Giulio C. Spagnoli

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

BackgroundMonitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals.MethodsWB (300 μl) was incubated at 37°C with specific antigens, in the form of peptides or commercial vaccines for 5–16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4°C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes.ResultsSpiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 μl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1β (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-γ gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination.ConclusionThis method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in field studies with difficult access to laboratory facilities.

Active Specific Immunotherapy Phase III Trials for Malignant Melanoma: Systematic Analysis and Critical Appraisal

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MAGE-A Antigens and Cancer Immunotherapy

MAGE-A antigens are expressed in a variety of cancers of diverse histological origin and germinal cells. Due to their relatively high tumor specificity, they represent attractive targets for active specific and adoptive cancer immunotherapies. Here, we (i) review past and ongoing clinical studies targeting these antigens, (ii) analyze advantages and disadvantages of different therapeutic approaches, and (iii) discuss possible improvements in MAGE-A-specific immunotherapies.

Expression of indoleamine 2,3-dioxygenase induced by IFN-γ and TNF-α as potential biomarker of prostate cancer progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested “in vitro” by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

Abstract 3384: High ALDH activity is associated with high tumorigenicity in prostate cancer

OBJECTIVE: Tumor initiation and progression might be driven by rare populations of cells endowed with stem-properties, and therefore defined as cancer stem cells (CSC). High Aldehyde dehydrogenase (ALDH) activity has been suggested to selectively identify CSC in several tumor types including breast cancer, and, possibly, prostate cancer (PCA). In this study, we investigated presence and potential CSC characteristics of cells with high ALDH activity (ALDH bright) in PCA cell lines, fresh surgical specimens, and primary cultures. MATERIALS AND METHODS: PC3, Du145, VCaP, and LNCaP PCA cell lines were evaluated. Surgical specimens, including Benign Prostate Hyperplasia (BPH) and PCA, were used directly for gene expression studies. Surgical samples were also enzymatically digested for functional analysis and the establishment of primary cultures. ALDH activity was tested using ALDEFLUOR® technology. Cells were sorted from individual cell lines by flow cytometry and evaluated for CSC properties, including spheroid formation ability, clonogenicity, stemness-related gene expression, ALDH specific isoforms expression, and tumorigenicity upon injection in NOD/SCID mice. RESULTS: PCA cell lines and primary cultures displayed heterogeneous ALDH activity and expression of specific ALDH isoforms. Despite a higher expression of Oct4A and Klf4 stemness associated genes, ALDH bright cells isolated from Du145 and PC3 did not show improved spheroid formation or clonogenic capacity, as compared to their dim counterparts. Interestingly, however, ALDH bright cells were associated with a significantly higher tumorigenic capacity in vivo as compared to ALDH low cells. Nevertheless, ALDH bright cells lost their higher tumorigenic capacity following serial “in vivo” passages. Most importantly, a well defined ALDH bright population could also be detected in cells isolated from PCA specimens (n>20). ALDH activity was consistent with a strong expression of several ALDH specific isoforms in PCA tissues. Notably, a significant increase of defined ALDH isoforms such as ALDH1A3 (p=0.001) was detectable in tissues obtained from patients with PCA as compared to BPH or normal specimens. CONCLUSIONS: ALDH bright subsets can be detected in cells isolated from fresh PCA tissue samples, PCA cell lines and primary cultures. These populations appear to be associated with increased “in vivo” tumorigenicity rather than enhanced “in vitro” stem properties in the cell lines investigated. Importantly, expression of ALDH specific isoforms appears to be increased in clinical PCA as compared to BPH and “normal” samples, thereby suggesting a putative role of ALDH in PCA tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3384. doi:1538-7445.AM2012-3384

Immunkompetenz bei Melanompatienten des Stadiums III–IV: Konsequenzen für eine aktiv spezifische Immuntherapie

Introduction: Patients with advanced cancer display some immunodepression. We assess here humoral and cytotoxic T lymphocyte responsiveness in melanoma patients undergoing active specific immunotherapy trials and correlate immunocompetency with the response rate to immunotherapy. Methods: 30 melanoma patients (TNM stages III and IV; n = 20 intradermal vaccination [1, 2]; n = 10 ongoing intranodal vaccination trial) were admitted to immunization trials with a recombinant vaccinia (rVV) virus encoding 3 tumor associated epitopes (TAA: gp100280–288, Mart-127–35 and tyrosinase1–9) and CD80/CD86 costimulatory molecules. Booster immunizations were performed with the same TAA peptides. Frequencies of CTL precursor (CTLp) specific for TAA or influenza matrix 58–66 (IM) control epitope were evaluated by limiting dilution analysis prior and after immunization. Humoral response against rVV vector was measured by ELISA prior and after vaccination. Depending on specific responses to immunization (CTLp > 2fold pre-treatment) patients were ranked as good responders (responsive to 3 or 2 antigens) or poor responders (unresponsive or responsive to 1 antigen only). Results: All stage III patients in both trials showed CTL responses against all 3 antigens. In contrast (p < 0.05), 6/13 stage IV patients were good responders. Specific humoral response for the rVV was increased in all stage III patients, but only in 6/13 stage IV patients (p < 0.05). A significant correlation (p < 0.05) was noted between a high humoral response against the rVV and the good responder status. The CTLp for influenza in melanoma stage IV patients was 4 times lower than in healthy volunteers, but for a patient who showed a long lasting complete response with a CTLp for influenza in normal range. The currently ongoing intranodal vaccination trial shows more good responders than the intradermal protocol (6/7 Vs 6/13 good responders, p < 0.05). Conclusion: Stage III melanoma patients and immunocompetent stage IV patients are more likely to respond to active specific immunotherapy. Immunocompromission in stage IV melanoma patients hampers the induction of tumor specific CTL responses. Conversely, induction of rVV specific humoral responses does not prevent but correlates with the generation of CTL specific for TAA. The intranodal immunization shows a higher response rate than the intradermal protocol. In summary, cellular and humoral immunocompetency are a marker for the response to immunotherapy and might therefore be an inclusion criterium in immunotherapy trial.

Verbesserung der immunstimulatorischen Wirkung von Tumorantigenen durch liposomale Vektoren

Introduction: Immunodominant epitopes from tumor associated antigens (TAA) are currently used as soluble peptides (SP) in clinical trials in spite of an acknowledged low immunogenicity. We asked whether encapsulation into liposomes (LP) could improve their immunogenic capacity. Materials and Methods: The HLA-A2 restricted melanoma associated epitope Mart26–35 was synthesized and included into sterically stabilized liposomes, characterized by prolonged bio-availability in biological fluids in comparison with conventional reagents. Proliferation, cytokine production and cytotoxicity induced by either LP or SP in Mart26-35 specific CTL clones were evaluated by 3H-thymidine incorporation, ELISA and 51Cr release assays. To assess the induction of specific CTL, peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with autologous antigen presenting cells and LP or SP. Cytotoxic activity was evaluated by 51Cr release. Results: Proliferation of CTL clones could be comparably stimulated by saturating doses (> 100 ng/ml) of either SP or LP. At limiting concentrations (< 50 ng/ml), however, only LP were effective. IFN-γ production by specific CTL was also enhanced by LP as compared to SP stimulation. Furthermore, LP displayed a significantly higher capacity of sensitizing target cells to the cytotoxic effects of specific CTL clones as compared to SP. Most importantly, LP were able to induce specific cytotoxic activity in healthy donors PBMC, following a limited number of restimulation courses (n = 2). In the same conditions SP was totally ineffective. Conclusion: These data indicate that the specific immunostimulatory capacity of Mart26-35 melanoma associated epitope can be significantly improved by encapsulation into sterically stabilized liposomes.

Induktion Tumor spezifischer zytotoxischer T-Lymphozyten durch nicht replizierende, Melan-A27–35 exprimierende rekombinante Vaccinia Viren in vitro

Heute sind Immunogene Epitope von humanen Tumor-assoziierten Antigenen (TAA) bekannt, die fur eine adjuvante Tumorvakzination genutzt werden konnen. Die Anwendung antigener Molekule und die Entwicklung von immuntherapeutischen Strategien, mit denen spezifische zytotoxische T-Lymphozyten gegen TAA induziert werden, konnte eine erfolgreiche spezifische Immuntherapie von Tumorleiden darstellen. Wir haben deshalb gepruft, ob mit einem rekombinanten Vaccinia Virus (recVV), welches das HLA-A2.1 restringierte immunodominante TAA Melan-A kodiert, Antigen-spezifische zytotoxische T-Lymphozyten (CTL) in vitro induziert werden konnen.