Folate Receptor ß (FRß) Expression on Myeloid Cells and the Impact of Reticuloendothelial System on Folate-Functionalized Nanoparticles' Biodistribution in Cancer.

Folate uptake is largely mediated by folate receptor (FR)ß, encoded by FOLR2 gene, in myeloid immune cells such as granulocytes, monocytes, and especially in macrophages that constitute the reticuloendothelial system (RES) and infiltrate the tumor microenvironment. Since the myeloid immune compartment dynamically changes during tumorigenesis, it is critical to assess the infiltration status of the tumors by FRß-expressing myeloid cells to better define the targeting efficacy of folate-functionalized drug delivery systems. On the other hand, clearance by RES is a major limitation for the targeting efficacy of nanoparticles decorated with folate. Therefore, the aims of this study are (i) to determine the amount and subtypes of FRß+ myeloid cells infiltrating the tumors at different stages, (ii) to compare the amount and subtype of FRß+ myeloid cells in distinct organs of tumor-bearing and healthy animals, (iii) to test if the cancer-targeting efficacy and biodistribution of a prototypic folate-functionalized nanoparticle associates with the density of FRß+ myeloid cells. Here, we report that myeloid cell infiltration was enhanced and FRß was upregulated at distinct stages of tumorigenesis in a mouse breast cancer model. The CD206+ subset of macrophages highly expressed FRß, prominently both in tumor-bearing and healthy mice. In tumor-bearing mice, the amount of all myeloid cells, but particularly granulocytes, was remarkably increased in the tumor, liver, lungs, spleen, kidneys, lymph nodes, peritoneal cavity, bone marrow, heart, and brain. Compared with macrophages, the level of FRß was moderate in granulocytes and monocytes. The density of FRß+ immune cells in the tumor microenvironment was not directly associated with the tumor-targeting efficacy of the folate-functionalized cyclodextrin nanoparticles. The lung was determined as a preferential site of accumulation for folate-functionalized nanoparticles, wherein FRß+CD206+ macrophages significantly engulfed cyclodextrin nanoparticles. In conclusion, our results demonstrate that the tumor formation augments the FR levels and alters the infiltration and distribution of myeloid immune cells in all organs which should be considered as a major factor influencing the targeting efficacy of nanoparticles for drug delivery.

Effect of Follicular T Helper and T Helper 17 Cells-Related Molecules on Disease Severity in Patients with Myasthenia Gravis.

INTRODUCTION: Contribution of T helper 1 and 2 cells-related cytokines to pathogenesis of myasthenia gravis (MG) is well known. Recently, the contribution of follicular T helper (Tfh) and T helper 17 cells-related molecules to the pathogenesis has gained importance. In this study, we aimed to evaluate the changes in Tfh- and Th17-related molecules before and after rescue therapy in patients with myasthenic crisis (cMG) and to reveal the molecular differences between stable MG and cMG patients. METHODS: Patients with stable generalized MG (gMG) and cMG were classified according to Myasthenia Gravis Foundation of America (MGFA) classification. Serum samples were collected from cMG patients both before and after rescue therapy (plasmapheresis or intravenous immunoglobulin [IVIg]). Serum levels of Tfh- and selected Th17-related molecules (IL-22, IL-17A, CXCL13, sPD-L1, sICOSLG, and sCD40L) were analyzed by commercial ELISA kits. RESULTS: Twelve cMG (6 for IVIg, 6 for plasmapheresis) and 10 gMG patients were included in the study. A decrease in serum sPD-L1 and CXCL13 levels was observed in cMG patients after treatment, regardless of the treatment modality (p < 0.05). In contrast, serum sICOSLG levels decreased only in patients treated with IVIg (p < 0.05) and serum IL-22 levels increased in patients receiving plasmapheresis (p < 0.05). cMG patients had higher serum IL-17A levels compared to stable patients (p < 0.001) and its level was positively correlated with disease severity (r = 0.678, p = 0.001). CONCLUSION: Our results confirm the contribution of Tfh- and Th17-related cell pathways to MG pathogenesis. Both IVIg and plasmapheresis appear to be effective in reducing Tfh- and Th17-related cytokine/molecule levels in cMG patients. Increased serum IL-17A levels may contribute to disease severity.

An all-in-one nanoparticle for overcoming drug resistance: doxorubicin and elacridar co-loaded folate receptor targeted PLGA/MSN hybrid nanoparticles.

Overexpression of permeability-glycoprotein (P-gp) transporter leads to multidrug resistance (MDR) through cellular exclusion of chemotherapeutics. Co-administration of P-gp inhibitors and chemotherapeutics is a promising approach for improving the efficacy of therapy. Nevertheless, problems in pharmacokinetics, toxicity and solubility limit the application of P-gp inhibitors. Herein, we developed a novel all-in-one hybrid nanoparticle system to overcome MDR in doxorubicin (DOX)-resistant breast cancer. First, folic acid-modified DOX-loaded mesoporous silica nanoparticles (MSNs) were prepared and then loaded into PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles along with a P-gp inhibitor, elacridar. This hybrid nanoparticle system had high drug loading capacity, enabled both passive and active targeting of tumour tissues, and exhibited sequential and pH-triggered release of drugs. In vitro and in vivo studies in DOX-resistant breast cancer demonstrated the ability of the hybrid nanoparticles to reverse P-gp-mediated drug resistance. The nanoparticles were efficiently taken up by the breast cancer cells and delivered elacridar, in vitro. Biodistribution studies demonstrated substantial accumulation of the folate receptor-targeted PLGA/MSN hybrid nanoparticles in tumour-bearing mice. Moreover, deceleration of the tumour growth was remarkable in the animals administered with the DOX and elacridar co-loaded hybrid nanoparticles when compared to those treated with the marketed liposomal DOX (Caelyx®) or its combination with elacridar.

Acquired immune resistance is associated with interferon signature and modulation of KLF6/c-MYB transcription factors in myeloid leukemia.

Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute myeloid leukemia (AML) and isolated the residual subpopulations as "immune-experienced" AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T-cell-mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c-MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients' bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous "immune experience" is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.

Characterization of serum extracellular vesicles and their differential level of miR-197-3p in familial Mediterranean fever patients.

OBJECTIVES: The aim of this study was to analyze the existence of miRNAs derived from serum extracellular vesicles (EVs) in familial Mediterranean fever (FMF) patients. Our group has previously shown the association of certain miRNAs with FMF. METHODS: Serum samples of adult and pediatric FMF patients and their age matched controls were used in the study. Serum EVs were characterized by transmission electron microscopy (TEM) and flow cytometry. RNAs were isolated from EVs and levels of miR-197-3p and miR-20a-5p were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: EV characterization using TEM demonstrated fraction of 30-120 nm-sized particles with cup-shaped morphology. Flow cytometry results revealed the CD63 and CD81 positive populations as 53.3% in serum EVs. We showed that miR-197-3p and miR-20a-5p were "circulating miRNAs" and carried in EVs of FMF patients and controls. In FMF patients, level of miR-197-3p was significantly decreased. There was no significant alteration in the level for miR-20a-5p between patients and controls. CONCLUSION: We showed the differential level of miR-197-3p in serum EVs of the FMF patients. miR-197-3p's potential as a biomarker and therapeutic target in FMF pathogenesis warrants further investigation.

EVs and EV-miRNAs can be identified in FMF patients' sera.Serum EV-miR-197-3p is dysregulated in FMF patients.Serum EV-miR-197-3p might have both diagnostic and therapeutic potentials.

Immune checkpoint status and exhaustion-related phenotypes of CD8+ T cells from the tumor-draining regional lymph nodes in breast cancer.

BACKGROUND: Functional status of T cells determines the responsiveness of cancer patients to immunotherapeutic interventions. Even though T cell-mediated immunity is inaugurated in the tumor-adjacent lymph nodes, peripheral blood has been routinely sampled for testing the immunological assays. The purpose of this study is to determine the immune checkpoint molecule expression and the exhaustion-related phenotype of cytotoxic T cells in the regional lymph nodes from breast cancer patients. PATIENTS AND METHODS: Multicolor immunophenotyping was used to determine the expression of PD-1, TIM-3, LAG3, CTLA-4, CCR7, CD45RO, CD127, CD25, CXCR5, and ICOS molecules on CD3+ CD4- CD56- CD8+ cytotoxic T cells freshly obtained from the lymph nodes and the peripheral blood samples of the breast cancer patients. The results were assessed together with the clinical data. RESULTS: A population of cytotoxic T cells was noted with high PD-1 and CXCR5 expression in the lymph nodes of the breast cancer patients. Co-expression of PD-1, CXCR5, TIM-3, and ICOS indicated a follicular helper T cell (Tfh)-like, exhaustion-related immunophenotype in these cytotoxic T cells. Only a minor population with CTLA-4 and LAG3 expression was noted. The PD-1+ CXCR5+ cytotoxic T cells largely displayed CD45RO+ CCR7+ central memory markers. The amount of CXCR5-expressing PD-1- cytotoxic T cells was elevated in the lymph nodes of the patients. CONCLUSION: The regional lymph nodes of breast cancer patients harbor Tfh-like exhausted cytotoxic T lymphocytes with high PD-1 and TIM-3 checkpoint molecule expression. The immunological conditions in the regional lymph nodes should be implicated for immune checkpoint immunotherapy (ICI) of cancer.

Pro-fibrogenic and adipogenic aspects of chronic muscle degeneration are contributed by distinct stromal cell subpopulations.

Chronic skeletal muscle degeneration is characterized by fiber atrophy accompanied by deposition of extracellular matrix (ECM) components and fatty infiltration. Excessive accumulation of ECM leads to fibrosis via the contribution of fibro-adipogenic precursors (FAPs). Fibrosis also accompanies disuse atrophy and sarcopenia without significant inflammation. The present study aimed to comparatively analyze heterogeneous population of FAPs during acute injury and immobilization (tenotomy and denervation). The comparative analysis was accomplished based on the following 3 stromal cell subpopulations: i) CD140a(+)/Sca1(+); ii) CD140a(+)/Sca1(-); iii) CD140a(-)/Sca1(+). RNASeq analysis was employed to characterize and compare their quiescent and activated states. Whereas CD140a(-)/Sca1(+) was the most predominant activated subpopulation in tenotomy, denervation stimulated the CD140a(+)/Sca1(+) subpopulation. Immobilization models lacked myofiber damage and exhibited a minute increase in CD45(+) cells, as compared to acute injury. Transcriptome analysis showed common and discordant regulation of ECM components, without profound proliferative activation. Herein, we suggest unique surface markers for further identification of the investigated cell subpopulations. FAP subpopulations show similar activation kinetics in an inflammatory environment but the present findings highlight the fact that inflammation may not be a prerequisite for FAP activation. Delayed proliferation kinetics indicate that signals beyond inflammation might trigger FAP activation, leading to irreversible stromal changes.

Liver resection modulates hepatic chemokine levels in breast cancer.

BACKGROUND: Resection of metastatic hepatic tumors of breast cancer may result in the acceleration of hepatic and extrahepatic tumor progression due to the microenvironmental circulation of chemokines. This study aimed to investigate the effect of hepatectomy on a large panel of chemokines, liver regeneration, and myeloid cell levels in an experimental breast cancer model. METHODS: The 4T1 breast cancer cells were inoculated, and 30% to 40% hepatectomy was performed. Mice without tumors or only laparotomy (no hepatectomy) served as control groups. After 14 days (short-term) and 21 days (long-term), tissue samples were obtained from the regions near and distant from the resection site. Chemokine levels were evaluated by enzyme-linked immunosorbent assay arrays. Myeloid infiltration in the liver and the primary tumor and hepatic regeneration status were also histopathologically evaluated. RESULTS: The levels of pro-tumorigenic chemokines such as CCL2, CCL3, CCL4, and CCL5 were elevated in hepatectomized tumor-bearing animals. This observation was consistent with the presence of hepatic metastases. Liver regeneration and myeloid cell infiltration showed significant differences between the tumor-bearing hepatectomized groups followed in the short and long term. CONCLUSION: Our study showed elevation and variations in chemokines after hepatectomy, with a prominent increase in pro-tumorigenic chemokines. These results can be associated with the acceleration of metastasis after liver resection. However, further prospective studies are required to better define the impact of resection, which may transform the liver into a favorable site for metastasis.

Human memory T cell dynamics after aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccination.

This study evaluates the functional capacity of CD4+ and CD8+ terminally-differentiated effector (TEMRA), central memory (TCM), and effector memory (TEM) cells obtained from the volunteers vaccinated with an aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac). The volunteers were followed for T cell immune responses following the termination of a randomized phase III clinical trial. Seven days and four months after the second dose of the vaccine, the memory T cell subsets were collected and stimulated by autologous monocyte-derived dendritic cells (mDCs) loaded with SARS-CoV-2 spike glycoprotein S1. Compared to the placebo group, memory T cells from the vaccinated individuals significantly proliferated in response to S1-loaded mDCs. CD4+ and CD8+ memory T cell proliferation was detected in 86% and 78% of the vaccinated individuals, respectively. More than 73% (after a short-term) and 62% (after an intermediate-term) of the vaccinated individuals harbored TCM and/or TEM cells that responded to S1-loaded mDCs by secreting IFN-γ. The expression of CD25, CD38, 4-1BB, PD-1, and CD107a indicated a modulation in the memory T cell subsets. Especially on day 120, PD-1 was upregulated on CD4+ TEMRA and TCM, and on CD8+ TEM and TCM cells; accordingly, proliferation and IFN-γ secretion capacities tended to decline after 4 months. In conclusion, the combination of inactivated whole-virion particles with aluminum adjuvants possesses capacities to induce functional T cell responses.

Targeted siRNA lipid nanoparticles for the treatment of KRAS-mutant tumors.

K-RAS is a highly relevant oncogene that is mutated in approximately 90% of pancreatic cancers and 20-25% of lung adenocarcinomas. The aim of this work was to develop a new anti-KRAS siRNA therapeutic strategy through the engineering of functionalized lipid nanoparticles (LNPs). To do this, first, a potent pan anti-KRAS siRNA sequence was chosen from the literature and different chemical modifications of siRNA were tested for their transfection efficacy (KRAS knockdown) and anti-proliferative effects on various cancer cell lines. Second, a selected siRNA candidate was loaded into tLyp-1 targeted and non-targeted lipid nanoparticles (LNPs). The biodistribution and antitumoral efficacy of selected siRNA-loaded LNP-prototypes were evaluated in vivo using a pancreatic cancer murine model (subcutaneous xenograft CFPAC-1 tumors). Our results show that tLyp-1-tagged targeted LNPs have an enhanced accumulation in the tumor compared to non-targeted LNPs. Moreover, a significant reduction in the pancreatic tumor growth was observed when the anti-KRAS siRNA treatment was combined with a classical chemotherapeutic agent, gemcitabine. In conclusion, our work demonstrates the benefits of using a targeting approach to improve tumor accumulation of siRNA-LNPs and its positive impact on tumor reduction.

A positive feedback loop driven by fibronectin and IL-1ß sustains the inflammatory microenvironment in breast cancer.

Inflammatory alterations of the extracellular matrix shape the tumor microenvironment and promote all stages of carcinogenesis. This study aims to determine the impact of cellular fibronectin on inflammatory facets of tumor-associated macrophages (TAMs) in breast cancer. Cellular fibronectin (FN) harboring the alternatively spliced extra domain A (FN-EDA) was determined to be a matrix component produced by the triple-negative breast cancer (TNBC) cells. High levels of FN-EDA correlated with poor survival in breast cancer patients. The proinflammatory cytokine IL-1ß enhanced the expression of cellular fibronectin including FN-EDA. TAMs were frequently observed in the tumor areas rich in FN-EDA. Conditioned media from TNBC cells induced the differentiation of CD206+CD163+ macrophages and stimulated the STAT3 pathway, ex vivo. In the macrophages, the STAT3 pathway enhanced FN-EDA-induced IL-1ß secretion and NF-κB signaling. In conclusion, our data indicate a self-reinforcing mechanism sustained by FN-EDA and IL-1ß through NF-κB and STAT3 signaling in TAMs which fosters an inflammatory environment in TNBC.

Synergistic Antitumor Potency of a Self-Assembling Cyclodextrin Nanoplex for the Co-Delivery of 5-Fluorouracil and Interleukin-2 in the Treatment of Colorectal Cancer.

Chemotherapy is the most used method after surgery in the treatment of colon cancer. Cancer cells escape the recognition mechanism of immune system cells to survive and develop chemoresistance. Therefore, the use of immunotherapy in combination with chemotherapy can increase the effectiveness of the treatment. Nanoparticles have been used clinically to increase the accumulation of therapeutics in target tissues and reduce toxicity. In this paper, nanoplexes were formed via cationic cyclodextrin polymer, 5-Fluorouracil, and Interleukin-2 based on the opposite charge interaction of macromolecules without undergoing any structural changes or losing the biological activity of Interleukin-2. Anticancer activities of nanoplexes were determined in two-dimensional and three-dimensional cell culture setups. The dual drug-loaded cyclodextrin nanoplexes diffused deeper into the spheroids and accelerated apoptosis when compared with 5-FU solutions. In the colorectal tumor-bearing animal model, survival rate, antitumor activity, metastasis, and immune response parameters were assessed using a cyclodextrin derivative, which was found to be safe based on the ALT/AST levels in healthy mice. Histomorphometric analysis showed that the groups treated with the nanoplex formulation had significantly fewer initial tumors and lung foci when compared with the control. The dual drug-loaded nanoplex could be a promising drug delivery technique in the immunochemotherapy of colorectal cancer.

Effects of Particle Geometry for PLGA-Based Nanoparticles: Preparation and In Vitro/In Vivo Evaluation.

The physicochemical properties (size, shape, zeta potential, porosity, elasticity, etc.) of nanocarriers influence their biological behavior directly, which may result in alterations of the therapeutic outcome. Understanding the effect of shape on the cellular interaction and biodistribution of intravenously injected particles could have fundamental importance for the rational design of drug delivery systems. In the present study, spherical, rod and elliptical disk-shaped PLGA nanoparticles were developed for examining systematically their behavior in vitro and in vivo. An important finding is that the release of the encapsulated human serum albumin (HSA) was significantly higher in spherical particles compared to rod and elliptical disks, indicating that the shape can make a difference. Safety studies showed that the toxicity of PLGA nanoparticles is not shape dependent in the studied concentration range. This study has pioneering findings on comparing spherical, rod and elliptical disk-shaped PLGA nanoparticles in terms of particle size, particle size distribution, colloidal stability, morphology, drug encapsulation, drug release, safety of nanoparticles, cellular uptake and biodistribution. Nude mice bearing non-small cell lung cancer were treated with 3 differently shaped nanoparticles, and the accumulation of nanoparticles in tumor tissue and other organs was not statistically different (p > 0.05). It was found that PLGA nanoparticles with 1.00, 4.0 ± 0.5, 7.5 ± 0.5 aspect ratios did not differ on total tumor accumulation in non-small cell lung cancer.

A novel combination for the treatment of small cell lung cancer: Active targeted irinotecan and stattic co-loaded PLGA nanoparticles.

Polymeric nanoparticles are widely used drug delivery systems for cancer treatment due to their properties such as ease of passing through biological membranes, opportunity to modify drug release, specifically targeting drugs to diseased areas, and potential of reducing side effects. Here, we formulated irinotecan and Stattic co-loaded PLGA nanoparticles targeted to small cell lung cancer. Nanoparticles were successfully conjugated with CD56 antibody with a conjugation efficiency of 84.39 ± 1.01%, and characterization of formulated nanoparticles was conducted with in-vitro and in-vivo studies. Formulated particles had sizes in the range of 130-180 nm with PDI values smaller than 0.3. Encapsulation and active targeting of irinotecan and Stattic resulted in increased cytotoxicity and anti-cancer efficiency in-vitro. Furthermore, it was shown with ex-vivo biodistribution studies that conjugated nanoparticles were successfully targeted to CD56-expressing SCLC cells and distributed mainly to tumor tissue and lungs. Compliant with our hypothesis and literature, the STAT3 pathway was successfully inhibited with Stattic solution and Stattic loaded nanoparticles. Additionally, intravenous injection of conjugated co-loaded nanoparticles resulted in decreased side effects and better anti-tumor activity than individual solutions of drugs in SCLC tumor-bearing mice. These results may indicate a new treatment option for clinically aggressive small cell lung cancer.

Tailored modulation of stemness and drug resistance marker characteristics in K-Ras mutant lung cancer cells via PD-L1 gene suppression.

AIMS: We aimed to analyze the association of tumor cell-specific Programmed Cell Death Ligand 1 (PD-L1) expression with stemness markers and multi-drug resistance genes in non-small cell lung cancer. MAIN METHODS: ATP Binding Cassette Subfamily G Member 2 (ABCG2), Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), CD44, Epithelial cell adhesion molecule (EPCAM), Kruppel-like factor 4 (KLF4), MYC Proto-Oncogene (MYC), Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX2) as well as ATP Binding Cassette Subfamily C Member (ABCC) 1-6, ABCC10-12 with ATP Binding Cassette Subfamily B Member (ABCB) 1 and ABCB4-9 belongs to ABC transporters family were analyzed and their correlation with PD-L1 was evaluated using Cancer Cell Line Encyclopedia and The Cancer Genome Atlas data sets. We validated potential lung cancer stemness markers harboring ABCG2, CD44, ALDH1A1 and SOX-2, which are affected as a result of siRNA-mediated suppression of PD-L1 via flow cytometry. K-Ras downstream signaling proteins as well as multidrug resistance proteins were also investigated. KEY FINDINGS: PD-L1 was found to be positively correlated with CD44, whereas negatively correlated with ALDH1A1 (Pearson r = 0.44, r = -0.48; respectively) in 45 K-Ras mutated NSCLC cells based on CCLE database. While ABCC5 was dominantly decreased in K-Ras mutant lung cancer cells affected by PD-L1 gene suppression, expression changes were observed in the activation of distinct signaling pathways in a cell line-dependent manner. SIGNIFICANCE: The evaluation of markers in lung adenocarcinoma with different types of K-Ras mutations in terms of both stemness and drug resistance markers will contribute to the development of personalized immunotherapy regimens.

Effector Th1 cells under PD-1 and CTLA-4 checkpoint blockade abrogate the upregulation of multiple inhibitory receptors and by-pass exhaustion.

Immune checkpoint inhibitor (ICI) immunotherapy relies on the restoration of T-cell functions. The ICI receptors are not only found on exhausted T cells but also upregulated upon activation and reach high levels on effector T cells. In an ex vivo model, this study explored the consequences of PD-1 and cytotoxic T-lymphocyte antigen (CTLA-4) blockade applied during specific time frames of T-cell stimulation that coincide with distinct functional phases in type 1 helper T (Th1) cells. When applied at an early stimulation stage, the checkpoint blockade interfered with the upregulation of multiple inhibitory receptors such as PD-1, LAG3, TIM-3 and CTLA-4. Moreover, extension of the blockade period restricted the hyporesponsiveness in T cells. Alternatively, a short-term ICI treatment was advantageous when applied at late time frames of Th1 cell stimulation. Here, a transition phase from effector to exhausted state, which coincided with the late time frames of Th1 stimulation, was clearly determined together with the transcriptomics data demonstrating the initiation of significant alterations in metabolic pathways, genetic information processes, effector and exhaustion specific pathways. Applied in this transition phase, PD-1 and/or CTLA-4 blockade downregulated the inhibitory receptors which were already present on the effector Th1 cells, potentially through endocytic pathways. Therefore, the efficacy of ICI therapy was modulated by the functional status of T cells and can be improved by modifying the timing and duration of PD-1 and CTLA-4 blockade. In conclusion, the ICI therapy not only supports the reactivation of T cells but can also constrain de novo exhaustion.

Memory T cell responses in seronegative older adults following SARS-CoV-2 vaccination.

Generating memory T cell responses besides humoral immune responses is essential when it comes to the efficacy of a vaccine. In this study, the presence of memory T cell responses after aluminum-adjuvanted inactivated whole-virion SARS-CoV-2 vaccine (CoronaVac) in seronegative and seropositive elderly individuals were examined. CD4+ and CD8+ memory T cell proliferation and IFN-γ production capacities were evaluated. Additionally, clinical frailty scale (CFS) and FRAIL scales of the individuals were scored. CD4+ memory T cell responses more prominent than CD8+ memory T cells. In seronegative individuals, 80% of them had memory CD4+ and IFN-γ, whereas 50% of them had memory CD4+ and all of them had IFN-γ responses. Additionally, 40% of seronegative patients and 50% of seropositive patients had memory CD8+ responses. To sum up, humoral immune responses are not associated with memory T cell responses, and in seronegative individuals, memory T cell responses can be detected.

Small cell lung cancer stem cells display mesenchymal properties and exploit immune checkpoint pathways in activated cytotoxic T lymphocytes.

Small cell lung cancer (SCLC) is an aggressive tumor type with early dissemination and distant metastasis capacity. Even though optimal chemotherapy responses are observed initially in many patients, therapy resistance is almost inevitable. Accordingly, SCLC has been regarded as an archetype for cancer stem cell (CSC) dynamics. To determine the immune-modulatory influence of CSC in SCLC, this study focused on the characterization of CD44+CD90+ CSC-like subpopulations in SCLC. These cells displayed mesenchymal properties, differentiated into different lineages and further contributed to CD8+ cytotoxic T lymphocytes (CTL) responses. The interaction between CD44+CD90+ CSC-like cells and T cells led to the upregulation of checkpoint molecules PD-1, CTLA-4, TIM-3, and LAG3. In the patient-derived lymph nodes, CD44+ SCLC metastases were also observed with T cells expressing PD-1, TIM-3, or LAG3. Proliferation and IFN-γ expression capacity of TIM-3 and LAG3 co-expressing CTLs are adversely affected over long-time co-culture with CD44+CD90+ CSC-like cells. Moreover, especially through IFN-γ secreted by the T cells, the CSC-like SCLC cells highly expressed PD-L1 and PD-L2. Upon a second encounter with immune-experienced, IFN-γ-stimulated CSC-like SCLC cells, both cytotoxic and proliferation capacities of T cells were hampered. In conclusion, our data provide evidence for the superior potential of the SCLC cells with stem-like and mesenchymal properties to gain immune regulatory capacities and cope with cytotoxic T cell responses. With their high metastatic and immune-modulatory assets, the CSC subpopulation in SCLC may serve as a preferential target for checkpoint blockade immunotherapy .

Therapeutic efficacy and gastrointestinal biodistribution of polycationic nanoparticles for oral camptothecin delivery in early and late-stage colorectal tumor-bearing animal model.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world and is the second leading cause of cancer related deaths. New cases are increasingly diagnosed every day, but current therapeutic options are still insufficient for an effective treatment. In CRC treatment, there is a significant need for alternative treatment approaches that can both prevent relapse and provide strong antimetastatic effects as the intestines and colon are prone to metastasis to neighboring organs and tissues as well as the liver and the lung. In this study, optimized polycationic cyclodextrin (CD) nanoparticles for oral Camptothecin (CPT) delivery were comprehensively examined for in vivo performance in early and late stage tumor bearing mouse model in terms of antitumoral and antimetastatic efficacy of CPT bound to polycationic CD nanoparticles in comparison to free CPT. In addition, the gastrointestinal localization of a single administration of fluorescent dye loaded polycationic CD nanoparticles in the gastrointestinal tract at the end of 24 h after oral administration was also imaged and evaluated by in vivo imaging system against fluorescent dye intensity. Results showed that survival percentage was significantly improved in CRC-bearing mice compared to oral CPT solution, with significantly reduced colorectal tumor masses and number of liver metastatic foci (p < 0.05). It was also possible to differentiate between the effectiveness of nanoparticles in early or late stages of CRC. In vivo imaging studies have also confirmed that polycationic CD nanoparticles are able to deliver the therapeutic load up to the colon and tend to accumulate especially in tumor foci, indicating an effective local treatment strategy. In addition number of liver metastases were significantly decreased with the CPT-loaded polycationic CD nanoparticle formulation in both early and late stage tumor models. These findings indicated that CPT-loaded polycationic CD nanoparticles could be an efficient oral nanocarrier formulation for anticancer molecules that have limited application because of oral bioavailability and stability problems.

Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature.

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71-CD34+ CD38mo/lo], megakaryocyte-erythroid progenitor cells (MEP) [CD71-CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.