Francesca Kalli

Alteration of Th17 and Treg cell subpopulations co-exist in patients affected with systemic sclerosis.

Aim of the study has been to understand the relationship between TH17 and Treg cell subsets in patients affected with systemic sclerosis (SSc). Phenotypes and functions of Th17 and Treg cell subsets were analyzed in a series of 36 SSc patients. Th17 cell concentration in the peripheral blood was found to be increased in SSc patients with respect to healthy controls independently from type or stage of disease. After PBMC stimulation with a polyclonal stimulus or Candida albicans antigens the frequency of Th17 T cell clones was significantly higher in SSc patients with respect to controls suggesting the skewing of immune response in SSc patients toward Th17 cell generation/expansion. Concerning the Treg compartment, both CD4+CD25+ and CD8+CD28- Treg subsets showed quantitative and qualitative alteration in the peripheral blood of SSc patients. Collectively, these data highlight the existence of an imbalanced ratio between Th17 and Treg cell subsets in SSc patients.

Th17 and regulatory T lymphocytes in primary biliary cirrhosis and systemic sclerosis as models of autoimmune fibrotic diseases

Fibrotic autoimmune diseases are characterized by an inflammatory process in which fibrogenic cytokines, such as TGFβ and IL6, have a major role. Interestingly, these cytokines are also involved in the generation and function of both an effector T lymphocyte subpopulation, the Th17 cells, and the regulatory T lymphocytes (Treg). These evidences raised the hypothesis that an unbalanced equilibrium induced by the overproduction of the fibrogenic cytokines may have pathogenic relevance in fibrotic autoimmune diseases. On this basis, this review analyzes the available data concerning Th17 and Treg generation and function in two representative fibrotic autoimmune diseases, primary biliary cirrhosis (PBC) and systemic sclerosis (SSc), as models for organ-specific and systemic diseases, respectively. With regard to the Th17 cells, their expansion was found to be a common feature associated with a relative contraction of Th1 immune responses. Concerning to the regulatory T cell compartment, quantitative and qualitative alterations were observed in both diseases. However, while PBC patients showed defects only in the CD8+ Treg subset, SSc patients demonstrated abnormalities regarding to both the CD4+CD25+ and the CD8+ Treg subpopulations. Hence, the CD8+ Treg subset seems to be the most involved in the pathogenic cascade leading to fibrotic disease onset and maintenance. Collectively, the reviewed data support the concept that altered homeostasis between effector and regulatory T cell circuits is present in fibrotic autoimmune diseases and that the major factors responsible for such disequilibrium are Th17 cells in the effector arm and CD8+ Treg in the regulatory arm.

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.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

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.

Immunogenicity of GX301 cancer vaccine: Four (telomerase peptides) are better than one

Peptide540–548, peptide611–626, peptide672–686 and peptide766–780, which are derived from human telomerase, constitute the immunogenic component of the GX301 cancer vaccine. The relative immunogenicity of these peptides is unknown, thus it is unsure whether their combined use offers real advantages over single peptide stimulation. Hence, this study compared the number of specific immune responses and responders to each peptide, as well as to their mixture (meaning the co-presence of the 4 peptides in the same culture well), achieved after ex vivo stimulation of PBMC from 21, HLA-A2+ (n.11) or HLA-A2- (n.10), healthy donors. The study was performed on freshly collected PBMC (T0) and on PBMC stimulated for 10 d with single peptides or their mixture (T1). Peptide-specific immune responses were analyzed by Elispot and cytokine intracellular staining by flow cytometry. The results showed that each peptide induced specific immune responses in some subjects, with different panels of responders among the peptides. Moreover, the numbers of responses and responders to the single peptides or their mixture were comparable. Importantly, the overall number of responders to the 4 peptides was higher than to each single peptide, or to their mixture, both at T0 and T1. These data demonstrate the immunogenicity of each of the 4 GX301 telomerase peptides. Moreover, they show the advantage of multi-peptide over single peptide stimulation, providing a clear support to their combined administration in vaccination protocols. However, the data pose a warning against peptide administration as a mixture due to possible interference phenomena during antigen presentation processes.

Generation of more effective cancer vaccines

Cancer vaccines represent a promising therapeutic approach for which prime time is imminent. However, clinical efficacy must be improved in order for cancer vaccines to become a valid alternative or complement to traditional cancer treatments. Considerable efforts have been undertaken so far to better understand the fundamental requirements for clinically-effective cancer vaccines. Recent data emphasize that important requirements, among others, are (1) the use of multi-epitope immunogens, possibly deriving from different tumor antigens; (2) the selection of effective adjuvants; (3) the association of cancer vaccines with agents able to counteract the regulatory milieu present in the tumor microenvironment; and (4) the need to choose the definitive formulation and regimen of a vaccine after accurate preliminary tests comparing different antigen formulations. The first requirement deals with issues related to HLA restriction of tumor antigen presentation, as well as usefulness of tumor antigen spreading and counteraction of immune escape phenomena, linked to tumor antigen down-modulation, for an effective anti-cancer immune response. The second point underscores the necessity of optimal activation of innate immunity to achieve an efficient adaptive anti-cancer immune response. The third point focuses on the importance to inhibit subsets of regulatory cells. The last requirement stresses the concept that the regimen and formulation of the vaccine impacts profoundly on cancer vaccine efficacy. A new generation of cancer vaccines, provided with both immunological and clinical efficacy, will hopefully soon address these requirements.