Samuele Tardito

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes.

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25hi Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25hi Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.

Comparative analysis of cancer vaccine settings for the selection of an effective protocol in mice

BackgroundCancer vaccines are considered a promising therapeutic approach. However, their clinical results are not yet satisfactory. This may be due to the the difficulty of selection of an efficient tumor associated antigen (TAA) and immunization protocol. Indeed, the weak antigenicity of many TAA impairs the design of robust procedures, therefore a systematic analysis to identify the most efficient TAA is mandatory. Here, we performed a study to compare different gp100 vaccination strategies to identify the best strategy to provide a 100% protection against experimental melanoma in a reproducible manner.MethodsC57BL/6J mice were challenged subcutaneously with B16F10 melanoma cells, after vaccination with: a) mouse or human gp10025-33 peptide plus CpG adjuvant; b) mouse or human gp100 gene; c) mouse or human gp10025-33 peptide-pulsed dendritic cells (DC). Alternatively, a neutralizing anti-IL-10 monoclonal antibody (mAb) was subcutaneously administered at the site of tumor challenge to counteract regulatory cells. Finally, combinatorial treatment was performed associating human gp10025-33 peptide-pulsed DC vaccination with administration of the anti-IL-10 mAb.ResultsVaccination with human gp10025-33 peptide-pulsed DC was the most effective immunization protocol, although not achieving a full protection. Administration of the anti-IL-10 mAb showed also a remarkable protective effect, replicated in mice challenged with a different tumor, Anaplastic Large Cell Lymphoma. When immunization with gp10025-33 peptide-pulsed DC was associated with IL-10 counteraction, a 100% protective effect was consistently achieved. The analysis on the T-cell tumor infiltrates showed an increase of CD4+granzyme+ T-cells and a decreased number of CD4+CD25+Foxp3+ Treg elements from mice treated with either gp10025-33 peptide-pulsed DC vaccination or anti-IL-10 mAb administration. These data suggest that processes of intratumoral re-balance between effector and regulatory T cell subpopulations may play a critical protective role in immunotherapy protocols.ConclusionsHere we demonstrate that, in the setting of a cancer vaccine strategy, a comparative analysis of different personalized approaches may favour the unveiling of the most effective protocol. Moreover, our findings suggest that counteraction of IL-10 activity may be critical to revert the intratumoral environment promoting Treg polarization, thus increasing the effects of a vaccination against selected TAA.

Indoleamine 2,3 dioxygenase gene polymorphisms correlate with CD8+ Treg impairment in systemic sclerosis

Systemic sclerosis (SSc) is characterized by tissue fibrosis, vasculopathy and autoimmunity. Indoleamine 2,3 dioxygenase (IDO) plays a pivotal role in immunological tolerance modulating regulatory T cell (Treg) generation and function. Single nucleotide polymorphisms (SNPs) of IDO gene could impact on Treg function and predispose to autoimmunity. Here, the existence of an association between specific IDO SNPs and SSc was analyzed. Five specific SNPs in IDO gene were searched in 31 SSc patients and 37 healthy controls by gene sequencing or restriction fragment length polymorphism. The function of both CD4+CD25+ and CD8+ Treg from SSc patients was analyzed by proliferation suppression assay. SNP rs7820268 was statistically more frequent in SSc patients than in controls. Notably, SSc patients bearing the T allelic variant of the rs7820268 SNP showed impaired CD8+ Treg function. Our unprecedented data show that a specific IDO gene SNP is associated with an autoimmune disease such as SSc.

Cyclophosphamide inhibits the generation and function of CD8+ regulatory T cells

CD8(+) regulatory T cells (Treg) and CD4(+)CD25(+) Treg infiltrate human cancers, thus favoring tumor immune escape. Therefore, in the setting of antitumor therapeutic protocols, it is important to associate antitumor treatment with agents that are able to inhibit Treg function. Cyclophosphamide (CY) has been demonstrated to be effective in counteracting CD4(+)CD25(+) Treg activity. Hence, we tested its inhibitory efficacy on human CD8(+) Treg. Because CY is a prodrug, 4-hydroperoxycyclophosphamide (4-HC), a derivative of CY that in aqueous solution is converted to 4-hydroxycyclophosphamide, an active metabolite of CY, was used. 4-HC significantly inhibited CD8(+) Treg generation and function but only at the higher tested concentration (0.5 μg/mL), that is, in the therapeutic range of the drug. The lower 4-HC concentration tested (0.1 μg/mL) was almost ineffective. 4-HC inhibitory effects were related to apoptosis/necrosis induction. When CD8(+)CD28(+) non-Treg were analyzed for comparative purposes, significantly lower cytotoxic rates among these cells were observed than among CD8(+) Treg, which were differentiated because they did not express the CD28 molecule. These data demonstrate that CD8(+) Treg are inhibited through cytotoxic phenomena by CY, thus supporting the use of this drug at adequate concentrations and schedules of administration as a Treg inhibitor in combinatorial chemo- or immunotherapeutic anticancer protocols.

Residual tumor micro-foci and overwhelming regulatory T lymphocyte infiltration are the causes of bladder cancer recurrence

Bladder cancer has an unexplained, high recurrence rate. Causes of recurrence might include the presence of sporadic tumor micro-foci in the residual urothelial tissue after surgery associated with an inverted ratio between intratumoral effector and regulatory T cell subsets. Hence, surgical specimens of both tumors and autologous, macroscopically/histologically free-of-tumor tissues were collected from 28 and 20 patients affected by bladder or renal cancer, respectively. The frequencies of effector (IFNγ+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127lo and CD8+CD28-CD127loCD39+ Treg) T cell subpopulations among tumor infiltrating lymphocytes were analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor-associated antigens was studied by RT-PCR. The results show that both the T cell infiltrate and the frequency of MAGE-A1/A2 gene expression were comparable in tumors and in autologous free-of-tumor tissues in bladder cancer, while the autologous free-of-tumor renal tissues showed reduced T cell infiltrate and frequency of MAGE gene expression as compared to the autologous tumors. Importantly, the intra-tumor T effector/Treg cell ratio was consistently <1 in bladder cancer patients (n. 7) who relapsed within two years, while it was always >1 in patients (n. 6) without recurrence (regardless of tumor stage) (P = 0.0006, Odds ratio = 195). These unprecedented findings clarify the pathogenic mechanism of bladder cancer recurrence and suggest that microscopically undetectable micro-foci of tumor may predispose to recurrence when associated with an inverted intratumoral T effector/Treg cell ratio.

Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis, vasculopathy, and autoimmunity. Although the exact pathogenetic mechanisms behind SSc remain to be fully elucidated, a great deal of evidence suggests the existence of an unbalanced ratio between the effector and regulatory arms of the immune system. With regard to the T regulatory (Treg) compartment, we observed that CD8+ Treg subsets display functional defects in SSc-affected patients. Since CD127 down-modulation and CD39 upregulation have been observed on Treg subsets, the phenotypic expression of these molecules was analyzed on the CD8+CD28− Treg precursors and on CD8+ Treg cells generated in vitro through interleukin-10 commitment. Immunophenotypic data from SSc patients were compared to those obtained from healthy subjects. The analyses performed on ex vivo-isolated CD8+CD28− Treg precursors did not show any significant differences in CD39 or CD127 expression as compared to values obtained from healthy donors. On the contrary, in vitro-generated CD8+ Tregs obtained from SSc patients displayed reduced expression of the CD39 molecule as compared to controls. Moreover, the percentage of CD127+ cells was significantly higher in in vitro-generated CD8+ Tregs from SSc patients compared to CD8+ Tregs obtained from healthy donors. Taken together, these findings may indicate an impairment of maturation processes affecting CD8+ Treg cells in SSc patients. This impairment of maturation involves phenotypic alterations that are mainly characterized by a deficient CD39 upregulation and a lack of down-modulation of the CD127 molecule.

AIRE polymorphism, melanoma antigen-specific T cell immunity, and susceptibility to melanoma

AIRE is involved in susceptibility to melanoma perhaps regulating T cell immunity against melanoma antigens (MA). To address this issue, AIRE and MAGEB2 expressions were measured by real time PCR in medullary thymic epithelial cells (mTECs) from two strains of C57BL/6 mice bearing either T or C allelic variant of the rs1800522 AIRE SNP. Moreover, the extent of apoptosis induced by mTECs in MAGEB2-specific T cells and the susceptibility to in vivo melanoma B16F10 cell challenge were compared in the two mouse strains. The C allelic variant, protective in humans against melanoma, induced lower AIRE and MAGEB2 expression in C57BL/6 mouse mTECs than the T allele. Moreover, mTECs expressing the C allelic variant induced lower extent of apoptosis in MAGEB2-specific syngeneic T cells than mTECs bearing the T allelic variant (p < 0.05). Vaccination against MAGEB2 induced higher frequency of MAGEB2-specific CTL and exerted higher protective effect against melanoma development in mice bearing the CC AIRE genotype than in those bearing the TT one (p < 0.05). These findings show that allelic variants of one AIRE SNP may differentially shape the MA-specific T cell repertoire potentially influencing susceptibility to melanoma.

White matter microstructure alterations correlate with terminally differentiated CD8+ effector T cell depletion in the peripheral blood in mania: Combined DTI and immunological investigation in the different phases of bipolar disorder

BACKGROUND White matter (WM) microstructural abnormalities and, independently, signs of immunological activation were consistently demonstrated in bipolar disorder (BD). However, the relationship between WM and immunological alterations as well as their occurrence in the various phases of BD remain unclear. METHOD In 60 type I BD patients - 20 in manic, 20 in depressive, 20 in euthymic phases - and 20 controls we investigated: (i) diffusion tensor imaging (DTI)-derived fractional anisotropy (FA), radial diffusivity (RD) and axial diffusivity (AD) using a tract-based spatial statistics (TBSS) approach; (ii) circulating T cell subpopulations frequencies, as well as plasma levels of different cytokines; (iii) potential relationships between WM and immunological data. RESULTS We found: (i) a significant widespread combined FA-RD alteration mainly in mania, with involvement of the body of corpus callosum (BCC) and superior corona radiata (SCR); (ii) significant increase in CD4+ T cells as well as significant decrease in CD8+ T cells and their subpopulations effector memory (CD8+ CD28-CD45RA-), terminal effector memory (CD8+ CD28-CD45RA+) and CD8+ IFNγ+ in mania; (iii) a significant relationship between WM and immunological alterations in the whole cohort, and a significant correlation of FA-RD abnormalities in the BCC and SCR with reduced frequencies of CD8+ terminal effector memory and CD8+ IFNγ+ T cells in mania only. CONCLUSIONS Our data show a combined occurrence of WM and immunological alterations in mania. WM abnormalities highly correlated with reduction in circulating CD8+ T cell subpopulations that are terminally differentiated effector cells prone to tissue migration, suggesting that these T cells could play a role in WM alteration in BD.

Abstract B127: Free DNA and tolerance

Soluble DNA circulates in the peripheral blood of healthy subjects, and, at increased concentrations, of pregnant women and patients affected by tumors and vasculitis. Since the discovery of intracellular DNA sensors, inducing inflammatory responses after binding to DNA, DNA has been included among danger signals. However, this view does not explain the presence of abundant circulating DNA in conditions characterized by tolerance (pregnancy) or immunodeficiency (tumor). In order to verify whether DNA mediates also an immunoregulating activity, a 20 base-pair long oligonucleotide (poly-CG) was synthesized and tested for its capacity to: 1) bind MHC class II molecules; 2) exert immunoregulatory activity. The oligo binding to PMJ2-PC macrophages was inhibited by anti-MHC monoclonal antibodies (mAb). The specific binding of DNA to MHC class II molecules was confirmed by western blot analysis. Proliferation of OVA-specific splenocytes was inhibited by incubation with the oligo as well as by circulating DNA from cancer patients; moreover, fluorescent oligo, injected intravenously to NZB/W F1 mice, bound only on MHC class II positive cells (macrophages and B lymphocytes). Finally, nephritic NZB/W F1 mice, administered weekly with the oligo, showed delayed onset of protenuria and improved survival with respect to untreated animals, a phenomenon associated with increased frequencies of Treg. Interestingly, in vitro incubation of PMJ2-PC macrophages with the oligo induced increased CCL22 secretion. These data demonstrate for the first time that free DNA may mediate immunoregulatory activities. Citation Format: Gilberto Filaci, Francesca Ferrera, Samuele Tardito, Tiziana Altosole, Cinzia Bernardi, Alessia Parodi, Daniela Fenoglio. Free DNA and tolerance [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B127.

Abstract A020: The analyses of immune infiltrate and gene expression of MAGE antigens in bladder cancer allow to explain and predict recurrence

Bladder cancer is characterized by a high rate of recurrence following surgical treatments. This phenomenon, not observed in other urogenital malignancies such as renal cancer, is still unexplained. Moreover, there are not markers allowing to predict recurrence in bladder cancer patients after the treatment. We faced these problems hypothesizing that: 1) recurrence in bladder cancer could be related to the existence of sporadic micro-foci of tumor in the residual (after surgery), macroscopically healthy urothelial tissue; 2) the ratio between intratumoral effector and regulatory T cell subsets could represent an efficient biomarker of bladder cancer recurrence. Hence, surgical specimens of both the tumor and the autologous, macroscopically healthy tissue were collected from 24 and 20 patients affected with bladder or renal cancer, respectively. The composition of the intratumoral T cell infiltrate, in terms of effector (IFNγ+ and IL17+ T cells) and regulatory (CD4+CD25hiCD127low and CD8+CD28-CD127lowCD39+ Treg) T cell subpopulations, was analyzed by immunofluorescence, while the gene expression of MAGE-A1 and MAGE-A2 tumor associated antigens was studied by RT-PCR. The T cell infiltrate of bladder and renal cancers were comparable in terms of frequencies of IFNγ+ and IL17+ T cells and CD8+CD28-CD127lowCD39+ Treg, while the CD4+CD25hiCD127low Treg predominated in bladder cancers (P = 0.0006). Importantly, the macroscopically healthy bladder tissue showed a T cell subset composition comparable with that of the autologous tumor, as if a similar immune reaction were at play. Instead, significantly lower frequencies of each of the tested T cell subsets were present in the macroscopically healthy renal tissue than in the autologous tumor. When the molecular gene expression of two tumor associated antigens, MAGE-A1 and MAGE-A2 (frequently expressed by both bladder and renal cancers), was analyzed, similar frequencies of expression were found in both tumors. On the contrary, the macroscopically healthy bladder tissue showed a frequency of MAGE gene expression remarkably higher (and almost comparable to that of the autologous tumor) than the macroscopically renal healthy tissue (P = 0.03) (where MAGE gene expression was almost negligible), supporting for the presence of sporadic micro-foci of tumor in the apparently healthy bladder tissue at the time of surgical intervention. In order to identify a reliable marker for recurrence, the Teff/Treg intratumoral T cell ratio was analyzed in 13 bladder cancer patients showing (6 patients) or not (7 patients) recurrence after a 2 year follow up. The Teff/Treg intratumoral T cell ratio demonstrated to be a consistent biomarker for recurrence in bladder cancer since a ratio 1 were invariably (100% of patients in each group) associated with recurrence and not recurrence, respectively. These unprecedented findings unveil the possible pathogenic mechanism for recurrence in bladder cancer and suggest that the Teff/Treg intratumoral T cell ratio is a reliable biomarker of recurrence in this disease. If confirmed, they may impact on both prognostic and the therapeutic approaches for bladder cancer. Citation Format: Daniela Fenoglio, Paolo Traverso, Alessia Parodi, Francesca Kalli, Giuseppina Conteduca, Samuele Tardito, Monica Curto, Francesca Ferrera, Federica Grillo, Luca Mastracci, Francesco Minaglia, Alchiede Simonato, Giorgio Carmignani, Gilberto Filaci. The analyses of immune infiltrate and gene expression of MAGE antigens in bladder cancer allow to explain and predict recurrence. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A020.