GM-CSF is crucial for mitochondrial maintenance in mononuclear phagocytes, as modeled in murine HIV studies (77). Furthermore, gene knockout animals reveal that GM-CSF influences mitochondrial turnover, function, and fatty acid β-oxidation (78). GM-CSF increases mitochondrial tricarboxylic acid cycle activity, oxidative phosphorylation, ATP production, and regulation of key metabolic pathways, such as glycolysis, pentose phosphate pathway, and amino acid synthesis. Magmas (mitochondria-associated granulocyte-macrophage CSF signaling molecule) is rapidly induced in vitro when murine myeloid-cell-line PGMD1 cells in culture are switched from IL-3 to GM-CSF in the medium (79). Also known as Tim16 in mammals, Pam16 in yeast, and Blp in drosophilia, Magmas is conserved across species, essential for cell growth, and anti-apoptotic when over-expressed (80–83). Magmas gene knockout mice die as embryos. RNAi-mediated knockdown of Blp resulted in mitochondrial membrane depolarization, 60% decreased ATP levels, 3.5-fold higher reactive oxygen species (ROS), cell-cycle arrest, autophagy activation, and 65% reduced cytochrome c oxidase activity in the mitochondrial electron transport chain (84). Magmas additionally functions as a ROS sensor and regulator, leading to reduced cellular ROS production (85).

In addition to other effects that induce phagocyte populations (12, 86–90), GM-CSF supports efferocytosis, an energy intensive process in which macrophages engulf and digest apoptotic cells, such as short-lived tissue-infiltrating neutrophils, thereby preventing release or accumulation of necrotic inflammatory material (13). Decreased efferocytosis is associated with tissue necrosis and autoimmune disease (91). Opsonizing milk fat globule epidermal growth factor 8 (MFG–E8) bridges the “eat me” signal of phosphatidylserine displayed on apoptotic cell membranes with integrins αVβ5 and potentially αVβ3 on efferocytotic phagocytes (92). GM-CSF is required for expression of MFG-E8 by efferocytotic antigen-presenting cells (APCs) (93, 94) and induces integrins αVβ3 and αVβ5 (95, 96).

In addition, growth arrest-specific protein 6 (GAS6) bridges MerTK receptors on efferocytotic phagocytes to phosphatidylserine on the apoptotic cell external plasma membrane (91). Murine GM-CSF-induced bone marrow-derived macrophages express MerTK receptors and exhibit high phagocytic ability (97). Human macrophages differentiated with GM-CSF from healthy adult monocyte samples, and which were cultured either under growth/serum factor deficiency, or with subsequent treatment of IL-4 or incubation with apoptotic cells, result in M2-polarized macrophages that exhibit increased MerTK expression (98). Finally, activated peroxisome proliferator-activated receptor (PPAR)-γ and liver X receptor (LXR)-α drive anti-inflammatory macrophage engulfment of apoptotic cells (91). GM-CSF induces expression of LXRα in human blood mononuclear cells (99) and induces PPAR-γ expression in multiple myeloid cell types (100, 101).

GM-CSF broadly affects neutrophil biology via neutrophil induction (2), in particular by enhancing pro-survival effects (102). Oddly and reflective of cytokine pleiotropy, GM-CSF also facilitates auto-phagocyte-like neutrophil cell death (103). Also, GM-CSF downregulates chemotaxis via loss of signaling in response to Interleukin-8 (IL-8), the primary neutrophil chemotactic factor, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP) chemotaxis is maintained (104). Finally, GM-CSF down-regulates neutrophil IL-8 receptor expression (105). In summary, GM-CSF might either facilitate or inhibit neutrophil chemotaxis depending on local environmental influences.

Separately, GM-CSF prevents blood neutrophil extravasation into tissues. L-Selectin mediates neutrophil trans-endothelial migration and is rapidly shed after activation and during the rolling phase of extravasation (106). ADAM17 (a disintegrin and metalloproteinase 17), also known as tumor necrosis factor-alpha-converting enzyme (TACE), is the principal “sheddase” that cleaves surface L-Selectin. Interestingly, ADAM17 sheddase activity acts on neutrophils but not monocytes. Consistent with its stimulation of ADAM17 expression, GM-CSF induces rapid, complete loss of L-Selectin, also known as leukocyte adhesion molecule-1 (LAM-1), from neutrophils, monocytes, and marrow cells but not lymphocytes (107).

Although its receptors are not expressed on lymphocytes, GM-CSF indirectly induces regulatory T cells (Tregs) in multiple autoimmune and chronic inflammatory disease models (108, 109). GM-CSF-deficient APCs exposed to MFG-E8-opsonized apoptotic cells produce altered cytokine profiles, resulting in decreased Tregs and increased inflammatory Th1 cells (93). GM-CSF also induces myeloid-derived suppressor cells (MDSCs) that suppress pro-inflammatory cytokine production, inhibit T cell proliferation, mediate chemotaxis, and activate Tregs (110–112).

In summary, GM-CSF influences a myriad of primarily myeloid cells, in part due to maturation and maintenance of metabolic capacity both systemically and locally, although the specific concentration that drives this change is yet to be determined (113). Follow-on effects include enhancement of phagocytosis and efferocytosis, as well as modulation of other immune cells including neutrophils and Tregs. Together, these data suggest that therapeutic rhu GM-CSF (e.g., sargramostim) might generate benefit in diseases characterized by mononuclear phagocyte dysfunction or dysregulation.

High titers of neutralizing GM-CSF autoantibodies in aPAP lead to multiple effects that drive pathophysiology of this disease. These effects include reduced alveolar macrophage cholesterol clearance, impaired surfactant homeostasis, dysfunctional immune defense, and in a subset of patients, pulmonary fibrosis and end stage lung disease (6, 7, 114, 115). As reviewed above in Emerging biology of GM-CSF, GM-CSF deficiency impairs the expression of PPAR-γ, a key cholesterol regulator, leading to surfactant lipid accumulation within foamy alveolar macrophages (116, 117). GM-CSF autoantibodies also diminish neutrophil phagocytic antimicrobial functions and may lessen alveolar epithelial cell-derived GM-CSF activation and recruitment of alveolar macrophages, dendritic cells, and T cells (118). Reduced immune cell signaling and impaired gas exchange from surfactant accumulation contribute to increased incidence (13-25%) of opportunistic infections from organisms including Aspergillus, Cryptococcus, Nocardia, or atypical mycobacteria (7, 115, 119, 120). Because aPAP is a very rare disorder, the true prevalence of infection in this patient population and its associated mortality remain unclear.

Fibrosis, an uncommon but severe complication of aPAP, probably results from multiple mechanisms (114). Type II alveolar epithelial cells produce GM-CSF that aids in alveolar epithelial cell repair, leading to epithelial proliferation and barrier restoration (11, 118). In the presence of neutralizing GM-CSF autoantibodies, these homeostatic processes are impaired (6). Also, absence of GM-CSF results in lipid composition changes within the alveolar space that may lead to reduced synthesis of antifibrotic prostaglandin PGE2, which may enhance fibrogenesis, especially in the presence of additional insults (121, 122). The relationship between GM-CSF deficiency and fibrogenesis has been studied in murine models to date; human studies are needed yet are challenging in this rare disease (122). With the progression of pulmonary fibrosis, patients may develop severe, irreversible lung dysfunction for which the only known effective treatment is lung transplantation.

Inhaled rhu GM-CSF is a potentially disease-modifying therapy with promising applications in aPAP, a mononuclear phagocyte disease. The inhaled route of administration delivers high drug concentrations directly to the disease site in the lung (123, 124). Clinical studies of inhaled rhu GM-CSF in aPAP have focused on achieving disease control or slowing or preventing disease progression. Trial endpoints have included measures of lung gas exchange, in particular diffusing capacity of the lungs for carbon monoxide (DLCO), exercise capacity, symptoms, and health-related quality of life. Inhaled rhu GM-CSF has been reported in clinical trials to improve clinical outcomes (42, 125). Table 2 summarizes phase 2-3 studies of sargramostim and molgramostim that report benefit in achieving disease control for patients with aPAP. The adverse event data are reported in Supplement Table S1 .

Trapnell et al. and Tazawa et al. reported benefits utilizing inhaled molgramostim and sargramostim, respectively, compared to placebo in aPAP patients (38, 39). Campo et al. reported that sargramostim combined with whole lung lavage (WLL; the current standard of care for therapy in aPAP) was safe and more effective than WLL alone (40, 41). Also, in a case series of 5 patients with aPAP, Ohkouchi et al. (126) reported that inhaled sargramostim given after WLL significantly improved disease severity score parameters. These parameters included biomarkers such as mucin-like glycoprotein KL-6, carcinoembryonic antigen (CEA), and lactate dehydrogenase (LDH), as well as markers of oxygenation including alveolar-arterial oxygen gradient (A-aDO₂) and partial pressure of oxygen (PaO₂). Sargramostim given only before WLL did not improve these parameters. Although optimal dosing and duration of therapy have yet to be established, results of current ongoing research and real-world evidence are eagerly awaited (127). A disease-modifying therapy for aPAP is a high unmet need to slow disease progression, reduce infectious complications, and prevent pulmonary fibrosis and death.

Of the three rhu GM-CSF formulations described in Introduction (sargramostim, molgramostim, and regramostim), sargramostim is the only form that is currently commercially available. Sargramostim can be sourced from the United States and obtained globally through a “named patient program” per each nation’s healthcare governing body (128). Based on data mentioned here and additional case reports (126), sargramostim use may decrease healthcare utilization in this rare lung disease population. In a study evaluating 15 million people in the US from 2008 to 2012, patients with PAP were determined to have more outpatient visits (17.30 ± 13.77 vs 10.40 ± 11.38; p < 0.01), more emergency room visits (1.49 ± 1.17 vs 1.08 ± 0.27; p = 0.014), and longer hospital stays (15.96 ± 20.71 vs 5.40 ± 5.07 inpatient days; p = 0.027), compared with non-PAP controls (129). Annual per-patient healthcare costs were also 5-fold higher (approximately $40,000 more annually) for PAP patients than for non-PAP controls. Increased costs were attributed to disease-related treatments, including prescriptions, hospitalizations, and outpatient visits. In another retrospective cohort study of 500 U.S. patients admitted with a primary diagnosis of PAP between 2012 and 2014, mean actual cost per admission was $29,932 (CI: 13,739-46,124) with an overall annual cost burden of approximately $5 million (130).

Timely, accurate aPAP diagnosis also remains an issue due to disease rarity, low physician awareness, and limited access to the blood test for serum GM-CSF antibodies, which is performed in few centers worldwide. Testing centers include the CAP/CLIA certified lab at Cincinnati Children’s Hospital, National Jewish Health, the National Institutes of Health, and Cincinnati Children’s Hospital Pulmonary Alveolar Proteinosis Clinical Research Lab (131–133). Additional testing centers can be found in Japan, Germany, and China. Similar to other rare diseases, a patient advocacy organization has emerged (www.papfoundation.org) with the goal to unite the patient community and to connect patients with the specialist physician community for access to appropriate testing and relevant clinical trials.

Sargramostim is not approved by the FDA for use in aPAP, which limits access, reimbursement, and manufacturer ability to provide label information on safe and effective use in this setting. Also, broader sargramostim use in aPAP is limited by the absence of aPAP clinical consensus guidelines. Important questions to be addressed include impact and timing of treatment for asymptomatic or mildly symptomatic patients, as well as ideal dose and treatment duration for those with more severe disease. An ongoing international, multi-center, placebo-controlled trial of molgramostim will provide more information on dosing, efficacy, and safety data for the rhu GM-CSF agent class (127). Other aPAP treatment agents that upregulate PPAR-γ (e.g., thiazolidinediones) or lower cholesterol (e.g., statins) show preclinical promise and could deploy additional repurposed therapeutics available with known safety (134–136). Answers to these clinical questions and more are critical to patients and providers and will hopefully be elucidated via continued investigation of the potential of sargramostim and other therapies to modulate disease and prevent infection and fibrosis.

Mononuclear phagocyte dysfunction due to GM-CSF insufficiency can contribute to disease (e.g., sepsis) precipitated by various events including trauma, major surgery, and hematopoietic cell transplant (HCT) (137, 138). Viral, bacterial, or fungal opportunistic infections all can cause sepsis and life-threatening organ dysfunction (139, 140). The focus of this section is viral respiratory pathogens and the delicate balance between an effective host response to eliminate respiratory viral infections versus an inadequate or even an overactive immune response to sepsis. The subset of patients who experience these inadequate or overactive immune responses may suffer serious or even fatal adverse events (8, 141, 142).

Insights garnered across dysfunctional mononuclear phagocyte disease states such as COVID-19, pneumonia, sepsis, and intensive care unit (ICU)-related critical illness may potentially be applied to many types of infections. Immunomodulatory agents (e.g., sargramostim) that orchestrate the immune system and behaviors of immune cells for optimal host immune response to different pathogens may be beneficial in improving outcomes for many patients (58, 61, 62, 64, 65). While the hematopoietic growth factor rhu G-CSF (discussed in the Introduction) is more widely prescribed and comprises more than 95% of recombinant growth factor use, its use in infection has not demonstrated a mortality benefit in pneumonia when used in combination with antibiotic therapy (1, 4, 5, 143). G-CSF recruits and increases the number of neutrophils, whereas GM-CSF orchestrates the behavior of many innate and adaptive immune cells to combat pathogens while avoiding an overwhelming systemic response (4, 5). Viral infections and sepsis can be viewed as examples of mononuclear phagocyte dysfunction sequelae and will serve as models for further investigation of pathology, immune responses, and novel treatment strategies across patient populations to decrease morbidity and mortality.

Immunomodulatory agents that boost host immune function against different pathogens may be beneficial to many patients by improving oxygenation, preventing ARDS, and reversing immunoparalysis. When given via various routes (inhalation, intravenous, and subcutaneous), rhu GM-CSF has been reported to improve outcomes for patients who are critically ill, immunocompromised, and suffering from respiratory infection ( Table 3 ) (58–65, 67). The adverse event data are reported in Supplement Table S1 . Optimal rhu GM-CSF dosing, route of administration, and duration of therapy, however, have yet to be established. Several authors report sargramostim therapy benefited patients with these conditions. The studies, however, were small, used varying routes of rhu GM-CSF administration, and included infections from multiple or unknown pathogens; hence further investigations are needed.

There is enthusiasm for developing innovative therapies to improve patient outcomes after respiratory viral infections, ARDS, and sepsis-induced immunoparalysis. Beyond antimicrobial therapy, strategies include enhancing host defense by either replacing deficient cells (such as neutrophils in cases of cancer/chemotherapy-induced neutropenia) or potentially providing specially activated immune/inflammatory cells, akin to CAR-T cell therapy (179–181). Such treatments require enormous resources and in many instances (such as white blood cell transfusion for infection) have not shown clear benefit (182, 183). In contrast, sargramostim may safely target and modulate specific cells and cellular behavior to achieve an effective and efficient immune response (58, 59, 61, 62, 64, 65). Sargramostim’s actions on alveolar macrophages, dendritic cells, and T cells may allow for complementary immune response changes versus increases in cell numbers as seen with other agents such as rhu G-CSF ( Figure 1 ) (5, 11, 12, 161, 162). Other novel products, such as hematopoietc stem cell-derived ex vivo-expanded myeloid progenitor cells and phenotypically typed functional granulocytes, have been shown to be efficacious in animal models (184, 185).

Biomarkers to predict and measure treatment effects in immunoparalysis, SIRS, CARS, and ARDS are urgently needed. Current surrogate biomarkers of inflammation (C-reactive protein [CRP], procalcitonin [PCT], ferritin) are used in practice, but further investigations of more sensitive and specific biomarkers are warranted (8). Definitions for immunoparalysis vary, for example monocyte HLA-DR levels less than 8,000 mAb/cell or TNF-α response assay to ex vivo stimulation results and should be standardized (64, 65). Standardized biomarkers would help stratify patients to better anticipate those at increased infection risk and hence should receive antimicrobial prophylaxis or treatment. Prospective immunophenotyping and/or patient stratification based on blood cell counts, immune function assays, cytokine levels, GM-CSF auto-antibody levels [e.g., in Cryptococcus gattii cryptococcosis (186)], or other markers of inflammation would help define optimal timing of drug administration (e.g., sargramostim) to prevent organ failure and death (9). Vulnerable patient populations such as immunocompromised HCT recipients, are at higher risk for all types of infection and should be a continued focus of future studies (138, 157).

Trials in pediatric sepsis [NCT03769844 (187), NCT05266001 (188)] aim to better understand the potential of sargramostim in modulating the immune system to enhance the pulmonary host defense capacity to eliminate pathogens, maintain alveolar homeostasis, and prevent disease progression. These trials will add to the existing evidence for sargramostim including attenuation of epithelial cell injury, epithelial repair, and improved barrier function and gas exchange in ARDS (59).

As the world continues to endure the COVID-19 pandemic, many trials investigating variant-independent treatment options are ongoing. Using single RNA-sequencing of MAFB and MAF transcription factors, 3 main lung macrophage populations expressing associated markers have been identified: FCN1 (ficolin-1; macrophages derived from circulating monocytes), SPP1 (secreted phosphoprotein 1; macrophage origin unknown), and FABP4 (fatty acid binding protein 4, an intracellular lipid chaperone and adipokine; found in GM-CSF-dependent alveolar macrophages) (118). These macrophage population ratios have shown to be altered in association with COVID-19 severity. Analysis of bronchoalveolar lavage fluid from patients infected with COVID-19 showed increased FCN1^high and SPP1^high macrophages and decreased FABP4^high macrophages correlated with disease progression (189). Altering the ratios of these lung macrophage populations towards an increased proportion of the FABP4^high macrophage subset, may promote pathogen clearance and epithelial repair while limiting an overly inflammatory response seen at the later stages of COVID-19 and other respiratory viral infections (189). Potential therapeutic intervention targeting the macrophage-activating upstream Jun N-terminal kinases (JNK) via MAPK inhibitors may alter macrophage population ratios, hence making these subsets both potential biomarkers and biopredictors (190).

The rapid SARS-CoV-2 genetic modifications created new variants that circumvented vaccine efforts and made it challenging to keep up with therapeutic targets due to changing resistance patterns. A trial in outpatients with COVID-19 is investigating using sargramostim to rebalance lung homeostasis to prevent disease progression to severe COVID-19 [NCT04707664 (191)]. Influenza shares many characteristics with SARS-CoV-2 as both pathogens invade and damage alveolar epithelial cells and have circumvented annual vaccine efforts, previously to an epidemic proportion. Although anticipated every year, seasonal influenza still causes significant morbidity and mortality, especially in high-risk HCT recipients (192). Prior to the COVID-19 pandemic, one study attributed influenza (seasonal A and B) to 30% of all respiratory viral infections in this population with up to 35% progressing to lower respiratory tract infections (193). In addition to HCT recipients, patients with other high risk factors (the very young, nursing home residence, chronic lung or heart disease, history of smoking) (194) contribute to the unmet need for further investigations to build upon the preclinical and clinical insights from sargramostim studies in COVID-19, pneumonia-associated lung injury, ARDS, and sepsis. Targeting these patient populations in future studies based on disease etiology, disease severity, and ideally key disease pathways per the individual patient will hone effective and efficient disease treatments and minimize side effects.

Spontaneous acute wound healing in a normal host involves complex immune system interactions over time to restore the skin barrier after injury (17). Wound healing is comprised of four sequential and overlapping phases including hemostasis, inflammation, growth, and re-epithelization ( Figure 2 ). Due to stresses both internal (e.g., aging, genetics, nutrition, chronic disease) and external (e.g., bacteria, medications), normal wound healing may be delayed or arrested at any stage (195). With concomitant diseases, such as diabetes, pathophysiologically inherent disease factors might further impair immune response, worsen peripheral arterial disease, and generate repetitive trauma due to neuropathic desensitization, all of which halt normal wound healing and contribute to development of chronic, non-healing wounds (i.e., diabetic foot ulcer) (16, 17). Diabetic foot ulcers typically stall at the inflammation phase, partly attributable to accumulation of advanced glycation end products (AGEs) (196). Presence of AGEs leads to increased oxidative stress and inflammation, stiffer skin, and reduced innate immune cell adhesion. Also, inhibition of immune-cell-signaling p38/MAPK pathway results in decreased damaged cell removal and reduced primary skin cell (keratinocyte) migration (197).

Within hours of a spontaneous skin injury in an intact host, blood vessels constrict, and platelets form a fibrinogenic plug to stop bleeding to start the hemostasis phase (17). Local neutrophils and macrophages extravasate to the injury to defend against invading bacteria with cell recruitment increasing over 2 to 3 days (198, 199). Injured cells and mast cells release cytokines and other bioactive molecules to attract leukocytes, including Langerhans cells, dendritic cells, T cells, neutrophils, and monocytes (17, 195). Keratinocytes release endogenous GM-CSF that promotes local myeloid proliferation and supports additional inflammatory signaling (200, 201). A day after injury, neutrophils constitute half of all cells in the wound (17). In human diabetic foot ulcers, impairment in GM-CSF activation of signal transducer and activator of transcription 3 (STAT3) expression results in decreased immune cell recruitment (202). In a murine diabetes model, proinflammatory cytokines, such as interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and GM-CSF, were reduced in wounds, compared to wounds in non-diabetic mice (15). Reduced signaling led to reduced neutrophil and macrophage recruitment and delaying healing. Mouse diabetic wound healing was almost completely restored by 2 weeks with perilesional exogenous rhu GM-CSF injected intradermally. In non-diabetic mice, exogenous rhu GM-CSF did not enhance wound healing.

In the inflammatory phase (3-20 days duration), immune cell recruitment continues, leading to pathogen, debris, and necrotic tissue removal (14). With an impaired wound healing environment, i.e. pressure ulcers in the elderly, decreased GM-CSF signaling leads to lower expression of nucleotide-binding domain-like receptor protein 3 (NLRP3) and reduced neutrophil interleukin-1 beta (IL-1β), resulting in impaired innate immune responses (203–205). In a normal host, GM-CSF signaling facilitates recruited monocyte differentiation into various immune response cells, including macrophages (206). Macrophages are often considered the most important immune cells in wound healing (19). They recognize and engulf pathogens, as well as eliminate expended neutrophils within 3 to 4 days of skin injury by efferocytosis (17). Decreased macrophage infiltration from impaired GM-CSF signaling in diabetes reduces neutrophil clearance, causing additional tissue damage from lysed neutrophils that prolongs the inflammatory phase. As mentioned in Emerging biology of GM-CSF, GM-CSF also induces PPAR-γ expression which is key to transitioning macrophages from a pro-inflammatory M1 phenotype to a pro-healing M2 phenotype (18). Impaired macrophage PPAR-γ activity and elevated M1 macrophages in diabetic wounds generate negative downstream effects. Decreased growth factor release, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), reduces granulation tissue formation and prolongs inflammation both ultimately resulting in delayed wound closure (18, 207).

Within days to weeks from injury in a normal host, the growth phase begins in which granulation tissue formation and neovascularization occur (17, 208). Pro-healing M2 macrophages deposit extracellular matrix components, induce myofibroblasts, and phagocytose excess cells/matrix (17, 18). VEGF released by GM-CSF-stimulated macrophages acts as a key growth factor in early angiogenesis to promote micro-vessel sprouting (17, 209). VEGF and PDGF promote proliferation of keratinocytes, fibroblasts, and epithelial cells to create granulation tissue (207). GM-CSF also promotes maturation and stabilization of newly developed micro-vessels (capillaries) to establish new tissue blood supply (15). In diabetic foot ulcers, the necessary M2 macrophage population is decreased due to a dysregulated M1 to M2 phenotype shift, thereby delaying granulation and blood vessel formation (18).

In weeks to months, normal host re-epithelization occurs, and the wound closes (208). Normal connective tissue replaces granulation tissue while epidermal stem cells give rise to epidermal layers, hair follicles, and glands (17). GM-CSF-dependent M2 macrophages induce fibroblast differentiation into contractile myofibroblasts for wound closure (17, 19, 20, 210). Conversely, for patients with diabetes, fewer M2 macrophages result in decreased fibroblast differentiation and slower wound closure.

Clinical studies to date of rhu GM-CSF (e.g., sargramostim or molgramostim) used diverse dosages, dosage forms, and durations of study therapy in small trials of diverse wound etiologies. Clinical studies of rhu GM-CSF in wound healing are summarized in Table 4 . The adverse event data are reported in Supplement Table S1 . Routes of administration have included both perilesional and topical, wound size and duration have varied considerably, and the affected subjects have varying degrees of immunocompromise. Hence, larger, randomized controlled trials that provide guidance on dose, route, frequency, and duration of therapy for definitive wound closure, specific to diabetes and other current etiologies, are needed to further confirm benefit of rhu GM-CSF.

Other conditions have also been the subject of wound healing investigations. Case reports describe improved chronic wound healing with rhu GM-CSF treatment of patients with leukocyte or vascular dysfunction disorders, including glycogen storage disease (211), chronic granulomatous disease (211), common variable immunodeficiency (212), Klippel-Trénaunay-Weber syndrome (213), and cutaneous polyarteritis nodosa (214). Important issues to be addressed include optimal dose, route, schedule, and therapy duration within each wound etiology.

Deficiency of autocrine and paracrine GM-CSF activity in every stage of chronic wound healing illustrates a vital role for this cytokine. In a young, healthy host, endogenous GM-CSF is necessary for immune cell recruitment and maturation, phenotype shifts, keratinocyte proliferation, and angiogenesis. Successful wound repair and regeneration may well rely on timely activity of GM-CSF within healing wounds, especially its impact on macrophages, fibroblasts, and endothelial cells in rebuilding skin architecture.

Additional biomarkers to inform wound healing are needed. Vatankhah and colleagues (215) reported correlation of blood neutrophil-to-lymphocyte ratio with likelihood of diabetic foot ulcer nonhealing. Sawaya and associates (202) demonstrated that the diabetic foot ulcer immune cell landscape featured diminished GM-CSF activity with high and low proportions of monocytes and macrophages, respectively, indicating successful monocyte recruitment but deficient activation. Similar to blood monocytes, dermal stem cells of patients with diabetes mellitus also express low human leukocyte antigen-DR (HLA-DR), the receptor responsible for antigen presentation to CD4+ helper T lymphocytes and initiation of adaptive immune responses (216). As sargramostim is known to restore HLA-DR expression in post-surgery patients and sepsis-associated immunosuppression (as mentioned in Immune responses to infections and risk of GM-CSF insufficiency (65, 217), this receptor might become a clinically useful wound healing biomarker.

It soon may be possible to improve wound prevention of diverse etiologies, importantly including diabetic foot ulcers. Noninvasive Terahertz screening of diabetic foot skin dehydration estimates the amount of water that evaporates through skin to the external environment (218). Alternatively, thermometry (219) and point of care ultrasound imaging (220) are proposed to quantify risk of wound development. Topical barrier gels often serve as prophylaxis for potential post-spinal-cord injury pressure ulcers (221, 222). As mentioned in Emerging biology of GM-CSF, GM-CSF also may have protective neural cell effects (76, 223), hence a sargramostim gel could be a potentially interesting prophylactic approach.

Patients with chronic non-healing wounds have high morbidity and mortality, require multimodal healing treatment, and present an unmet need for novel therapies to improve outcomes (224). In the future, approaching all chronic wounds as a disease state, instead of as an underlying component of disease or the aging process, would establish a fresh viewpoint for management and prevention. Additionally, it is important to include elderly patients in future clinical trials as they are often excluded, as evidenced in cancer clinical trials (225, 226). Sargramostim’s immunotherapeutic potential should be further studied in at-risk patients and address wound healing outcomes that optimize ulcer-free, hospital-free, and activity-rich days. Sargramostim’s preventative therapeutic potential should be further studied, especially in combination with improved noninterventional diagnostic practices.

In a healthy immune system, immune checkpoint receptors dampen immune responses to prevent prolonged T cell activation and autoimmunity (227). Immune checkpoint inhibitors (ICIs) block checkpoint-originated immune suppression both in the tumor, to enable a desired antitumor T cell response, and potentially in healthy organ systems such as the gastrointestinal (GI) tract and lungs, causing unwanted tissue damage. Immune-related toxicity can occur at any time during treatment, is a frequent cause of ICI discontinuation, and may persist after cessation of therapy (228).The GI and pulmonary organ systems provide dual immune protection via physical barriers and cell-mediated immune responses. Immune-mediated colitis and checkpoint-induced pneumonitis can resemble infectious or spontaneous autoimmune disorders, and may have a delayed clinical presentation creating a diagnostic challenge for clinicians (228).

Immunotherapy aims to mobilize the immune system and restore antitumor immunity that is actively suppressed either by tumors cells themselves or by other immune cells in the tumor microenvironment (21). In an intact immune system, immune checkpoint receptors like CTLA-4, LAG-3, and PD-1 inhibit T cell activity at different steps of the immune response (229, 230). These receptors assist in limiting and preventing autoimmunity that could occur with unencumbered activated T cells. Blocking these receptors with ICIs inhibits inactivation, allowing the activated immune system to overcome cancer escape mechanisms and eliminate tumor cells (230–232).

Current research in cancer immunotherapy includes the activities of endogenous cytokines, such as GM-CSF, and their potential to influence an antitumor immune response (21). While sargramostim has been used for 30 years for other purposes, recent research has focused on its immunomodulatory properties as discussed in the preceding sections (1). Intralesional rhu GM-CSF therapy increased tumor-infiltrating CD4⁺ T cell numbers and localized tumoricidal macrophages (233). CD4⁺ T cells play a major role in CD8⁺ T cell-mediated responses. An expanded macrophage response may lead to increased tumor-cell phagocytosis, antigen presentation and T-cell stimulation. Proposed mechanisms for increased survival in clinical studies with sargramostim include increased cytotoxic CD8⁺ T cell and dendritic cell recruitment to the tumor and sentinel lymph node, respectively (22–24). In addition, GM-CSF stimulation of increased metabolic capacity of mononuclear phagocytes may counteract the immunosuppressive potential of tumor associated myeloid cells (25). Interactions among these cells may lead to enhanced antitumor T cell priming and activation via increased dendritic cell tumor-associated antigen presentation ( Figure 3 ).

ICI and sargramostim therapy link blockade of immune inhibitor mechanisms with increased immune cell activation which might boost antitumor responses in solid tumors (29). Correspondingly, in prostate cancer, sargramostim and ipilimumab combination therapy augmented tumor-reactive cytotoxic circulating CD8⁺ T cell responses (54). Furthermore, in melanoma, sargramostim and ipilimumab combination therapy increased overall survival (OS) and enhanced expression of inducible T-cell co-stimulator (ICOS) on CD4⁺ and CD8⁺ T cells over ipilimumab alone (28). Hence, addition of sargramostim appears to promote synergistic activation of key adaptive immune cells in antitumor immune responses.

Until recently, the mechanisms of immune-mediated colitis were unclear. Biopsies from patients with colitis, in contrast to control patients, revealed a large population of proliferating, cytotoxic CD8⁺ T cells (234). Further analyses of inflamed tissue revealed that ICI colitis is characterized by expanded populations of IFN-γ- and granzyme B⁺-producing, cytotoxic CD8⁺ T cells, and to a lesser extent—expanded populations of Th1 skewed CD4⁺ T cells, and inflammatory macrophages (235–237). Based on clonal rearrangement analysis used to identify T cells and their progeny, these cytotoxic CD8⁺ T cells were determined to be derived from colon-resident memory cells, likely held in check by CLTA-4 and/or PD-1 receptors (238). These tissue-resident memory T cells are likely reactivated due to CLTA-4 and/or PD-1 blockade (237). It is hypothesized that reactivated tissue-resident memory T cells proliferate, become cytotoxic, and release IFN-γ. IFN-γ then may signal myeloid cells to amplify the inflammatory response and recruit T cells from the circulation, overwhelming Treg-mediated immunosuppression to damage colon tissue and impair barrier integrity.

In animal models, antitumor action of CTLA-4 blocking antibodies occurs via Treg depletion, cells that express high CTLA-4 levels (237). Human immune-mediated colitis biopsies indicate CTLA-4 Treg depletion is likely not a major mechanism of action of anti-CTLA-4 antibodies as the Treg population was preserved or expanded in post-CLTA-4 blockade biopsies.

Based on immune cells present in organs at homeostasis, the lungs share the same functional predictors of inflammatory toxicities as the GI tract (26). Both GI tract and lungs contain epithelial barriers colonized with microbiota, as well as a large population of tissue-resident memory T cells. Therefore, PD-1 pneumonitis may share mechanisms seen in colitis, including upregulation of inflammatory immune alveolar cells (TNF-α, IFN-γ, CD8+ cytotoxic T cells) and downregulation of important suppressor regulatory cells such as Tregs and alveolar macrophages that express PD-1 (27). Expression of CLTA-4 and PD-1 differs among different tissue cells and immune cells, which might explain why adverse event prevalence varies for immune checkpoint inhibitors between GI tract and lungs (229). Importantly, the addition of sargramostim to CTLA-4 blockade with ipilimumab reduced the incidence of GI and pulmonary adverse events, compared to ipilimumab alone in a phase 2 study of patients with metastatic melanoma (28).

Clinical combination studies of ICIs and sargramostim have shown benefit in metastatic melanoma and prostate cancer (only melanoma studies detailed here) (28, 29, 54–56, 240). The adverse event data are reported in Supplement Table S1 . There were 3 author-reported grade 5 adverse events (death) in these clinical trials. All studies are of sargramostim in combination with ICI therapy.

In a phase 2 study, Hodi et al. (28) reported longer survival (17.5 months vs 12.7 months, p=0.01) and reduced grade 3-5 adverse events (45% vs 58%, p=0.04), for patients with unresectable stage III or IV melanoma treated with ipilimumab 10mg/kg plus sargramostim 250 μg subcutaneously days 1 to 14 of 21-day cycles (n=123) vs ipilimumab alone (n=122). Efficacy results were similar in two much smaller, non-randomized ipilimumab/sargramostim melanoma treatment studies wherein most patients had poor prognostic characteristics such as brain and liver metastases (55, 56). Patients with brain metastases are often excluded from clinical trials, however, they were included in the following studies which reported combination treatment benefit. A single institution, retrospective analysis (N=32) examined combination ipilimumab 3 mg/kg and sargramostim 250 μg/day for 14 days in 21-day cycles for 4 cycles (55). These authors reported overall disease control ≥ 12 weeks in 50% by response evaluation criteria in solid tumors (RECIST) and 44% by immune-related response criteria. Median OS was 41 weeks with an overall incidence of immune-related adverse events of 31.3% with 9.4% grade 3-4 events. A separate prospective, single-arm phase 2 trial (N=22) examined ipilimumab 10 mg/kg with sargramostim 125 μg/m²/day for 14 days in 21-day cycles, followed by sargramostim alone, then maintenance therapy of ipilimumab with sargramostim every 3 months for up to 2 years (56). Authors reported disease control in 41% of patients at 24 weeks. Median OS was 21.1 months, and grade 3-4 adverse events occurred in 41% of patients.

The ongoing ECOG-ACRIN EA6141 phase 2-3 trial is evaluating the combination of ipilimumab, nivolumab and sargramostim, compared with ipilimumab and nivolumab, in patients with unresectable stage III and stage IV melanoma. The trial advanced to the phase 3 portion after meeting prespecified efficacy and safety thresholds in the lead-in phase 2 portion (57). Trials continue to demonstrate the benefit of combination immunotherapy in melanoma and other diseases. However, this effect comes at a high toxicity cost for patients, making this exploration of triplet therapy incredibly important to the oncology field.

In addition to efficacy, sargramostim can act as an immune modulator to potentially attenuate or avoid immune-related adverse events ( Figure 3 ). Randomized controlled trials primarily focusing on immune-related adverse event prevention and/or treatment are lacking. In patients with dysregulated immunity of the GI tract or lung (Crohn’s disease or aPAP), sargramostim has been shown to be more effective than placebo at inducing disease control (39, 241). In the phase 2 trial in melanoma discussed above, Hodi et al. (28) reported combination sargramostim and ipilimumab therapy led to fewer GI and pulmonary toxicities and improved survival compared to ipilimumab alone. Treatment paradigms have shifted since completion of this study and ipilimumab doses less than 10mg/kg are now used due to treatment-limiting toxicity of higher ipilimumab doses; thus, additional studies are warranted to verify these findings with the current treatment regimens.

Guidelines are evolving to include more combination ICI therapy options. Investigators for the phase 3 CheckMate 067 trial recently reported 6.5-year OS data for patients with stage III/IV melanoma (242). 49% of patients treated with nivolumab/ipilimumab (n=314) versus 42% with nivolumab alone (n=316), or 23% with ipilimumab alone (n=315) achieved a durable OS response. Duration of response for study patients at 6.5 years for nivolumab/ipilimumab dramatically exceeded that of single agent use of ipilimumab or nivolumab (61.9-NR months vs 8.8,-47.4 months vs 45.7-NR, respectively). However, patients assigned to the nivolumab/ipilimumab arm experienced higher rates of autoimmune toxicities than patients treated with the single agent immunotherapy agents. The ongoing study EA6141 will assess if the addition of sargramostim to the nivolumab/ipilimumab combination corroborates and improves results obtained from CheckMate 067 (57). The study will assess the triplet combination of sargramostim/nivolumab/ipilimumab also in the alleviation of toxicities which may make therapy more tolerable and greatly expand the patient population able to benefit from this therapy.

EA6141 and CheckMate 067 exclude melanoma patients with poor prognostic factors like active or untreated brain metastases (57, 242). CheckMate 204 focused on the effects of nivolumab/ipilimumab combination therapy on melanoma brain metastases (243). Follow-up CheckMate 204 report noted a 3-year OS of 71.9% for asymptomatic (n=101) and 36% for symptomatic (n=18) patients (244). An important clinical question to answer for patients with poor prognostic factors would be the impact of adding sargramostim to combination ICI therapy (i.e., nivolumab/ipilimumab) on patient outcomes and rates of severe toxicity.

Investigations are underway to identify biomarkers with predictive utility for benefit from sargramostim. One strategy examines blood monocyte HLA-DR (human leukocyte antigen-DR isotype, a humanized major histocompatibility complex [MHC] II) expression on CD14⁺ immunosuppressive peripheral blood mononuclear cells, also known as MDSCs (25). This cell population is continuously renewed and reflects functional capacity of these cells, including their ability to phagocytose, to digest and present tumor antigens to T lymphocytes, as well as to support those lymphocytes with cytokines. Low HLA-DR expression has been proposed to be a surrogate marker of immunoparalysis in multiple clinical conditions, including sepsis where it is associated with worse patient outcomes (245). Equally, elevated monocytes with low HLA-DR expression have been correlated with worse outcomes for patients with melanoma treated with ipilimumab, among other cancers and therapies (246). In a number of different disorders as mentioned in Wound healing and risk of GM-CSF deficiency and Immune responses to infection and risk of GM-CSF insufficiency, sargramostim therapy increases HLA-DR expression and reverses immunoparalysis (65, 217) justifying further investigation in the context of cancer immunotherapy.

Additional trials are needed to evaluate immune-related adverse event risk factor prediction and prevention/reduction strategies, as well as the impact of immune-related adverse event management on ICI efficacy. Lozano et al. (247) reported, regardless of organ system, increased circulating activated CD4⁺ memory T cell numbers and increased T cell receptor diversity in melanoma correlate with an increased risk for severe immune-related adverse event development. Uncovering mechanisms responsible for organ-specific immune-related adverse events may enable expanded use of immunomodulatory agents in other disease states (e.g., GM-CSF autoantibodies in aPAP or Crohn’s disease) (125, 248). Future study designs might incorporate serial tissue biopsies to map toxicity development, identify potential biomarkers, and define the immune cell signaling involved in checkpoint-induced pneumonitis and colitis. These research goals could be incorporated into randomized controlled trials that measure immune-related adverse events with immunomodulatory agents like sargramostim.

Supported by Hodi et al. (28), the anti-cancer potential of sargramostim may be additive to or synergistic with ICIs to boost efficacy in patients with metastatic melanoma. Results from ICI combination studies are encouraging and may help answer the unmet need of low cure rates in melanoma and other cancers. Combination sargramostim and the PD-1 inhibitor, pembrolizumab, is being studied in melanoma [NCT04703426 (249)], biliary cancer [NCT02703714 (250)], and NSCLC [NCT04856176 (251)]. These trials and other investigations in renal cell carcinoma, and head and neck cancers aim to better understand the role that sargramostim plays in the generation of antitumor immune responses as well as attenuating toxicity.