Silvia Dalla Santa

Predicting Tumor Outcome following Cancer Vaccination by Monitoring Quantitative and Qualitative CD8+ T Cell Parameters

Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.

Survivin expression impacts prognostically on NSCLC but not SCLC

Survivin is expressed in lung cancer and in most cancer tissues and has a significant impact on prognosis. This work aimed to comparatively assess survivin expression and significance in Non-Small (NSCLC) and Small Cell Lung Cancers (SCLC). Sixty-five NSCLC and 35 SCLC samples were analyzed by semi-quantitative real-time RT-PCR. Survivin mRNA levels were significantly higher in tumors than in normal tissue, and in SCLC than in NSCLC samples. Immunohistochemistry and FISH analyses were performed in 59 and 26 tumor specimens, respectively. In SCLC survivin was only present in cytoplasm, while in some NSCLC cases it also showed nuclear or mixed patterns. FISH analysis did not disclose survivin gene amplification, except for one NSCLC case. Finally, 90 samples were genotyped for the -31G/C SNP of survivin promoter by direct sequencing; the -31G/C SNP genotype status showed a significant association only with nodal NSCLC metastasis, but not with survivin expression in any tumor group. A better prognosis was correlated to higher levels of survivin mRNA and to the presence of at least one G allele at -31 SNP in NSCLC, while these parameters did not correlate with overall survival in SCLC. Moreover, this SNP would appear to have no effect on the risk of lung cancer in our samples. The different prognostic role played by survivin in NSCLC and SCLC highlights the biological differences between these lung tumor histotypes and stresses the need to clarify the molecular pathways leading to their neoplastic transformation.

Individual Analysis of Mice Vaccinated against a Weakly Immunogenic Self Tumor-Specific Antigen Reveals a Correlation between CD8 T Cell Response and Antitumor Efficacy

The weakly immunogenic murine P1A Ag is a useful experimental model for the development of new vaccination strategies that could potentially be used against human tumors. An i.m. DNA-based immunization procedure, consisting of three inoculations with the P1A-coding pBKCMV-P1A plasmid at 10-day intervals, resulted in CTL generation in all treated BALB/c mice. Surprisingly, gene gun skin bombardment with the pBKCMV-P1A vector did not induce CTL, nor was it protective against a lethal challenge with the syngeneic P1A-positive J558 tumor cell line. To speed up the immunization procedure, we pretreated the tibialis anterior muscles with cardiotoxin, which induces degeneration of myocytes while sparing immature satellite cells. The high muscle-regenerative activity observable after cardiotoxin inoculation was associated with infiltration of inflammatory cells and expression of proinflammatory cytokines. A single pBKCMV-P1A plasmid inoculation in cardiotoxin-treated BALB/c mice allowed for sustained expansion of P1A-specific CTL and the induction of strong lytic activity in <2 wk. Cardiotoxin adjuvanticity could not be replaced by another muscle-degenerating substance, such as bupivacaine, or by MF59, a Th1 response-promoting adjuvant. Although this vaccination schedule failed to induce tumor rejection in all immunized mice, the analysis of CD8 T cell responses at an individual mouse level disclosed that the cytotoxic activity of P1A-specific CTL was correlated to the antitumor efficacy. These results highlight the critical need to identify reliable, specific immunological parameters that may predict success or failure of an immune response against cancer.

A Large Number of T Lymphocytes Recognize Moloney-Murine Leukemia Virus-Induced Antigens, but a Few Mediate Long-Lasting Tumor Immunosurveillance

The CD8+ T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRVβ chain rearrangements. In mice lacking T cells expressing these TCRVβ, we demonstrate that alternative TCRVβ can substitute for the lack of the dominant TCRVβ in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2b-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant Vβ rescues the alternative Vβ response. The mechanism of clonal T cell “immunodomination” that guides the preferential Vβ expansion is likely the result of a proliferative advantage of T cells expressing dominant Vβ, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, Vβ gene rearrangements. The ability of T cells expressing alternative TCRVβ rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative Vβ chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.

Contribution of susceptibility gene variants to melanoma risk in families from the Veneto region of Italy

to take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Contribution of susceptibility gene variants to melanoma risk in families from the Veneto region of italy Chiara menin, antonella Vecchiato, maria Chiara scaini, lisa Elefanti, gloria Funari, gian luca de salvo, monica Quaggio, silvia tognazzo, simona agata, silvia dalla santa, marco montagna, mauro alaibac, Vanna Chiarion-sileni6 and Emma d’andrea

Functional Avidity–Driven Activation-Induced Cell Death Shapes CTL Immunodominance

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8+ T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional “immune kamikazing.” Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma

In the Sp6 mouse plasmacytoma model, a whole‐cell vaccination with Sp6 cells expressing de novo B7‐1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL) ‐mediated protection against wild‐type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down‐regulated expression of MHC H‐2 Ld. Increase of H‐2 Ld expression by cDNA transfection (Sp6/B7/Ld) raised tumour immune protection and shifted most CTL responses towards H‐2 Ld‐restricted antigenic epitopes. The tumour‐protective responses were not specific for the H‐2 Ld‐restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL‐lysed Sp6 cell cultures, containing damage‐associated molecular patterns (DAMPs). It has been shown that Toll‐like receptor triggering induces gp70 expression. Damage‐associated molecular patterns are released by CTL‐mediated killing of Sp6/B7‐Sp6/B7/Ld cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen‐presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6‐cytosol, discharged by dying cells and superinfecting antigen‐presenting cells. The outcome of such a massive gp70 cross‐presentation would probably be tolerogenic for the high‐affinity AH1‐gp70‐specific CTL clones. In this scenario, autologous whole‐tumour‐cell vaccines rescue tumour‐specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost.

Fighting viral infections and virus-driven tumors with cytotoxic CD4+ T cells

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors.

Identification of a HLA-A*0201-restricted immunogenic epitope from the universal tumor antigen DEPDC1

ABSTRACT The identification of universal tumor-specific antigens shared between multiple patients and/or multiple tumors is of great importance to overcome the practical limitations of personalized cancer immunotherapy. Recent studies support the involvement of DEPDC1 in many aspects of cancer traits, such as cell proliferation, resistance to induction of apoptosis and cell invasion, suggesting that it may play key roles in the oncogenic process. In this study, we report that DEPDC1 expression is upregulated in most types of human tumors, and closely linked to a poorer prognosis; therefore, it might be regarded as a novel universal oncoantigen potentially suitable for targeting many different cancers. In this regard, we report the identification of a HLA-A*0201 allele-restricted immunogenic DEPDC1-derived epitope, which is able to induce cytotoxic T lymphocytes (CTL) exerting a strong and specific functional response in vitro toward not only peptide-loaded cells but also triple negative breast cancer (TNBC) cells endogenously expressing the DEPDC1 protein. Such CTL are also therapeutically active against human TNBC xenografts in vivo upon adoptive transfer in immunodeficient mice. Overall, these data provide evidence that this DEPDC1-derived antigenic epitope can be exploited as a new tool for developing immunotherapeutic strategies for HLA-A*0201 patients with TNBC, and potentially many other cancers.