It is in the lymph node that antigen is processed and the initial immune response occurs

It is in the lymph node that antigen is processed and the initial immune response occurs. different categories of B cell sub populations with flow cytometry. Hybridomas were generated from the lymph node cells after CD40L-stimulation. Cells were fused to murine plasmacytoma P3X63.Ag8.653 using Helix electrofusion chamber. ELISA was used to evaluate hybridoma derived antibody binding to vaccine peptides. == Results == Viable MNCs were satisfactorily recovered from lymph nodes cryopreserved from six vaccine study patients 814 years previously. B cell ELISPOT demonstrated responses for each patient to multiple vaccine peptides. CD40L stimulation of lymph node cells increased the proportion of CD19+CD27+cells from 12 to 65% of the sample and increased the proportion of class-switched cells. Screening of IgG secreting clones demonstrated binding to melanoma vaccine peptides. == Conclusions == B cells were successfully recovered and expanded from human cryopreserved vaccine-draining lymph nodes. Individual B cells were identified that secreted antibodies that bound to cancer vaccine peptides. The ability to reliably generate in vitro the same antibodies observed in the blood of vaccinated patients will facilitate research to understand mechanisms of human antibody activity and possibly lead to therapeutic antibodies. Keywords:Cancer vaccine, B cells, Antibodies, Melanoma, Human == Background == Our research is focused on using lymph nodes receiving drainage from a cancer or a vaccine to better understand the immune response and to generate new anti-cancer reagents. Debris from a tumor or interstitial injection of antigens are absorbed into lymphatic ducts and transported to regional lymph nodes. It is in the lymph node that antigen is processed and the initial immune response occurs. The development of lymphatic mapping with radioactive tracers allows precise localization of the draining lymph nodes from almost any tissue in the body [1]. In a series of clinical vaccine studies at the University of Virginia (UVA), a draining lymph node was used for evaluating the T cell response [2,3]. Radiotracer lymphatic mapping was used to identify the lymph node receiving drainage from the vaccine and the lymph node was removed with minimal morbidity [4]. Analysis of the T cell response in the vaccine-draining lymph nodes was more sensitive than analysis of the circulating T cell response in the peripheral blood [4,5]. Findings related to T cells in the node correlated with systemic findings. For example, CD4+T cell responses were positively associated with serum antibody titers to the vaccine peptides [2]. It was prescient for the investigators of the UVA vaccine AQ-13 dihydrochloride study to cryopreserve residual lymph node mononuclear cells (MNCs) and other clinical material for future research. In the UVA study as well as other vaccine studies, positive clinical outcomes were associated with elevated serum titers of anti-vaccine antibodies [2,6,7]. In animals, vaccine-induced antibodies can prevent tumor growth and even eradicate existing bulky tumors [8]. Characterization of these antibodies shows that they exhibit an extensive range of functional activity. Further study of vaccine-induced antibodies will be valuable to better understand how antibodies interact with other immune effector mechanisms to inhibit the tumor. Unfortunately, there is not yet a reliable method to produce antibodies generated by human cancer patients that have received a cancer vaccine. The data presented here is focused on antibody secreting cells present in vaccine-draining lymph nodes. We hypothesized that B cells producing antibodies to the vaccine peptides may be detected ex vivo from the cryopreserved lymph node MNCs. These cryopreserved MNCs archived from the UVA vaccine trial were used for the study presented here to identify individual B cells that secreted anti-vaccine AQ-13 dihydrochloride antibodies. == Methods == == Subjects and vaccination == The patients were enrolled on a vaccine study (FDA, BB-IND No. 10825, and UVA institutional review board HIC No. 10464,NCT00089219) and had received a series of vaccinations with 6 different melanoma related peptides. Patients with stage IIIB to IV melanoma and expressed at least one of the five HLADR alleles Rabbit Polyclonal to PPM1K by which CD4 T-cell recognition of the vaccine peptides had been defined were included in the study. Patients received vaccine comprised of 6 melanoma peptides namely, AQN (AQNILLSNAPLGPQFP), FLL (FLLHHAFVDSIFEQWLQRHRP), RNG (RNGYRALMDKSLHVGTQCALTRR), TSY (TSYVKVLHHMVKISG), LLK (LLKYRAREPVTKAE), WNR (WNRQLYPEWTEAQRLD) and tetanus helper peptide (AQYIKANSKFIGITEL). Melanoma peptides were derived from Tyrosinase (AQ and FLL), Melan-MART1 (RNG), MAGE-1, MAGE-2, MAGE-3, MAGE-6 (TSY and LLK), AQ-13 dihydrochloride and gp100 (WNR) proteins..