Cytotoxic T-lymphocyte Immune Response Research Articles

Mycobacterium tuberculosis-associated synthetic mycolates differentially exert immune stimulatory adjuvant activity.

Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl β-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis (Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group.

Abstract SY03-01: Macrophage PI3Kgamma drives cancer immune suppression

Abstract Innate immune cells can switch from a proinflammatory state that defends against pathogens and to an anti-inflammatory state that repairs damaged tissue. Proinflammatory myeloid cells activate cytotoxic T cells to eliminate pathogens, but anti-inflammatory myeloid cells inhibit T-cell-mediated immunity, stimulate angiogenesis, and stimulate fibrosis, all of which promote tumor progression. The predominant isoform of PI(3)Kinase in myeloid cells, PI3Kgamma, controls this switch between immune stimulation and immune suppression during inflammation and cancer. Myeloid cell PI3Kgamma and its effectors Akt1, mTor, and S6Kinase stimulate C/EBPbeta-dependent expression of immunosuppressive factors, including Arginase and TGFbeta, while also suppressing NFkappaB-dependent expression of proinflammatory cytokines such as IL12 and IFNgamma, thereby inducing tumor immune suppression and consequent tumor growth and metastasis. In contrast, inhibition of PI3Kgamma activity by genetic or pharmacological means suppresses C/EPBbeta-mediated transcription of anti-inflammatory factors and stimulates NFkappaB-mediated transcription of the proinflammatory cytokines IL12 and IFNgamma, thereby restoring CD8+ T-cell-dependent cytotoxicity that inhibits tumor growth and metastasis. Therefore, inhibitory targeting of PI3Kgamma indirectly restores cytotoxic T-cell immune responses that elicit tumor suppression without unwanted side effects. Our studies highlight the strong therapeutic potential of targeting this kinase to control inflammation and cancer. This work was supported by grants to JAV from the NIH (5R01CA126820), the Landon Foundation-AACR (12-60-27-VARN), the Lustgarten Foundation for Pancreatic Research and the Whitworth Foundation for Cancer Research. Citation Format: Judith A. Varner. Macrophage PI3Kgamma drives cancer immune suppression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY03-01.

Global Properties of a HBV Infection Model with HBV DNA-Containing Capsids and CTL Immune Response

In this paper, the global dynamical behavior of a hepatitis B virus (HBV) infection model with HBV DNA-containing capsids and cytotoxic T lymphocytes (CTLs) immune response is investigated. We derive the conditions for global asymptotic stability of the steady states of the model in terms of the basic reproduction number $$R_{0}$$ and the immune response reproduction number $$R_{{ CTL}}$$ . By constructing appropriate Lyapunov functions, it is shown that the disease-free steady state is globally asymptotically stable when $$R_{0}\le 1$$ , the immune-free steady state is globally asymptotically stable when $$R_{{ CTL}}\le 1<R_{0}$$ and the endemic steady state is globally asymptotically stable when $$R_{{ CTL}}>1$$ . Further, we incorporate two discrete delays in the model to account for the intracellular delays in the production of productively infected hepatocytes and capsids. We also derive the global properties of this two-delay model in terms of $$R_{0}$$ and $$R_{{ CTL}}$$ . Finally, illustrative numerical simulations are presented to support our theoretical findings.

Structural and Biochemical Analyses of Swine Major Histocompatibility Complex Class I Complexes and Prediction of the Epitope Map of Important Influenza A Virus Strains.

The lack of a peptide-swine leukocyte antigen class I (pSLA I) complex structure presents difficulties for the study of swine cytotoxic T lymphocyte (CTL) immunity and molecule vaccine development to eliminate important swine viral diseases, such as influenza A virus (IAV). Here, after cloning and comparing 28 SLA I allelic genes from Chinese Heishan pigs, pSLA-3*hs0202 was crystalized and solved. SLA-3*hs0202 binding with sβ2m and a KMNTQFTAV (hemagglutinin [HA]-KMN9) peptide from the 2009 pandemic swine H1N1 strain clearly displayed two distinct conformations with HA-KMN9 peptides in the structures, which are believed to be beneficial to stimulate a broad spectrum of CTL immune responses. Notably, we found that different HA-KMN9 conformations are caused, not only by the flexibility of the side chains of residues in the peptide-binding groove (PBG), but also by the skewing of α1 and α2 helixes forming the PBG. In addition, alanine scanning and circular-dichroism (CD) spectra confirmed that the B, D, and F pockets play critical biochemical roles in determining the peptide-binding motif of SLA-3*hs0202. Based on biochemical parameters and comparisons to similar pockets in other known major histocompatibility complex class I (MHC-I) structures, the fundamental motif for SLA-3*hs0202 was determined to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) by refolding in vitro and multiple mutant peptides. Finally, 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains, and two of them have been found in humans as HLA-A*0201-specific IAV epitopes. Structural and biochemical illumination of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine. We crystalized and solved the first SLA-3 structure, SLA-3*hs0202, and found that it could present the same IAV peptide with two distinct conformations. Unlike previous findings showing that variable peptide conformations are caused only by the flexibility of the side chains in the groove, the skewing of the α1 and α2 helixes is important in the different peptide conformations in SLA-3*hs0202. We also determined the fundamental motif for SLA-3*hs0202 to be X-(M/A/R)-(N/Q/R/F)-X-X-X-X-X-(V/I) based on a series of structural and biochemical analyses, and 28 SLA-3*hs0202-restricted epitope candidates were identified from important IAV strains. We believe our structure and analyses of pSLA-3*hs0202 can benefit vaccine development to control IAV in swine.

Specific cytotoxic T-cell immune responses against autoantigens recognized by chronic lymphocytic leukaemia cells.

Mounting evidence suggests that autoreactivity and inflammatory processes are involved in the pathogenesis of chronic lymphocytic leukaemia (CLL). Cytoskeletal proteins, including non-muscle myosin heavy chain IIA (MYHIIA), vimentin (VIM) and cofilin-1 (CFL1), exposed on the surface of apoptotic cells have been identified as autoantigens that are recognized by the specific B-cell receptors of the CLL cells. In 212 CLL patients analysed with quantitative reverse transcriptase-polymerase chain reaction we found CFL1 overexpression and low expression of MYH9 in comparison with healthy volunteers. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 66·7%, VIM in 87·5% and CFL1 in 62·5% CLL patients in an Enzyme-Linked ImmunoSpot assay. Low frequencies of autoreactive peptide-specific T cells were detected against MYHIIA, VIM and CFL1 in CLL patients exvivo; most of the detected cells had an effector-memory phenotype. Our findings support the existence of cytotoxic immune responses against three autoantigens that have been identified as targets of CLL clonotypic B-cell receptors. The presence of autoreactive CD8(+) T cells against MYHIIA, VIM and CFL1 in CLL patients indicates the involvement of antigen-specific autoreactive T cells in the pathogenesis of CLL.

An observational study evaluating the expression of HER2 (1+, 2+, and 3+) with HLA A2/A3+ in gastric adenocarcinoma patients.

18 Background: Gastric cancer remains a major health issue and results in 800,000 annual deaths worldwide. Despite approximately 50% of the gastric and gastroesophageal (GE) junction adenocarcinomas are diagnosed with resectable disease, 40% of disease recurrence is observed in 24 months. While trastuzumab has shown to increase overall survival in HER2 IHC3+ metastatic disease, no current therapies are available for low expressing, or IHC 1+ or 2+ patients. NeuVax (nelipepimut-S) is an immunogenic peptide epitope derived from the extracellular domain of HER2/nu protein administered in combination with rhGM-CSF. NeuVax binds to HLA-A2 and A3 on tumor and antigen presenting cells (APC) and elicits a proliferation of CD8+ (cytotoxic) T-cell immune response against HER2 expressing cancer cells. A proof of concept clinical study will be conducted to assess the effectiveness of nelipepimut-S to prevent recurrence and increase disease free survival in gastric cancer patients with all levels of HER2 expression with HLA A2+/A3+. This prospective observational study was undertaken to estimate the HLA A2/A3+ and HER2 expression status in the Indian population. Methods: Tumor tissue samples from gastric or GE junction adenocarcinoma patients were collected from S. L. Raheja Hospital, Mumbai, India. A serum sample was collected for HLA testing by quantitative PCR. IHC (DAKO, 1:600 dilution) was employed for detection of HER2. All patients were consented for the study. Results: Of the 50 patients evaluated, 12 (24%) were positive for HER2 (1+, 2+, 3+) with HLA2/A3 alleles, meeting the defined projected clinical protocol population of all levels of expression of HER2 and HLA A2+ and/or A3+. Of the 12 patients, 9 (75%) and 3 (25%) expressed HLA A2 and A3, respectively, with no patient demonstrating HER2 3+ expression. Conclusions: Results from this prospective observational study suggest patients with gastric and GE junction adenocarcinoma have HLA A2+ and/or A3+ expressing tumors with mostly HER2 1+, 2+ expression. The estimated incidence of HER2 3+ expression in this population may be limited by size of current study. Results will inform the screen failure rate of the planned Phase 2 clinical study.

Global Stability of Delayed Viral Infection Models with Nonlinear Antibody and CTL Immune Responses and General Incidence Rate.

The dynamical behaviors for a five-dimensional viral infection model with three delays which describes the interactions of antibody, cytotoxic T-lymphocyte (CTL) immune responses, and nonlinear incidence rate are investigated. The threshold values for viral infection, antibody response, CTL immune response, CTL immune competition, and antibody competition, respectively, are established. Under certain assumptions, the threshold value conditions on the global stability of the infection-free, immune-free, antibody response, CTL immune response, and interior equilibria are proved by using the Lyapunov functionals method, respectively. Immune delay as a bifurcation parameter is further investigated. The numerical simulations are performed in order to illustrate the dynamical behavior of the model.

Immune response in virus model structured by cell infection-age.

This paper concerns modeling the coupled within-host population dynamics of virus and CTL (Cytotoxic T Lymphocyte) immune response. There is substantial evidence that the CTL immune response plays a crucial role in controlling HIV in infected patients. Recent experimental studies have demonstrated that certain CTL variants can recognize HIV infected cells early in the infected cell lifecycle before viral production, while other CTLs only detect viral proteins (epitopes) presented on the surface of infected cells after viral production. The kinetics of epitope presentation and immune recognition can impact the efficacy of the immune response. We extend previous virus models to include cell infection-age structure in the infected cell compartment and immune response killing/activation rates of a PDE-ODE system. We characterize solutions to our system utilizing semigroup theory, determine equilibria and reproduction numbers, and prove stability and persistence results. Numerical simulations show that ' early immune recognition' precipitates both enhanced viral control and sustained oscillations via a Hopf bifurcation. In addition to inducing oscillatory dynamics, considering immune process rates to be functions of cell infection-age can also lead to coexistence of multiple distinct immune effector populations.

P70: Construction and studying expression in eukaryotic cells of allel-specific HLA-A∗0201 DNA-vaccine against breast cancer

Background Breast cancer ranks the first among female oncological diseases and ranks the second after lung cancer among causes of cancer related deaths. Use of contemporary anticancer drugs significantly enhanced possibilities of cancer therapy. However, most of medicines against cancer have such disadvantages as toxicity, narrowness of therapeutic action, and emergence of drug resistance. Recently, an approach based on potential of dendritic cells (DCs) to induce formation of cytotoxic T-cell immune response against cancer cells is widely used. Priming of dendritic cells by DNA-constructs encoding immunogenic epitopes of tumor antigens is regarded as a rather promising approach to design cancer vaccines. The aim of this study is to construct, obtain and study expression in eukaryotic cells of recombinant plasmid – candidate DNA-vaccines inducing T-cell immune response against breast cancer. Materials and methods Using the PolyCTLDesigner software, DNA-vaccine construct was made encoding polyepitope T-cell immunogen BC-A ∗ 0201. Gene encoding target immunogen was synthesized and cloned in the composition of plasmid pcDNA3.1 that is eukaryotic expression vector. Accuracy of gene insertion encoding polyepitopeimmunogen was verified using restriction analysis. Obtained target plasmid pBC-A ∗ 0201 – candidate DNA-vaccine was used for evaluation of gene expression in transfected eukaryotic cells 293T and dendritic cells by means of flow cytofluorometry and immunohistochemistry using MAT to p24 epitope-marker inserted into the structure of target plasmid. Dendritic cells were obtained from peripheral blood precursors by 2-h plastic adhesion. The nonadherent cells were removed 2 h later and the adherent monolayer was cultivated in the medium containing growth factors (GM-CSF and IL-4) according to (Naik, 2010; Ganul and Khranovskaya, 2012). Transfection of immature DCs (iDC) was performed by using the reagents purchased from Promokine. Further cultivation of iDCs was carried out in the medium containing TNF- α to obtain fully mature DCs (mDC). Analysis of mDCsurface markers expression was made using MAO (HLA-DR, CD83, CD86) and flow cytofluorometer (BD FACSCalibur). Results Plasmid pBC-A ∗ 0201 encoding epitopes of breast cancer antigens was constructed and produced in preparative amount. Using the method of intracellular staining of product of expression by specific MAO 29F2 to epitope p24, we showed expression of gene BC-A ∗ 0201 in eukaryotic cells 293T and dendritic cells transfected by plasmid pBC-A ∗ 0201. Maturity of dendritic cells was also proved. Conclusion Thus, obtained candidate DNA-vaccine encoding target polyepitopeimmunogen provides synthesis of relevant protein in the eukaryotic cells culture. In future, we plan to study biological activity of DNA-vaccine to evaluate level of induction of cytotoxic response against SKBR3 breast cancer cells.

Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme.

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

Abstract 4053: Misregulation of dicer miRNA processing correlates with antigen presentation and survival in colorectal cancer

Abstract Background Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. The downregulation of the MHC I complex reduces the immunogenicity of tumors. We hypothesized that the expression of Tapasin may predict immune infiltration of colorectal cancer (CRC) by CD8+ cytotoxic T lymphocytes (CTLs) and overall survival. In vitro, the expression of Tapasin can be rescued by cytokine treatment, indicating that the misregulation is neither genetic nor structural and may occur on an epigenetic level. We therefore tested the possible involvement of the RNA silencing protein Dicer in the post-transcriptional regulation of Tapasin expression. Methods Resection specimens from 219 CRC patients with full clinicopathological information were included in this study. ngTMA (next generation tissue microarray) slides containing 8 punches from each case (tumor and normal tissue) were immunostained for MHC I pathway components (MHC I, beta-2-microglobuline, Calnexin, Calreticulin, Erp57 TAP1, TAP2 and Tapasin), Dicer and CD8. Marker expression was analyzed for immune activation, prediction of patient survival and TNM-staging. Results High Tapasin expression strongly correlated with lack of venous invasion (AUC: 0.682, p = 0.044, OR: 0.37; 95% CI: 0.2-0.69), lymphatic invasion (AUC: 0.727, p = 0.040, OR: 0.5; 95% CI: 0.3-0.8), absence of distant metastasis (AUC: 0.727, p = 0.040, OR: 0.35; 95% CI: 0.17-0.71) as well as a lack of infiltrative tumor border configuration (AUC: 0.621 p = 0.020, OR: 0.446; 95% CI: 0.23-0.87). Further, Tapasin expression correlated strongly with higher intra- and peritumoral CD8+ CTL invasion (AUC: 0.729, p&amp;lt;0.001, OR: 5.4; 95% CI: 2.6-11 and AUC: 0.650, p = 0.040, OR: 2.4 95% CI: 1.4-4.2, respectively). The prediction of CD8+ infiltration based on Tapasin was independent of the presence of other MHC I components. Furthermore, Tapasin expression was significantly associated with favorable overall survival (p = 0.004, OR: 0.6, 95% CI: 0.42-0.85). This effect was independent from the expression of the other MHC I components Calnexin, Calreticulin, Erp57, TAP1 and TAP2, but not independent of MHC I itself (p = 0.054) or Dicer expression (p = 0.068). Testing for a possible epigenetic regulation mechanism, we found that Dicer and Tapasin were positively correlated (r = 0.34, p&amp;lt;0.001). Conclusion Loss of Tapasin is an adverse prognostic factor in CRC. This may result from a reduced anti-tumoral CTL immune response driven by loss of Tapasin expression. Our results additionally suggest that Tapasin may be directly or indirectly regulated by RNA silencing and the Dicer protein. This may allow for further insight into the mechanism of epigenetic regulation of tumor immune evasion. Citation Format: Lena Sokol, Viktor H. Koelzer, Eva Karamitopoulou, Alessandro Lugli, Inti Zlobec. Misregulation of dicer miRNA processing correlates with antigen presentation and survival in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2015-4053

Vaccination with ubiquitin-hepatitis B core antigen-cytoplasmic transduction peptide enhances the hepatitis B virus-specific cytotoxic T-lymphocyte immune response and inhibits hepatitis B virus replication in transgenic mice.

Chronic hepatitis B virus (HBV) infection is characterized by functionally impaired type 1 T-helper cell (Thl) immunity and poor HBV‑specific T‑cell responses. Ubiquitin (Ub), a highly conserved small regulatory protein, commonly serves as a signal for target proteins that are recognized and degraded in proteasomes. The rapid degradation of Ub‑mediated antigens results in efficient stimulation of cell‑mediated immune responses. Thus, the Ub‑HBV core antigen (HBcAg)‑cytoplasmic transduction peptide (CTP) fusion protein was designed for specific delivery of a foreign modified antigen to the cytoplasm of antigen‑presenting cells. HBV transgenic mice were used to determine whether Ub‑HBcAg‑CTP would restore HBV‑specific immune responses and anti‑viral immunity in these animals. The results demonstrated that synthesized Ub‑HBcAg‑CTP not only significantly increased the levels of interleukin‑2 and interferon (IFN)‑γ compared with those in the HBcAg‑CTP, IFN‑α, Ub‑HBcAg, HBcAg and phosphate‑buffered saline groups, but additionally induced the highest IFN‑γ+ CD8+ T‑cell numbers and HBV‑specific cytotoxic T lymphocyte (CTL) responses, indicating a strong immune response. In addition, enhancement of specific CTL activity provoked by the fusion protein reduced hepatitis B surface antigen (HBsAg) and HBV DNA serum levels and diminished the expression of HBsAg and HBcAg in liver tissue of HBV transgenic mice, suggesting that there was a therapeutic effect. In conclusion, the present study provided evidence that Ub‑HBcAg‑CTP activated the Th1‑dependent immunity, triggered functional T cell responses and subsequently inhibited viral replication in HBV transgenic mice. These observations suggested that the fusion protein may represent an innovative and promising candidate for active immunotherapy during chronic and persistent HBV.

A class of delayed viral infection models with general incidence rate and adaptive immune response

To model the role of the adaptive immune response in viral infection, take account the time needed for infected cells to produce new virions after viral entry and the time necessary for the newly produced virions to become mature and infectious, we propose three generalized models that describe the interactions between virus, host cells, antibodies and cytotoxic T lymphocytes (CTL) cells. The incidence rate of the infection into three models is given by a general function which includes many special cases and depends on the uninfected cells, infected cells and virus. Our main results show that the global dynamics of the three models under certain hypotheses on the incidence function is fully determined by five threshold parameters called the reproduction numbers for viral infection, for antibody immune response, for CTL immune response, for antibody immune competition and for CTL immune competition. Several viral infection models with discrete, finite and infinite distributed delays existing in other previous studies are extended and generalized. Furthermore, we give some biological findings of our analytical results.

HIV-1 vaccine immunogen design strategies.

An effective human immunodeficiency virus type 1 (HIV-1) vaccine is expected to have the greatest impact on HIV-1 spread and remains a global scientific priority. Only one candidate vaccine has significantly reduced HIV-1 acquisition, yet at a limited efficacy of 31%, and none have delayed disease progression in vaccinated individuals. Thus, the challenge remains to develop HIV-1 immunogens that will elicit protective immunity. A combination of two independent approaches - namely the elicitation of broadly neutralising antibodies (bNAb) to prevent or reduce acquisition of infection and stimulation of effective cytotoxic T lymphocyte (CTL) responses to slow disease progression in breakthrough infections (recent evidence suggests that CTLs could also block HIV-1 from establishing persistent infection) – is the current ideal. The purpose of this review is to summarise strategies and progress in the design and testing of HIV-1 immunogens to elicit bNAb and protective CTL immune responses. Recent advances in mimicking the functional native envelope trimer structure and in designing structurally-stabilised bNAb epitope forms to drive development of germline precursors to mature bNAb are highlighted. Systematic or computational approaches to T cell immunogen design aimed at covering viral diversity, increasing the breadth of immune responses and/or reducing viable viral escape are discussed. We also discuss a recent novel vaccine vector approach shown to induce extremely broad and persistent T cell responses that could clear highly pathogenic simian immunodeficiency virus (SIV) early after infection in the monkey model. While in vitro and animal model data are promising, Phase II and III human clinical trials are ultimately needed to determine the efficacy of immunogen design approaches.

Caspase-1 independent viral clearance and adaptive immunity against mucosal respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is recognized by the innate immune system through Toll like receptors (TLRs) and retinoic acid inducible gene I. These pathways lead to the activation of type I interferons and resistance to infection. In contrast to TLRs, very few studies have examined the role of NOD-like receptors in viral recognition and induction of adaptive immune responses to RSV. Caspase-1 plays an essential role in the immune response via the maturation of the proinflammatory cytokines IL-1β and IL-18. However, the role of caspase-1 in RSV infection in vivo is unknown. We demonstrate that RSV infection induces IL-1β secretion and that caspase-1 deficiency in bone marrow derived dendritic cells leads to defective IL-1β production, while normal RSV viral clearance and T cell responses are observed in caspase-1 deficient mice following respiratory infection with RSV. The frequencies of IFN-γ producing or RSV specific T cells in lungs from caspase-1 deficient mice are not impaired. In addition, we demonstrate that caspase-1 deficient neonatal or young mice also exhibit normal immune responses. Furthermore, we find that IL-1R deficient mice infected with RSV exhibit normal Th1 and cytotoxic T lymphocytes (CTL) immune responses. Collectively, these results demonstrate that in contrast to TLR pathways, caspase-1 might not play a central role in the induction of Th1 and CTL immune responses to RSV.

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.

Schistosoma mansoni soluble egg antigens enhance T cell responses to a newly identified HIV-1 Gag H-2b epitope.

Schistosome infection induces significant T helper type 2 (Th2) and anti-inflammatory immune responses and has been shown to negatively impact vaccine efficacy. Our goal was to determine if the administration of schistosome soluble egg antigens (SEA) would negatively influence the induction of cytotoxic T lymphocyte (CTL) and Th1-type T cell responses to an HIV candidate vaccine in the Th1-biased C57BL/6 mouse strain. Initial experiments failed, as we were unable to detect any response to the defined class I epitope for HIV-1 IIIB Gag. Therefore, we initiated an epitope mapping study to identify C57BL/6 (H-2(b)) T cell epitopes in HIV-1 IIIB Gag in order to perform the experiments. This analysis defined two previously unreported minimal class I H-2(b) and class II I-A(b) epitopes for HIV-1 IIIB Gag. The newly defined HIV-1 IIIB Gag epitopes were used to evaluate the influence of SEA on the generation of CTL and Th1-type HIV-1 IIIB Gag responses. Surprisingly, in contrast to our hypothesis, we observed that the coadministration of SEA with a Listeria monocytogenes vector expressing HIV-1 IIIB Gag (Lm-Gag) led to a significantly increased frequency of gamma interferon (IFN-γ)-producing CD8(+) and CD4(+) T cells in C57BL/6 mice compared to mice immunized with Lm-Gag only. These observations suggest that SEA contains, in addition to Th2-type and immune-suppressive molecules, substances that can act with the Lm-Gag vaccine to increase CTL and Th1-type vaccine-specific immune responses.

Using HDAC Inhibitors to Eradicate Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with a poor prognosis caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL cannot be cured with conventional chemotherapy thus urging the development of new therapeutic strategies. Histone deacetyalse (HDAC) inhibitors are broadly active anti-neoplastic agents, which have shown efficacy in T-cell lymphomas. At our institution we encounter a relatively high number of ATLL cases as compared to other regions in the U.S. We have conducted a pilot trial using AZT/interferon-α(IFNα) plus the HDAC inhibitor valproic acid (VPA) during maintenance therapy in acute (leukemia-type) ATLL. We hypothesized that VPA would reactivate HTLV-I leading to a cytotoxic T-cell immune response followed by reduction of residual blood circulating ATLL clones that normally persist during AZT/IFNα therapy despite its suppressive effects. Supporting this notion, the addition of VPA to AZT/IFNα resulted in reduction of HTLV-I proviral loads in treated patients and a sustained molecular response in one subject who is still alive >4.5 years later. More recently, we have tested newly available and more potent HDAC inhibitors using fresh primary leukemic ATLL cells and low-passage cell lines. Through HDAC inhibition, these agents reactivated HTLV-I Tax gene expression and induced apoptosis in a dose-dependent manner. Belinostat, which is currently FDA-approved for the treatment of relapsed or refractory peripheral T-cell lymphoma, augmented ATLL cell death when added to AZT/IFNα at nanomolar concentrations. Therefore, we have proposed a new pilot clinical trial using belinostat for the treatment of ATLL during post-induction therapy. The role of HDAC inhibitors in ATLL, pre-clinical laboratory studies involving these agents, and clinical trial design will be discussed at the meeting DisclosuresOff Label Use: The use of HDAC inhibitors in adult T-cell Leukemia-lymphoma.

Metastases in immune-mediated dormancy: a new opportunity for targeting cancer.

The aim of any anticancer treatment is to avoid, control, or eliminate disseminated tumor cells. Clinical and experimental evidence has revealed that metastases can remain in a latency state, that is, metastasis dormancy. Three mechanisms are thought to be involved in cancer dormancy: cellular dormancy, angiogenic dormancy, and immune-mediated dormancy. Here, we review the mechanisms and cells involved in immune-mediated cancer dormancy and discuss current and future immunotherapeutic strategies. Recent results indicate that the immune system can restrain disseminated cancer cells, promoting their permanent dormancy. CD8(+) T lymphocytes play a relevant role in maintaining immune equilibrium with metastatic dormant cells, and MHC class I surface expression on tumor cells may also be involved. Natural killer (NK) cells have an activator function that triggers a cytotoxic T lymphocyte (CTL) response. Furthermore, immune dormancy promotes cancer cell growth arrest and angiogenic control. Immunotherapeutic interventions in metastatic dormancy may help to control or eradicate cancer disease. Treatments that activate or increase the CTL immune response or reverse cancer cell-induced CTL immunosuppression might be useful to restrain or destroy metastatic cells. These objectives may be achieved by recovering or increasing MHC class I surface expression on cancer cells or even by activating NK cells. Immune-mediated metastasis dormancy provides an opportunity for targeting cancer in novel immune treatments.

Stability Analysis of an Improved HBV Model with CTL Immune Response.

To better understand the dynamics of the hepatitis B virus (HBV) infection, we introduce an improved HBV model with standard incidence function, cytotoxic T lymphocytes (CTL) immune response, and take into account the effect of the export of precursor CTL cells from the thymus and the role of cytolytic and noncytolytic mechanisms. The local stability of the disease-free equilibrium and the chronic infection equilibrium is obtained via characteristic equations. Furthermore, the global stability of both equilibria is established by using two techniques, the direct Lyapunov method for the disease-free equilibrium and the geometrical approach for the chronic infection equilibrium.