Concise Review: Next-Generation Cell Therapies to Prevent Infections in Neutropenic Patients

Bacterial and fungal infections are the leading cause of morbidity and mortality in neutropenic patients. Transfusing the patient with neutrophil-like cells manufactured in the laboratory from hematopoietic stem and progenitor cells enriched from umbilical cord blood can potentially prevent fulminant infections. The authors review the rationale for focusing research efforts toward ex vivo neutrophil production.

Abstract

High-dose chemotherapy is accompanied by an obligate period of neutropenia. Resulting bacterial and fungal infections are the leading cause of morbidity and mortality in neutropenic patients despite prophylactic antimicrobials and hematopoietic growth factor supplements. Replacing neutrophils in the patient through transfusion of donor cells is a logical solution to prevent fulminant infections. In the past, this strategy has been hampered by poor yield, inability to store collected cells, and possible donor morbidity caused by granulocyte colony-stimulating factor injections and apheresis. Today, neutrophil-like cells can be manufactured in the laboratory at the clinical scale from hematopoietic stem and progenitor cells enriched from umbilical cord blood. This article reviews the rationale for focusing research efforts toward ex vivo neutrophil production and explores clinical settings for future trials.

Preventing Infections Is Vital for Neutropenic Patients

Neutropenia is a disorder characterized by an abnormally low number of circulating blood neutrophils. Patients are considered neutropenic when their absolute neutrophil count (ANC) falls below 1,500 cells per microliter of peripheral blood. Subclassifications are reported based on the depth of neutropenia, which range from mild (1,000–1,500 cells per microliter) to moderate (500–1,000 per microliter) to severe (<500 cells per microliter) [1]. In their landmark study, Bodey et al. observed that the rate of infections increases with the depth of neutropenia, and the severity of infections correlates with duration of neutropenia [2]. About one third of severely neutropenic patients develop life-threatening infections within a week. Intensive chemotherapy schedules cause obligatory neutropenic periods and are associated with a higher risk of infections [3]. Therefore, patients treated for leukemias and myeloproliferative disorders are particularly at risk. Moreover, neutropenia frequently dictates dose reduction or treatment interruptions, thereby worsening outcomes, especially in pediatric and elderly patients [4–6].

Established infections threaten the lives of neutropenic patients and considerably burden the health care system. Early diagnosis of infection is vital but difficult because these patients cannot mount potent and specific inflammatory responses [7, 8]. Improvement of diagnostic tools has ameliorated patient prognosis, but 10% of admitted febrile neutropenic patients still die of infections [9]. Therefore, robust prophylactic strategies are crucial to prevent the onset of infections and related complications. Antibacterials such as fluoroquinolones were initially successful at preventing gram-negative bacterial infections. However, their systematic use progressively led to the emergence of resistant strains and frequent shifts between gram-negative and gram-positive pathogens [10]. In addition, the use of antibacterial agents eliminates niche competition for yeast and molds, which may enhance the risk for fungal infections [11]. Prophylactic antifungals reduce infection-related mortality, but the associated toxicity, emergence of resistant strains, and possible drug reactions render this option a solution for small patient cohorts only. Autopsy studies in multiple institutions reveal that the rate of fungal infections is increasing among patients treated for leukemias, with up to 78% positive cases. Within this group, a dramatic 30% of deaths can be attributed to fungal infections in spite of antifungal prophylactic use [12, 13]. Moreover, Viscoli et al. suggested a correlation between patients receiving antifungal prophylaxis and a higher risk of bacteremia [14]. The available literature demonstrates that the success of antibiotics in prophylactic, empiric, or pre-emptive strategies depends on an educated guess of the most probable pathogen for a particular neutropenic patient. This practice can cause toxicity and resistance, with associated negative impact for the patient and the broader community.

Although antibiotics reduce morbidity by preventing the spread of infecting pathogens, the prevailing determinant for the onset of infection remains blood ANC. Neutrophils being short-lived cells, the maintenance of a healthy blood ANC relies on constant neutrophil production from hematopoietic stem and progenitor cells (HSPCs). Chemotherapy-induced niche exhaustion is the underlying cause for neutropenia. A strategy to dampen the myelosuppressive effect of chemotherapy is to maintain HSPCs in a quiescent state transiently during the anticancer treatment. The crucial role of HSPC binding to E-selectins on the epithelium is exemplified by a significant decrease in proliferation and self-renewal of HSPCs in E-selectin knockout mice [15]. Injections of the E-selectin antagonist GMO-1017 during chemotherapy or irradiation protect the HSPC pool from exhaustion, which significantly accelerates the recovery of ANC in wild-type mice [15]. Similarly, Lucas et al. demonstrated that protecting adrenergic nerves during chemotherapy by administration of 4-methylcatechol or glial-derived neurotrophic factor maintained the HSPC niche, which significantly improved hematopoietic recovery [16]. Although these results are promising, the current procedure is to facilitate neutrophil recovery from the remaining HSPC pool to restore patient immunity. Granulocyte colony-stimulating factor (G-CSF) promotes neutrophil production and release from the bone marrow. In patients in whom myeloablation is incomplete, support with G-CSF achieves significant reduction in neutropenic days and related complications [17, 18]. In contrast, for patients receiving high-dose chemotherapy that damages the HSPC niche, such as for treating acute myeloid leukemia (AML), restoring protective blood ANC takes too long to significantly affect infection-related morbidity and mortality despite G-CSF support [19, 20]. Furthermore, although G-CSF may shorten the neutropenic window, complete abrogation cannot be achieved as neutrophil production takes several days. A constant pursuit for alternatives or complementing factors to G-CSF has been motivated by these pitfalls. Among these agents, recent efforts have proposed prostaglandin E2 (PGE2). Administration of PGE2 agonists, such as dimethyl PGE2, increases the stem cell pool in zebrafish and mice [21]. A recent study showed that injections of dimethyl PGE2 to irradiated mice enhanced hematopoietic recovery and survival [22]. These results propose that, similar to G-CSF, administration of PGE2 agonists to neutropenic patients may boost healthy ANC recovery. However, the effect of PGE2 is biphasic as continued exposure eventually inhibits expansion of the progenitor pool [23]. Therefore, clinical trials are expected in the future to confirm the role of early PGE2 injections in neutropenic patients and to contrast with results currently obtained with G-CSF. Meanwhile, despite some benefits provided by combinations of G-CSF and antimicrobial prophylactics, neutropenic infections remain the leading cause of chemotherapy-associated death.

In contrast to G-CSF support, neutrophil transfusions can potentially restore protective ANC immediately. Similar to red blood cell and platelet transfusions used to treat anemia and thrombocytopenia, respectively, neutrophils can be collected from healthy donors for transfusion into neutropenic patients. Neutrophils are harvested using density sedimentation during apheresis. Although neutrophils usually make up >70% of collected cells, the product also contains the other granulocytes, eosinophils and basophils. Hence, neutrophil transfusions have been commonly named granulocyte transfusions (GTx) [24]. The first GTx trials were conducted in the early 1960s, but the low cell yields possible at the time barely impacted host ANC upon transfusion. Transfusions of granulocytes harvested from chronic myelogenous leukemia patients did provide proof of concept as recipients showing blood ANC increment demonstrated rapid clinical improvement post-transfusion [25]. G-CSF administration to healthy donors has enabled collection of clinically meaningful neutrophil numbers and several small trials have suggested some degree of efficacy. Additional large-scale trials of therapeutic GTx have been proposed in light of the current knowledge of neutrophil biology and transfusion [26].

Even if efficacy is demonstrated in large-scale trials, significant logistic and donor safety issues remain. Neutrophils have a short half-life; therefore, GTx is usually performed daily or every second day during the neutropenic period. Neutrophils do not store well and must be infused within 24 hours of collection. Finally, whereas G-CSF is usually well tolerated in healthy donors, it can cause bone pain, flu-like symptoms, and spleen enlargement that in rare cases leads to spleen rupture [27, 28]. There are also long-term safety concerns for G-CSF-mobilized donors [29]. Finally, steroids such as prednisone and dexamethasone, frequently used in combination with G-CSF to mobilize donor neutrophils, may increase the risk of developing subcapsular cataracts later in life [30, 31]. Accordingly, there is a clear need for alternative cell products.

Stem Cell-Derived Therapies to Treat Neutropenia: Rationale and Options

In order to mitigate neutropenia, possible strategies are to regenerate the hematopoietic niche or to immediately replace functional cells in the blood (Fig. 1). Autologous transplantation of mobilized peripheral blood (mPB) HSPCs coupled with cytokine support promotes engraftment and blood cell recovery [32]. Clinically significant delays to protective blood ANC recovery are common and have prompted efforts to expand HSPCs ex vivo prior to transfusion [33]. The development of the Amgen-defined serum-free medium containing a key cocktail of the three cytokines stem cell factor (SCF), thrombopoietin (TPO), and G-CSF eased large-scale ex vivo expansion of HSPCs, with bias toward differentiation into a neutrophil phenotype [34]. Transfusion of ex vivo-expanded HSPCs (eHSPCs) to chemotherapy-treated patients caused significantly fewer neutropenic days, febrile episodes, and shorter hospital stays [35, 36]. Furthermore, the reduction in time to engraftment corresponded to the transfused dose, reinforcing the need for better ex vivo expansion protocols [36, 37]. Subsequent studies have assessed a wide range of parameters for increasing eHSPC yields, including different culture vessels, seeding densities, feeding schedules, oxygen tensions, addition of small molecules to culture medium, and genetic manipulations of developmental factors, all of which have been extensively discussed in recent reviews of the literature [38–40].

Figure 1.

Simplified representation of granulopoiesis. (A): Location of cell types in the human adult. (B): The known successive maturation phenotypes and the corresponding composition of existing cell products proposed to mitigate neutropenia. Abbreviations: CMP, common myeloid progenitors; eHSPC, ex vivo-expanded hematopoietic stem and progenitor cell; HSPC, hematopoietic stem and progenitor cell; eNeut, ex vivo-manufactured neutrophils.

A fundamentally distinct approach promotes HSPC expansion by preventing intercellular communication and ensuing repressive feedback. Csaszar et al. [41] and Kirouac et al. [42] have modeled and validated the hypothesis that secreted factor-mediated intercellular communication regulates the fate of HSPCs. Preventing autologous feedback through a dilution strategy caused an 11-fold expansion of umbilical cord blood (UCB)-enriched HSPCs without impacting self-renewal or differentiation potential [41]. A remaining concern is the concomitant mobilization of tumor cells during autologous HSPC collection that has caused relapse in transfused patients [43, 44]. Therefore, this strategy may be restricted to nonhematopoietic cancer treatments only, although some studies suggest that purging the graft from malignant cells using chemotherapy is possible [45]. When autologous transplantations are not advised, patients may turn to allogeneic options.

Allogeneic mPB HSPC transfusions are an alternative if human leukocyte antigen (HLA)-matched donors are available. Successful neutrophil engraftment relies on further expansion of eHSPCs in vivo; therefore, the product cannot be irradiated before transfusion (Fig. 1). Hence, a significant concern with allogeneic transfusions is the risk of developing acute and chronic graft-versus-host-disease (GVHD) due to engrafting lymphoid cells. Cellerant Therapeutics, Inc. (San Carlos, CA, www.cellerant.com) has demonstrated that the protocol for producing the off-the-shelf cell therapy CLT-008 overcomes this problem [46]. mPB HSPC are expanded in a myeloid-driving, defined medium consisting of X-VIVO 15 (Lonza, Basel, Switzerland, www.lonza.com) supplemented with SCF, Fms-related tyrosine kinase 3 ligand, interleukin-3, and TPO. Elimination of lymphoid cells is achieved passively, alongside expansion of myeloid progenitors (Fig. 1). Over 8 days of culture, this process averages a 40-fold expansion. The CLT-008 product is cryopreserved and it is suggested that HLA matching is not required, facilitating access to patients. At the time of writing, Cellerant Therapeutics, Inc. is recruiting patients to investigate the safety and efficacy of CLT-008 to abrogate neutropenia in chemotherapy-treated patients [47]. Despite clear improvements over the current therapies, a major issue with the CLT-008 strategy remains the source of stem cells. Cellerant Therapeutics, Inc. uses mobilized healthy donors to source a sufficient number of HSPCs. Furthermore, the average fold expansion of the myeloid compartment is similar to that obtained by Paquette et al. a decade ago [36, 37]; therefore, several mobilized blood donations may be required per patient. UCB has been used as a source of HSPCs for transplantations for the past 25 years and is a promising alternative to using mPB [48]. Historically, UCB HSPCs have been transfused in patients for whom autologous transplants are not advised and matched donors are unavailable. Advantages of UCB as a source of HSPCs include the ease of procurement and decreased incidence of GVHD; however, delays in engraftment remain an issue. Ex vivo expansion protocols have been developed to transfuse a larger number of cells, and downstream transplantations in vivo have been performed successfully [49, 50]. In specific settings, UCB eHSPCs have yielded better expansion than mPB eHSPCs [51].

The delay to engraftment of transfused eHSPCs can be clinically significant. A natural extension to the eHSPC protocol consists of expanding and differentiating a fully mature neutrophil product from HSPCs. Our group has developed a protocol to differentiate UCB HSPCs toward a neutrophil-like phenotype [52]. Over 15 days of culture, an average 5,800-fold expansion of UCB HSPCs is reached. Accounting for the mean CD34⁺ HSPC yield from a single donation of cord blood (2–5 × 10⁶ CD34⁺ cells), this expansion is estimated sufficient for a single prophylactic dose (10¹⁰ cells per day) [53]. However, during the culture process, the entire HSPC pool inevitably enters differentiation, so that the ex vivo-manufactured neutrophils (eNeut) yield is currently finite. These eNeut can be produced under good manufacturing practice conditions at the clinical scale. The mature product is composed in its majority of postmitotic neutrophils that exhibit bactericidal functions in vitro. eNeut are poorly immunogenic as assessed by the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination test (GAT), endorsing the product for allogeneic use. eNeut transfusions should cause immediate increments in ANC, as observed post-GTx with donor neutrophils (Fig. 2) [54]. Interestingly, the culture process used in this work merges a dilution feeding strategy to the key culture medium protocol to enable the highest yield of eNeut to date from expanded UCB-enriched HSPCs. Again, a major challenge is to produce large quantities of cells. Current studies are attempting clever combinations of culture protocols to further enhance eNeut yields [55].

Figure 2.

Venn diagrams of the current main advantages and disadvantages of existing cell products proposed to mitigate neutropenia. Abbreviations: ANC, absolute neutrophil count; CMP, common myeloid progenitors; eHSPC, ex vivo-expanded hematopoietic stem and progenitor cell; eNeut, ex vivo-manufactured neutrophils; HLA, human leukocyte antigen; HNA, human neutrophil antigen; mPB, mobilized peripheral blood; UCB, umbilical cord blood.

Approval of eNeut by regulatory bodies will be crucial for implementation in the clinic. The U.S. Food and Drug Administration (FDA) is evolving its regulatory processes on regenerative medicine products, with a handful of ex vivo-generated cellular therapies recently approved for the market. These therapies include autologous cultured cells like chondrocytes (Carticel) and allogeneic solutions like Gintuit, a product containing keratinocytes and fibroblasts cultured in bovine collagen. FDA approval for biologic licensing has doubled in the last 2 years, which suggests the imminent infiltration of cellular therapies in the clinic [56]. Nonetheless, emerging therapies must be fully supported by clinical data before licensure, especially in the booming field of ex vivo-cultured hematopoietic cells. Although the clinical potential of eNeut is promising, controlled randomized trials remain to be implemented (Fig. 2).

A major determinant holding preliminary human trials is the high cost of manufacture for cell therapies (Fig. 2). For instance, a unit of platelet pool, which is considered a blood product by the FDA, costs ∼450 Euros to the receiving hospital [57]. By comparison, Provenge (sipuleucel-T), an autologous dendritic cell therapy for prostate cancer, is sold by its manufacturer for U.S. $93,000 per patient [58]. The preparation of a platelet unit involves donor apheresis, leukocyte depletion, resuspension in a platelet additive solution, storage, and irradiation prior to infusion. This process evokes the culture protocol for eNeut, although the differentiation part of the protocol may suggest that eNeut be considered under the “cell therapy” scheme. Although the regulatory format framing eNeut production will dramatically impact product cost, other factors may contribute to lowering manufacture expenses, such as automation and implementation of closed systems, discussed in detail elsewhere [38]. A direct comparison of the cost of eNeut treatment to the mean patient cost of neutropenia-associated hospitalization, estimated at U.S. $19,100 per episode of febrile neutropenia [9], may not be sufficient as cost-benefit analysis should also consider patients’ reduced morbidity and increased survival.

Lessons Learned From GTx and Their Relevance for eNeut Production and Clinical Use

Lesson 1: Keeping the Transfusion Recipient Safe

In transfusion medicine, part of the “non-self” nature of the transfused product is dealt with through lymphoreduction and irradiation to prevent GVHD. A remaining source of concern is alloimmunization, which occurs when antibodies in the transfusion recipient target their cognate antigen present on the recipient cells. Consequently, the infused product survives poorly in the recipient who becomes refractory to future transfusions [59, 60]. Alloimmunization against human leukocyte antigens usually causes transplant rejection, and if the involved antibodies target human neutrophil antigens (HNAs), the transfused neutrophils may become activated, pool in the lungs, and exhibit limited chemotaxis to infection sites [61, 62]. In addition to limiting transfusion efficacy, alloimmunization can initiate a cascade of events leading to life-threatening complications such as transfusion-related acute lung injury (TRALI) [62]. Lastly, transfusion of incompatible neutrophils causes delayed HSPC engraftment [63]. These issues prompt HLA and HNA typing prior to granulocyte collections, further burdening the health care system and restricting potential donor availability [64]. On the other hand, mature eNeut cultures have been tested by GIFT and GAT techniques using a positive serum pool. Mild or complete absence of reaction confirmed that eNeut are not immunogenic and therefore may present a lower risk of transfusion-associated complications compared with donor neutrophils [52]. Also, eNeut are generated in a chemically defined medium, devoid of animal products, potential pathogens, or blood contaminants responsible for adverse reactions. These precautions further suggest that eNeut may be a safe donor granulocyte alternative for transfusions (Fig. 2), which may not require frequent, time-consuming, and expensive immunotyping and serotyping.

Despite these measures, eNeut remain allogeneic in nature. γ irradiation of blood products is an obligatory practice to prevent engraftment and GVHD. γ-Irradiated myeloid progenitor cells display dose-dependent viability over 6 days, which suggests arrest of expansion of mitotic cells [65]. Although further studies are necessary to assess the effects of the 25-Gy irradiation dose required for blood product transfusions, these data demonstrate that irradiation does not prevent final maturation of progenitor cells. This feature is attractive if one considers that current neutrophil differentiation protocols do not use synchronized cells. It will be interesting to see whether irradiated eNeut transfusions confer longer lasting protection than donor GTx through post-transfusion late-phase maturation in vivo. In addition, if nonsynchronized HSPCs are used to initiate eNeut cultures, the mature cells will appear in waves. As a result, the product phenotype can be characterized as soon as the first mature eNeut are available. This feature is important to avoid delays in patient transfusions given that typing assays can be time consuming [66].

Lesson 2: Timing of GTx Is Key to Benefit Recipients

To date, no randomized controlled trial has clearly demonstrated the benefit of GTx. However, most clinical studies set to assess GTx have been performed in the context of febrile or septic neutropenic patients for whom the pathogen replication has not been contained by innate immunity. Arguably, choosing the setting of an established infection, it is inappropriate to expect improvement using GTx as there is a large discrepancy between the dose of neutrophils needed to abrogate the infection and the dose of neutrophils actually transfused. In a healthy individual, 10¹¹ neutrophils are released every day from the bone marrow to maintain homeostasis in tissues and steady blood ANC, and the bone marrow reserve contains approximately 6 times more neutrophils [67]. Following a microbial insult, neutrophils are mobilized from the bone marrow reserve, which creates a dramatic increment in blood ANC [68]. Apheresis of G-CSF-mobilized neutrophils typically yields only 4–8 × 10¹⁰ neutrophils, leaving donors in a state of transient neutropenia [54, 69]. It is unreasonable to expect that a lower-than-homeostatic neutrophil dose should be able to treat an established infection. Therefore, the apparent lack of benefit of GTx may be attributable to trials in inadequate settings rather than fundamental misconceptions of GTx antimicrobial potential.

A more favorable situation is prophylactic GTx in at-risk populations, such as AML patients who receive aggressive myeloablative chemotherapy. The proposed objective of prophylactic GTx would be to maintain peripheral immunological surveyance. This suggestion is supported by successful prevention of infection relapse in patients receiving GTx as secondary prophylaxis [70]. In addition, a landmark meta-analysis of GTx prophylaxis trials indicates that a transfusion dose of as few as 10¹⁰ neutrophils is sufficient to improve prognosis [53].

Lesson 3: Patient Outcome Depends on the Phenotype of Transfused Granulocytes

The immunological phenotype of donor neutrophils is likely to change during harvest and ex vivo procedures, which may have a dramatic impact on transfusion efficacy. Cell purification processes can inadvertently damage or activate neutrophils. For instance, neutrophil enrichment using filtration leukapheresis leads to neutrophil phenotype modification including activation. Furthermore, enriched concentrates may contain apoptotic neutrophils and other damage-associated molecular patterns, which cause activation of neutrophils. Activated neutrophils exhibit an altered phenotype of apoptosis, decreased deformity potential, and spontaneous degranulation, all of which promote tissue damage in the transfused host and may participate in the initiation of TRALI [43]. Determining factors for activation are diverse and include the type of anticoagulant used, temperature and length of storage period, and contamination by other blood products. Mobilization agents also have an impact on the phenotype of collected neutrophils: collection using G-CSF plus dexamethasone increased CD11b and CD18 surface receptors, which suggests cell activation. [71]. G-CSF-mobilized neutrophils are larger than homeostatic blood neutrophils, have a different surface molecule phenotype, and some reports suggest reduced efficacy in vivo.

Similar to G-CSF-elicited neutrophils, eNeut display a larger surface area compared with unstimulated donor neutrophils. However, eNeut are maintained under consistent conditions throughout their production, which may limit prospects for activation. Furthermore, mature eNeut can be harvested quickly and directly prior to transfusion, while the lengthy apheresis process and associated temperature variations may contribute to donor neutrophil activation. Current expansion strategies used for eNeut production grant the equivalent of at least one protective dose from a single UCB donation, eliminating the need for invasive procedures on donors. Defined durations of cultures to a mature product promise definite availability of protective neutrophil doses at a particular time without relying on donors. Because standard protocols lead to a uniform eNeut phenotype, transfusing patients using eNeut standardizes the transfused product by eliminating the naturally occurring variability within donor neutrophil phenotype and subpopulations. Therefore, from a research perspective, transfusing eNeut instead of donor neutrophils may emphasize correlations between patient conditions and GTx success without being confounded by inconsistencies in donor neutrophil characteristics and numbers. Furthermore, using a single homogenous product devoid of contaminants, such as antibodies and serum proteins, reduces donor exposure and possibly the rate of complications (Fig. 2).

Conclusion

Despite obvious advantages of eNeut over donor neutrophils, critical milestones must be met before clinical implementation can be suggested. Quality control guidelines, automation of bioreactors, and costs consideration are examples of current optimization concerns, discussed recently elsewhere [38]. The ultimate optimization strategy for eNeut production would be conditional immortalization of UCB HSPCs. In addition to solving the yield issue, this outcome would standardize the manufactured product into a well-characterized, off-the-shelf solution for neutropenic patients. Although neutrophil-like cell lines are currently available, they originally derive from human leukemic samples, which may hamper safe clinical use. Lin et al. have shown that HL-60-differentiated neutrophil-like cells, named ATAK, improve overall outcome in a chemotherapy-treated neutropenic mouse model challenged with Candida albicans and Aspergillus fumigatus [72]. Their effort to engineer a “suicide trap” within these cells points toward the significant safety concern of transplanting immortalized cells into patients. Conditional immortalization of primary cells through targeted genetic manipulation may therefore be preferred. Wang et al. have demonstrated unlimited production of neutrophils through conditional regulation of Hoxb8 in HSPCs from mouse bone marrow [73]. Further studies are warranted in human cells, however, as similar manipulations of HSPCs may lead to different outcomes in different species [74]. Regardless of the used strategy, the risk of tumorigenesis post-transplant must be carefully examined before implementing a human trial. We feel that assessments of phenotypic stability and long-term survival of transplanted cells must also be performed for nonimmortalized but in vitro-manipulated eHSPCs.

It is relevant to consider the extreme condition of common candidates for GTx. Chemotherapies damage the gut mucosa dramatically, facilitating dissemination of microbes. Therefore, transfused neutrophils must be highly mobile, respond to the finest chemotactic signals, and must be able to kill microbes efficiently. Hence, it may be appropriate to investigate prospects of producing more competent eNeut by culture manipulation. Li et al. showed that SF1670 enhances neutrophil function and GTx efficacy in vivo [75]. Similarly, addition of the retinoid agonist Am80 to eNeut cultures gives rise to neutrophils with enhanced bactericidal abilities compared with G-CSF-derived neutrophils [76]. We recommend extreme caution in the exploration of this field, however, as there is a fine balance between functional superiority and promotion of inflammation that might culminate in patient tissue damage.

Successful studies of GTx highlight specific patient subpopulations and treatment context. Exploration of this niche is critical in the emerging era of personalized medicine to confirm and reveal new degrees of responses. Using eNeut instead of donor-mobilized neutrophils to facilitate future GTx studies is motivated by their practical and physiological advantages including relatively weak immunogenicity. Using a more generic and readily available eNeut product may eliminate issues associated with donor safety and accessible dose of neutrophils. Finally, research in the field of eNeut production and transfusion should attract maximal allocation of resources as the rate of cancers and associated neutropenic disorders is increasing dramatically with general population aging. Therefore, we foresee an increase in the neutropenic population requiring neutrophil support.

Author Contributions

M.E.G.B.: conception and design, manuscript writing; L.K.N.: conception and design, manuscript writing, financial support.

Disclosure of Potential Conflicts of Interest

L.K.N. is an inventor of a patent involving eNeut (US patent 8173427).