PHENOTYPIC AND FUNCTIONAL MODULATION OF REGULATORY T CELLS IN MELANOMA PATIENTS

Recent studies indicated that regulatory T cells (Tregs) are implicated in the suppression of the immune response against tumors. Accumulation of Tregs in peripheral blood and in tumor microenvironment has been described for patients with various types of cancer. The balance between T effector cells and Tregs, both at tumor site or at local draining lymph nodes is subverted thus limiting the immune response against cancer and restraining the effect of immunotherapy. As concern melanoma, data till now available confirm the presence of Tregs at tumor site, however no in deep analysis on phenotypic or functional characterization of these cells have been provided. This thesis is aimed at evaluating the role of CD4+ Tregs in the immune response against melanoma either by in vivo or in vitro approaches. Among molecules found in mice as expressed by Tregs, lymphocytes activation gene-3 (LAG-3) has been described to be expressed by activated Tregs and more in general as a molecule involved in the control of T cell expansion and homeostasis. In this thesis I explored the expression of LAG-3 in human CD4+ T cells and found that LAG-3 identifies a discrete subset of CD4+CD25highFoxp3+ T cells. This CD4+CD25highFoxp3+LAG-3+ population is preferentially expanded in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral and sub cute metastasis of melanoma patients. Ex-vivo analysis showed that CD4+CD25highFoxp3+LAG-3+ T cells are functionally active cells that release the immunosuppressive cytokines interleukin-10 (IL-10) and transforming growth factor beta (TGF-\uf0621). An in vitro suppression assay using CD4+CD25highLAG-3+ T cells sorted from in vitro expanded CD4+CD25high Tregs showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. All together, our data show that LAG-3 defines an active CD4+CD25high Tregs subset in melanoma patients whose frequency is expanded at tumor sites. My data showed that Tregs are accumulated in different immunological districts of patients with melanoma and they also stress the notion that in melanoma patients these Tregs are preferentially in an activation status. However, the real impact of these cells on tumor progression has not been totally clarified. To get insights on the relevance of Tregs in the immunological response to tumor, a phase II randomized trial of multipeptide vaccination in stage IIB-C/III melanoma patients has been designed to include the administration of low dose cyclophosphamide (CTX; 300mg/m2) and low dose interleukin-2 (IL-2; 3x106). CTX has been described as limiting the expansion of Tregs, while IL-2 was given with the aim of expanding tumor-specific responses. The modulation that these drugs exert on different T cell compartment, namely Tregs and conventional T cells was evaluated for its impact on patients\u2019 immunological response. Careful ex-vivo immunological monitoring has been performed aimed at assessing the status of vaccine-induced immune response and the levels of Tregs. Importantly, Treg frequency was defined combining physical and functional markers trying to take into account the plasticity of this population. We observed that CTX has a limited efficacy and a transient effect on Tregs modulation; frequency of Tregs identified by multiparametric fluorescence-activated cell sorting (FACS) analysis as CD4+CD25highFoxp3+ dropped in peripheral blood mononuclear cells (PBMCs) collected 4-7 days after CTX administration, with 6 out of 13 patients displaying a reduction ranging from 20 to 65 %. IL-2 showed higher immunomodulatory effects, expanding both circulating conventional activated CD4+ T cells and Tregs; interestingly, a fraction of these Tregs displayed a Th-1 like phenotype, expressing ex-vivo T-bet (Th1 specific T-box transcription factor) and interferon-\uf067\uf020\uf028INF-\uf067\uf029. Importantly, this enhanced frequency of Tregs does not significantly affect patients\u2019 immunization assessed ex-vivo by human leukocyte antigen (HLA)-A*0201/peptide multimer staining and IFN-\uf067 ELISpot assays

TLR3 expression induces apoptosis in human non‐small‐cell lung cancer

The prognostic value of Toll\u2010like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early stage non\u2010small\u2010cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3\u2019s favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase\u20103 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re\u2010activate local innate immune response

A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases. In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease

In Situ Prior Proliferation of CD4+ CCR6+ Regulatory T Cells Facilitated by TGF-β Secreting DCs Is Crucial for Their Enrichment and Suppression in Tumor Immunity

BACKGROUND: CD4(+)CD25(+) regulatory T cells (Tregs), a heterogeneous population, were enrichment in tumor mass and played an important role in modulating anti-tumor immunity. Recently, we reported a Treg subset, CCR6(+) Tregs but not CCR6(-)Tregs, were enriched in tumor mass and closely related to poor prognosis of breast cancer patients. However, the underlying mechanism remains elusive. Here, we carefully evaluate the enrichment of CCR6(+)Tregs in tumor mass during progression of breast cancer and explore its possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The frequency of CCR6(+)Tregs in tumor infiltrating lymphocytes (TILs ) was analyzed at early stage and at late stage of tumor in a murine breast cancer model by FACS respectively. The expansion of CCR6(+)Tregs and their CCR6(-) counterpart in tumor mass were determined by BrdU incorporation assay. The effect and its possible mechanism of tumor-resident antigen presenting cells (APCs) on the proliferation of CCR6(+)Tregs also were evaluated. The role of local expansion of CCR6(+)Tregs in their enrichment and suppression in vivo also was evaluated in adoptive cell transfer assay. We found that the prior enrichment of CCR6(+)Tregs but not CCR6(-)Tregs in tumor mass during progression of murine breast cancer, which was dependent on the dominant proliferation of CCR6(+) Tregs in situ. Further study demonstrated that tumor-resident DCs triggered the proliferation of CCR6(+)Treg cells in TGF-β dependent manner. Adoptive transfer of CCR6(+)Tregs was found to potently inhibit the function of CD8(+)T cells in vivo, which was dependent on their proliferation and subsequently enrichment in tumor mass. CONCLUSIONS/SIGNIFICANCE: Our finding suggested that CCR6(+) Tregs, a distinct subset of Tregs, exert its predominant suppressive role in tumor immunity through prior in situ expansion, which might ultimately provide helpful thoughts for the designing of Treg-based immunotherapy for tumor in the future

Validity of Anti-PSMA ScFvD2B as a Theranostic Tool: A Narrative-Focused Review

Prostate cancer (PCa) is the second leading cause of cancer among men, and its diagnosis and adequate staging are fundamental. Among the biomarkers identified in recent years for PCa management, prostate-specific-membrane-antigen (PSMA), physiologically expressed at a low level on healthy prostate and in other normal tissues and highly overexpressed in PCa, represents a reliable marker ideal for imaging and therapy. The development of anti-PSMA antibodies, such as D2B, demonstrated slow clearance of intact antibodies compared with fragments resulting in low tumor-to-blood ratios; however, the modular structural and functional nature of antibodies allowed the generation of smaller fragments, such as scFvs. In this review of the anti-PSMA antibody fragment scFvD2B, we combined further characterization of its biomolecular and tissue cross-reactivity characteristics with a comprehensive summary of what has already been performed in preclinical models to evaluate imaging and therapeutic activities. A molecular dynamics study was performed, and ScFvD2B occupied a limited conformational space, characterized by low-energy conformational basins, confirming the high stability of the protein structure. In the cross-reactivity study, the weak/absent immunoreactivity in non-tumor tissues was comparable to the PSMA expression reported in the literature. Biodistribution studies and therapeutic treatments were conducted in different animal models obtained by subcutaneous or locoregional injection of PSMA-positive-versus-negative xenografts. The maximum tumor uptake was observed for (123)I(SPECT), (124)I(PET), and optical imaging, which avoids kidney accumulation (compared with radiometals) and leads to an optimal tumor-to-kidney and tumor-to-background ratios. Regarding its possible use in therapy, experimental data suggested a strong and specific antitumor activity, in vitro and in vivo, obtained using CAR-T or NK-92/CAR cells expressing scFvD2B. Based on presented/reviewed data, we consider that scFvD2B, due to its versatility and robustness, seems to: (i) overcome some problems observed in other studied scFvs, very often relatively unstable and prone to form aggregates; (ii) have sufficient tumor-to-background ratios for targeting and imaging PSMA-expressing cancer; (iii) significantly redirect immune killing cells to PSMA-positive tumors when inserted in second-generation CAR-T or NK-92/CAR cells. These data suggest that our product can be considered the right reagent to fill the gap that still exists in PCa diagnosis and treatment