Immunotherapeutic Protocols Research Articles

Does surgery affect systemic immune response? a perioperative analysis of TGF-β, IL-8 and CD45RO.

The options of (neo-)adjuvant immunotherapy in addition to surgery in the treatment of oral squamous cell carcinoma (OSCC) are steadily increasing, but patients do not always respond to therapy as intended. The objectives of this study were to investigate the systemic perioperative course of the biomarkers CD45RO, TGF-β, and IL-8 in non-tumor-related minor and tumor-related major maxillofacial surgery and to perform association analyses with demographic and histomorphologic parameters. A deeper understanding of surgery-related changes in various of different immune biomarkers could help to better understand the immunologic consequences of surgery which could influence immunotherapeutic protocols. Peripheral whole blood from 38 patients was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) at five different timepoints before and after maxillofacial surgery to detect changes in mRNA expression of the biomarkers TGF-β, IL-8 and CD45RO. All patients underwent general anesthesia to undergo either resection and free flap reconstruction for OSCC or minor maxillofacial surgery (controls). Statistical analysis was done using Mann-Whitney-U test, Wilcoxon test, and Spearman's correlation. Compared to the preoperative expression, there was a significant postoperative downregulation of CD45RO, TGF-β and IL-8 until the 4th postoperative day (p ≤ 0.003) in OSCC patients. For TGF-β and IL-8, the reduction in expression was significant (p ≤ 0.004) compared to controls. By postoperative day 10, all analyzed parameters converged to baseline levels. Only CD45RO still showed a significant downregulation (p=0.024). Spearman analysis revealed a significant correlation between increased duration of surgery and perioperative reduction in peripheral blood expression of CD45RO, TGF-β and IL-8 (p ≤ 0.004). Perioperative changes in TGF-β and PD-L1 expression were shown to be not correlated. Preoperative TGF-β expression was significantly lower in patients with lymph node metastases (p=0.014). With regard to the analyzed parameters, major oncologic head-and-neck surgery does not seem to have long-lasting systemic immunologic effects. Reduced CD45RO might be an expression of transient systemic immunosuppression in response to major surgery. The association of duration of surgery with expression changes of immunologic markers supports efforts to keep the duration of surgery as short as possible. As perioperative TGF-β and PD-L1 expression changes are not associated, these results support further investigation of a combined perioperative anti-PD-1 and anti-TGF-β immunotherapy.

Expression of CD 20 B-Lymphocyte in oral epithelial dysplasia and oral squamous cell carcinoma: A comparative immunohistochemistry study.

As the progressive trends in the field of immunotherapy, it is very favourable to reconsider the role played by B lymphocytes in the tumor microenvironment. Both the protumorogenic and antitumorogenic responses have to be evaluated to formulate an effective immunotherapeutic protocol. The study was primarily conducted to assess the qualitative expression of B lymphocytes in pretumorogenic (oral epithelial dysplasia) and tumorogenic environment (oral squamous cell carcinoma). The differential immunohistochemical staining of CD 20 immune marker was assessed in about 60 cases that included 30 cases of oral epithelial dysplasia and 30 cases of oral squamous cell carcinoma. The study found significant correlation between CD 20 IHC immune expression and histopathological diagnosis along with significant correlation between the subject's age group and histopathological diagnosis. Modulating the immune response in a precancerous state can be highly beneficial in implementing better immunotherapeutic strategies to treat or prevent malignancy at an early stage.

Expression of down-regulated ERV LTR elements associates with immune activation in human small-cell lung cancers

Small-cell lung cancer (SCLC) is an aggressive cancer characterized by immunosuppressive features leading to poor responses to current immunotherapies. Activation of transposable elements (TE) can trigger an innate immune response, which can synergize with immunotherapeutic protocols in patients. However, TE activity in relation to immune gene response is not fully known in human SCLC. Here, we compared TE expression in 104 human SCLC and 24 normal tissues and established their involvement in innate immune responses. We observed that different intergenic TEs, mainly endogenous retroviral (ERV) families, are deregulated in SCLC. Similarly to other cancers, we detected a subset of LTRs that correlate with innate immune gene signatures and cytosolic RNA sensors, such as RIG-I. These LTRs are downregulated in SCLC tumors vs. normal tissues, and are mainly located at transcriptional repressed regions, marked with H3K4me2 in different cell lines. Analyses of different genomic datasets show that chromatin repression is likely due to de-methylase LSD1 activity. Moreover, high expression levels of ERV LTRs predict a better survival upon chemotherapy of SCLC patients. The findings reveal a specific pattern of TE-mediated activation of innate immune genes in SCLC, which can be exploited to establish more effective immunotherapeutic combinations.

PD-1 blockade augments CD8+ T cell dependent antitumor immunity triggered by Ad-SGE-REIC in Egfr-mutant lung cancer

ObjectivesNo immunotherapeutic protocol has yet been established in never-smoking patients with lung cancer harboring driver oncogenic mutations, such as epidermal growth factor receptor (EGFR) mutations. The immunostimulatory effect of Ad-REIC, a genetically engineered adenovirus vector expressing a tumor suppressor gene, reduced expression in immortalized cells (REIC), has been investigated in clinical trials for various solid tumors. However, the immunostimulatory effect of the Ad-REIC in EGFR-mutant lung cancer with a non-inflamed tumor microenvironment (TME) has not been explored. Materials and methodsWe used a syngeneic mouse model developed by transplanting Egfr-mutant lung cancer cells into single or double flanks of C57BL/6J mice. Ad-SGE-REIC, a 2nd-generation vector with an enhancer sequence, was injected only into the tumors from one flank, and its antitumor effects were assessed. Tumor-infiltrating cells were evaluated using immunohistochemistry or flow cytometry. The synergistic effects of Ad-SGE-REIC and PD-1 blockade were also examined. ResultsInjection of Ad-SGE-REIC into one side of the tumor induced not only a local antitumor effect but also a bystander abscopal effect in the non-injected tumor, located on the other flank. The number of PD-1+CD8+ T cells increased in both injected and non-injected tumors. PD-1 blockade augmented the local and abscopal antitumor effects of Ad-SGE-REIC by increasing the number of CD8+ T cells in the TME of Egfr-mutant tumors. Depletion of CD8+ cells reverted the antitumor effect, suggesting they contribute to antitumor immunity. ConclusionAd-SGE-REIC induced systemic antitumor immunity by modifying the TME status from non-inflamed to inflamed, with infiltration of CD8+ T cells. Additionally, in Egfr-mutant lung cancer, this effect was enhanced by PD-1 blockade. These findings pave the way to establish a novel combined immunotherapy strategy with Ad-SGE-REIC and anti-PD-1 antibody for lung cancer with a non-inflamed TME.

Combined immunotherapy and radiation induce cellular and humoral immune responses to tumor-associated antigens and prolong survival of patients with advanced cancers.

Immunotherapy is effective in treating patients with a variety of cancers We developed and employed an immunotherapeutic protocol that combined intra-tumor injection of autologous immature dendritic cells , with standard of care radiation to treat patients with a variety of different advanced solid tumors. Pre and post treatment peripheral blood mononuclear cells ( PBMC) were assayed for their capacity to kill autologous tumor cell lines that had been established from patients with different advanced solid tumors. PBMC obtained post treatment were cytotoxic to autologous but not allogeneic tumor cell lines. Cytotoxic activity increased as relative numbers of CD8+-CD56+ dual positive lymphoid cells increased in PBMC obtained post treatment. The autologous tumor cell line directed cytotoxicity was shown to be mediated by both innate and adaptive immune mechanisms. Cytotoxicity of PBMC obtained post treatment was enhanced after co-culture with autologous and not allogeneic tumor cell line lysates. Antibody activity to mesothelin, a tumor associated antigen expressed in a variety of solid tumors, increased in post treatment serum samples from lung cancer patients. These sera mediated antibody directed cellular cytotoxicity ( ADCC) to mesothelin expressing tumor cell lines. SSurvival of 96 patients with a variety advanced cancers patients treated on this protocol over a 3 year period and followed for 1000+ days was significantly increased in patients with low tumor burden ( lesion at <5 sites with diameters < 3cm) (Km= <0.0001) regardless of malignancy. These findings support application of this combination of immunotherapy and radiation in the treatment of cancer patients with advanced disease

Tumor-Stroma Ratio and Programmed Cell Death Ligand 1 Expression in Preoperative Biopsy and Matched Laryngeal Carcinoma Surgical Specimen.

Programmed cell death ligand 1 (PD-L1) seems to rely on close relations between neoplastic and immune cells in the tumor microenvironment. Tumor to stroma ratio (TSR) has been associated with prognosis in different malignancies. The aims of this exploratory investigation were to analyze for the first time the: (i) association between TSR, PD-L1 expression and other clinical–pathological features in laryngeal squamous cell carcinoma (LSCC) biopsies and paired surgical specimens; (ii) prognostic and predictive role of TSR and PD-L1. TSR, PD-L1 expression (in terms of combined positive score [CPS]), and other clinical–pathological features were analyzed in biopsies and surgical specimens of 43 consecutive LSCC cases. A CPS < 1 evaluated on surgical specimens was associated with a low TSR (stroma rich) on both biopsies and surgical specimens (p = 0.0143 and p = 0.0063). Low TSR showed a significant negative prognostic value when evaluated on both biopsies and surgical specimens (HR = 8.808, p = 0.0003 and HR = 11.207, p = 0.0002). CPS ≥ 1 appeared to be a favorable prognostic factor (HR = 0.100, p = 0.0265). The association between bioptic and surgical specimen TSR and PD-L1 expression should be further investigated for a potential impact on targeted treatments, also with regard to immunotherapeutic protocols.

IFN-γ-dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis.

Tumor-associated lymphatic vessels promote metastasis and regulate antitumor immune responses. Here, we assessed the impact of cytotoxic T cells on the local lymphatic vasculature and concomitant tumor dissemination during an antitumor response. Interferon-γ (IFN-γ) released by effector T cells enhanced the expression of immunosuppressive markers by tumor-associated lymphatic endothelial cells (LECs). However, at higher effector T cell densities within the tumor, T cell–based immunotherapies induced LEC apoptosis and decreased tumor lymphatic vessel density. As a consequence, lymphatic flow was impaired, and lymph node metastasis was reduced. Mechanistically, T cell–mediated tumor cell death induced the release of tumor antigens and cross-presentation by tumor LECs, resulting in antigen-specific LEC killing by T cells. When LECs lacked the IFN-γ receptor expression, LEC killing was abrogated, indicating that IFN-γ is indispensable for reducing tumor-associated lymphatic vessel density and drainage. This study provides insight into how cytotoxic T cells modulate tumor lymphatic vessels and may help to improve immunotherapeutic protocols.

Oxygen therapy in traditional and immunotherapeutic treatment protocols of cancer patients: current reality and future prospects

Introduction The metabolic environment in ischemic and hypoxic tumors is known to contribute to cancer progression. Importantly, peculiar metabolic changes occurring in malignant cells (the increased glycolysis and the hampered Krebs cycle) may contribute to decreased antioxidant-dependent defence in ischemic and hypoxic tumors. Areas covered In the clinic, oxygen saturation of tumours is usually achieved by the application of water-soluble ozone and hyperbaric oxygen therapy. Tumour oxygenation has been shown to inhibit tumour growth and potentiate anti-tumour effects of chemoradiotherapy in animal experiments and the clinical setting. Tumour oxygenation could enhance anti-tumour effects achieved by tumor blood vessel occlusion or angiostatic therapy. Expert opinion Owing to a profound influence of ROS on both the innate and adaptive immunity, oxygen therapy, when combined simultaneously or sequentially with immunotherapeutic interventions (such as immune checkpoint inhibition, drug-induced immunostimulation, adoptive cell therapy, hyperthermia, etc.), could be considered as a novel highly-effective clinical biological approach to cancer treatment.

HLA-G and Other Immune Checkpoint Molecules as Targets for Novel Combined Immunotherapies.

HLA-G is an HLA-class Ib molecule that is involved in the establishment of tolerance at the maternal/fetal interface during pregnancy. The expression of HLA-G is highly restricted in adults, but the de novo expression of this molecule may be observed in different hematological and solid tumors and is related to cancer progression. Indeed, tumor cells expressing high levels of HLA-G are able to suppress anti-tumor responses, thus escaping from the control of the immune system. HLA-G has been proposed as an immune checkpoint (IC) molecule due to its crucial role in tumor progression, immune escape, and metastatic spread. We here review data available in the literature in which the interaction between HLA-G and other IC molecules is reported, in particular PD-1, CTLA-4, and TIM-3, but also IDO and TIGIT. Clinical trials using monoclonal antibodies against HLA-G and other IC are currently ongoing with cancer patients where antibodies and inhibitors of PD-1 and CTLA-4 showed encouraging results. With this background, we may envisage that combined therapies using antibodies targeting HLA-G and another IC may be successful for clinical purposes. Indeed, such immunotherapeutic protocols may achieve a better rescue of effective anti-tumor immune response, thus improving the clinical outcome of patients.

Cancer-Immunity Cycle and Therapeutic Interventions- Opportunities for Including Pet Dogs With Cancer.

The tumor-immune interplay represents a dynamic series of events executed by cellular and soluble participants that either promote or inhibit successful tumor formation and growth. Throughout a tumor’s development and progression, the host organism’s immune system reacts by generating anti-cancer defenses through various incremental and combinatorial mechanisms, and this reactive orchestration is termed the cancer-immunity cycle. Success or failure of the cancer-immunity cycle dictates the fate of both host and tumor as winner or loser. Insights into how the tumor and host immune system continuously adapt to each other throughout the lifecycle of the tumor is necessary to rationally develop new effective immunotherapies. Additionally, the evolving nature of the cancer-immunity cycle necessitates therapeutic agility, requiring real-time serial assessment of immunobiologic markers that permits tailoring of therapies to the everchanging tumor immune microenvironment. In order to accelerate advances in the field of immuno-oncology, this review summarizes the steps comprising the cancer-immunity cycle, and underscores key breakpoints in the cycle that either favor cancer regression or progression, as well as shaping of the tumor microenvironment and associated immune phenotypes. Furthermore, specific large animal models of spontaneous cancers that are deemed immunogenic will be reviewed and proposed as unique resources for validating investigational immunotherapeutic protocols that are informed by the cancer-immunity cycle. Collectively, this review will provide a progressive look into the dynamic interplay between tumor and host immune responses and raise awareness for how large animal models can be included for developing combinatorial and sequenced immunotherapies to maximizing favorable treatment outcomes.

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy.

The advent of T-cell-based immunotherapy has remarkably transformed cancer patient treatment. Despite their success, the currently approved immunotherapeutic protocols still encounter limitations, cause toxicity, and give disparate patient outcomes. Thus, a deeper understanding of the molecular mechanisms of T-cell activation and inhibition is much needed to rationally expand targets and possibilities to improve immunotherapies. Protein ubiquitination downstream of immune signaling pathways is essential to fine-tune virtually all immune responses, in particular, the positive and negative regulation of T-cell activation. Numerous studies have demonstrated that deregulation of ubiquitin-dependent pathways can significantly alter T-cell activation and enhance antitumor responses. Consequently, researchers in academia and industry are actively developing technologies to selectively exploit ubiquitin-related enzymes for cancer therapeutics. In this review, we discuss the molecular and functional roles of ubiquitination in key T-cell activation and checkpoint inhibitory pathways to highlight the vast possibilities that targeting ubiquitination offers for advancing T-cell-based immunotherapies.

Glioblastoma cells potentiate the induction of the Th1-like profile in phosphoantigen-stimulated γδ T lymphocytes.

γδ T lymphocytes are non-conventional T cells that participate in protective immunity and tumor surveillance. In healthy humans, the main subset of circulating γδ T cells express the TCRVγ9Vδ2. This subset responds to non-peptide prenyl-pyrophosphate antigens such as (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP). This unique feature of Vγ9Vδ2 T cells makes them a candidate for anti-tumor immunotherapy. In this study, we investigated the response of HMBPP-activated Vγ9Vδ2 T lymphocytes to glioblastoma multiforme (GBM) cells. Human purified γδ T cells were stimulated with HMBPP (1µM) and incubated with GBM cells (U251, U373 and primary GBM cultures) or their conditioned medium. After overnight incubation, expression of CD69 and perforin was evaluated by flow cytometry and cytokines production by ELISA. As well, we performed a meta-analysis of transcriptomic data obtained from The Cancer Genome Atlas. HMBPP-stimulated γδ T cells cultured with GBM or its conditioned medium increased CD69, intracellular perforin, IFN-γ, and TNF-α production. A meta-analysis of transcriptomic data showed that GBM patients display better overall survival when mRNA TRGV9, the Vγ9 chain-encoding gene, was expressed in high levels. Moreover, its expression was higher in low-grade GBM compared to GBM. Interestingly, there was an association between γδ T cell infiltrates and TNF-α expression in the tumor microenvironment. GBM cells enhanced Th1-like profile differentiation in phosphoantigen-stimulated γδ T cells. Our results reinforce data that have demonstrated the implication of Vγ9Vδ2 T cells in the control of GBM, and this knowledge is fundamental to the development of immunotherapeutic protocols to treat GBM based on γδ T cells.

Parvovirus-Based Combinatorial Immunotherapy: A Reinforced Therapeutic Strategy against Poor-Prognosis Solid Cancers.

Simple SummaryOncolytic virotherapy using oncolytic viruses with natural or engineered cancer-destroying capacities has emerged as a promising treatment concept in modern oncology. Rodent protoparvoviruses, in particular the rat H-1 parvovirus (H-1PV), have demonstrated their broad-range tumor-suppressive properties in both preclinical models and clinical studies. In addition to inducing selective tumor cell death, these viruses are also able to exert immunostimulating effects and reverse tumor-driven immune suppression. Parvovirotherapy holds therefore a potential for enhancing the efficacy of other cancer immunotherapies. The aim of this review is to provide an overview of all H-1PV-based combinatorial immunotherapeutic approaches against poor-prognosis human solid cancers that have been tested so far. Current challenges and future prospects of parvoviro-immunotherapy, notably parvovirus inclusion into various immunotherapeutic protocols against glioblastoma, pancreatic cancer, among other standard therapy-refractory solid malignancies, are also discussed in the light of H-1PV further clinical development.Resistance to anticancer treatments poses continuing challenges to oncology researchers and clinicians. The underlying mechanisms are complex and multifactorial. However, the immunologically “cold” tumor microenvironment (TME) has recently emerged as one of the critical players in cancer progression and therapeutic resistance. Therefore, TME modulation through induction of an immunological switch towards inflammation (“warming up”) is among the leading approaches in modern oncology. Oncolytic viruses (OVs) are seen today not merely as tumor cell-killing (oncolytic) agents, but also as cancer therapeutics with multimodal antitumor action. Due to their intrinsic or engineered capacity for overcoming immune escape mechanisms, warming up the TME and promoting antitumor immune responses, OVs hold the potential for creating a proinflammatory background, which may in turn facilitate the action of other (immunomodulating) drugs. The latter provides the basis for the development of OV-based immunostimulatory anticancer combinations. This review deals with the smallest among all OVs, the H-1 parvovirus (H-1PV), and focuses on H-1PV-based combinatorial approaches, whose efficiency has been proven in preclinical and/or clinical settings. Special focus is given to cancer types with the most devastating impact on life expectancy that urgently call for novel therapies.

4th Summer School in Immuno-Oncology, July 1st-3rd, 2021, Athens, Greece.

The 4th Summer School in Immuno-Oncology was held from July 1st-July 3rd as a web meeting. Many eminent researchers and leading oncologists from Europe and the USA working on basic, translational and clinical cancer research participated, presented, and discussed the most recent advances in cancer immunology and immunotherapy. Besides sharing the newest information in the field of cancer immunology and immunotherapy, the meeting also focused on the actual translation of new knowledge acquired in the lab to the clinical setting; particular emphasis was given to the mode of action of novel therapeutic modalities and to biomarkers helpful for treatment decision-making, as well as to means that may improve cancer immunotherapeutic protocols used for the treatment of a variety of malignancies. The main topics presented by the speakers included: (1) mechanisms of tumor immune evasion and resistance; (2) host-tumor interactions and means to regulate antitumor immunity; (3) exploitation of new biomarkers and tumor or immune signatures able to potentially guide therapeutic interventions; (4) emerging therapeutic modalities for cancer treatment and specific immunotherapeutics for thoracic, genito-urinary, gastrointestinal, skin and breast cancers; and (5) innovative treatment options and alternatives to minimize the toxic adverse events of cancer immunotherapy.

Prognostic impact of neutrophils-to-lymphocytes ratio (NLR), PD-L1 expression, and tumor immune microenvironment in laryngeal cancer

PurposeIn laryngeal carcinoma (LSCC), tumor immune microenvironment is attracting increasing interest, given the recent progresses in immunotherapy. Immune cells migrate to tumors as a result of a tumor antigen-induced immune reaction and cancer cells recruit immune regulatory cells to induce an immunosuppressive network, resulting in the escape from host immunity. This interaction reflects both on tumor microenvironment and systemic inflammatory status. Blood neutrophil-to-lymphocyte ratio (NLR), reflecting a highly pro-inflammatory status, has been related to worse oncological survival outcomes.The aim of this study was to analyze in LSCC the relationship between circulating inflammatory cells (also in terms of NLR) and tumor immune microenvironment histopathological features (programmed cell death ligand 1 [PD-L1] expression, and tumor-infiltrating lymphocytes [TILs]), also investigating their clinical-pathological and prognostic significance. MethodsBlood pre-operative NLR, and, at pathology, PD-L1 (in terms of combined positive score [CPS]) and TILs were assessed on 60 consecutive cases of LSCC. ResultsBlood NLR, neutrophils, and lymphocytes counts showed a significant value in predicting DFS and recurrence risk. Moreover, PD-L1 CPS ≥ 1 and TILs count rate ≥30% were associated with higher disease-free survival (DFS) and reduced recurrence risk. A logistic regression model found a significant positive association between increasing NLR values, and PD-L1 CPS < 1 and TILs count rate <30%. ConclusionsFurther studies are needed to better characterize the role of pre-operative blood NLR in association with PD-L1 expression and tumor immune microenvironment features as prognostic factors and markers of anti-tumor immune response in LSCCs, also with regard to the effectiveness of immunotherapeutic protocols.

Prognostic role of programmed death ligand 1 (PD-L1) and the immune microenvironment in laryngeal carcinoma.

The immune system is crucial in the evolution of head and neck cancer. Programmed cell death ligand 1 (PD-L1) seems to rely on close relations between neoplastic cells and immune cells in the tumor microenvironment. The main aim of this study was to apply univariate/multivariate analysis to investigate the prognostic significance of PD-L1, tumor-infiltrating lymphocytes (TILs), and tertiary lymphoid structures (TLS) in laryngeal carcinoma (LSCC). PD-L1 (in terms of combined positive score [CPS]), TILs and TLS were assessed at pathology on 70 consecutive samples of LSCC. A CPS≥1 coincided with a lower recurrence rate (RR) (p=0.007) and longer disease-free survival (DFS) than a CPS<1 (p=0.0027). Cases with higher TIL counts showed a lower RR (p=0.036) and longer DFS than those with lower TIL counts (p=0.0062). Cases revealing TLS had a lower RR (p=0.004) and longer DFS (p=0.0034) than those with no TLS. On multivariate analysis, the presence of TLS retained its positive prognostic value (p=0.024), while CPS remained significant as regards disease recurrence (p=0.050). PD-L1 seems to be an indirect marker of effective anti-tumor response in LSCC, possibly being expressed as a result of a greater immune pressure on cancer cells. The presence of TLS emerged as a positive prognostic factor. Further prospective studies are needed to characterize the role of PD-L1 as a marker of anti-tumor immune response and prognostic factor in LSCC, also with regard to the effectiveness of immunotherapeutic protocols.

Gamma delta T cells as novel immunotherapeutic tool to cure drug-resistant viral infections in transplanted pediatric patients: development and validation of procedures for a new ATMP (advanced therapy medicinal product)

Background & Aim Viral infections remain a leading cause of morbidity and mortality after transplantation. Although prophylactic pharmacotherapy is effective in reducing the risk for some viral infections, therapeutic options for breakthrough infections are still unsatisfactory. Conventional pharmacologic agents against Adenovirus (AdV) and Epstein-Barr virus (EBV) have limited efficacy and relevant toxicity, whereas in the case of cytomegalovirus (CMV) are documented better response rates, but toxicity and reactivation after treatment stop are frequent. T-cell reconstitution is a key requirement for effective antiviral control following transplantation, and factors that influence the speed of T-cell recovery impact also the risk of viral infection in this period. The use of lymphocyte infusions derived from seropositive donors is an effective salvage therapy for viral infections in transplanted recipients prior to T-cell recovery, but the risk of severe graft-versus-host disease (GVHD) is a critical concern. Thus, we propose a novel immunotherapeutic approach based on gd T cells specific for AdV, CMV and EBV. Many studies highlighted the importance of gd T cells in antiviral immunity, uniquely combining conventional adaptive features with rapid, innate-like responses. These lymphocytes are effector cells that: i) recognize target cells in a MHC-independent manner, thus do not cause GvHD; ii) participate CMV and EBV responses, particularly when conventional adaptive immunity is insufficient to clear the viral infection; iii) may develop memory responses, and iv) cross-talk with other immune cells, including NK lymphocytes, alfa beta T cells and dendritic cells. These features render gd T cells extremely appealing for clinical purposes. We believe that an ATMP containing Vd1 and Vd2 gd T cells will be the ideal immunotherapeutic approach to prevent and treat viral infections after transplantation. Methods, Results & Conclusion To this end, we are developing a new method to obtain a cell product containing expanded Vd1 and Vd2 gd T cells using exclusively GMP-graded reagents, including zoledronic acid, cytokines, viral peptides (related to AdV, CMV and EBV) and ab depletion techniques. Such cell product has been tested for i) phenotype, ii) allo-reactivity, iii) cytotoxicity and iv) specificity. Our preliminary results strongly support the feasibility for development of new immunotherapeutic protocols using gd T cells, with the aim of preventing and treating viral infections after transplantation.

T-cell and B-cell intratumoural interactions affect the progression of oropharyngeal squamous cell carcinoma

Abstract Background Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with strong morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, the development of a complex therapeutic protocol combining checkpoint inhibition with other targets is needed for better responses. The significance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC. Methods In this study, we evaluated the density and distribution of TIL-Bs in FFPE samples from 2 cohorts of OPSCC patients. Than we assessed the phenotype of TIL-Bs by means of flow cytometry in fresh tumor samples immediately after radical surgery. The acquired data were corelated with the clinical outcome of the patients. Results We observed that CD20+ TIL-Bs and CD8+ T cells formed non-organized clusters with clearly interacting cells and the densities of both intraepithelial CD20+ B cells and B/CD8+ T cell interactions have prognostic significance for the overall survival of OPSCC patients. Furthermore, a high density of TIL-Bs was associated with an activated B cell phenotype and a high density of direct B cell/CD8+ T cell interactions significantly correlated with the abundance of HPV16-specific CD8+ T cells. Conclusion Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can preselect patients with excellent prognosis who would profit from less invasive treatment. Furthermore, including B cells as an additional target in novel immunotherapeutic protocols may help to establish the desired sustained antitumor T cell responses in OPSCC patients. Legal entity responsible for the study Kamila Hladikova. Funding Sotio a.s. Disclosure All authors have declared no conflicts of interest.

Human Papillomavirus Epitope Mimicry and Autoimmunity: The Molecular Truth of Peptide Sharing

Objective: To define the cross-reactivity potential and the consequent autoimmunity intrinsic to viral versus human peptide sharing. Methods: Using human papillomavirus (HPV) infection/active immunization as a research model, the experimentally validated HPV L1 epitopes catalogued at the Immune Epitope DataBase were analyzed for peptide sharing with the human proteome. Results: The final data show that the totality of the immunoreactive HPV L1 epi­topes is mostly composed by peptides present in human proteins. Conclusions: Immunologically, the high extent of peptide sharing between the HPV L1 epitopes and human proteins invites to revise the concept of the negative selection of self-reactive lymphocytes. Pathologically, the data highlight a cross-reactive potential for a spectrum of autoimmune diseases that includes ovarian failure, systemic lupus erythematosus (SLE), breast cancer and sudden death, among others. Therapeutically, analyzing already validated immunoreactive epitopes filters out the peptide sharing possibly exempt of self-reactivity, defines the effective potential for pathologic autoimmunity, and allows singling out peptide epitopes for safe immunotherapeutic protocols.

Γδ T Cell-Based Immunotherapy in Melanoma: State of the Art.

Metastatic melanoma is still associated with a poor prognosis, and there is increasing interest in immunotherapy alone or in combination with other adjuvant therapies. Γδ T lymphocytes play a pivot role in the immune response against cancer, but while γδ-based immunotherapy is already a clinical reality for several solid tumors, data on melanoma are still limited and fragmented. This systematic review presents preclinical and clinical evidence for a role of γδ T lymphocytes in immunotherapeutic strategies for advanced melanoma and discusses research state of the art and future perspectives. Current strategies focus on in vivo stimulation, and ex vivo adoptive therapy and vaccination; results are promising, but further studies are needed to better investigate the interactions in tumoral microenvironment and to improve clinical efficacy of immunotherapeutic protocols.