Feasibility study of personalized peptide vaccination for hepatocellular carcinoma patients refractory to locoregional therapies.

Overall survival of patients with hepatocellular carcinoma (HCC) refractory to locoregional therapy is dismal, even following treatment with sorafenib, a multikinase inhibitor. To develop a more efficacious treatment, we undertook a feasibility study of personalized peptide vaccination (PPV) for HCC, in which the peptides were selected from 31 peptide candidates based on the pre-existing immunity. Twenty-six HCC patients refractory to locoregional therapies (cohort 1) and 30 patients refractory to both locoregional and systemic therapies (cohort 2) were entered into the study. There were no severe adverse events related to PPV except for one injection site reaction. At the end of the first cycle of six vaccinations, successful CTL or IgG boosting was observed in 57% or 46% of patients in cohort 1 and in 54% or 52% of patients in cohort 2, respectively. Successful IgG boosting at the end of the second cycle was observed in the majority of patients tested. Median overall survival was 18.7 months (95% confidence interval, 12.2-22.5 months) in cohort 1, and 8.5 months (95% confidence interval, 5.9-12.2 months) in cohort 2. Based on the higher rates of immune boosting and the safety profile of PPV, further clinical studies of PPV would be warranted for patients with HCC refractory to not only locoregional therapy but also both locoregional and systemic therapies. The protocol of this study was registered with the UMIN Clinical Trials Registry (UMIN000001882 and UMIN000003590).

Duality of the murine CD8 compartment.

CD8αß plays crucial roles in the thymic selection, differentiation, and activation of some, but not all, CD8(+) T cells, whereas CD8αα does not. To investigate these roles, we produced mice that expressed transgene P14 T-cell receptor ß (TCRß) chain and CD8ß or did not (WT and KO mice, respectively). The primary CD8(+) T-cell response to acute lymphocytic choriomeningitis virus (LCMV) infection was predominantly D(b)/GP33 specific and CD8 independent in KO mice and was mostly CD8 dependent in WT mice. Cytotoxic T lymphocytes (CTL) from KO mice failed to mobilize intracellular Ca(2+) and to kill via perforin/granzyme. Their strong Fas/FasL-mediated cytotoxicity and IFN-γ response were signaled via a Ca(2+)-independent, PI3K-dependent pathway. This was also true for 15-20% of CD8-independent CTL found in WT mice. Conversely, the perforin/granzyme-mediated killing and IFN-γ response of CD8-dependent CTL were signaled via a Ca(2+), p56(lck), and nuclear factor of activated T cells-dependent pathway. Deep sequencing of millions of TCRα chain transcripts revealed that the TCR repertoires of preimmune CD8(+) T cells were highly diverse, but those of LCMV D(b)/GP33-specific CTL, especially from KO mice, were narrow. The immune repertoires exhibited biased use of Vα segments that encoded different complementary-determining region 1α (CDR1α) and CDR2α sequences. We suggest that TCR from WT CD8-independent T cells may engage MHC-peptide complexes in a manner unfavorable for efficient CD8 engagement and Ca(2+) signaling but permissive for Ca(2+)-independent, PI3K-dependent signaling. This duality of the CD8 compartment may provide organisms with broader protective immunity.

G1-4 A, an arabinogalactan polysaccharide from Tinospora cordifolia increases dendritic cell immunogenicity in a murine lymphoma model.

The immunogenicity of dendritic cells (DC) is known to increase with their maturation state and both are induced by microbial products like LPS. In this study, we have investigated the effect of G1-4A, a polysaccharide isolated from Indian medicinal plant, Tinospora cordifolia on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDC) and its ability to be used as an adjuvant in immunotherapy. G1-4A, enhanced surface expression of CD40, CD80, CD86, MHCII by BMDC in vitro and splenic DC in vivo. T cell allostimulatory activity and secretion of IL-12 and TNFα by BMDC were also increased. Treatment with G1-4A resulted in decreased phagocytosis and increased antigen processing that are characteristic of mature DC. G1-4A treated DC cross presented exogenous antigens on a MHC I background which resulted in the activation of cytotoxic T cells. These cells thus activated could cause lysis of target tumor cells in vitro. Administration of tumor lysate pulsed G1-4A treated DC resulted in decreased tumor burden in preventive as well as therapeutic tumor challenge experiments in a murine lymphoma model. These results thus confirm that G1-4A could be a promising nontoxic maturation agent to be potentially used in DC based immunotherapy of tumor.

Dual effects of hyperbaric oxygen on proliferation and cytotoxic T lymphocyte activity of rat splenic lymphocytes.

Contradictory reports exist regarding the effects of hyperbaric oxygen (HBO2) on immune functions. We hypothesized that the intensity of exposure is a key factor, which leads to the different effects of HBO2 on immunity. In this study, we determined the function of rat splenic lymphocytes after in vitro exposure to different pressure-durations of HBO2. The proliferation rate and total cytotoxic T lymphocyte (CTL) activity stimulated by concanavalin A were enhanced by exposure to low (100 kPa for 60 or 90 minutes and 150 kPa for 30 or 60 minutes) HBO2 pressure-durations, but were inhibited by exposure to higher pressure-durations. We conclude that HBO2 exerts dual effects on lymphocyte function, as appropriate exposure promotes and excessive exposure inhibits the functions.

Black tea-induced decrease in IL-10 and TGF-beta of tumor cells promotes Th1/Tc1 response in tumor bearer.

Several lines of evidence support that impairment of host immune function by tumor may be related to several strategies of tumor escape from immunosurveillance. We found that in Ehrlich's ascites carcinoma (EAC)-bearing mice, the tumor cells secrete immunosuppressive cytokines, transforming growth factor beta (TGF-beta) and interleukin-10 (IL-10) that induce a general T helper cells type 2 (Th2) dominance dampening the T cytotoxic cells type 1 (Tc1) population. Interestingly, black tea at the antitumor dose of 2.5% significantly reduced TGF-beta and IL-10 in tumor cells in vivo, thereby preventing Th2 dominance in the tumor bearers and initiating a Th1/Tc1 response. Thus, apart from its anticancer activity, this popular beverage also rejuvenates cancer immunosurveillance by modulating cytokine profiles and establishing Th1/Tc1 dominance in the tumor-bearing host.

Ganoderma lucidum polysaccharides enhance the function of immunological effector cells in immunosuppressed mice.

The present study was designed to determine in vivo efficacy of Ganoderma lucidum polysaccharides (Gl-PS) for enhancing the activity of immunological effector cells in immunosuppressed mice. Mice were injected intraperitoneally (i.p.) once daily with low-dose (2.5mg/kg), intermediate-dose (25mg/kg), and high-dose (250 mg/kg) of Gl-PS, respectively, for 7 consecutive days 24h after i.p. injection of a immunosuppressing anti-tumor agent cyclophosphamide (Cy, 300 mg/kg). In Cy-treated mice, compared to vehicle, low-dose Gl-PS accelerated recovery of bone marrow cells, red blood cells and white blood cells, as well as splenic natural killer cells and natural killer T cells, and enhanced T and B cell proliferation responses on day 8, cytotoxic T lymphocyte activity on day 5, as well as NK cell and lymphokine activated killer cell activity on days 7-9. Furthermore, it promoted phagocytosis and cytotoxicity of macrophages on day 12. The above beneficial effects induced by the low-dose Gl-PS treatment did not result in any side effects. These results demonstrate the efficacious effects of low-dose Gl-PS treatment for enhancing the activity of immunological effector cells in immunosuppressed mice, and may provide a basis for applying this herb as an efficacious adjacent immunopotentiating therapy against cancer chemotherapy-induced immunosuppression.

Inhibition of ex vivo-expanded cytotoxic T-lymphocyte function by high-dose cyclosporine.

BACKGROUND: Donor-derived, ex vivo-expanded cytotoxic T lymphocytes (CTL) can provide stem-cell transplantation (SCT) patients with a renewed capacity for virus-specific immune surveillance. Because SCT patients are often treated with cyclosporine (CsA), we questioned whether ex vivo-expanded CTL were susceptible to inhibition by this immunosuppressive drug. METHODS: Human Epstein-Barr virus (EBV)-specific CTL were established by cultivating T cells for at least 5 weeks with interleukin (IL)-2 and irradiated, autologous EBV-transformed B-lymphoblastoid cell lines (LCL). In some cases, CsA was added during the last week of T-cell expansion. Effectors were then tested for cytotoxicity toward their targets in a chromium-release assay or by coculture with viable, unlabeled targets, in the presence or absence of CsA. Alloreactive CTL were similarly expanded and tested against major histocompatibility complex-mismatched stimulator cells. RESULTS: CsA had a marginal effect on CTL function when added at concentrations greater than or equal to 250 ng/mL during the 4- to 6-hour chromium release assay. However, exposure of CTL to CsA for 1 week before assay reduced lytic function significantly. When the CTL lines were cocultured with viable targets in the presence of CsA, effectors were unable to eliminate their targets, which ultimately dominated the culture. CONCLUSIONS: We suggest that the activity of ex vivo-expanded CTL may be significantly compromised in the presence of high-dose CsA in vivo, particularly if CTL are administered for the purpose of long-term virus-specific immune surveillance.

Selective stimulation of murine cytotoxic T cell and antibody responses by particulate or monomeric hepatitis B virus surface (S) antigen.

In the murine system, we tested in vivo the immunogenicity of different preparations of the yeast-derived surface antigen (S-antigen or S-protein) of hepatitis B virus (HBV). Native S-protein molecules self-assemble into stable 22-nm particles. BALB/c mice immunized with low doses of native S-particles without adjuvants efficiently generated an H-2 class I-restricted CD8+ cytotoxic T lymphocyte (CTL) response, and developed easily detectable serum antibody titers against conformational determinants of the native S-particle or linear epitopes of the denatured S-protein. Disruption of S-particles with sodium dodecyl sulfate and beta-2-mercaptoethanol generated p24 S-monomers. Injection of an equal dose of S-monomers into mice efficiently primed CTL, but did not stimulate an antibody response against conformational or linear epitopes of the native or denatured S-protein. In vivo priming of CTL by S-particles or S-monomers required "endogenous" processing of the antigen because the injection of an equimolar (or higher) dose of an antigenic, S-derived 12-mer peptide into mice did not prime CTL. Native (particulate) or denatured (monomeric) S-antigen injected with mineral oil (incomplete Freund's adjuvant) or aluminum hydroxide failed to stimulate a CTL response. Hence, different preparations can be produced from a small protein antigen which specifically stimulate selected compartments of the immune system.

The impact of acemannan on the generation and function of cytotoxic T-lymphocytes.

Acemannan, an antiviral agent with immune enhancement capabilities, was studied for its impact on cytotoxic T-lymphocyte (Tc) function generated in response to alloantigen. To investigate whether acemannan directly stimulated the generation of Tc from primary mixed lymphocyte cultures (MLC), the drug was added at the initiation of the MLC. There was a dose-related, statistical increase in killer T-cell generation produced by acemannan in the clinically relevant dose range. The lowest test dose of the drug (2.6 x 10(-9) M) increased chromium release nearly two-fold; the 2.6 x 10(-8) M dose gave a maximal 3.5 fold increase in cytotoxic T-cells. To study whether acemannan enhanced the capacity of Tc once generated to alloantigen to destroy targets bearing the sensitizing antigens, MLR were established in the absence of any drug. Acemannan at the two highest doses increased the functional capacity of Tc to destroy target cells to which they had been sensitized in the MLR. To control for the possibility that acemannan was directly cytotoxic to target cells, targets were incubated alone with drug and without sensitized killer T-cells. No dose of acemannan was found to be cytotoxic to these cells. In conclusion, acemannan did enhance the generation of cytotoxic T-cells when added at the initiation of the MLR. When acemannan was added at the completion of allostimulation, an increase of almost 50% killing by Tc was also observed. These effects can not be explained by direct drug related toxicity and suggest a functional correlate to the previously described immune enhancing properties of the agent. As this drug is being tested for the treatment of HIV infections, these data provide at least one immunologic mechanism by which acemannan may be clinically salutory.