecular groups of CLL patients. These data suggest that MYHIIA, VIM and CFL-1 might be considered as a novel constitutive genes, highly expressed in CLL. In summary, our findings suggest presence of cytotoxic immune responses against three autoantigens recognized by BCR of CLL cells. The existence of autoreactive CD8+ T cells against MYHIIA, vimentin, cofilin-1 in CLL patients points to the involvement of antigen-specific autoreactive T cells in pathogenesis of CLL.