Impact of Cryopreservation of Peripheral Blood Stem Cells (PBSC) in Transplantation from Matched Unrelated Donor (MUD).

Background: Cryopreservation of PBSC for allogenic hematopoietic stem cell transplantation (allo-HSCT) was implemented due to the current Coronavirus 2019 pandemic. The impact of match unrelated donor (MUD) graft freezing on the outcome of allo-HSCT in terms of hematological recovery, graft versus host disease (GVHD), and survival are still controversial. Methods: In this study, we compared graft composition, clinical characteristics, and outcome of 31 allo-HSCT from MUD cryopreserved PBSC (Cryo Group) with 23 matched-pair allo-HSCT from fresh MUD PBSC (Fresh Group) performed in our center between January 2020 and July 2021. Results: No significant differences were recognized in clinical characteristics of patients, donors, and transplants between the Cryo and Fresh groups except for a better prognostic comorbidity index (HCT-CI) of the Cryo group. In the Cryo Group, the median time from apheresis to cryopreservation was 46.0 h (range 23.8-53.5), while the median time from cells collection and reinfusion was 13.9 days (range 5.8-28.1). In the Fresh Group, median time from apheresis to reinfusion was 35.6 h (range 21.4-51.2). The number of viable (7-AAD negative) CD34+ cells per kg patient infused was significantly lower in the Cryo Group (5.2 ± 1.9 × 106/kg vs. 7.0 ± 1.3 × 106/kg; p < 0.001). Indeed, there was a 36% (11-70) median loss of viable CD34+/kg cells after freezing. All patients engrafted: median time to neutrophil engraftment (>0.5 × 109/L) was 13.5 days (range 12-15) for Cryo Group and 14 days (range 13-16) days for Fresh Group (p = 0.522), while the median time to platelet engraftment (>20 × 109/L) was, respectively, 14 (range 12-18) and 15 (range 12-17) days (p = 0.904). The incidence of grade ≥ 2 acute GVHD was similar in the two groups (56.5% Cryo Group vs. 60.0% Fresh Group; p = 0.832) and no differences in terms of OS (p = 0.090), PFS (p = 0.200) and TRM (p = 0.970) were observed between the Cryo and Fresh groups. Conclusions: In our series, no differences between the Cryo and Fresh groups were found in engraftment, grade ≥ 2 acute GVHD incidence, OS, PFS, and TRM despite a lower CD34+ infused dose in the Cryo Group. Frozen PBSCs could be considered a safe option also for allo-HSCT from MUD but a higher amount of PBSC should be collected to warrant an adequate viable CD34+ post-thawing.

The long and winding road: homeostatic and disordered haematopoietic microenvironmental niches: a narrative review.

Haematopoietic microenvironmental niches have been described as the 'gatekeepers' for the blood and immune systems. These niches change during ontogeny, with the bone marrow becoming the predominant site of haematopoiesis in post-natal life under steady state conditions. To determine the structure and function of different haematopoietic microenvironmental niches, it is essential to clearly define specific haematopoietic stem and progenitor cell subsets during ontogeny and to understand their temporal appearance and anatomical positioning. A variety of haematopoietic and non-haematopoietic cells contribute to haematopoietic stem and progenitor cell niches. The latter is reported to include endothelial cells and mesenchymal stromal cells (MSCs), skeletal stem cells and/or C-X-C motif chemokine ligand 12-abundant-reticular cell populations, which form crucial components of these microenvironments under homeostatic conditions. Dysregulation or deterioration of such cells contributes to significant clinical disorders and diseases worldwide and is associated with the ageing process. A critical appraisal of these issues and of the roles of MSC/C-X-C motif chemokine ligand 12-abundant-reticular cells and the more recently identified skeletal stem cell subsets in bone marrow haematopoietic niche function under homeostatic conditions and during ageing will form the basis of this research review. In the context of haematopoiesis, clinical translation will deal with lessons learned from the vast experience garnered from the development and use of MSC therapies to treat graft versus host disease in the context of allogeneic haematopoietic transplants, the recent application of these MSC therapies to treating emerging and severe coronavirus disease 2019 (COVID-19) infections, and, given that skeletal stem cell ageing is one proposed driver for haematopoietic ageing, the potential contributions of these stem cells to haematopoiesis in healthy bone marrow and the benefits and challenges of using this knowledge for rejuvenating the age-compromised bone marrow haematopoietic niches and restoring haematopoiesis.

Allogeneic hematopoietic stem cell transplantation: lessons learned by a molecular immunologist/transplant patient.

Much has been learned about the genes and pathways that contribute to a diverse array of hematopoietic malignancies and other hematopoietic diseases. However, for many of these diseases, an allogeneic hematopoietic stem cell (HSC) transplant remains the preferred treatment option. This opinion article provides the perspective of a molecular immunologist who became a transplant patient after many years studying basic mechanisms of blood cell development. Among many lessons learned were the magnitude of racial and ethnic disparities in donor registries, the substantial improvement in outcomes over time that were due to the collective impact of numerous advances, the benefits and limitations of genetic and clinical data, and the remarkably intricate balance between promoting graft-versus-disease activity of donor cells while suppressing graft-versus-host disease (GVHD).

The Role of Fecal Microbiota Transplantation in the Treatment of Acute Graft-versus-Host Disease.

The number of allogeneic hematopoietic stem cell transplantations conducted worldwide is constantly rising. Together with that, the absolute number of complications after the procedure is increasing, with graft-versus-host disease (GvHD) being one of the most common. The standard treatment is steroid administration, but only 40-60% of patients will respond to the therapy and some others will be steroid-dependent. There is still no consensus regarding the best second-line option, but fecal microbiota transplantation (FMT) has shown encouraging preliminary and first clinically relevant results in recent years and seems to offer great hope for patients. The reason for treatment of steroid-resistant acute GvHD using this method derives from studies showing the significant immunomodulatory role played by the intestinal microbiota in the pathogenesis of GvHD. Depletion of commensal microbes is accountable for aggravation of the disease and is associated with decreased overall survival. In this review, we present the pathogenesis of GvHD, with special focus on the special role of the gut microbiota and its crosstalk with immune cells. Moreover, we show the results of studies and case reports to date regarding the use of FMT in the treatment of steroid-resistant acute GvHD.

New Applications of JAK/STAT Inhibitors in Pediatrics: Current Use of Ruxolitinib.

Janus kinases (JAK) are a family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that transduce cytokine-mediated signals through the JAK-STAT metabolic pathway. These kinases act by regulating the transcription of specific genes capable of inducing biological responses in several immune cell subsets. Inhibition of Janus kinases interferes with the JAK-STAT signaling pathway. Besides being used in the treatment of cancer and inflammatory diseases, in recent years, they have also been used to treat inflammatory conditions, such as graft-versus-host disease (GVHD) and cytokine release syndrome as complications of allogeneic hematopoietic stem cell transplantation and cell therapy. Recently, the FDA approved the use of ruxolitinib, a JAK1/2 inhibitor, in the treatment of acute steroid-refractory GVHD (SR-aGVHD), highlighting the role of JAK inhibition in this immune deregulation. Ruxolitinib was initially used to treat myelofibrosis and true polycythemia in a high-dose treatment and caused hematological toxicity. Since a lower dosage often could not be effective, the use of ruxolitinib was suspended. Subsequently, ruxolitinib was evaluated in adult patients with SR-aGVHD and was found to achieve a rapid and effective response. In addition, its early low-dose use in pediatric patients affected by GVHD has proved effective, safe, and reasonably preventive. The review aims to describe the potential properties of ruxolitinib to identify new therapeutic strategies.

Mesenchymal Stromal Cells for Enhancing Hematopoietic Engraftment and Treatment of Graft-Versus-Host Disease, Hemorrhages and Acute Respiratory Distress Syndrome.

Mesenchymal stromal cells (MSCs) possess profound immunomodulatory and regenerative properties that are of clinical use in numerous clinical indications with unmet medical need. Common sources of MSCs include among others, bone marrow (BM), fat, umbilical cord, and placenta-derived decidua stromal cells (DSCs). We here summarize our more than 20-years of scientific experience in the clinical use of MSCs and DSCs in different clinical settings. BM-MSCs were first explored to enhance the engraftment of autografts in hematopoietic cell transplantation (HCT) and osteogenesis imperfecta around 30 years ago. In 2004, our group reported the first anti-inflammatory use of BM-MSCs in a child with grade IV acute graft-versus-host disease (GvHD). Subsequent studies have shown that MSCs appear to be more effective in acute than chronic GvHD. Today BM-MSC-therapy is registered for acute GvHD in Japan and for GvHD in children in Canada and New Zeeland. MSCs first home to the lung following intravenous injection and exert strong local and systemic immunomodulatory effects on the host immune system. Thus, they were studied for ameliorating the cytokine storm in acute respiratory distress syndrome (ARDS). Both, MSCs and DSCs were used to treat SARS-CoV-2 coronavirus-induced disease 2019 (COVID-19)-induced ARDS. In addition, they were also used for other novel indications, such as pneumomediastinum, colon perforation, and radiculomyelopathy. MSC and DSCs trigger coagulation and were thus explored to stop hemorrhages. DSCs appear to be more effective for acute GvHD, ARDS, and hemorrhages, but randomized studies are needed to prove superiority. Stromal cell infusion is safe, well tolerated, and only gives rise to a slight fever in a limited number of patients, but no major side effects have been reported in multiple safety studies and metaanalysis. In this review we summarize current evidence from in vitro studies, animal models, and importantly our clinical experience, to support stromal cell therapy in multiple clinical indications. This encloses MSC's effects on the immune system, coagulation, and their safety and efficacy, which are discussed in relation to prominent clinical trials within the field.

Addition of a Single Low Dose of Anti T-Lymphocyte Globulin to Post-Transplant Cyclophosphamide after Allogeneic Hematopoietic Stem Cell Transplant: A Pilot Study.

Correlation between risk of graft-versus-host disease (GvHD) and CD3+ counts within the peripheral blood stem cell graft has recently been reported in the setting of post-transplant cyclophosphamide (PT-Cy). We aimed to investigate the benefit of the addition of a single dose of anti-T lymphocyte globulin (ATLG 5 mg/kg) to PT-Cy in this setting. Starting in 2019, all patients receiving PBSC transplant containing CD3+ counts above 300 × 106/kg (study group) received a post-transplant dose of ATLG in addition to standard PT-Cy. The study was designed as a real-life analysis and included all consecutive Hematopoietic Stem Cell Transplantation (HSCT) recipients according to the above-mentioned inclusion criterion (n = 21), excluding cord blood and bone marrow donors. Using a 1:2 matched-pair analysis, we compared the outcomes with a historical population who received PT-Cy only (control group). We found a delayed platelet engraftment (29% vs. 45% at 30 days, p = 0.03) and a non-significant trend toward higher risk of poor graft function (29% vs. 19%, p = 0.52). The addition of ATLG impacted long-term immune reconstitution on the CD4+ subsets, but this did not translate into higher rate of relapse or viral infection. Acute GvHD was not significantly impacted, but 1-year cumulative incidence of chronic GvHD was significantly lower in the study group (15% vs. 41%, p = 0.04). Survival outcomes were comparable. In conclusion PT-Cy and ATLG was overall safe and translated into a low rate of chronic GvHD incidence.

Bone Marrow versus Peripheral Blood Grafts for Haploidentical Hematopoietic Cell Transplantation with Post-Transplantation Cyclophosphamide.

In the coronavirus disease 19 (COVID-19) pandemic era, the number of haploidentical hematopoietic cell transplantations (HCTs) with peripheral blood (PB) grafts increased significantly compared with HCTs with bone marrow (BM) grafts, which may be associated with adverse outcomes. We compared outcomes of HCT in BM graft and PB graft recipients age ≥18 years with hematologic malignancies who underwent T cell- replete haploidentical HCT and received graft-versus-host disease (GVHD) prophylaxis with post-transplantation cyclophosphamide, tacrolimus, and mycophenolate mofetil. Among the 264 patients, 180 (68%) received a BM graft and 84 (32%) received a PB graft. The median patient age was 50 years in both groups. The majority (n = 199; 75%) received reduced-intensity conditioning. The rate of acute leukemia or myelodysplastic syndrome was higher in the BM graft recipients compared with the PB graft recipients (85% [n = 152] versus 55% [n = 46]; P < .01). The median times to neutrophil and platelet engraftment and the incidence of grade II-IV and grade III-IV acute GVHD (aGVHD) were comparable in the 2 groups. Among the patients with grade II-IV aGVHD, the rate of steroid-refractory aGVHD was 9% (95% confidence interval [CI], 5% to 18%) in the BM group versus 32% (95% CI, 19% to 54%) in the PB group (hazard ratio [HR], 3.7, 95% CI, 1.5 to 9.3; P = .006). At 1 year post-HCT, the rate of chronic GVHD (cGVHD) was 8% (95% CI, 4% to 13%) in the BM group versus 22% (95% CI, 14% to 36%) in the PB group (HR, 3.0; 95% CI, 1.4-6.6; P = .005), and the rate of systemic therapy-requiring cGVHD was 2.5% (95% CI, 1% to 7%) versus 14% (95% CI, 7% to 27%), respectively (HR, 5.6; 95% CI, 1.7 to 18; P = .004). The PB group had a significantly higher risk of bacterial and viral infections, with no appreciable advantage in the duration of hospitalization, immune reconstitution, relapse, nonrelapse mortality, or survival. Our data suggest a benefit of the use of BM grafts over PB grafts for haplo-HCT.

Mesenchymal Stromal Cell Secretome for the Treatment of Immune-Mediated Inflammatory Diseases: Latest Trends in Isolation, Content Optimization and Delivery Avenues.

Considering the high prevalence and the complex pharmacological management of immune-mediated inflammatory diseases (IMIDs), the search for new therapeutic approaches for their treatment is vital. Although the immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSCs) have been extensively studied as a potential therapy in this field, direct MSC implantation presents some limitations that could slow down the clinical translation. Since the beneficial effects of MSCs have been mainly attributed to their ability to secrete a plethora of bioactive factors, their secretome has been proposed as a new and promising pathway for the treatment of IMIDs. Formed from soluble factors and extracellular vesicles (EVs), the MSC-derived secretome has been proven to elicit immunomodulatory effects that control the inflammatory processes that occur in IMIDs. This article aims to review the available knowledge on the MSC secretome, evaluating the advances in this field in terms of its composition, production and application, as well as analyzing the pending challenges in the field. Moreover, the latest research involving secretome administration in IMIDs is discussed to provide an updated state-of-the-art for this field. Finally, novel secretome delivery alternatives are reviewed, paying special attention to hydrogel encapsulation as one of the most convenient and promising strategies.

The CoV-2 outbreak: how hematologists could help to fight Covid-19.

COVID-19 is a medical emergency, with 20 % of patients presenting with severe clinical manifestations. From the pathogenetic point of view, COVID-19 mimics two other well-known diseases characterized by cytokine storm and hyper-activation of the immune response, with consequent organ damage: acute graft-versus-host disease (aGVHD) and macrophage activation syndrome (MAS). Hematologists are confident with these situations requiring a prompt therapeutic approach for switching off the uncontrolled cytokine release; here, we discuss pros and cons of drugs that are already employed in hematology in the light of their possible application in COVID-19. The most promising drugs might be: Ruxolitinib, a JAK1/2 inhibitor, with a rapid and powerful anti-cytokine effect, tyrosine kinase inhibitors (TKIs), with their good anti-inflammatory properties, and perhaps the anti-Cd26 antibody Begelomab. We also present immunological data from gene expression experiments where TKIs resulted effective anti-inflammatory and pro-immune drugs. A possible combined treatment algorithm for COVID-19 is here proposed.

The Unique Immunomodulatory Properties of MSC-Derived Exosomes in Organ Transplantation.

Methods for suppressing the host immune system over the long term and improving transplantation tolerance remain a primary issue in organ transplantation. Cell therapy is an emerging therapeutic strategy for immunomodulation after transplantation. Mesenchymal stem cells (MSCs) are adult multipotent stem cells with wide differentiation potential and immunosuppressive properties, which are mostly used in regenerative medicine and immunomodulation. In addition, emerging research suggests that MSC-derived exosomes have the same therapeutic effects as MSCs in many diseases, while avoiding many of the risks associated with cell transplantation. Their unique immunomodulatory properties are particularly important in the immune system-overactive graft environment. In this paper, we review the effects of MSC-derived exosomes in the immune regulation mechanism after organ transplantation and graft-versus-host disease (GvHD) from various perspectives, including immunosuppression, influencing factors, anti-inflammatory properties, mediation of tissue repair and regeneration, and the induction of immune tolerance. At present, the great potential of MSC-derived exosomes in immunotherapy has attracted a great deal of attention. Furthermore, we discuss the latest insights on MSC-derived exosomes in organ transplantation and GvHD, especially its commercial production concepts, which aim to provide new strategies for improving the prognosis of organ transplantation patients.

Mesenchymal stem/stromal cells as a valuable source for the treatment of immune-mediated disorders.

Over recent years, mesenchymal stem/stromal cells (MSCs) and their potential biomedical applications have received much attention from the global scientific community in an increasing manner. Firstly, MSCs were successfully isolated from human bone marrow (BM), but in the next steps, they were also extracted from other sources, mostly from the umbilical cord (UC) and adipose tissue (AT). The International Society for Cellular Therapy (ISCT) has suggested minimum criteria to identify and characterize MSCs as follows: plastic adherence, surface expression of CD73, D90, CD105 in the lack of expression of CD14, CD34, CD45, and human leucocyte antigen-DR (HLA-DR), and also the capability to differentiate to multiple cell types including adipocyte, chondrocyte, or osteoblast in vitro depends on culture conditions. However, these distinct properties, including self-renewability, multipotency, and easy accessibility are just one side of the coin; another side is their huge secretome which is comprised of hundreds of mediators, cytokines, and signaling molecules and can effectively modulate the inflammatory responses and control the infiltration process that finally leads to a regulated tissue repair/healing or regeneration process. MSC-mediated immunomodulation is a direct result of a harmonic synergy of MSC-released signaling molecules (i.e., mediators, cytokines, and chemokines), the reaction of immune cells and other target cells to those molecules, and also feedback in the MSC-molecule-target cell axis. These features make MSCs a respectable and eligible therapeutic candidate to be evaluated in immune-mediated disorders, such as graft versus host diseases (GVHD), multiple sclerosis (MS), Crohn's disease (CD), and osteoarthritis (OA), and even in immune-dysregulating infectious diseases such as the novel coronavirus disease 2019 (COVID-19). This paper discussed the therapeutic applications of MSC secretome and its biomedical aspects related to immune-mediated conditions. Sources for MSC extraction, their migration and homing properties, therapeutic molecules released by MSCs, and the pathways and molecular mechanisms possibly involved in the exceptional immunoregulatory competence of MSCs were discussed. Besides, the novel discoveries and recent findings on immunomodulatory plasticity of MSCs, clinical applications, and the methods required for their use as an effective therapeutic option in patients with immune-mediated/immune-dysregulating diseases were highlighted.

Clinical course of severe COVID19 treated with tocilizumab and antivirals post-allogeneic stem cell transplant with extensive chronic GVHD.

Recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are an immunocompromised group who are likely to develop severe complications and mortality because of coronavirus disease 2019 (COVID-19). We report here a 61-year-old male patient of primary myelofibrosis who underwent an allo-HSCT 6 years earlier, had chronic graft-versus-host disease (cGVHD) involving the liver, lung, eyes, and skin, (with recurrent episodes of pulmonary infections) who developed severe COVID-19. The patient was treated with tocilizumab, and a combination of lopinavir/ritonavir, ribavirin, interferon-ß1b. He was discharged after 31 days with full recovery. Tocilizumab, a humanized monoclonal antibody against IL6, has been shown to benefit respiratory manifestations in severe COVID19. However, this is first report, to our knowledge, of its use and benefit in a post HSCT recipient.

Logistics of an advanced therapy medicinal product during COVID-19 pandemic in Italy: successful delivery of mesenchymal stromal cells in dry ice.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. METHODS: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. RESULTS: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. CONCLUSIONS: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

Current approaches used in treating COVID-19 from a molecular mechanisms and immune response perspective.

Coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared by the World Health Organization (WHO) as a global pandemic on March 11, 2020. SARS-CoV-2 targets the respiratory system, resulting in symptoms such as fever, headache, dry cough, dyspnea, and dizziness. These symptoms vary from person to person, ranging from mild to hypoxia with acute respiratory distress syndrome (ARDS) and sometimes death. Although not confirmed, phylogenetic analysis suggests that SARS-CoV-2 may have originated from bats; the intermediary facilitating its transfer from bats to humans is unknown. Owing to the rapid spread of infection and high number of deaths caused by SARS-CoV-2, most countries have enacted strict curfews and the practice of social distancing while awaiting the availability of effective U.S. Food and Drug Administration (FDA)-approved medications and/or vaccines. This review offers an overview of the various types of coronaviruses (CoVs), their targeted hosts and cellular receptors, a timeline of their emergence, and the roles of key elements of the immune system in fighting pathogen attacks, while focusing on SARS-CoV-2 and its genomic structure and pathogenesis. Furthermore, we review drugs targeting COVID-19 that are under investigation and in clinical trials, in addition to progress using mesenchymal stem cells to treat COVID-19. We conclude by reviewing the latest updates on COVID-19 vaccine development. Understanding the molecular mechanisms of how SARS-CoV-2 interacts with host cells and stimulates the immune response is extremely important, especially as scientists look for new strategies to guide their development of specific COVID-19 therapies and vaccines.