CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

IFN-Alpha-Mediated Differentiation of Dendritic Cells for Cancer Immunotherapy: Advances and Perspectives.

The past decade has seen tremendous developments in novel cancer therapies through targeting immune-checkpoint molecules. However, since increasing the presentation of tumor antigens remains one of the major issues for eliciting a strong antitumor immune response, dendritic cells (DC) still hold a great potential for the development of cancer immunotherapy. A considerable body of evidence clearly demonstrates the importance of the interactions of type I IFN with the immune system for the generation of a durable antitumor response through its effects on DC. Actually, highly active DC can be rapidly generated from blood monocytes in vitro in the presence of IFN-α (IFN-DC), suitable for therapeutic vaccination of cancer patients. Here we review how type I IFN can promote the ex vivo differentiation of human DC and orientate DC functions towards the priming and expansion of protective antitumor immune responses. New epigenetic elements of control on activation of the type I IFN signal will be highlighted. We also review a few clinical trials exploiting IFN-DC in cancer vaccination and discuss how IFN-DC could be exploited for the design of effective strategies of cancer immunotherapy as a monotherapy or in combination with immune-checkpoint inhibitors or immunomodulatory drugs.

Advances and perspectives of dendritic cell-based active immunotherapies in follicular lymphoma.

Follicular lymphoma (FL) is a remarkably immune-responsive malignancy, which is still considered incurable. As, standard immunochemotherapy is complex, toxic and not curative, improvement in FL care is now a crucial topic in hemato-oncology. Recently, we and others have shown that dendritic cell (DC)-based therapies allow a specific immune response associated with sustained lymphoma regression in a proportion of low-tumor burden FL patients. Importantly, the rate of objective clinical response (33-50%) and of sustained remission is remarkably higher compared to similar studies in solid tumors, corroborating the assumption of the immune responsiveness of FL. Our experimental intra-tumoral strategy combined injection with rituximab and interferon-α-derived dendritic cells (IFN-DC), a novel DC population particularly efficient in biasing T-helper response toward the Th1 type and in the cross-priming of CD8 + T cells. Noteworthy, intra-tumoral injection of DC is a new therapeutic option based on the assumption that following the induction of cancer-cell immunogenic death, unloaded DC would phagocytize in vivo the tumor associated antigens and give rise to a specific immune response. This approach allows the design of easy and inexpensive schedules. On the other hand, advanced and straightforward methods to produce clinical-grade antigenic formulations are currently under development. Both unloaded DC strategies and DC-vaccines are suited for combination with radiotherapy, immune checkpoint inhibitors, immunomodulators and metronomic chemotherapy. In fact, studies in animal models have already shown impressive results, while early-phase combination trials are ongoing. Here, we summarize the recent advances and the future perspectives of DC-based therapies in the treatment of FL patients.

Lenalidomide improves the therapeutic effect of an interferon-α-dendritic cell-based lymphoma vaccine.

The perspective of combining cancer vaccines with immunomodulatory drugs is currently regarded as a highly promising approach for boosting tumor-specific T cell immunity and eradicating residual malignant cells. The efficacy of dendritic cell (DC) vaccination in combination with lenalidomide, an anticancer drug effective in several hematologic malignancies, was investigated in a follicular lymphoma (FL) model. First, we evaluated the in vitro activity of lenalidomide in modulating the immune responses of lymphocytes co-cultured with a new DC subset differentiated with IFN-α (IFN-DC) and loaded with apoptotic lymphoma cells. We next evaluated the efficacy of lenalidomide and IFN-DC-based vaccination, either alone or in combination, in hu-PBL-NOD/SCID mice bearing established human lymphoma. We found that lenalidomide reduced Treg frequency and IL-10 production in vitro, improved the formation of immune synapses of CD8 + lymphocytes with lymphoma cells and enhanced anti-lymphoma cytotoxicity. Treatment of lymphoma-bearing mice with either IFN-DC vaccination or lenalidomide led to a significant decrease in tumor growth and lymphoma cell spread. Lenalidomide treatment was shown to substantially inhibit tumor-induced neo-angiogenesis rather than to exert a direct cytotoxic effect on lymphoma cells. Notably, the combined treatment with the vaccine plus lenalidomide was more effective than either single treatment, resulting in the significant regression of established tumors and delayed tumor regrowth upon treatment discontinuation. In conclusion, our data demonstrate that IFN-DC-based vaccination plus lenalidomide exert an additive therapeutic effect in xenochimeric mice bearing established lymphoma. These results may pave the way to evaluate this combination in the clinical ground.

Clinical and Antitumor Immune Responses in Relapsed/Refractory Follicular Lymphoma Patients after Intranodal Injections of IFNα-Dendritic Cells and Rituximab: a Phase I Clinical Trial.

PURPOSE: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. PATIENTS AND METHODS: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. RESULTS: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. CONCLUSIONS: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.

NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients.

Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity.

Epstein-Barr virus infection induces miR-21 in terminally differentiated malignant B cells.

The association of Epstein-Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR-21 is induced after in vitro EBV infection. The PU.1 binding site in miR-21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR-21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR-21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR-21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.

IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing.

Cross-presentation allows antigen-presenting cells to present exogenous antigens to CD8(+) T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8(+) T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing-dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.

Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12.

IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.

Anti-tumor CD8+ T cell immunity elicited by HIV-1-based virus-like particles incorporating HPV-16 E7 protein.

Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.

In vitro and in vivo efficacy of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on human melanoma.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. In the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC(50) values in the low micromolar range (IC(50) of 1.2+/-0.1microM and 2.0+/-0.2 microM for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, JNK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of tumour inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies.

IFN-alpha in the generation of dendritic cells for cancer immunotherapy.

Dendritic cells (DCs) play a crucial role in linking innate and adaptive immunity, by virtue of their unique ability to take up and process antigens in the peripheral blood and tissues and, upon migration to draining lymph nodes, to present antigen to resting lymphocytes. Notably, these DC functions are modulated by cytokines and chemokines controlling the activation and maturation of these cells, thus shaping the response towards either immunity or tolerance.An ensemble of recent studies have emphasized an important role of type I IFNs in the DC differentiation/activation, suggesting the existence of a natural alliance between these cytokines and DCs in linking innate and adaptive immunity. Herein, we will review how type I IFNs can promote the ex vivo differentiation of human DCs and orient DC functions towards the priming and expansion of protective antitumor immune responses. We will also discuss how the knowledge on type I IFN-DC interactions could be exploited for the design of more selective and effective strategies of cancer immunotherapy.

IFN-alpha-conditioned dendritic cells are highly efficient in inducing cross-priming CD8(+) T cells against exogenous viral antigens.

Dendritic cells (DC) generated after a short-term exposure of monocytes to IFN-alpha and GM-CSF (IFN-DC) are highly effective in inducing cross-priming of CD8(+ )T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN-DC and IL-4-derived DC. Both DC types efficiently cross-presented soluble HCV NS3 protein to the specific CD8(+) T cell clone, even though IFN-DC were superior in cross-presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV-1 and injected into hu-PBL-SCID mice, the generation of virus-specific CD8(+ )T cells was markedly higher in animals immunized with IFN-DC than in mice immunized with CD40L-matured IL-4-DC. Of interest, in experiments with purified CD8(+ )T cells, IFN-DC were superior with respect to CD40L-matured IL-4-DC in inducing in vitro cross-priming of HIV-specific CD8(+ )T cells. This property correlated with enhanced potential to express the specific subunits of the IL-23 and IL-27 cytokines. These results suggest that IFN-DC are directly licensed for an efficient CD8(+) T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL-12 family cytokines.

Type I interferons as regulators of the differentiation/activation of human dendritic cells: methods for the evaluation of IFN-induced effects.

Recent studies have revealed that type I interferons (IFNs) are powerful inducers of the differentiation and activation of dendritic cells (DCs). These findings emphasize the importance of these cytokines in linking innate and adaptive immunity, suggesting that effects of type I IFN on DCs can play a role in the antitumor and antiviral activity observed in some IFN-treated patients. Thus, the evaluation of the effects of IFN on the differentiation/activation of DCs has become an important approach for testing novel biologically important IFN activities, and the description of some reference methods are urgently needed. In this chapter, we describe some methods for testing the effects of IFNs on the differentiation and activation of human DCs from the peripheral blood monocytes and for the characterization of the DCs generated after IFN treatment.

Anti-nerve growth factor Ab abrogates macrophage-mediated HIV-1 infection and depletion of CD4+ T lymphocytes in hu-SCID mice.

Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+ T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate the in vivo effect of HIV-1-infected macrophages on virus spread and CD4+ T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+ T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast, in vivo treatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustaining in vivo HIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.

The natural alliance between type I interferon and dendritic cells and its role in linking innate and adaptive immunity.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and thus play a pivotal role in induction of the immune response. Recent studies in both human and mouse models have shown that type I IFN, cytokines originally characterized for their antiviral activity and exerting multiple biologic effects, efficiently promote the differentiation and activation of DCs. These observations, together with the findings that DCs can express biologically relevant levels of type I interferon (IFN) and, in particular, that high amounts of these cytokines are released by specialized DC precursors (i.e., plasmacytoid DCs) in response to viral infections, strongly suggest the existence of a natural alliance between type I IFN and DCs, which is instrumental in ensuring an efficient immune response to both infectious agents and tumors. Further recent knowledge on the interactions between type I IFN and DCs emphasizes the importance of these cytokines in linking innate and adaptive immunity and may lead to new perspectives in their use as vaccine adjuvants as well as in strategies for the development of DC-based vaccines.