Extracellular vesicles at the crossroad between cancer progression and immunotherapy: focus on dendritic cells.

Extracellular vesicles (EVs) are nanosized heat-stable vesicles released by virtually all cells in the body, including tumor cells and tumor-infiltrating dendritic cells (DCs). By carrying molecules from originating cells, EVs work as cell-to-cell communicators in both homeostasis and cancer but may also represent valuable therapeutic and diagnostic tools. This review focuses on the role of tumor-derived EVs (TEVs) in the modulation of DC functions and on the therapeutic potential of both tumor- and DC-derived EVs in the context of immunotherapy and DC-based vaccine design. TEVs were originally characterized for their capability to transfer tumor antigens to DCs but are currently regarded as mainly immunosuppressive because of the expression of DC-inhibiting molecules such as PD-L1, HLA-G, PGE2 and others. However, TEVs may still represent a privileged system to deliver antigenic material to DCs upon appropriate engineering to reduce their immunosuppressive cargo or increase immunogenicity. DC-derived EVs are more promising than tumor-derived EVs since they expose antigen-loaded MHC, costimulatory molecules and NK cell-activating ligands in the absence of an immunosuppressive cargo. Moreover, DC-derived EVs possess several advantages as compared to cell-based drugs such as a higher antigen/MHC concentration and ease of manipulation and a lower sensitivity to immunosuppressive microenvironments. Preclinical models showed that DC-derived EVs efficiently activate tumor-specific NK and T cell responses either directly or indirectly by transferring antigens to tumor-infiltrating DCs. By contrast, however, phase I and II trials showed a limited clinical efficacy of EV-based anticancer vaccines. We discuss that the future of EV-based therapy depends on our capability to overcome major challenges such as a still incomplete understanding of their biology and pharmacokinetic and the lack of standardized methods for high-throughput isolation and purification. Despite this, EVs remain in the limelight as candidates for cancer immunotherapy which may outmatch cell-based strategies in the fullness of their time.

Extracellular microRNAs induce dendritic cell-dependent joint inflammation and potentiate osteoclast differentiation via TLR7/8 engagement.

OBJECTIVES: Monocyte-derived dendritic cells (DCs) are key players in the induction of inflammation, autoreactive T cell activation and loss of tolerance in rheumatoid arthritis (RA), but the precise mechanisms underlying their activation remain elusive. Here, we hypothesized that extracellular microRNAs released in RA synovial fluids may represent a novel, physiological stimulus triggering unwanted immune response via TLR8-expressing DC stimulation. METHODS: Human monocyte-derived DCs were stimulated with a mixture of GU-rich miRNAs upregulated in RA tissues and released in synovial fluids (Ex-miRNAs). Activation of DCs was assessed in terms of NF-κB activation by Western blot, cytokine production by ELISA, T cell proliferation and polarization by allogeneic mixed lymphocyte reaction. DC differentiation into osteoclasts was evaluated in terms of tartrate-resistant acid phosphatase production and formation of resorption pits in dentine slices. Induction of joint inflammation in vivo was evaluated using a murine model of DC-induced arthritis. TLR7/8 involvement was assessed by specific inhibitors. RESULTS: Ex-miRNAs activate DCs to secrete TNFα, induce joint inflammation, start an early autoimmune response and potentiate the differentiation of DCs into aggressive osteoclasts. CONCLUSIONS: This work represents a proof of concept that the pool of extracellular miRNAs overexpressed in RA joints can act as a physiological activator of inflammation via the stimulation of TLR8 expressed by human DCs, which in turn exert arthritogenic functions. In this scenario, pharmacological inhibition of TLR8 might offer a new therapeutic option to reduce inflammation and osteoclast-mediated bone destruction in RA.

CD8-positive memory T cells in tumor-draining lymph nodes of patients with breast cancer.

BACKGROUND: Human immunological memory is a hallmark of the adaptive immune system and plays an important role in the development of effective immune responses against tumors. In the present study, we aimed to determine the frequencies of CD8+ memory T cell subsets including T stem cell memory (TSCM) in tumor-draining lymph nodes of patients with breast cancer (BC). METHODS: Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with BC and stained for CD8, CCR7, CD45RO, CD95 markers to detect different subtypes of memory cells in the CD8+ lymphocyte population. Data were acquired on four-color flow cytometer and analyzed with CellQuest Pro software. RESULTS: We observed that 47.65 ± 2.66% of CD8+ lymphocytes expressed the CD45RO, a marker for memory T cells. Statistical analysis showed that the total frequency of central memory T cells (TCM) and their subset with low CD45RO expression was significantly higher in tumor-involved nodes compared to tumor-free ones (P = 0.024 and P = 0.017, respectively). The level of CD95 expression (based on mean fluorescence intensity) on the surface of TCM, their CD45ROhi and CD45ROlow subsets, and TSCM was higher in patients with stage II compared to those in stage I (P < 0.05). In addition, the percentage of naive CD8+ T cells was significantly lower in tumor-involved lymph nodes compared to tumor-free ones (P = 0.025). CONCLUSIONS: Our data collectively indicate no significant differences in the frequencies of CD8+ lymphocytes or their memory subsets in tumor-draining lymph nodes of patients with BC. However, the frequency of CD45low TCM was higher in tumor-involved nodes. Along with a decrease in the frequency of naive T cells, the higher frequency of CD45low TCM suggests that despite the immune reaction to provide a pool of effective memory cells, it is blocked in early-stage of memory cells' differentiation (CD45ROlow), probably by tumor-derived suppressive factors. Identifying the molecular and cellular mechanisms behind this suppression can provide invaluable tools for adoptive T cell therapies in cancer.

Bibliometric analysis of global scientific research on Coronavirus (COVID-19).

Background: Since the outbreak of the novel coronavirus disease from Wuhan, China, in early December 2019, many scientists focused on this infection to find a way to deal with it. Due to the dramatic scientific growth in this field, we conducted a scientometric study to gain a better understanding of the scientific literature on COVID-19. Methods: We extracted all COVID-19 documents indexed in the Scopus from December 1, 2019, to April 1, 2020, without any language limitation and determined their bibliometric characteristics, including document type, open accessibility status, citation counting, H-index, top cited documents, the most productive countries, institutions and journals, international collaboration, the most frequent terms and keywords, journal bibliographic coupling and cocitations. Results: A total of 923 documents on COVID-19 were retrieved, of which 418 were original articles. All documents had received 2551 citations with an average citation of 2.76 per document and an h-index of 23. China ranked first with 348 documents, followed by the United States (n = 160). The Lancet and BMJ Clinical Research Ed published the most documents (each with 74 documents) and 2 institutions (University of Hong Kong and Huazhong University of Science and Technology) ranked first in this regard. In addition, the present study analyzed the top 25 highly-cited documents (those that had received 70% of all citations). Conclusion: This study highlighted the focused subjects on various aspects of COVID-19 literature such as pathogenesis, epidemiology, transmission, diagnosis, treatment, prevention, and its complications.

Clinical and prognostic significance of follicular helper and regulatory T cells in bladder cancer draining lymph nodes.

Follicular helper and regulatory T cells (Tfh/TFR) cells are distinct subsets of CD4+ cells that have been recognized for their critical role in regulating cellular reactions within the germinal centers of lymphoid follicles. In the present study, we aimed to determine the presence and the frequency of these cells in draining lymph nodes of patients with bladder cancer (BC). Forty-six patients with BC who had undergone radical cystectomy and pelvic lymph node dissection were enrolled. Following routine pathological examination, a portion of the dissected lymph nodes was minced to obtain a single-cell suspension. Mononuclear cells were then separated using Ficoll-Hypaque gradient centrifugation, and the samples with proper viability (> 95%) were subjected to further analysis. To phenotype the follicular subsets, cells were stained with appropriate fluorochrome-conjugated antibodies specific for CD4, CXCR5, BCL6, and FOXP3. The cells were then acquired on a four-color flow cytometer. The data were analyzed with the FlowJo software version 10.8.1 package. Our analysis indicated that, on average 37.89 ± 16.36% of CD4+ lymphocytes in draining lymph nodes of patients with BC expressed CXCR5. The majority of them were negative for FOXP3, representing helper subsets (28.73 ± 13.66). A small percent simultaneously expressed BCL6 transcription factor (1.65% ± 1.35), designated as Tfh (CD4+BCL6+CXCR5+FOXP3-). While less than 10% of CD4+ lymphocytes expressed CXCR5 and FOXP3, 1.78 ± 2.54 were also positive for BCL6, known as TFR. Statistical analysis revealed that the frequency of both Tfh and TFR cells was higher in draining lymph nodes of patients with tumor-infiltrated nodes (P = 0.035 and P = 0.079, respectively) compared to those with negative ones. The percentage of these cells was also higher in high-grade tumors compared to low-grade ones (P = 0.031 for both). Our data collectively indicated that however approximately one third of CD4+ lymphocytes expressed CXCR5 and accordingly had the capacity to enter the follicles, less than 2% of them represented Tfh and TFR phenotypes. The percentage of these cells increased in progressed tumors and showed an association with negative prognostic factors.

GM-CSF-producing lymphocytes in tumor-draining lymph nodes of patients with bladder cancer.

Bladder cancer (BC) is the tenth common cancer worldwide. Despite progress in treatment and the use of chemotherapoeutic drugs, the survival rate of BC patients is still low. Manipulation of the immune system was recently introduced as an interesting alternative treatment for this immunogenic cancer with fewer side effects. Accordingly, in the present study, we assessed the frequency of GM-CSF-producing lymphocytes in tumor-draining lymph nodes (TDLNs) of BC patients and evaluated their relationship with clinicopathological factors and survival rate. Fifty-four patients with BC who had received no treatment were recruited. Mononuclear cells were isolated from fresh homogenized lymph nodes by centrifugation over Ficoll-Hypaque, activated and subsequently analyzed by flow cytometry for the cell surface expression of CD4 and CD8 and the intracellular production of GM-CSF. Flow cytometric analysis revealed that 4.97 ± 2.7% of lymphocytes in TDLNs of patients with BC produced GM-CSF. The mean frequency of GM-CSF-producing cells was 5.5% among CD4+ lymphocytes and 11.7% in the CD8+ population. Elevated frequencies of GM-CSF-producing lymphocytes, as well as a higher production of GM-CSF by CD4+ lymphocytes was observed in the patients with tumor-free lymph nodes, as compared to those with at least one tumor-infiltrated lymph node (p < 0.05). On the other hand, the lower frequency of GM-CSF-producing CD4+ lymphocytes (ThGM) was associated with improved overall, but not one-year, survival. No other significant relationship was observed between clinicopathological parameters and the frequency of GM-CSF-producing subsets. Collectively, our findings suggest a protective role for GM-CSF in the early stages of BC; however, the unfavorable association of ThGM frequency with survival rate may imply a more complex role for this cytokine in BC.

Prognostic significance of CD4-positive regulatory T cells in tumor draining lymph nodes from patients with bladder cancer.

INTRODUCTION AND METHODS: To clarify the role of CD4+ regulatory T cells in bladder cancer, we investigated the frequency of these cells in tumor draining lymph nodes of 50 patients with bladder cancer who underwent radical cystectomy using flow cytometry method. We also assessed their association with prognosis and survival. RESULTS: On average, 30.13 ± 2.17% of lymphocytes in draining lymph nodes from patients with bladder cancer were positive for both CD4 and FOXP3 molecules. Analyses also showed that 9.92 ± 0.8% of CD4+ lymphocytes had a regulatory phenotype (CD4+CD25+FOXP3+CD127low/neg). The frequency of total CD4+FOXP3+ lymphocytes as well as regulatory T cells was significantly greater in patients with at least one tumor-involved lymph node compared to those with tumor-free nodes (P = 0.026 and P = 0.036, respectively). Mean FOXP3 expression in CD4+ lymphocytes was greater in patients with stage IV compared with those in stage III (P = 0.046). No other significant associations were found between the frequency of regulatory T cells and other clinicopathological characteristics or patient survival. CONCLUSIONS: The increased frequency of regulatory T cells in patients with involved lymph nodes suggests that these cells may negatively regulate antitumor immune responses in draining lymph nodes. Our findings may have implications for immunotherapy-based treatments for bladder cancer.

Memory CD4+ T cell subsets in tumor draining lymph nodes of breast cancer patients: A focus on T stem cell memory cells.

BACKGROUND: The compartments of memory T cells play a fundamental role in the immune system by substantiating specific and acquired immunity. A new subset of memory cells, T stem cell memory (TSCM) cells, with stem cell-like properties, a high capacity to proliferate, a long survival, and an ability to differentiate into all effector and memory cells has recently been introduced. In the present study, we aimed to determine the frequency of CD4+ TSCM and other T memory cell subsets in tumor draining lymph nodes of breast cancer patients. MATERIALS AND METHODS: Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with breast cancer (BC) and stained with fluorochrome conjugated anti-CD4, -CCR7, -CD45RO and -CD95 antibodies to detect different subtypes of memory cells in CD4+ lymphocyte populations. Data were acquired using a four-color FACSCalibur flow cytometer and analyzed using CellQuest Pro software. RESULTS: We found that >70% of CD4+ lymphocytes in draining lymph nodes of BC patients exhibited a memory phenotype of which 7.04 ± 1.04% had a TSCM phenotype (CD4+CCR7+CD45RO-CD95+). The frequency of TSCM cells was significantly higher in tumor positive lymph nodes compared to tumor negative lymph nodes (p = 0.026) as well as among those patients who had at least one affected lymph node (p = 0.012). Moreover, we found that the total frequency of central memory T cells (TCM) with a low expression of CD45RO was significantly higher among these patients. The percentage of CD45ROLow TCM cells was also found to increase with tumor progression from stage I to stage III (p = 0.020). On the other hand, we found that the percentage of CD95Hi effector memory T cells (TEM) was significantly decreased in involved lymph nodes (p = 0.009). CONCLUSION: Our data suggest that following long-term exposure to putative tumor antigens, TSCM cells proliferate to generate a pool of committed memory and effector T cells. As the tumor progresses, the immunosuppressive milieu induced by tumor cells may slow down the differentiation of CD45ROLow TCM cells to more functional sub-populations.