T-Cell manipulation strategies to prevent graft-versus-host disease in haploidentical stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (HSCT) from an human leukocyte antigen (HLA)-identical donor can be curative for eligible patients with non-malignant and malignant haematological disorders. HSCT from alternative donor sources, such as HLA-mismatched haploidentical donors, is increasingly considered as a viable therapeutic option for patients lacking HLA-matched donors. Initial attempts at haploidentical HSCT were associated with vigorous bidirectional alloreactivity, leading to unacceptably high rates of graft rejection and graft-versus-host disease (GVHD). More recently, new approaches for mitigating harmful T-cell alloreactivity that mediates GVHD, while preserving the function of tumour-reactive natural killer (NK) cells and γδ T cells, have led to markedly improved clinical outcomes, and are successfully being implemented in the clinic. This article will provide an update on in vitro strategies and in vivo approaches aimed at preventing GVHD by selectively manipulating key components of the adaptive immune response, such as T-cell receptor (TCR)- αβ T cells and CD45RA-expressing naive T cells

Granulocyte transfusions in children and adults with hematological malignancies: benefits and controversies

Bacterial and fungal infections continue to pose a major clinical challenge in patients with prolonged severe neutropenia after chemotherapy or hematopoietic stem cell transplantation (HSCT). With the advent of granulocyte colony-stimulating factor (G-CSF) to mobilize neutrophils in healthy donors, granulocyte transfusions have been broadly used to prevent and/or treat life-threatening infections in patients with severe febrile neutropenia and/or neutrophil dysfunction. Although the results of randomized controlled trials are inconclusive, there are suggestions from pilot and retrospective studies that granulocyte transfusions may benefit selected categories of patients. We will critically appraise the evidence related to the use of therapeutic granulocyte transfusions in children and adults, highlighting current controversies in the field and discussing complementary approaches to modulate phagocyte function in the host

The Asp272-Glu282 Region of Platelet Glycoprotein Ib Interacts with the Heparin-binding Site of -Thrombin and Protects the Enzyme from the Heparin-catalyzed Inhibition by Antithrombin III

Platelet glycoprotein Ib (GpIb) mediates interaction with both von Willebrand factor and thrombin. Thrombin binds to GpIb via its heparin-binding site (HBS) (De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thromb. Haemostasis 77, 735–740; De Cristofaro, R., De Candia, E., Croce, G., Morosetti, R., and Landolfi, R. (1998) Biochem. J. 332, 643–650). To identify the thrombin-binding domain on GpIbα, we examined the effect of GpIbα1–282, a GpIbα fragment released by the cobra venom mocarhagin on the heparin-catalyzed rate of thrombin inhibition by antithrombin III (AT). GpIbα1–282 inhibited the reaction in a dose-dependent and competitive fashion. In contrast, the GpIbα1–271 fragment, produced by exposing GpIbα1–282 to carboxypeptidase Y, had no effect on thrombin inhibition by the heparin-AT complex. Measurements of the apparent equilibrium constant of the GpIbα1–282 binding to thrombin as a function of different salts (NaCl and tetramethyl-ammonium chloride) concentration (0.1–0.2 M) indicated a large salt dependence (Γ± = −4.5), similar to that pertaining to the heparin binding to thrombin. The importance of thrombin HBS in its interaction with GpIbα was confirmed using DNA aptamers, which specifically bind to either HBS (HD22) or the fibrinogen recognition site of thrombin (HD1). HD22, but not HD1, inhibited thrombin binding to GpIbα1–282. Furthermore, the proteolytic derivative γT-thrombin, which lacks the fibrinogen recognition site, binds to GpIbα via its intact HBS in a reaction that is inhibited by HD22. Neither α- nor γT-thrombin bound to GpIbα1–271, suggesting that the Asp272–Glu282 region of GpIbα may act as a “heparin-like” ligand for the thrombin HBS, thereby inhibiting heparin binding to thrombin. It was also demonstrated that intact platelets may dose-dependently inhibit the heparin-catalyzed thrombin inhibition by AT at enzyme concentrations <5 nM. Altogether, these findings show that thrombin HBS binds to the region of GpIbα involving the Asp272–Glu282 segment, protecting the enzyme from the inactivation by the heparin-AT system

Fibrinogen-elongated Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

The expression of the elongated fibrinogen γ chain, termed γ′, derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how γ′ chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibα (GpIbα), protease-activated receptor (PAR) 1, and PAR4. Both synthetic γ′ peptide and fibrinogen fragment D*, containing the elongated γ′ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC50 values of 42 ± 3.5 and 0.47 ± 0.03 μm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic γ′ peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbα with a mean Ki ≈ 0.5 and ≈35 μm, respectively. Both these γ′ chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide d-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen γ′ chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation

Integrative systems medicine approaches to identify molecular targets in lymphoid malignancies

Although survival rates for lymphoproliferative disorders are steadily increasing both in the US and in Europe, there is need for optimizing front-line therapies and developing more effective salvage strategies. Recent advances in molecular genetics have highlighted the biological diversity of lymphoproliferative disorders. In particular, integrative approaches including whole genome sequencing, whole exome sequencing, and transcriptome or RNA sequencing have been instrumental to the identification of molecular targets for treatment. Herein, we will discuss how genomic, epigenomic and proteomic approaches in lymphoproliferative disorders have supported the discovery of molecular lesions and their therapeutic targeting in the clinic

Immune-phenotyping and transcriptomic profiling of peripheral blood mononuclear cells from patients with breast cancer: identification of a 3 gene signature which predicts relapse of triple negative breast cancer

Background: Interactions between the immune system and tumors are highly reciprocal in nature, leading to speculation that tumor recurrence or therapeutic resistance could be influenced or predicted by immune events that manifest locally, but can be detected systemically. Methods: Multi-parameter flow cytometry was used to examine the percentage and phenotype of natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), monocyte subsets and regulatory T (Treg) cells in the peripheral blood of of 85 patients with breast cancer (50 of whom were assessed before and after one cycle of anthracycline-based chemotherapy), and 23 controls. Transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) in 23 patients were generated using a NanoString gene profiling platform. Results: An increased percentage of immunosuppressive cells such as granulocytic MDSCs, intermediate CD14++CD16+ monocytes and CD127negCD25highFoxP3+ Treg cells was observed in patients with breast cancer, especially patients with stage 3 and 4 disease, regardless of ER status. Following neoadjuvant chemotherapy, B cell numbers decreased significantly, whereas monocyte numbers increased. Although chemotherapy had no effect on the percentage of Treg, MDSC and NK cells, the expression of inhibitory receptors CD85j, LIAR and NKG2A and activating receptors NKp30 and NKp44 on NK cells increased, concomitant with a decreased expression of NKp46 and DNAM-1 activating receptors. Transcriptomic profiling revealed a distinct group of 3 patients in the triple negative breast cancer (TNBC) cohort who expressed high levels of mRNA encoding genes predominantly involved in inflammation. The analysis of a large transcriptomic dataset derived from the tumors of patients with TNBC revealed that the expression of CD163, CXCR4, THBS1 predicted relapse-free survival. Conclusions: The peripheral blood immunome of patients with breast cancer is influenced by the presence and stage of cancer, but not by molecular subtypes. Furthermore, immune profiling coupled with transcriptomic analyses of peripheral blood cells may identify patients with TNBC that are at risk of relapse after chemotherapy

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by α-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibα also has a high affinity binding site for α-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbα may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by α-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparentk cat/K m value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 ± 0.1 × 107 m −1sec−1. The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbα on platelet membranes by mocarhagin treatment reduced the k cat/K m value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k cat/K m value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbα, reduced the apparentk cat/K m value by about 5-fold. 4) Displacement of α-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparentk cat/K m value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparentk cat/K m value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a “cofactor” for PAR-1 activation by thrombin

Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV

Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory

Immune reconstitution after autologous hematopoietic stem cell transplantation in Crohn's disease: current status and future directions. A review on behalf of the EBMT Autoimmune Diseases Working Party and the autologous stem cell transplantation in refractory CD—low intensity therapy evaluation study investigators

Patients with treatment refractory Crohn’s disease (CD) suffer debilitating symptoms, poor quality of life, and reduced work productivity. Surgery to resect inflamed and fibrotic intestine may mandate creation of a stoma and is often declined by patients. Such patients continue to be exposed to medical therapy that is ineffective, often expensive and still associated with a burden of adverse effects. Over the last two decades, autologous hematopoietic stem cell transplantation (auto-HSCT) has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies have provided proof of concept that auto-HSCT can restore immunological tolerance in chronic autoimmunity via the eradication of pathological immune responses and a profound reconfiguration of the immune system. Herein, we review current experience of auto-HSCT for the treatment of CD as well as approaches that have been used to monitor immune reconstitution following auto-HSCT in patients with ADs, including CD. We also detail immune reconstitution studies that have been integrated into the randomized controlled Autologous Stem cell Transplantation In refractory CD—Low Intensity Therapy Evaluation trial, which is designed to test the hypothesis that auto-HSCT using reduced intensity mobilization and conditioning regimens will be a safe and effective means of inducing sustained control in refractory CD compared to standard of care. Immunological profiling will generate insight into the pathogenesis of the disease, restoration of responsiveness to anti-TNF therapy in patients with recurrence of endoscopic disease and immunological events that precede the onset of disease in patients that relapse after auto-HSCT

Mobilization of healthy donors with plerixafor affects the cellular composition of T-cell receptor (TCR)-αβ/CD19-depleted haploidentical stem cell grafts

Background: HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is suitable for patients lacking related or unrelated HLA-matched donors. Herein, we investigated whether plerixafor (MZ), as an adjunct to G-CSF, facilitated the collection of mega-doses of hematopoietic stem cells (HSC) for TCR-αβ/CD19-depleted haploidentical HSCT, and how this agent affects the cellular graft composition. Methods: Ninety healthy donors were evaluated. Single-dose MZ was given to 30 ‘poor mobilizers’ (PM) failing to attain ≥40 CD34+ HSCs/μL after 4 daily G-CSF doses and/or with predicted apheresis yields ≤12.0x106 CD34+ cells/kg recipient’s body weight. Results: MZ significantly increased CD34+ counts in PM. Naïve/memory T and B cells, as well as natural killer (NK) cells, myeloid/plasmacytoid dendritic cells (DCs), were unchanged compared with baseline. MZ did not further promote the G-CSF-induced mobilization of CD16+ monocytes and the down-regulation of IFN-γ production by T cells. HSC grafts harvested after G-CSF + MZ were enriched in myeloid and plasmacytoid DCs, but contained low numbers of pro-inflammatory 6-sulfo-LacNAc+ (Slan)-DCs. Finally, children transplanted with G-CSF + MZ-mobilized grafts received greater numbers of monocytes, myeloid and plasmacytoid DCs, but lower numbers of NK cells, NK-like T cells and Slan-DCs. Conclusions: MZ facilitates the collection of mega-doses of CD34+ HSCs for haploidentical HSCT, while affecting graft composition