Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM1(58–66 )and CMVpp65(495–503 )peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization

Combination therapy of renal cell carcinoma or breast cancer patients with dendritic cell vaccine and IL-2: results from a phase I/II trial

<p>Abstract</p> <p>Background</p> <p>Ten cancer patients (Six renal cell carcinoma and four breast cancer patients) were treated in a phase I/II study with a vaccine composed of autologous dendritic cells (DCs) and IL-2 to evaluate the DC vaccine-related toxicity and antigen-specific immune alteration.</p> <p>Methods</p> <p>Cancer patients were treated twice with autologous CD34⁺hematopoietic stem cell-derived, GM-CSF/IFN-γ-differentiated DCs pulsed with autologous tumor lysate and KLH, by 4-week interval. Following each subcutaneous injection of therapeutic DCs, low-dose (200 MIU) IL-2 was introduced for 14 consecutive days as an immune adjuvant. To determine the DC vaccine-induced immunological alterations, the KLH-specific lymphocyte proliferation, number of IFN-γ secreting T cells (ELISPOT assay), NK activity and the cytokine modulation were measured.</p> <p>Results</p> <p>Cultured-DCs expressing HLA-DR, CD11c, CD83, and B7.1/B7.2 produced IL-12p70. After vaccination, the patients tolerated it. Clinical response was observed in one RCC patient as stable disease. However DC-vaccine related antigen-specific immune responses including peripheral blood lymphocyte proliferation and the number of IFN-r secreting cells were induced in six patients without clear correlation with clinical responses. Also NK activity was induced significantly in six patients after vaccination. DC vaccine-related decrease of TGF-β level or increase of IL-12p70 level and decline of CD4⁺CD25⁺T cells were observed in three patients. However only in the RCC patient whose disease stabilized, combination of stimulatory as well as inhibitory immune alterations including induction of IFN-γ secreting T cell with reduction of CD4⁺CD25⁺T cell were correlated with clinical responses.</p> <p>Conclusion</p> <p>Data indicated that DC vaccine combined with IL-2 is well tolerated without major side effects. DC vaccine induced the specific immunity against introduced antigen. Combinatorial alterations of immunological parameters indicating antigen-specific immune induction along with reduction of inhibitory immunity were correlated with clinical responses in DC vaccine treated patients.</p

Establishment of a national on-line registry for apheresis in Korea

National apheresis registries can be used to evaluate changes in technology, clinical indications, and applications over the years. Since the establishment of the Korean Society of Apheresis (KSFA) in 1999, basic data on the status of apheresis have been collected using letters and e-mails on a biennial basis. In February 2006, a KSFA homepage and an on-line apheresis registry were constructed (http://www.apheresis.or.kr/). The registry consists of two sub-registries, one addressing overall aspects, e.g., the numbers and types of apheresis machines, total numbers of apheresis procedures, and the other addressing therapeutic plasmapheresis, e.g., diagnoses, modes of treatment, instrument used, replacement fluids, volumes processed, vascular access, anticoagulants, complications, and clinical responses. Data registered on-line is presumed to represent about 95% of total apheresis procedures performed in Korea. In this report, we introduce our on-line registry system and compared the data obtained by on-line registry with the previous ones