Inhibition of mast cells: a novel mechanism by which nintedanib may elicit anti-fibrotic effects.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease which presents a grave prognosis for diagnosed patients. Nintedanib (a triple tyrosine kinase inhibitor) and pirfenidone (unclear mechanism of action) are the only approved therapies for IPF, but have limited efficacy. The pathogenic mechanisms of this disease are not fully elucidated; however, a role for mast cells (MCs) has been postulated. OBJECTIVES: The aim of this work was to investigate a role for MCs in IPF and to understand whether nintedanib or pirfenidone could impact MC function. METHODS AND RESULTS: MCs were significantly elevated in human IPF lung and negatively correlated with baseline lung function (FVC). Importantly, MCs were positively associated with the number of fibroblast foci, which has been linked to increased mortality. Furthermore, MCs were increased in the region immediately surrounding the fibroblast foci, and co-culture studies confirmed a role for MC-fibroblast crosstalk in fibrosis. Nintedanib but not pirfenidone inhibited recombinant stem cell factor (SCF)-induced MC survival. Further evaluation of nintedanib determined that it also inhibited human fibroblast-mediated MC survival. This was likely via a direct effect on ckit (SCF receptor) since nintedanib blocked SCF-stimulated ckit phosphorylation, as well as downstream effects on MC proliferation and cytokine release. In addition, nintedanib ablated the increase in lung MCs and impacted high tissue density frequency (HDFm) in a rat bleomycin model of lung fibrosis. CONCLUSION: Nintedanib inhibits MC survival and activation and thus provides a novel additional mechanism by which this drug may exert anti-fibrotic effects in patients with IPF.

Functional specific-T-cell expansion after first cytomegalovirus reactivation predicts viremia control in allogeneic hematopoietic stem cell transplant recipients.

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients might result in over-treatment, inducing drug-related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow-up, in combination with the use of streptamers (STs) and cytokine-intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV-specific CD8+ T-cell (CMV-CTL) populations, we monitored 25 patients who have undergone allo-HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV-specific T cells, determined by early interferon γ production or by significant T-cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV-CTLs does correlate with CMV recurrence. These results support that behavior of CMV-specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.

Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8+ T cells by flow cytometry.

BACKGROUND: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice. METHODS: CMVpp65495-503 -specific HLA-A*02:01 CD8+ T lymphocytes (CTLA *02:01 -CMVpp65495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry. RESULTS: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (rSpearman = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTLA *02:01 -CMVpp65495-503 (rSpearman = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTLA *02:01 -CMVpp65495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8+ T-cells. CONCLUSION: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTLA *02:01 -CMVpp65495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTLA *02:01 -CMVpp65495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTLA *02:01 -CMVpp65495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society.

The immune response to cytomegalovirus in allogeneic hematopoietic stem cell transplant recipients.

Approximately, up to 70 % of the human population is infected with cytomegalovirus (CMV) that persists for life in a latent state. In healthy people, CMV reactivation induces the expansion of CMV-specific T cells up to 10 % of the entire T cell repertoire. On the contrary, CMV infection is a major opportunistic viral pathogen that remains a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Due to the delayed CMV-specific immune recovery, the incidence of CMV reactivation during post-transplant period is very high. Several methods are currently available for the monitoring of CMV-specific responses that help in clinical monitoring. In this review, essential aspects in the immune recovery against CMV are discussed to improve the better understanding of the immune system relying on CMV infection and, thereby, helping the avoidance of CMV disease or reactivation following hematopoietic stem cell transplantation with severe consequences for the transplanted patients.

Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood.

BACKGROUND: Adoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional. METHODS: MHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers. RESULTS: CMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products. CONCLUSIONS: The translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.

CMV-specific T cell isolation from G-CSF mobilized peripheral blood: depletion of myeloid progenitors eliminates non-specific binding of MHC-multimers.

BACKGROUND: Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. METHODS: In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. RESULTS: After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. CONCLUSIONS: G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.

Manufacturing of highly functional and specific T cells for adoptive immunotherapy against virus from granulocyte colony-stimulating factor-mobilized donors.

BACKGROUND AIMS: Cytomegalovirus (CMV) reactivation remains an important risk after hematopoietic stem cell transplantation, which can be effectively controlled through adoptive transfer of donor-derived CMV-specific T cells (CMV-T). CMV-T are usually obtained from donor peripheral blood mononuclear cells (PBMCs) collected before G-CSF mobilization. Despite previous studies that showed impaired T-cell function after granulocyte colony-stimulating factor (G-CSF) mobilization, recent publications suggest that G-CSF-primed PBMCs retain anti-viral function and are a suitable starting material for CMV-T manufacturing. The objective of this study was to assess the feasibility of generating CMV-T from G-CSF-mobilized donors by use of the activation marker CD137 in comparison with conventional non-primed PBMCs. METHODS: CMV-T were isolated from G-CSF-mobilized and non-mobilized donor PBMCs on the basis of CMVpp65 activation-induced CD137 expression and expanded during 3 weeks. Functional assays were performed to assess antigen-specific activation, cytokine release, cytotoxic activity and proliferation after anti-genic re-stimulation. RESULTS: We successfully manufactured highly specific, functional and cytotoxic CMV-T from G-CSF-mobilized donor PBMCs. Their anti-viral function was equivalent to non-mobilized CMV-T, and memory phenotype would suggest their long-term maintenance after adoptive transfer. CONCLUSIONS: We confirm that the use of an aliquot from G-CSF-mobilized donor samples is suitable for the manufacturing of CMV cellular therapies and thereby abrogates the need for successive donations and ensures the availability for patients with unrelated donors.

Isolation of highly suppressive CD25+FoxP3+ T regulatory cells from G-CSF-mobilized donors with retention of cytotoxic anti-viral CTLs: application for multi-functional immunotherapy post stem cell transplantation.

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.

Impact of T cell selection methods in the success of clinical adoptive immunotherapy.

Chemotherapy and/or radiotherapy regular regimens used for conditioning of recipients of hematopoietic stem cell transplantation (SCT) induce a period of transient profound immunosuppression. The onset of a competent immunological response, such as the appearance of viral-specific T cells, is associated with a lower incidence of viral infections after haematopoietic transplantation. The rapid development of immunodominant peptide virus screening together with advances in the design of genetic and non-genetic viral- and tumoural-specific cellular selection strategies have opened new strategies for cellular immunotherapy in oncologic recipients who are highly sensitive to viral infections. However, the rapid development of cellular immunotherapy in SCT has disclosed the role of the T cell selection method in the modulation of functional cell activity and of in vivo secondary effects triggered following immunotherapy.

The abrogation of TCR-independent interactions with human serum ensures a selective capture of therapeutic virus-specific CD8+ T-cells by multimer technology in Adoptive Immunotherapy.

Multimers are complexes of recombinant MHC-class I molecules conjugated with antigenic immunodominant peptides and labeled with fluorescent molecules or magnetic microbeads that allow the quantification and selection of virus-specific cytotoxic T-cell subpopulations. Specific T-cell receptors recognize the immunodominant peptides and bind to the multimers. Although these complexes are only recognized by CD8(+) T cells with specific T-cell receptors for the particular antigen, it has been observed that multimers can also bind non-specifically to CD8- cells, such as B-cells and monocytes. Using PBMCs from CMV-seropositive healthy donors, we analyze the tendency of Pentamer and Streptamer multimers towards non-specific interactions and describe a method to avoid this unwanted event. We find that a notable proportion of multimer-positive cells are likely to represent cross-contamination by cells lacking a TCR specific for pp65. In addition, we demonstrate that this unspecific interaction can be overcome by the pre-incubation of multimer-stained PBMCs with human AB serum, without altering their capacity to bind specifically to the CD8(+) T cell population of interest. In conclusion, in this study we characterize a novel method to abrogate TCR-independent interactions of multimers to ensure a pure and safe therapeutic product for Adoptive Immunotherapy.

CD14 and TLR4 mediate cytokine release promoted by electronegative LDL in monocytes.

AIMS: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes. METHODS AND RESULTS: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells. CONCLUSIONS: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations.

The Induction of Cytokine Release in Monocytes by Electronegative Low-Density Lipoprotein (LDL) Is Related to Its Higher Ceramide Content than Native LDL.

Electronegative low-density lipoprotein (LDL(-)) is a minor modified LDL subfraction that is present in blood. LDL(-) promotes inflammation and is associated with the development of atherosclerosis. We previously reported that the increase of cytokine release promoted by this lipoprotein subfraction in monocytes is counteracted by high-density lipoprotein (HDL). HDL also inhibits a phospholipase C-like activity (PLC-like) intrinsic to LDL(-). The aim of this work was to assess whether the inhibition of the PLC-like activity by HDL could decrease the content of ceramide (CER) and diacylglycerol (DAG) generated in LDL(-). This knowledge would allow us to establish a relationship between these compounds and the inflammatory activity of LDL(-). LDL(-) incubated at 37 °C for 20 h increased its PLC-like activity and, subsequently, the amount of CER and DAG. We found that incubating LDL(-) with HDL decreased both products in LDL(-). Native LDL was modified by lipolysis with PLC or by incubation with CER-enriched or DAG-enriched liposomes. The increase of CER in native LDL significantly increased cytokine release, whereas the enrichment in DAG did not show these inflammatory properties. These data point to CER, a resultant product of the PLC-like activity, as a major determinant of the inflammatory activity induced by LDL(-) in monocytes.

Receptores implicados en la inducción de citoquinas promovida por la LDL electronegativa en monocitos / Receptors involved in cytokine induction promoted by electronegative LDL in monocytes

Introducción La LDL electronegativa (LDL[−]) es una fracción minoritaria de la LDL total que se encuentra en circulación y presenta diferentes propiedades inflamatorias, siendo una de las más relevantes la inducción de citoquinas en células endoteliales y mononucleares. Sin embargo, no se conoce el mecanismo por el cual la LDL(−) ejerce su acción (..) (AU)

Introduction Elecronegative LDL (LDL(−)) is a small fraction of the total circulating LDL and has different inflammatory properties, one of the most important being the induction of cytokines in endotelial cells and monocytes. However, the mechanism by which LDL (−) exercises its action at cellular level is not known. The objective of this study was to evaluate the receptors involved in LDL (−) binding in monocytes, and how the liberation of cytokines (..) (AU)

Aggregated electronegative low density lipoprotein in human plasma shows a high tendency toward phospholipolysis and particle fusion.

Aggregation and fusion of lipoproteins trigger subendothelial retention of cholesterol, promoting atherosclerosis. The tendency of a lipoprotein to form fused particles is considered to be related to its atherogenic potential. We aimed to isolate and characterize aggregated and nonaggregated subfractions of LDL from human plasma, paying special attention to particle fusion mechanisms. Aggregated LDL was almost exclusively found in electronegative LDL (LDL(-)), a minor modified LDL subfraction, but not in native LDL (LDL(+)). The main difference between aggregated (agLDL(-)) and nonaggregated LDL(-) (nagLDL(-)) was a 6-fold increased phospholipase C-like activity in agLDL(-). agLDL(-) promoted the aggregation of LDL(+) and nagLDL(-). Lipoprotein fusion induced by α-chymotrypsin proteolysis was monitored by NMR and visualized by transmission electron microscopy. Particle fusion kinetics was much faster in agLDL(-) than in nagLDL(-) or LDL(+). NMR and chromatographic analysis revealed a rapid and massive phospholipid degradation in agLDL(-) but not in nagLDL(-) or LDL(+). Choline-containing phospholipids were extensively degraded, and ceramide, diacylglycerol, monoacylglycerol, and phosphorylcholine were the main products generated, suggesting the involvement of phospholipase C-like activity. The properties of agLDL(-) suggest that this subfraction plays a major role in atherogenesis by triggering lipoprotein fusion and cholesterol accumulation in the arterial wall.