A radiolabeled dendrimer non-invasively identifies and tracks innate immune cell activation in a mouse model of experimental autoimmune encephalomyelitis

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: major_revision

Abstract
Multiple sclerosis (MS) is a chronic neurodegenerative disease driven by infiltration of activated innate immune cells into the central nervous system (CNS). Current imaging approaches for diagnosing and monitoring disease progression rely on structural lesions and cannot directly assess innate immune activity. Here, we describe a dendrimer positron emission tomography (PET) tracer, 18F-flurimedrimer (18F-FMD), for non-invasive, longitudinal tracking of activated myeloid cells. In an experimental autoimmune encephalomyelitis (EAE) murine model, 18F-FMD specifically detects myeloid activation at presymptomatic and symptomatic stages, with PET signal correlating with disease severity. Moreover, 18F-FMD sensitively captures therapeutic response to fingolimod (FTY720) and a CSF1R dendranib (H74DS3M8), both of which suppress immune cell activation and attenuate disease severity. These findings highlight the potential of 18F-FMD PET for specific, real-time monitoring of innate immune responses, and the applicability of the dendrimer in clinical settings for monitoring therapeutic efficacy, advancing the development of personalized, myeloid-targeted strategies for MS.

Introduction
Multiple sclerosis (MS) is the most prevalent chronic autoimmune disease of the central nervous system (CNS), affecting nearly 2.9 million people worldwide1. Characterized by inflammation, demyelination, and neurodegeneration, MS presents as a heterogeneous disease with clinical manifestations that vary significantly among individuals. Although its exact etiology is unclear, MS is driven by a complex interplay between the adaptive and innate immune systems. Within the adaptive immune response, autoreactive T and B cells are central to MS pathogenesis. Pro-inflammatory T helper 1 (Th1) and Th17 cell subsets secrete cytokines such as interleukin-17 (IL-17) and IL-21, promoting CNS infiltration and amplifying inflammation, while B cells contribute by producing autoantibodies and modulating T cell responses2,3. In parallel, the innate immune system – particularly myeloid cells including microglia, monocytes, macrophages, neutrophils, and dendritic cells – plays a crucial role in both the progression and potential remission of MS4,5. Activated microglia and infiltrating macrophages are the predominant immune cell types found in acute and chronic active MS lesions within the CNS6–9. These cells drive neuroinflammation through the release of pro-inflammatory cytokines, reactive oxygen species, and other neurotoxic mediators; they also phagocytose myelin and debris, and sustain lymphocyte activation through antigen presentation and cytokine polarization10–12. Notably, myeloid cell activation is present early in the disease course and persists throughout various clinical subtypes, including relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS), which together account for over 85% of MS cases13. Given their central involvement in MS pathology, activated microglia and macrophages can serve as clinically relevant biomarkers of chronic neuroinflammation and disease progression.

Currently, there is no cure for MS, but several disease-modifying therapies (DMT) are approved, primarily for managing RRMS and SPMS, with only one Food and Drug Administration (FDA)-approved option for PPMS14. These treatments mainly work by modulating immune responses or depleting specific immune cell populations to reduce disease activity and slow clinical progression15. While no FDA-approved MS therapies directly target activated myeloid cells, a growing number of investigational treatments aim to address this gap. Moreover, several existing immunomodulatory therapies indirectly affect myeloid cells through changes in cytokine signaling and adaptive immune cell-mediated pathways4. Despite these advancements, PPMS remains particularly challenging to manage. The complexity and heterogeneity of MS – driven by multiple interacting immune cell types – makes selecting effective therapies difficult4. Compounding this challenge is the lack of specific in vivo imaging biomarkers for activated myeloid cells, which limits our ability to fully elucidate their role in neurodegeneration, stratify patients for trials of emerging microglia-targeting treatments, or monitor therapeutic responses.

Structural magnetic resonance imaging (MRI) is the current standard of care for diagnosing and staging MS lesions, as well as for monitoring treatment response16. While MRI is effective for visualizing structural changes, it lacks the molecular specificity needed to differentiate different immune cell subsets. As a result, MRI provides limited insight into treatment efficacy at the cellular level and may lead to suboptimal therapeutic choices, increasing the risk of continued CNS damage. Since activated myeloid cells are an early and persistent feature of MS and play a critical role in disease progression and remission, there is a critical need for non-invasive imaging strategies to specifically identify and track maladaptive myeloid populations and their functional phenotypes in the CNS of MS patients.

Positron emission tomography (PET) is a functional molecular imaging modality that has the potential to profoundly enhance our understanding of the in vivo roles of activated myeloid cells in MS. Unfortunately, the current selection of PET tracers for imaging innate immune activation is limited. The most widely evaluated PET tracers for visualizing activated myeloid cells target the translocator protein 18 kDa (TSPO)17,18. Despite providing valuable insights into the spatiotemporal dynamics of neuroinflammation in MS patients in addition to showing potential for predicting disease progression19,20, TSPO-PET imaging has several critical limitations, including its lack of cellular specificity (i.e., it is expressed on microglia, peripheral myeloid cells, astrocytes, and vascular endothelium, thus making interpretation of images challenging), unclear functional role in MS, and a genetic polymorphism in the human TSPO gene resulting in variable binding affinity across patients for all second-generation TSPO-PET tracers21. Moreover, recent post-mortem studies of MS brain tissue revealed that TSPO expression does not increase in activated cells but rather in the number of TSPO-expressing cells, underscoring its limitations as a reliable biomarker for myeloid activation22. While alternative PET imaging targets, such as colony-stimulating factor 1 receptor (CSF1R), cluster of differentiation molecule 11b (CD11b), cyclooxygenase-2 (COX2), triggering receptor expressed on myeloid cells 1 (TREM1), and P2Y purinoceptor 12 (P2Y12R), are being investigated as potentially improved biomarkers for specific in vivo tracking of innate immune activation23, none have yet been validated as effective clinical biomarkers for neuroinflammation.

To address this unmet need for a PET tracer that enables highly specific detection of activated myeloid cells24, we recently developed 18F-flurimedrimer (18F-FMD; previously known as 18F-OP-801), a fluorine-18-labeled synthetic fourth-generation polyamidoamine (PAMAM) hydroxyl dendrimer25,26. Fourth-generation hydroxyl dendrimers have been demonstrated to penetrate regions of a dysfunctional, but not healthy, intact blood-brain barrier (BBB), where they are quickly and selectively taken up by activated myeloid cells via fluid-phase endocytosis and rapidly cleared from tissue that does not contain these cells27–34. In preclinical PET imaging, we have demonstrated that 18F-FMD sensitively detects neuroinflammation induced by systemic sepsis in a lipopolysaccharide mouse model, with signal intensity correlating with the murine sepsis severity score35. Moreover, the synthesis of 18F-FMD has recently been optimized and approved for clinical use26, positioning this PET tracer as a potentially valuable tool for highly specific, functionally relevant, and non-invasive imaging of activated myeloid cells in patients with MS and other inflammatory diseases.

In this study, we evaluate the ability of 18F-FMD to non-invasively detect activated myeloid cells in an experimental autoimmune encephalomyelitis (EAE) mouse model at both presymptomatic and acute disease stages (Fig. 1). We demonstrate that 18F-FMD selectively accumulates in activated myeloid cells and sensitively tracks therapeutic modulation of neuroinflammation by fingolimod (FTY720), an FDA-approved small-molecule immunosuppressant, and H74DS3M8, a recently developed hydroxyl dendrimer-based receptor tyrosine kinase inhibitor (referred to as “dendranib”). These findings establish 18F-FMD as a promising tool for in vivo monitoring of innate immune activation in the CNS and support its translational potential for evaluating therapeutic response and disease progression in neuroinflammatory disorders.

Results
18F-FMD PET imaging enables early disease detection in EAE mice before symptom onset
We first assessed the ability of 18F-FMD PET imaging to detect and track activated myeloid cells in vivo in an EAE mouse model characterized by a sustained progressive disease course with persistent inflammation, demyelination, and axonal degeneration36–38. PET/computed tomography (CT) imaging was performed at presymptomatic (pre-EAE; score 0, no paresis/paralysis, ≥1 g weight loss in 24–48 h) and symptomatic (EAE; score 0.5–3.5, hindlimb paresis/paralysis) stages of disease, compared to naïve controls, 50 min after tracer injection (Fig. 2a). Clinical signs of EAE, which progress rostrocaudally from the tail to hindlimbs and forelimbs, began to manifest by day 12 and peaked by day 15 with an average disease score of 2.3 ± 1.2 (Fig. 2b and Supplementary Table 2).

In naïve mice, 18F-FMD PET signal was predominantly observed in the kidneys and bladder, reflecting the tracer’s urinary excretion pathway. Conversely, pre-EAE and symptomatic EAE mice exhibited visible increases in spinal cord signal (Fig. 2c, white arrow). PET image quantification confirmed elevated 18F-FMD uptake in pre-EAE mice compared to naïve controls in both the cervical/thoracic (0.69 to 1.02 %ID/g [percent injected dose per gram]) and lumbar (2.04–2.71 %ID/g) spinal cord regions (p<0.05; Fig. 2d). The signal increased further upon symptom manifestation in EAE mice, reaching 2.92 %ID/g in the cervical/thoracic and 4.71 %ID/g in the lumbar spinal cord (p<0.05; Fig. 2e). While no significant differences in 18F-FMD PET signal were observed in the brain during the pre-EAE stage, symptomatic EAE mice showed elevated tracer uptake in white matter-rich brain regions, particularly the cerebellum (0.29 to 1.05 %ID/g) and pons (0.29 to 1.06 %ID/g; p<0.05).

Following PET/CT imaging, mice were perfused with phosphate-buffered saline (PBS) to remove residual tracer in the blood pool. Ex vivo gamma counting was then performed to validate in vivo PET findings in CNS tissues. Notably, in pre-EAE mice with no clinical symptoms, 18F-FMD signal in the lumbar spinal cord was significantly elevated compared to naïve controls (p<0.01; Fig. 3a). As the disease progressed, tracer accumulation markedly intensified, with symptomatic EAE mice showing a 2- to 4.5-fold increase in PET signal in the cervical/thoracic and lumbar spinal cords compared to pre-EAE mice, and a dramatic 9- to 12-fold increase compared to naïve controls (p<0.01; Fig. 3a). Additionally, tracer uptake in the blood pool of symptomatic EAE mice was significantly higher than in naïve mice (p<0.0001), with a trend toward significance in pre-EAE mice (p=0.29; Supplementary Fig. 3a). However, no significant differences were observed in peripheral organs such as spleen, which is known to exhibit fluctuations in immune cell populations like T cells, B cells, monocytes, and neutrophils during EAE39.

High-resolution autoradiography of lumbar spinal cords validated the in vivo PET and ex vivo gamma counting results. In naïve mice, 18F-FMD signal was minimal (Fig. 3b). In contrast, despite pre-EAE mice showed no clinical symptoms, they exhibited focal tracer uptake in regions corresponding to early immune cell infiltration. Strikingly, symptomatic EAE mice with hindlimb paralysis demonstrated even more pronounced tracer accumulation, forming punctuate hotspots that colocalized with multifocal mononuclear infiltrates and peripheral white matter demyelination in adjacent hematoxylin and eosin (H&E)-stained sections, which are hallmarks of inflammatory lesions in this EAE model40,41 (Fig. 3c). Quantitative analysis of autoradiography supported these findings, revealing a 2.5-fold and 3.5-fold increase in tracer uptake in the spinal cords of pre-EAE and symptomatic EAE mice, respectively, compared to naïve controls (p<0.05; Supplementary Fig. 3b). Furthermore, CD68 immunohistochemical staining in a subset of mice from the same PET imaging cohort confirmed the presence of activated myeloid cells in the lumbar spinal cords of both pre-EAE and symptomatic EAE mice (Fig. 3c and Supplementary Fig. 4). Together, these results provide compelling evidence for the specificity and sensitivity of 18F-FMD PET in detecting neuroinflammation.

CSF1R dendranib is effective in reducing EAE disease severity
H74DS3M8 is a hydroxyl dendrimer, conjugated with eight dasatinib payloads, engineered to selectively target activated myeloid cells via fluid-phase endocytosis and inhibit CSF1R with high potency42,43. Hydroxyl dendrimers of this class are metabolically stable and are eliminated intact via renal clearance44–46. Kinase profiling revealed its sub-nanomolar potency against CSF1R, ABL tyrosine kinase 1 (ABL1), cluster of differentiation 117 (c-Kit), and cellular Src (c-Src), with minimal off-target activity against epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor beta (PDGFRβ; Supplementary Table 3). In cell-based assays, H74DS3M8 inhibited proliferation in bellus verum 2 (BV2) microglial cells in a dose-dependent manner, with an IC50 of 343 nM at 52 h (Supplementary Fig. 1). Importantly, this effect on inhibition of proliferation was seen in the absence of cell death, differentiating the mechanism of action of H74DS3M8 from other CSF1R inhibitors whose primary mechanism is through microglial killing or depletion. Pharmacokinetic studies demonstrated sustained systemic exposure following both intraperitoneal (i.p.) and intravenous administration, supporting its suitability for in vivo applications (Supplementary Table 4). Noncompartmental analysis of sparse-sampling data revealed log-linear elimination kinetics with terminal plasma half-lives (HL_Lambda_z) of 3.44 h (i.p.) and 3.15 h (intravenous, i.v.), respectively (Supplementary Fig. 2), consistent with a clearance profile appropriate for therapeutic targeting of CNS inflammation.

To evaluate its therapeutic potential in a context of neuroinflammation and demyelination, we treated EAE mice daily with H74DS3M8 (60 mg/kg, i.p.). Disease severity was compared to vehicle-treated controls (4.4% [weight per volume] trehalose dihydrate/10 mM histidine via i.p. injection) and FTY720-treated mice (3 mg/kg via oral gavage; Fig. 4a). Treatment began at the presymptomatic stage on day 8 post-EAE induction, the earliest timepoint at which 18F-FMD PET imaging detected molecular changes in myeloid cell activation. By day 15, 100% of vehicle-treated mice developed mild to severe EAE symptoms (Fig. 4b). In contrast, only 52% of H74DS3M8-treated mice and 27% of FTY720-treated mice showed mild symptoms (e.g., tail weakness), with none progressing to severe EAE (e.g., hindlimb paralysis). Both treatments effectively reduced disease severity, with average scores reduced to 0.6 ± 0.9 (H74DS3M8) and 0.4 ± 0.6 (FTY720), compared to 2.7 ± 0.9 in vehicle-treated controls by day 15 (p<0.0001; Fig. 4b and Supplementary Table 5).

18F-FMD PET imaging sensitively detects therapeutic response of EAE mice to different MS therapies
To assess the ability of 18F-FMD PET to monitor the immunomodulatory effects of H74DS3M8 in vivo, we performed a treatment monitoring study comparing its therapeutic efficacy to FTY720 and vehicle control in EAE mice. On day 15 post-EAE induction, 18F-FMD PET imaging showed a marked reduction in spinal cord signal (white arrow) in both H74DS3M8- and FTY720-treated mice compared to vehicle-treated controls (Fig. 4c). Quantification of in vivo PET images revealed significant decreases in tracer uptake across multiple CNS regions, including the medulla (p<0.01), cerebellum (p<0.01), pons (p<0.05), cervical/thoracic spinal cord (p<0.01), and lumbar spinal cord (p<0.05), consistent with a reduction in innate immune activity. These results demonstrate that 18F-FMD PET imaging can sensitively detect the immunomodulatory effects of both treatments (Fig. 4d).

Ex vivo gamma counting and high-resolution autoradiography further confirmed in vivo PET findings. H74DS3M8- and FTY720-treated EAE mice showed significantly lower 18F-FMD signal in the cervical/thoracic and lumbar spinal cords, as well as blood, compared to vehicle controls. The cervical/thoracic spinal cords of treated mice exhibited 5- to 8-fold lower signal, while lumbar spinal cords and blood showed 2- to 3-fold decreases (p<0.05; Fig. 5a and Supplementary Fig. 3c). Autoradiography confirmed these results, with H74DS3M8 and FTY720 reducing tracer signal to near-naïve levels (Fig. 5b and Supplementary Fig. 3d), and H&E staining revealed significantly less demyelination in treated mice, closely resembling naïve controls (Fig. 5c). Immunohistochemistry staining for CD68+ cells in the same PET imaging cohort confirmed negligible presence of activated myeloid cells in H74DS3M8- and FTY720-treated mice, underscoring their efficacy in modulating neuroinflammation and potentially altering disease severity (Fig. 5c and Supplementary Fig. 4).

Flow cytometry confirmed therapeutic modulation of CNS and peripheral myeloid responses in EAE
To identify the immune cell subsets driving neuroinflammation and assess their modulation by therapeutic interventions in this EAE mouse model, we performed single-cell flow cytometry on spinal cord and peripheral blood samples from naïve, pre-EAE, vehicle-treated, H74DS3M8-treated, and FTY720-treated EAE mice (Fig. 6a and Supplementary Fig. 5). This approach enabled profiling of immune dynamics across disease stages and treatment responses. Tissues were harvested on day 8 post-induction for pre-EAE mice and on day 15 for EAE mice treated with H74DS3M8, FTY720, and vehicle at various disease stages (score 0.5–3.5, hindlimb paresis/paralysis), as well as from naïve mice.

Flow cytometric analysis revealed profound immune remodeling in the CNS of EAE mice compared to naïve controls. There was a dramatic reduction in the proportion of resting microglia (CD11b+CD45lowTMEM119+), which declined from 69.9% in naïve mice to 20.28% in EAE mice. In parallel, activated microglia (CD11b+CD45lowTMEM119−) expanded significantly, from 0.13% to 20.84%, representing a >160-fold increase and indicating a marked phenotypic shift. This transformation was accompanied by widespread infiltration of peripheral immune cells, including T cells (CD3+CD45+; 0.49% in naïve vs. 22.39% in EAE), infiltrating macrophages (CD11b+CD45hi; 0.88% vs. 37.53%), monocyte/dendritic cells (DC) (CD11b+CD45hiLy6G−; 1.01% vs. 17.42%), and neutrophils (CD11b+CD45hiLy6G+; 0.99% vs. 13.64%) (all comparisons p<0.0001; Fig. 6b). Collectively, these data reveal a profound shift from a predominantly homeostatic microglial environment to a highly inflammatory landscape, with infiltrating macrophages emerging as the most abundant immune cell type within the CNS of EAE mice.

Prior to symptom onset, pre-EAE mice showed a substantial reduction in resting microglia (28.24% vs. 69.86% in naïve; p<0.0001) in the spinal cord, alongside an early expansion of activated microglia (1.32% vs. 0.13% in naïve), suggesting that innate immune activation precedes peripheral immune infiltration and may play a key initiating role in EAE pathogenesis. Notably, B cells (CD19+CD45+) remained absent from the CNS across all groups and stages, indicating a minimal role of B cells in this model. In the periphery, a parallel pattern of immune activation was observed: circulating myeloid cells increased significantly (p<0.05; Fig. 6c), and T cells underwent a transient expansion during the preclinical phase, suggesting early systemic priming. B cells in blood modestly declined during the pre-EAE stage and returned to baseline levels during acute disease, mirroring levels seen in naïve mice.

Building on this data, we next evaluated the therapeutic potential of H74DS3M8 in modulating immune cell populations and compared its effects to the established immunomodulator FTY720. Both treatments significantly reduced pathogenic myeloid populations in the CNS and peripheral blood of EAE mice, including activated microglia, infiltrating macrophages, and monocytes (p<0.01; Fig. 6b, c). H74DS3M8 also markedly suppressed CNS-infiltrating T cells, DCs, and neutrophils, and decreased both T and B cell frequencies in the circulation. FTY720 similarly inhibited T cell infiltration into the CNS and significantly reduced circulating monocytes, neutrophils, and B cells (p<0.001). To further characterize the functional impact of treatment, we analyzed macrophage polarization states. Both therapies significantly decreased the proportion of pro-inflammatory M1-like microglia/macrophages (CD11b+CD45+CD86+; p<0.0001; Supplementary Fig. 6).

18F-FMD is selectively taken up by activated myeloid cells in EAE mice
To investigate the specific cell populations that endocytose 18F-FMD and thus gain an understanding of what the signal in our 18F-FMD PET scans truly represent, we used Cy5-labeled generation-4 hydroxyl dendrimer (Cy5-HD), a fluorescent analog that closely mirrors the structure, size, and uptake mechanism of 18F-FMD. Cy5-HD was administered intraperitoneally to all experimental groups 24 h prior to tissue collection for flow cytometry analysis. Quantitative analysis revealed a significant increase in Cy5-HD+ myeloid populations, including infiltrating macrophages, activated microglia, neutrophils, and monocyte/DCs, in both the spinal cord and peripheral blood of pre-EAE and symptomatic EAE mice compared to naïve controls (p<0.001, Fig. 7). Among microglia, Cy5-HD uptake was more than 9-fold higher in cells with a pro-inflammatory, activated phenotype compared to resting microglia, which showed negligible uptake (Supplementary Fig. 7). T cells and B cells lacked Cy5-HD uptake, further supporting the tracer’s specificity for activated myeloid subsets.

Following therapeutic intervention, Cy5-HD+ myeloid populations were markedly reduced by up to 19-fold in infiltrating macrophages and 46-fold in activated microglia, highlighting the ability of both treatments to suppress pro-inflammatory innate immune activity (Fig. 7). These findings align with previously published findings demonstrating the selective accumulation of hydroxyl dendrimers in activated myeloid cells under inflammatory conditions27–29. Notably, the spatial and temporal distribution of Cy5-HD+ cells observed by flow cytometry closely mirrored the trends captured by in vivo PET imaging, ex vivo gamma counting, and autoradiography. PET signal intensity decreased 2- to 2.5-fold following treatment, paralleling the observed reductions in Cy5-HD+ myeloid populations.

Methods
Pharmacokinetics of H74DS3M8
8–12-week-old male C57BL/6J mice (Jackson Laboratory, strain #: C57BL6J, RRID: 1MSR_JAX:000664) were dosed with 60 mg/kg H74DS3M8 in 6.3% Trehalose/10 mM histidine pH 5.5 (at a dose concentration of 12 mg/mL) by i.p. injection into the lower left or right quadrant of the abdomen or by i.v. injection into the lateral tail vein, at Meadowhawk Biolabs (Hayward, CA). Blood was collected from each animal at two time points (via submental vein for survival collection and cardiac puncture for terminal collection), of 0.5, 1, 4, 12, 24, 36 h, for a total of n=3 per time point. Samples were collected into K2 EDTA tubes and shipped to Alturas Analytics (Moscow, ID) for bioanalytical measurement using a previously established high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) bioanalytical method.

Microglia proliferation assay
BV2 cells (ATCC, Manassas, VA, #CRL-2467) were cultured in the presence of 100 ng/mL macrophage colony-stimulating factor (M-CSF, Thermo Fisher Scientific, Waltham, MA, #PMC2044) in serum-free Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, #11885084) supplemented with 1% Penicillin-Streptomycin (Thermo Fisher Scientific, #15140122). Cells were treated with various concentrations of H74DS3M8, and confluence was observed every 2 h in the Incucyte (Sartorius, Göttingen, Germany) maintained at 37 °C in 5% CO2. Brightfield images were obtained in one of four field-views per well at 10x magnification at 52 h post-plating and treatment. Concentration data from the 52-h time point were used to calculate the IC50.

Animals
Female 8-week-old C57BL/6J mice (Jackson Laboratory, strain #: C57BL6J, RRID: 1MSR_JAX:000664) were housed in a non-barrier, temperature-controlled environment (humidity 40–60%) under a 12-h light/dark schedule with unrestricted access to food and water. A total of 160 mice were used for this study. Treatment groups were randomly assigned by cage and co-housed in the same room as vehicle groups. All animal care and procedures complied with the Animal Welfare Act and were in accordance with institutional guidelines. Permission to perform all animal experiments was granted by the Stanford Administrative Panel on Laboratory Animal Care (APLAC), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

EAE induction
EAE was induced in female C57BL/6J WT mice (9 weeks; the Jackson Laboratory, #000664) using MOG35-55 emulsified in CFA (Hooke Laboratories, Lawrence, MA, #EK-0111). All vehicle and therapy-treated mice received two pertussis toxin i.p. injections (110 ng) 2 and 24 h after MOG induction. Naïve littermates were used as additional controls. EAE mice were weighed and scored daily from day 8 onward using a standard scoring protocol for levels of paresis/paralysis (Supplementary Table 1).

Drug treatments
Mice were randomly assigned to the following groups: naïve, vehicle-treated EAE, H74DS3M8-treated EAE, and fingolimod-treated EAE. All therapies were given starting from 8 days post disease-induction for 8 days, till day 15 post-induction. Vehicle-treated EAE mice received 4.4% (w/v) trehalose dihydrate/10 mM histidine via i.p. injection. H74DS3M8 (Ashvattha Therapeutics, Redwood City, CA, 60 mg/kg) was administered via i.p. injection. Fingolimod (Cayman Chemicals, Ann Arbor, MI, 3 mg/kg, #10006292) was administered via oral gavage.

Flow cytometry
Mice were euthanized under anesthesia (with inhaled isoflurane [2–3%]) and blood was collected by cardiac puncture in EDTA-treated tubes and lysed with ACK lysis buffer (Lonza, Walkersville, MD, #BP10-548E) for 10 min at room temperature. All cells were then resuspended in fluorescence-activated cell sorting (FACS) buffer (2% fetal bovine serum [Thermo Fisher Scientific, #A5670701] in PBS [Gibco, #10010023]). Mice were then anesthetized and exsanguinated by cardiac perfusion with PBS. Spinal cords were harvested, mechanically homogenized in ice-cold CNS buffer (2.5% Hepes [pH 7.5, Gibco, #15630080] in Hanks’ balanced salt solution without Ca/Mg/PSP [Thermo Fisher Scientific, #J67681.K2]), and resuspended in FACS buffer. Flow cytometry was performed to delineate immune cell populations using the following antibodies (Supplementary Table 6): AF700 CD45 (BioLegend, San Diego, CA, #147715), PerCP CD19 (BioLegend, #115531), BV421 CD3 (BioLegend, #100228), APC/Fire750 CD11b (BioLegend, #101261), BV570 Ly6G (BioLegend, 127629), PB CD86 (BioLegend, #105021), BUV737 CD80 (BD Biosciences, Franklin Lakes, NJ, #612773), PE CD163 (BioLegend, #156703), PerCP/Cy5.5 CD206 (BioLegend, #141715), PeCy7 TMEM119 (Thermo Fisher Scientific, #25-6119-82), and Cy5-HD (Ashvattha Therapeutics). Samples were washed with FACS buffer and resuspended in 2% paraformaldehyde (PFA, Thermo Fisher Scientific, J19943.K2) for 20 min at room temperature. After fixation, cells were washed twice and resuspended in FACS buffer until being analyzed on the Cytek Aurora (Cytek Biosciences, Fremont, CA). FlowJo software (v.10, BD Biosciences) was used for the analysis and depiction of the gating strategy.

18F-FMD radiosynthesis
All chemicals were acquired from commercial sources except for synthetic precursors 3-azidopropyl-4-methylbenzenesulfonate and PAMAM-G4-OH alkyne dendrimer precursor 10, which were synthesized to order by Snapdragon Chemistry. All chemicals were used without further purification. 18F-FMD was radiolabeled in two steps26: nucleophilic 18F-fluorination of 3-azidopropyl-4-methylbenzenesulfonate yielded 18F-3-fluoropropylazide, which was purified via high-performance liquid chromatography (HPLC). This was subsequently reacted with PAMAM-G4-OH alkyne dendrimer precursor 10 (0.5 mg) via copper catalyzed azide-alkyne cycloaddition to produce 18F-FMD, which was purified and reformulated in saline with 10% ethanol for injection. HPLC was used to confirm identity and determine specific activity. Specific activity for these experiments (n=4 syntheses) was 0.556 mCi/μg and the approximate injected mass of dendrimer per mouse was 0.270 μg for in vivo PET imaging studies and 1.345 µg for ex vivo autoradiography studies.

In vivo PET/CT imaging studies
Mice were anesthetized with isoflurane gas (2.0–3.0% for induction and 1.0–2.0% for maintenance) and intravenously injected with 18F-FMD (150 µCi). 10-min static PET images were acquired 50 min post-injection of 18F-FMD on days 8 and 15 after EAE induction using the GNEXT scanner (Sofie Biosciences, Dulles, VA) in list mode format. CT images were acquired after each PET scan to provide an anatomical reference frame in addition to scatter and attenuation correction for PET data. Isotropic resolution was achieved using OSEM3D/MAP reconstruction algorithms with 24 subsets, 3 iterations, and a matrix size of 240 × 240 × 191.

PET image analysis
PET images were analyzed using VivoQuant 4.0 (Perspective Therapeutics, Seattle, WA). Briefly, CT was overlaid on PET for anatomical reference. A three-dimensional mouse brain atlas was used along with total decay-corrected dose to each mouse to quantify tracer uptake in the whole brain. Cervical/thoracic and lumbar spinal cord quantification was obtained via segmentation and exclusion of the vertebral column using L1-L5 as landmarks77,78. Analysis was not conducted truly blind because the researchers have access to the mouse EAE scoring data. However, all PET/CT imaging data were assigned a scan number, and all mice were allocated unique ID numbers based on mouse positions in the scanner. All data were grouped for graphing data by experimental group after analysis.

Ex vivo gamma counting
Immediately after PET, mice were euthanized under anesthesia (with inhaled isoflurane [2–3%]) and cardiac puncture was performed. Mice were subsequently perfused with 20 mL of PBS and tissues of interest (heart, spleen, kidney, brain, spinal cord, and tail) were individually harvested, weighed and gamma-counted (Hidex automatic gamma counter, Hidex, Turku, Finland). Biodistribution results were calculated as %ID/g using the weight of each dissected organ.

Ex vivo autoradiography studies
A separate cohort of EAE and naïve mice were used to evaluate 18F-FMD distribution in the CNS tissues. Sixty minutes after intravenous injection of a higher dose of 18F-FMD (700 µCi), mice were euthanized under anesthesia (with inhaled isoflurane (2–3%)) and perfused with PBS (20 mL), and CNS tissues were harvested for autoradiography and immunohistochemistry. Spinal cord sections were analyzed via digital autoradiography using a Typhoon phophorimager (Amersham Biosciences, Amersham, United Kingdom) and stained with H&E (Hematoxylin Gills 3, Thermo Fisher Scientific, NC9964763; Eosin-Y Richard-Allan Scientific, Thermo Fisher Scientific, #22-110-637) for anatomical reference and visualization of the distribution of immune cells.

Immunohistochemistry
Dissected spinal cords were fixed in 4% PFA (Thermo Fisher Scientific) in PBS for 24 h, then switched to 30% sucrose (Thermo Fisher Scientific, #A15583.0E) and stored at 4 °C for at least 1 week before being immersed in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA, #4583). 12 μm sections were then cut and mounted onto slides, which were stored at −80 °C for one day before staining. For staining, slides were incubated overnight at 4 °C in rat anti-mouse CD68 primary antibody (1:500) (MCA1957, Bio-Rad Laboratories, Hercules, CA). The next day, slides were incubated with biotinylated anti-rat secondary antibody (1:1000; BA-4001, Vector Laboratories, Newark, CA) for 45 min. Peroxidase substrate solution 3,3’-diaminobenzidine tetrahydrochloride hydrate (DAB; #D5637, Sigma-Aldrich, St. Louis, MO) was used to intensify stain.

Statistics and reproducibility
Statistical analyses of flow cytometry, in vivo PET, ex vivo biodistribution, and autoradiography were performed using Prism (version 10, GraphPad Software, San Diego, CA). All data were assessed for normalization (D’Agostino-Pearson omnibus K2/Shapiro-Wilk tests). Statistical analyses were performed using two-tailed unpaired t tests, Mann-Whitney tests, or one-way analysis of variance (ANOVA) with multiple comparison tests as indicated in the figure legends for each dataset. Error bars represent standard deviation (SD). PET/CT and autoradiography results are from four experiments. Flow cytometry results are from three experiments.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a novel dendrimer-based positron emission tomography (PET) tracer, 18F-FMD, designed to detect and track activated myeloid cells in an experimental autoimmune encephalomyelitis murine model of MS. The authors demonstrated that 18F-FMD can detect myeloid cells during both pre-symptomatic and symptomatic stages of EAE, with signal intensity correlating with symptom severity. They also confirmed increased uptake of 18F-FMD in the spinal cord of EAE mice via gamma counting and autoradiography. Additionally, the study shows that treatment with fingolimod and a novel drug targeting the CSF1 receptor, dendranib, leads to reduced symptom severity, which is reflected in a decrease in 18F-FMD uptake across various CNS regions. Flow cytometry further reveals an increased number of innate immune cells in the spinal cord and blood of EAE mice, with reductions following treatment with fingolimod and dendranib. The authors conclude that this new tracer could enable earlier diagnosis, reduce misdiagnosis and allow more precise treatment choice in people with MS.\nOverall, the data presented convincingly support the claim that 18F-FMD detects and monitors myeloid cells in EAE mice, demonstrating increased tracer uptake from the early pre-symptomatic stages of EAE, which corresponds to a rise in innate immune cells, and a reduction in tracer uptake following disease modifying treatment.\nWhile the study offers important insights into this potentially very useful novel tracer, I have major concerns that should be addressed:\n1. The authors have clearly demonstrated the specificity of 18F-FMD for myeloid cells. However, the functional phenotype of the innate immune cells responsible for the increased uptake of the tracer remains unclear. Could the authors provide further details on the phenotype of these cells, particularly microglia? Specifically, what criteria define “active cells” imaged with 18F-FMD? Among microglial cells endocytosing Cy5-labeled hydroxyl dendrimers, what proportion presents a pro-inflammatory versus homeostatic phenotype? Does this proportion change over time after the onset of EAE or after DMT, and if so, is this reflected by changes in the 18F-FMD signal?\n2. The manuscript, in its current form, does not demonstrate the utility of this novel tracer in the context of progressive MS. I would recommend that the authors include post-mortem data from patients with progressive MS, demonstrating the ability of Cy5-labeled hydroxyl dendrimers to distinguish lesions characterized by activated microglia (e.g., smoldering lesions) from inactive lesions, and to identify regions of normal-appearing white matter with diffuse microglial activation, based on differential labeling intensity.\n3. The results presented do not support the claim that this tracer enables early diagnosis of MS, reduces misdiagnosis, or aids in treatment selection in clinical practice. With the new diagnostic criteria for MS introduced at the end of 2024, structural MRI has shown the ability to diagnose MS at an exceptionally early stage, even before the onset of clinical symptoms, thereby allowing for the initiation of disease-modifying therapies prior to symptom manifestation. The evidence showing that 18F-FMD uptake increases before symptom onset in the EAE model does not indicate that the tracer is sufficiently sensitive or specific to diagnose MS earlier or reduce misdiagnoses in patients. All references to these claims should be removed from the manuscript.\n4. EAE is an acute inflammatory disease model, not one that reflects the chronic progression seen in MS, which is defined as the gradual and irreversible accumulation of clinical disability driven by neurodegeneration and axonal loss developing over time. For this reason, I suggest the authors avoid referring to EAE as a model of chronic or progressive MS. The term “disease progression” should be replaced with “disease severity” in their experiment descriptions.\n5. I recommend that the authors revise the abstract, introduction, and discussion sections to avoid referring on the need for novel biomarkers to enable early diagnosis, reduce misdiagnoses, and guide DMT selection. Instead, the emphasis should be on the crucial need for non-invasive biomarkers to specifically measure activated microglia, whose role is critical in neurodegeneration in chronic MS. This need is particularly pressing as new treatments targeting innate immune cells, such as Tolebrutinib (Hercules clinical trial, ECTRIMS 2024), are emerging and showing promising results in slowing clinical progression in non-active secondary progressive MS. Novel tracers such as 18F-FMD could be employed to stratify patients for clinical trials of novel microglia-targeting treatments to monitor their effects, and to perform studies to explore further the role of innate immune cells in the physiopathology of neurodegeneration in MS.\nIn addition, I recommend the authors update their references, particularly those related to clinical forms of MS and alternative PET tracers to TSPO. Reference 28, which cites an unpublished manuscript, should be replaced with more appropriate references.", "reviewer_2": "In the present study, Kuo & Carlson et al. provide evidence of a PET imaging method by the use of radiolabeled dendrimer, namely, the 18F-FMD, to non-invasively track innate immune activation in EAE mice. The authors further investigate the potential of the method following administration of two immunomodulatory treatments in EAE. This is an original work with added value in the field, as it poses implication for imaging modalities with a focus on the CNS innate immune system, a key contributor in CNS immune-mediated pathologies, as in Multiple Sclerosis (MS). Overall the methodology is sound and reported in detail. Few issues arise:\n1. The authors used the EAE model induced by immunization by MOG in CFA and they state that they used the EAE model as a model of chronic/progressive MS. However, the MOG-EAE mouse model is not characterized by clinical progression rather, the mice exhibit an acute stage of disease (approximately 15-20 days post-induction) resembling an MS-like clinical relapse, usually followed by a chronic phase (established approximately 50 days post-induction), upon which the mice tend to exhibit a so-called “plateau” in terms of clinical disability (though in the absence of progression) [10.3791/51275]. In the present study, to my understanding, time-points of euthanization were 8- and 15-days post-immunization, therefore, the pre-clinical and the acute phase. In this respect, in my opinion, the authors should rephrase page 5 lines 102-103: “In this study, we evaluated the ability of 18F-FMD to detect activated myeloid cells non-invasively in an experimental autoimmune encephalomyelitis (EAE) mouse model of chronic/progressive MS”. The interpretation of the results should, in my opinion, take into account these limitations related to the experimental model used. Similarly, the term “progression”, in my opinion, should be replaced by, for example, by “deterioration” or “disability”, in order to better depict the EAE pathology and not to be interpreted as corresponding to the MS-related progression.\n2. Moreover, the authors, as stated in page 5 lines 111-112, “assessed the uptake of 18F-FMD using PET imaging in EAE mice to confirm its sensitivity for detecting activated myeloid cells at both presymptomatic and symptomatic disease”. However, a difference between MOG-EAE experimental model and MS is the induction of EAE via MOG-CFA immunization, a procedure that does not naturally occur upon MS pathogenesis. Innate immune cell activation upon MOG-EAE induction is strongly triggered by CFA in the periphery in order to secure effective antigen-presentation, and this procedure has been shown to actively influence the activation status of CNS innate cells as well [10.1002/eji.200939568]. It is, therefore, likely, that the CNS innate immune cells activation profile in the context of MS differs from the MOG-EAE CNS innate immune cells’ activation profile and tis difference may influence the tracer uptake. In this respect, the potential of the method to detect innate immune cell activation in the CNS at pre-clinical stage, as well as across disease stages and disease activity status in MS requires further evaluation, in the clinical setting. Please add a respective comment.\n3. Furthermore, the authors used flow cytometry by the use of a fluorescent analog of the tracer, in order to show the potential of 18F-FMD PET to detect activated myeloid cells in the CNS. However, although they used well-established markers for innate immune cells, such as CD11b, CD11c, CD45low expression levels, these markers are not selective for specific CNS innate immune cell populations, as is microglia, and they do not assess the innate immune cells’ activation status. The use of a more specific marker in order to study cell specific populations, for instance, Tmem 119 for microglia, may be more suitable in this respect. Of note, the CNS innate immune cell populations exhibit high complexity and pose challenge in terms of phenotypic characterization [10.1016/j.celrep.2024.114250]. Taking into account the potential novelty of the method and the added value in labelling activated CNS immune cells, a more detailed phenotypic and/or transcriptional characterization of the labeled CNS immune cells populations, following, for instance, cell sorting and further downstream investigations, is of interest. Such investigation, in my opinion, would be of importance, in order to better establish the potential of the method to detect activated myeloid cells in the CNS, and to further characterize these cellular populations, thus enhancing the mechanistic insight and supporting the translational value of the study. Please add a respective comment.", "reviewer_3": "I've reviewed the manuscript titled \"Radiolabeled dendrimer non-invasively tracks innate immune activation in multiple sclerosis mice after immunomodulatory therapy.\" This is an interesting study exploring the use of a novel PET tracer (18F-FMD) for detecting activated myeloid cells in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.\nThe study demonstrates that 18F-FMD can detect neuroinflammation even before clinical symptoms appear in the EAE model. Multiple validation methods (PET imaging, ex vivo gamma counting, autoradiography, flow cytometry) strengthen the findings.\nThe comparison of two different therapies (H74DS3M8 and fingolimod) provides insights into different mechanisms of action.\nThe selectivity of the tracer for activated myeloid cells is well-documented.\nHowever,because of the reasons below,I would recommend rejection of this paper:\nMethodological concerns: The study lacks appropriate controls for the specificity of 18F-FMD uptake. While flow cytometry with Cy5-HD is used as a surrogate, direct validation that 18F-FMD and Cy5-HD target identical cell populations was not adequately demonstrated. The authors should have included additional control experiments to rule out non-specific binding.\nInsufficient statistical power: The sample sizes in several experimental groups (n=4-7 per group) appear underpowered for the conclusions being drawn, particularly in the treatment response studies. This raises questions about the reproducibility of the findings.\nLimited translational relevance: While the EAE mouse model is well-established, it primarily recapitulates aspects of relapsing-remitting MS. The manuscript claims relevance for progressive forms of MS but provides insufficient evidence supporting this assertion. The direct applicability to human MS patients remains speculative.\nConfounding factors not addressed: The potential impact of BBB disruption on tracer uptake (common in EAE models) is not sufficiently addressed. The authors do not adequately demonstrate that 18F-FMD signal is specifically related to activated myeloid cells rather than passive accumulation due to increased vascular permeability.\nIncomplete characterization of H74DS3M8: The novel CSF1R dendranib is inadequately characterized pharmacologically. Basic properties such as plasma half-life, brain penetrance, and selectivity profiling are missing, making it difficult to interpret the therapeutic response data.\nOverlapping author affiliations with Ashvattha Therapeutics: Several authors are affiliated with the company developing both 18F-FMD and H74DS3M8, creating a potential conflict of interest that may influence the interpretation of results.\nI would suggest the authors address these concerns with additional experiments and resubmit a more comprehensive manuscript with expanded controls and improved statistical rigor."}, "author_response": "Response to Reviewers’ Comments:\nReviewer #1 (Remarks to the Author):\nGeneral comments:\nThis manuscript presents a novel dendrimer-based positron emission tomography (PET) tracer, 18F-FMD,\ndesigned to detect and track activated myeloid cells in an experimental autoimmune encephalomyelitis murine\nmodel of MS. The authors demonstrated that 18F-FMD can detect myeloid cells during both pre-symptomatic\nand symptomatic stages of EAE, with signal intensity correlating with symptom severity. They also confirmed\nincreased uptake of 18F-FMD in the spinal cord of EAE mice via gamma counting and autoradiography.\nAdditionally, the study shows that treatment with fingolimod and a novel drug targeting the CSF1 receptor,\ndendranib, leads to reduced symptom severity, which is reflected in a decrease in 18F-FMD uptake across\nvarious CNS regions. Flow cytometry further reveals an increased number of innate immune cells in the spinal\ncord and blood of EAE mice, with reductions following treatment with fingolimod and dendranib. The authors\nconclude that this new tracer could enable earlier diagnosis, reduce misdiagnosis and allow more precise\ntreatment choice in people with MS.\nOverall, the data presented convincingly support the claim that 18F-FMD detects and monitors myeloid cells in\nEAE mice, demonstrating increased tracer uptake from the early pre-symptomatic stages of EAE, which\ncorresponds to a rise in innate immune cells, and a reduction in tracer uptake following disease modifying\ntreatment.\nWhile the study offers important insights into this potentially very useful novel tracer, I have major concerns that\nshould be addressed:\nComment 1:\nThe authors have clearly demonstrated the specificity of 18F-FMD for myeloid cells. However, the functional\nphenotype of the innate immune cells responsible for the increased uptake of the tracer remains unclear. Could\nthe authors provide further details on the phenotype of these cells, particularly microglia? Specifically, what\ncriteria define “active cells” imaged with 18F-FMD? Among microglial cells endocytosing Cy5-labeled hydroxyl\ndendrimers, what proportion presents a pro-inflammatory versus homeostatic phenotype? Does this proportion\nchange over time after the onset of EAE or after DMT, and if so, is this reflected by changes in the 18F-FMD\nsignal?\nResponse: Thank you for asking these very important questions. We agree that further characterizing the\nfunctional phenotype of myeloid cells that predominantly endocytose 18F-FMD is essential for interpreting tracer\nsignal in the EAE model. To address this, we performed additional flow cytometry studies and added new data\nand graphs for infiltrating macrophages and activated microglia to Figure 7A and updated our flow gating\nstrategy in Supplementary Figure 5.\nSpecially, we evaluated Cy5-labeled hydroxyl dendrimer (Cy5-HD) signal in spinal cord myeloid subsets–\nspecifically resting microglia (CD11b+CD45lowTMEM119+), activated microglia (CD11b+CD45lowTMEM119-), and\ninfiltrating macrophages (CD11b+CD45hi)–across naïve, pre-EAE, vehicle-treated EAE, and treated EAE mice.\nTo distinguish resting from activated microglia, we used TMEM119, which is a validated homeostatic marker of\nmicroglia in the context of both EAE mouse models and human MS. Prior studies (Bennett et al., Proc Natl Acad\nSci 2016; Satoh et al., Neuropathology 2016; Ruan et al., Brain Behav Immun 2020) have shown that TMEM119\nis selectively expressed in homeostatic human and murine microglia but not in other brain-resident cells or in\ninfiltrating macrophages in the context of MS pathology. Specifically, these publications show that in the MOG\n35-\nmouse model of EAE that we use in our paper, and in MS post-mortem tissue, TMEM119 expression is\n55\ndownregulated in chronic active lesions. This supports its utility as a marker of homeostatic microglia in our\nstudies.\nIn naïve spinal cords, Cy5-HD+ myeloid cells were rare: 0.29% of infiltrating macrophages, 0.13% of activated\nmicroglia, and 1.02% of resting microglia. In contrast, EAE spinal cords showed a marked increase in the\nproportion of Cy5-HD+ myeloid cells: 33.4% of infiltrating macrophages and 16.3% of activated microglia, while\nonly 1.76% of resting microglia endocytosed Cy5-HD. These data indicate that 18F-FMD was selectively\nendocytosed by myeloid lineage cells and among microglia, there was >9-fold higher levels of Cy5-HD in those\nwith a pro-inflammatory phenotype compared to negligible levels in those with a resting phenotype.\nWe further examined changes in Cy5-HD+ myeloid subsets during early disease and after DMTs. In spinal cords\nfrom pre-EAE (prior to symptom onset) mice, Cy5-HD+ macrophages and activated microglia increased modestly\nto 1.32% (4.5-fold higher than naïve) and 0.66% (5-fold higher than naïve), respectively. Upon treatment with\nH74DS3M8 or FTY720, these populations decreased substantially: Cy5-HD+ infiltrating macrophages fell to\n7.46% and 1.70%, and activated microglia to 0.90% and 0.35%, respectively, representing up to 19-fold\nreductions in the proportion of infiltrating macrophages and 46-fold decrease in activated microglia compared to\nvehicle-treated EAE mice. Notably, the proportion of resting microglia remained relatively constant across\nconditions.\nThese treatment-induced reductions in Cy5-HD+ myeloid cells mirrored changes observed in 18F-FMD PET\nsignal, which showed a 2- to 2.5-fold decrease following DMT. Together, these results support the conclusion\nthat 18F-FMD uptake reflects both the abundance and activation state of CNS-resident and infiltrating myeloid\ncells, particularly those with a pro-inflammatory phenotype.\nSee below for the new graph we added to the main paper and supplementary and the text we inserted in the\nresults section of our manuscript describing these findings.\n“Flow cytometric analysis revealed profound immune remodeling in the CNS of EAE mice compared to\nnaïve controls. There was a dramatic reduction in the proportion of resting microglia\n(CD11b+CD45lowTMEM119+), which declined from 69.9% in naïve mice to 20.28% in EAE mice. In parallel,\nactivated microglia (CD11b+CD45lowTMEM119-) expanded significantly, from 0.13% to 20.84%, representing a\n>160-fold increase and indicating a marked phenotypic shift. This transformation was accompanied by\nwidespread infiltration of peripheral immune cells, including T cells (CD3+CD45+; 0.49% in naïve vs. 22.39% in\nEAE), infiltrating macrophages (CD11b+CD45hi; 0.88% vs. 37.53%), monocyte/DCs (CD11b+CD45hiLy6G-;\n1.01% vs. 17.42%), and neutrophils (CD11b+CD45hiLy6G+; 0.99% vs. 13.64%) (all comparisons p<0.0001; Fig.\n6b). Collectively, these data reveal a profound shift from a predominantly homeostatic microglial environment to\na highly inflammatory landscape, with infiltrating macrophages emerging as the most abundant immune cell type\nwithin the CNS of EAE mice.\nPrior to symptom onset, pre-EAE mice showed a substantial reduction in resting microglia (28.24% vs.\n69.86% in naïve; p<0.0001) in the spinal cord, alongside an early expansion of activated microglia (1.32% vs.\n0.13% in naïve), suggesting that innate immune activation precedes peripheral immune infiltration and may play\na key initiating role in EAE pathogenesis.”\n“Both treatments significantly reduced pathogenic myeloid populations in the CNS and peripheral blood of EAE\nmice, including activated microglia, infiltrating macrophages, and monocytes (p<0.01; Fig. 6b and c).”\n“Quantitative analysis revealed a significant increase in Cy5-HD+ myeloid populations, including infiltrating\nmacrophages, activated microglia, neutrophils, and monocyte/DCs, in both the spinal cord and peripheral blood\nof pre-EAE and symptomatic EAE mice compared to naïve controls (p<0.001, Fig 7). Among microglia, Cy5-HD\nuptake was more than 9-fold higher in cells with a pro-inflammatory, activated phenotype compared to resting\nmicroglia, which showed negligible uptake (Supplementary Fig. 7). T cells and B cells lacked Cy5-HD uptake,\nfurther supporting the tracer’s specificity for activated myeloid subsets.\nFollowing therapeutic intervention, Cy5-HD+ myeloid populations were markedly reduced by up to 19-fold\nin infiltrating macrophages and 46-fold in activated microglia, highlighting the ability of both treatments to\nsuppress pro-inflammatory innate immune activity (Fig. 7). These findings align with previously published\nfindings demonstrating the selective accumulation of hydroxyl dendrimers in activated myeloid cells under\ninflammatory conditions28–30. Notably, the spatial and temporal distribution of Cy5-HD+ cells observed by flow\ncytometry closely mirrored the trends captured by in vivo PET imaging, ex vivo gamma counting, and\nautoradiography. PET signal intensity decreased 2- to 2.5-fold following treatment, paralleling the observed\nreductions in Cy5-HD+ myeloid populations.”\nFIGURE 7. Cy5-labeled hydroxyl dendrimers are specifically endocytosed by activated myeloid cells in\nEAE mice and extent of uptake reflects PET imaging findings using 18F-FMD. Percentage of Cy5-HD+cells\nin the a spinal cord and b blood of naïve (n=11), pre-EAE (n=9), EAE with vehicle (n=16), EAE with H74DS3M8\n(n=9), and EAE with FTY720 mice (n=5). H74 = H74DS3M8. Statistical analyses were performed using one-way\nANOVA with Tukey’s multiple comparisons tests. + versus naïve mice, # versus H74DS3M8-treated EAE mice,\nand * denotes direct comparison of groups (+p<0.05, ++p<0.01, ++++p<0.0001, #p<0.05, ####p<0.0001, **p<0.01,\n****p<0.0001). Data are means ± SD.\nSUPPLEMENTARY FIGURE 7. Hydroxyl dendrimers are not endocytosed by resting microglia, T cells, or\nB cells in EAE mice. Cy5-HD+ cells as a proportion of all live single cells in a spinal cord and b blood. H74 =\nH74DS3M8. Data are means ± SD.\nSUPPLEMENTARY FIGURE 5. Flow cytometry gating strategy for immune cells. Representative flow\ncytometry plots from a vehicle-treated EAE mouse showing the sequential gating strategy used to identify Cy5-\nHD+ immune cell populations in the spinal cord. Live, single cells were gated and differentiated into infiltrating\nmacrophages (CD11b+CD45hi), resting microglia (CD11b+CD45lowTMEM119+), activated microglia\n(CD11b+CD45lowTMEM119-), T cells (CD45+CD3+), and B cells (CD45+CD19+). Myeloid cells (CD11b+CD45+)\nwere further subdivided into neutrophils (CD11b+CD45hiLy6G+) and monocytes/dendritic cells (Mo/DC;\nCD11b+CD45hiLy6G-). Cy5-HD signal was gated using fluorescence-minus-one (FMO) controls, and uptake was\nassessed across all defined immune cell subsets.\nComment 2:\nThe manuscript, in its current form, does not demonstrate the utility of this novel tracer in the context of\nprogressive MS. I would recommend that the authors include post-mortem data from patients with progressive\nMS, demonstrating the ability of Cy5-labeled hydroxyl dendrimers to distinguish lesions characterized by\nactivated microglia (e.g., smoldering lesions) from inactive lesions, and to identify regions of normal-appearing\nwhite matter with diffuse microglial activation, based on differential labeling intensity.\nResponse: Thank you for raising this consideration. We agree that validating PET tracers in human tissue is an\nimportant step. However, due to the biophysical mechanism of uptake of our PET tracer, via fluid-phase\nendocytosis by activated myeloid cells, in vitro evaluation in tissue sections is not possible. Our imaging agents,\nincluding Cy5- and 18F-labeled versions, are selectively internalized by living, metabolically active cells. This\nprocess does not occur in post-mortem tissue (loss of endocytic activity), making it technically not possible to\nassess tracer specificity or uptake patterns in vitro using fresh frozen or fixed specimens.\nThat said, we have extensively characterized our PET tracer in an EAE mouse model of MS with two different\ntherapies as described in this manuscript. Furthermore, we are actively investigating the question of clinical utility\nin patients with progressive MS in our ongoing phase 1/2 clinical trial (NCT05395624). Preliminary data have\nrecently been presented at the Society of Nuclear Medicine and Molecular Imaging 2025 (Moradi et al., J Nuc\nMed 2024, reference 77). Additional human data will be reported in detail in future publications.\n[77] Moradi, F. et al. [18F]Flurimedrimer, a Novel Nanomedicine Radiotracer, Selectively Targeting Activated\nMicroglia in Human Brain. J. Nucl. Med. 66, 251092 (2025).\nComment 3:\nThe results presented do not support the claim that this tracer enables early diagnosis of MS, reduces\nmisdiagnosis, or aids in treatment selection in clinical practice. With the new diagnostic criteria for MS introduced\nat the end of 2024, structural MRI has shown the ability to diagnose MS at an exceptionally early stage, even\nbefore the onset of clinical symptoms, thereby allowing for the initiation of disease-modifying therapies prior to\nsymptom manifestation. The evidence showing that 18F-FMD uptake increases before symptom onset in the\nEAE model does not indicate that the tracer is sufficiently sensitive or specific to diagnose MS earlier or reduce\nmisdiagnoses in patients. All references to these claims should be removed from the manuscript.\nResponse: Thank you for this important point. We have now removed any mention of our tracer as a potential\ntool for earlier MS diagnosis. Instead, we have added new information about the revised diagnostic criteria for\nMS and the potential of our PET tracer to provide complementary insights to the current gold standard.\nThe following text has been added to the discussion section:\n“These preclinical results highlight the potential utility of 18F-FMD in a clinical setting, particularly as the field\nmoves toward earlier diagnosis of MS. The revised McDonald criteria introduced at the 40th Congress of the\nEuropean Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) in 2024 have expanded the\nrole of clinical and imaging parameters to facilitate more timely diagnoses using structural MRI61. However,\ndiagnostic uncertainty remains a challenge in cases with ambiguous radiographic or clinical presentations.\nMoreover, current standard-of-care imaging tools lack the sensitivity and specificity to capture subtle changes in\nimmune activity, limiting their ability to guide treatment selection or assess responses to emerging therapies\ntargeting neuroinflammation.\nIn this context, PET imaging offers a complementary approach by enabling cell-specific, real-time\nvisualization of innate immune activation. We demonstrated that 18F-FMD imaging enabled monitoring of\ntherapeutic responses to both FTY720, an FDA-approved S1P receptor modulator, and H74DS3M8, a novel\nCSF1R-targeting dendranib, in EAE mice. In both treatment arms, PET signal in the CNS was significantly\nreduced compared to vehicle-treated controls, with findings confirmed by flow cytometry and ex vivo analyses.\nThese results demonstrate that 18F-FMD PET provides a sensitive and non-invasive method for monitoring innate\nimmune dynamics and therapeutic efficacy in vivo. As such, 18F-FMD PET has the potential to serve as a first-\nin-class imaging biomarker for quantifying activated myeloid cells and enabling precise monitoring of myeloid-\ntargeting treatments in both preclinical and clinical settings.”\nComment 4:\nEAE is an acute inflammatory disease model, not one that reflects the chronic progression seen in MS, which is\ndefined as the gradual and irreversible accumulation of clinical disability driven by neurodegeneration and axonal\nloss developing over time. For this reason, I suggest the authors avoid referring to EAE as a model of chronic or\nprogressive MS. The term “disease progression” should be replaced with “disease severity” in their experiment\ndescriptions.\nResponse: Thank you for this comment. We have replaced disease progression with disease severity\nthroughout the introduction and results section.\n“We also illustrate that 18F-FMD can sensitively capture the therapeutic effects of Food and Drug Administration\n(FDA)-approved disease-modifying MS drug fingolimod (FTY720) and a novel CSF1R dendranib (H74DS3M8),\nboth of which suppressed immune cell activation and attenuated disease severity.”\n“Novel CSF1R dendranib is effective in reducing EAE disease severity”\n“Disease severity was compared to vehicle-treated controls (4.4% [weight per volume] trehalose dihydrate/10mM\nhistidine via i.p. injection) and fingolimod (FTY720)-treated mice (3 mg/kg via oral gavage; Fig. 4a).”\n“Both treatments effectively reduced disease severity, with average scores reduced to 0.6 ± 0.9 (H74DS3M8)\nand 0.4 ± 0.6 (FTY720), compared to 2.7 ± 0.9 in vehicle-treated controls by day 15 (p<0.0001; Fig. 4b and\nSupplementary Table 5).”\n“Immunohistochemistry staining for CD68+ cells in the same PET imaging cohort confirmed negligible presence\nof activated myeloid cells in H74DS3M8- and FTY720-treated mice, underscoring their efficacy in modulating\nneuroinflammation and potentially altering disease severity (Fig. 5c and Supplementary Fig. 4).”\nComment 5:\nI recommend that the authors revise the abstract, introduction, and discussion sections to avoid referring to the\nneed for novel biomarkers to enable early diagnosis, reduce misdiagnoses, and guide DMT selection. Instead,\nthe emphasis should be on the crucial need for non-invasive biomarkers to specifically measure activated\nmicroglia, whose role is critical in neurodegeneration in chronic MS. This need is particularly pressing as new\ntreatments targeting innate immune cells, such as Tolebrutinib (Hercules clinical trial, ECTRIMS 2024), are\nemerging and showing promising results in slowing clinical progression in non-active secondary progressive MS.\nNovel tracers such as 18F-FMD could be employed to stratify patients for clinical trials of novel microglia-\ntargeting treatments to monitor their effects, and to perform studies to explore further the role of innate immune\ncells in the physiopathology of neurodegeneration in MS.\nIn addition, I recommend the authors update their references, particularly those related to clinical forms of MS\nand alternative PET tracers to TSPO. Reference 28, which cites an unpublished manuscript, should be replaced\nwith more appropriate references.\nResponse: Thank you for this very helpful suggestion. In response, we have revised the abstract, introduction,\nand discussion to shift the emphasis away from early diagnosis and misdiagnosis, and instead highlight the\nurgent need for non-invasive biomarkers that specifically measure activated microglia, particularly in the context\nof neurodegeneration in chronic MS. We now emphasize the translational relevance of ¹⁸F-FMD as a tool for\nevaluating emerging therapies targeting innate immune cells.\nWe have revised the following sentences in our abstract:\n“These findings highlight the potential of 18F-FMD PET for specific, real-time monitoring of innate immune\nresponses. Since we have translated 18F-FMD for clinical imaging, it holds promise for stratifying patients for\nclinical trials of myeloid-targeted therapies, evaluating treatment efficacy, and advancing our understanding of\nthe innate immune contribution to neurodegeneration in MS, paving the way toward more personalized, targeted\ntreatment strategies.”\nWe have modified the second introductory paragraph so that it now includes the following text:\n“Compounding this challenge is the lack of specific in vivo imaging biomarkers for activated myeloid cells, which\nlimits our ability to fully elucidate their role in neurodegeneration, stratify patients for trials of emerging microglia-\ntargeting treatments, or monitor therapeutic responses.”\nWe have added the following paragraphs to the discussion section:\n“These preclinical results highlight the potential utility of 18F-FMD in a clinical setting, particularly as the field\nmoves toward earlier diagnosis of MS. The revised McDonald criteria introduced at the 40th Congress of the\nEuropean Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) in 2024 have expanded the\nrole of clinical and imaging parameters to facilitate more timely diagnoses using structural MRI61. However,\ndiagnostic uncertainty remains a challenge in cases with ambiguous radiographic or clinical presentations.\nMoreover, current standard-of-care imaging tools lack the sensitivity and specificity to capture subtle changes in\nimmune activity, limiting their ability to guide treatment selection or assess responses to emerging therapies\ntargeting neuroinflammation.\nIn this context, PET imaging offers a complementary approach by enabling cell-specific, real-time\nvisualization of innate immune activation. We demonstrated that 18F-FMD imaging enabled monitoring of\ntherapeutic responses to both FTY720, an FDA-approved S1P receptor modulator, and H74DS3M8, a novel\nCSF1R-targeting dendranib, in EAE mice. In both treatment arms, PET signal in the CNS was significantly\nreduced compared to vehicle-treated controls, with findings confirmed by flow cytometry and ex vivo analyses.\nThese results demonstrate that 18F-FMD PET provides a sensitive and non-invasive method for monitoring innate\nimmune dynamics and therapeutic efficacy in vivo. As such, 18F-FMD PET has the potential to serve as a first-\nin-class imaging biomarker for quantifying activated myeloid cells and enabling precise monitoring of myeloid-\ntargeting treatments in both preclinical and clinical settings.”\n“Building on this translational potential, 18F-FMD could be integrated into trials of emerging therapies such as\ntolebrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor evaluated in the HERCULES trial, which has shown\npromise in slowing clinical progression in non-relapsing SPMS72. By enabling quantitative, real-time monitoring\nof myeloid cell activity, 18F-FMD PET could support pharmacodynamic assessments and facilitate patient\nstratification. Beyond therapeutic response monitoring, this tracer also offers a unique opportunity to deepen our\nunderstanding of the in vivo role of innate immune cells in neuroinflammation, thereby supporting both drug\ndevelopment and biomarker-guided clinical decision making.”\nWe have also replaced reference 28 (now reference 26) with a published manuscript in ACS Chemical\nNeuroscience on the clinical radiosynthesis and translation of 18F-FMD (previously known as 18F-OP-801).\n[26] Jackson, I. M. et al. Clinical Radiosynthesis and Translation of [18F]OP-801: A Novel Radiotracer for Imaging\nReactive Microglia and Macrophages. ACS Chem. Neurosci. 14, 2416–2424 (2023).\n-------------------------------------------------\nReviewer #2 (Remarks to the Author):\nIn the present study, Kuo & Carlson et al. provide evidence of a PET imaging method by the use of radiolabeled\ndendrimer, namely, the 18F-FMD, to non-invasively track innate immune activation in EAE mice. The authors\nfurther investigate the potential of the method following administration of two immunomodulatory treatments in\nEAE. This is an original work with added value in the field, as it poses implication for imaging modalities with a\nfocus on the CNS innate immune system, a key contributor in CNS immune-mediated pathologies, as in Multiple\nSclerosis (MS). Overall the methodology is sound and reported in detail. Few issues arise:\nComment 1:\nThe authors used the EAE model induced by immunization by MOG in CFA and they state that they used the\nEAE model as a model of chronic/progressive MS. However, the MOG-EAE mouse model is not characterized\nby clinical progression rather, the mice exhibit an acute stage of disease (approximately 15-20 days post-\ninduction) resembling an MS-like clinical relapse, usually followed by a chronic phase (established approximately\n50 days post-induction), upon which the mice tend to exhibit a so-called “plateau” in terms of clinical disability\n(though in the absence of progression) [10.3791/51275]. In the present study, to my understanding, time-points\nof euthanization were 8- and 15-days post-immunization, therefore, the pre-clinical and the acute phase. In this\nrespect, in my opinion, the authors should rephrase page 5 lines 102-103: “In this study, we evaluated the ability\nof 18F-FMD to detect activated myeloid cells non-invasively in an experimental autoimmune encephalomyelitis\n(EAE) mouse model of chronic/progressive MS”. The interpretation of the results should, in my opinion, take into\naccount these limitations related to the experimental model used. Similarly, the term “progression”, in my opinion,\nshould be replaced by, for example, by “deterioration” or “disability”, in order to better depict the EAE pathology\nand not to be interpreted as corresponding to the MS-related progression.\nResponse: Thank you for raising these important points. We have removed any mentions of using this EAE\nmodel as a representation for chronic/progressive MS and modified the representation of our imaging timepoints\nto include preclinical and acute phase of disease. We have removed all mentions of disease progression when\nreferring to the EAE pathology. We have also taken the limitations mentioned into account for interpretation of\nour data and only used words like deterioration or disability rather than progression to avoid misinterpretation of\nEAE pathology and MS-related progression.\nWe have modified the introduction to remove any reference to chronic/progressive MS:\n“In this study, we evaluated the ability of 18F-FMD to non-invasively detect activated myeloid cells in an\nexperimental autoimmune encephalomyelitis (EAE) mouse model induced by MOG at both preclinical and\n35-55\nacute stage of disease (Fig. 1).”\nWe have added the following text describing the limitations of the EAE model to the discussion section:\n“While animal models are invaluable for understanding disease mechanisms and evaluating diagnostic and\ntherapeutic approaches, they do not fully recapitulate the clinical progression observed in human MS. The\ninduction of EAE via MOG peptide and Complete Freund’s adjuvant (CFA) is an artificial process that does\n35-55\nnot naturally occur in human MS. CFA triggers strong peripheral innate immune activation to ensure antigen\npresentation, which in turn alters CNS-resident immune cell behavior76. This artificial immune environment likely\nlimits how directly findings can translate to patients. In addition, EAE models are associated with BBB disruption,\nwhich may enable some degree of tracer access to CNS parenchyma. However, since the selective uptake and\nretention of 18F-FMD is driven by fluid-phase endocytosis by activated myeloid cells, the tracer is not retained in\nresting or non-activated immune cells. In the absence of high endocytic activity, hydroxyl dendrimers do not\nexhibit sustained accumulation. Therefore, even in the context of increased BBB permeability, nonspecific\nleakage alone does not account for the tracer signal observed in our study.\nDespite these limitations, EAE models remain a powerful and well-characterized preclinical platform for\nstudying MS-like neuroinflammation. In our study, we leveraged the EAE model to evaluate the in vivo\nperformance of 18F-FMD and its ability to selectively monitor activated myeloid cells. While this model does not\ncapture the full spectrum of disease progression seen in clinical MS, it provides a robust system for assessing\nsystemic tracer distribution and uptake by activated myeloid populations within the CNS and throughout the\nbody. These preclinical findings serve as a foundation for translation to human studies.”\nWe have also modified this discussion paragraph to discuss the ongoing clinical trial for our tracer:\n“Notably, our team has recently translated 18F-FMD for clinical use. It is currently being evaluated in a phase 1/2\nclinical trial (NCT05395624)77 to assess its diagnostic performance across MS subtypes and other\nneuroinflammatory diseases26,78. This trial aims to validate the sensitivity and specificity of 18F-FMD for detection\nof neuroinflammation in patients and assess its potential as a tool for disease monitoring, therapeutic\nassessment, and patient stratification.”\nComment 2:\nMoreover, the authors, as stated in page 5 lines 111-112, “assessed the uptake of 18F-FMD using PET imaging\nin EAE mice to confirm its sensitivity for detecting activated myeloid cells at both presymptomatic and\nsymptomatic disease”. However, a difference between MOG-EAE experimental model and MS is the induction\nof EAE via MOG-CFA immunization, a procedure that does not naturally occur upon MS pathogenesis. Innate\nimmune cell activation upon MOG-EAE induction is strongly triggered by CFA in the periphery in order to secure\neffective antigen-presentation, and this procedure has been shown to actively influence the activation status of\nCNS innate cells as well [10.1002/eji.200939568]. It is, therefore, likely, that the CNS innate immune cells\nactivation profile in the context of MS differs from the MOG-EAE CNS innate immune cells’ activation profile and\nthis difference may influence the tracer uptake. In this respect, the potential of the method to detect innate\nimmune cell activation in the CNS at pre-clinical stage, as well as across disease stages and disease activity\nstatus in MS requires further evaluation, in the clinical setting. Please add a respective comment.\nResponse: The authors thank the reviewer for this helpful comment. As mentioned in our response to the\nprevious comment (comment 1), we have modified the discussion section to discuss the limitations:\n“While animal models are invaluable for understanding disease mechanisms and evaluating diagnostic and\ntherapeutic approaches, they do not fully recapitulate the clinical progression observed in human MS. The\ninduction of EAE via MOG peptide and Complete Freund’s adjuvant (CFA) is an artificial process that does\n35-55\nnot naturally occur in human MS. CFA triggers strong peripheral innate immune activation to ensure antigen\npresentation, which in turn alters CNS-resident immune cell behavior76. This artificial immune environment likely\nlimits how directly findings can translate to patients. In addition, EAE models are associated with BBB disruption,\nwhich may enable some degree of tracer access to CNS parenchyma. However, since the selective uptake and\nretention of 18F-FMD is driven by fluid-phase endocytosis by activated myeloid cells, the tracer is not retained in\nresting or non-activated immune cells. In the absence of high endocytic activity, hydroxyl dendrimers do not\nexhibit sustained accumulation. Therefore, even in the context of increased BBB permeability, nonspecific\nleakage alone does not account for the tracer signal observed in our study.”\nComment 3:\nFurthermore, the authors used flow cytometry by the use of a fluorescent analog of the tracer, in order to show\nthe potential of 18F-FMD PET to detect activated myeloid cells in the CNS. However, although they used well-\nestablished markers for innate immune cells, such as CD11b, CD11c, CD45low expression levels, these markers\nare not selective for specific CNS innate immune cell populations, as is microglia, and they do not assess the\ninnate immune cells’ activation status. The use of a more specific marker in order to study cell specific\npopulations, for instance, Tmem 119 for microglia, may be more suitable in this respect. Of note, the CNS innate\nimmune cell populations exhibit high complexity and pose challenge in terms of phenotypic characterization\n[10.1016/j.celrep.2024.114250]. Taking into account the potential novelty of the method and the added value in\nlabelling activated CNS immune cells, a more detailed phenotypic and/or transcriptional characterization of the\nlabeled CNS immune cells populations, following, for instance, cell sorting and further downstream\ninvestigations, is of interest. Such investigation, in my opinion, would be of importance, in order to better establish\nthe potential of the method to detect activated myeloid cells in the CNS, and to further characterize these cellular\npopulations, thus enhancing the mechanistic insight and supporting the translational value of the study. Please\nadd a respective comment.\nResponse: Thank you for this thoughtful and constructive comment. Please see our above response to\nReviewer 1, comment 1 regarding the inclusion of TMEM119 to differentiate resting from activated microglia. To\nfurther address your comment, we performed additional flow cytometry studies to characterize the functional\npolarization of microglia/macrophages in vehicle- and therapy-treated EAE mice. Within the population of\nactivated microglia/macrophages (CD11b+CD45+TMEM119-), we gated for CD86+ cells to identify M1-like\nmicroglia/macrophages and CD86-CD163+CD206+ cells to identify M2-like microglia/macrophages. CD86 is a\nwell-established marker of pro-inflammatory (M1) polarization and is commonly used to define M1-activated\nmicroglia/macrophages in both human and murine studies (Oshi et al., Scientific Reports 2020, Furgiuele et al.,\nInt J Mol Sci 2022, and Bertani et al., Scientific Reports 2017). CD163 and CD206 are widely recognized markers\nfor anti-inflammatory (M2) polarization and are frequently used to characterize reparative or regulatory myeloid\nphenotypes in CNS inflammation (Hu et al., Oncotarget 2017 and Xu et al., Oncoimmunology 2019).\nThese additional data are now included in the revised manuscript (Supplementary Figure 6 - new; modified\nFigure 7 and Supplementary Figures 5 and 7 in our response to Reviewer 1, comment 1) and provide a more\ngranular phenotypic characterization of the CNS myeloid populations labeled by Cy5-HD and, by extension,\nimaged with 18F-FMD PET. We have also added a respective paragraph in the discussion section addressing\nthe complexity and challenges of CNS innate immune cell phenotypic characterization, including the limitations\nof the M1/M2 classification framework. While this dichotomy is thought to be an oversimplification, particularly in\nthe context of neuroinflammation, it highlights the critical need for imaging tools that can resolve the functional\nheterogeneity of immune cells in vivo.\nNew relevant text has also been added to the results section:\n“Flow cytometric analysis revealed profound immune remodeling in the CNS of EAE mice compared to naïve\ncontrols. There was a dramatic reduction in the proportion of resting microglia (CD11b+CD45lowTMEM119+),\nwhich declined from 69.9% in naïve mice to 20.28% in EAE mice. In parallel, activated microglia\n(CD11b+CD45lowTMEM119-) expanded significantly, from 0.13% to 20.84%, representing a >160-fold increase\nand indicating a marked phenotypic shift. This transformation was accompanied by widespread infiltration of\nperipheral immune cells, including T cells (CD3+CD45+; 0.49% in naïve vs. 22.39% in EAE), infiltrating\nmacrophages (CD11b+CD45hi; 0.88% vs. 37.53%), monocyte/DCs (CD11b+CD45hiLy6G-; 1.01% vs. 17.42%),\nand neutrophils (CD11b+CD45hiLy6G+; 0.99% vs. 13.64%) (all comparisons p<0.0001; Fig. 6b). Collectively,\nthese data reveal a profound shift from a predominantly homeostatic microglial environment to a highly\ninflammatory landscape, with infiltrating macrophages emerging as the most abundant immune cell type within\nthe CNS of EAE mice.”\n“To further characterize the functional impact of treatment, we analyzed macrophage polarization states. Both\ntherapies significantly decreased the proportion of pro-inflammatory M1-like microglia/macrophages\n(CD11b+CD45+CD86+; p<0.0001; Supplementary Fig. 6).”\nThe following text has also been added to the discussion section:\n“These complementary mechanisms underscore the importance of understanding the functional plasticity of\nmyeloid cells–an area of growing interest in MS research. While macrophages were traditionally categorized into\nM1 (pro-inflammatory) and M2 (anti-inflammatory) subsets, recent studies suggest that this dichotomy is overly\nsimplistic. Histological analyses of active MS lesions reveal that up to 70% of macrophages co-express both M1\nand M2 markers, reflecting intermediate or hybrid activation states shaped by the surrounding cytokine milieu,\noxidative stress, and tissue-derived signals 37,66–68. This context-dependent activation is exemplified by\nTMEM119, a marker commonly used to identify homeostatic microglia. In the MOG EAE model, TMEM119\n35-55\nexpression is significantly downregulated in the spinal cord, mirroring observations in postmortem MS tissue,\nwhere TMEM119 immunoreactivity is lost in white matter lesions but preserved in gray matter69–71. These shifts\nreflect the complex, region-specific immune landscape in MS and emphasize the need for tools like 18F-FMD\nPET that can capture the dynamic and spatially heterogenous nature of myeloid activation in vivo. Such a tool\nholds great promise not only for understanding disease biology but also for enhancing clinical trial design.”\nSUPPLEMENTARY FIGURE 6. Treatment with CSF1R dendranib and fingolimod affects activated\nmicroglia and macrophage phenotypes in EAE mice. a Summarized proportion of M1 and M2\nmicroglia/macrophages in the spinal cord of naïve (n=5), pre-EAE (n=5), EAE with vehicle (n=5), EAE with\nH74DS3M8 (n=5), and EAE with FTY720 mice (n=5). b Flow cytometry gating strategy in the spinal cord of a\nrepresentative EAE mouse. Live single cells were gated for TMEM119- microglia/macrophages, which were\nfurther differentiated into M1-like microglia/macrophages (CD11b+CD45+CD86+) and M2-like\nmicroglia/macrophages (CD11b+CD45+CD86-CD163+CD206+). Cy5-HD uptake was investigated on both\nmicroglia/macrophage phenotypes. Statistical analyses were performed using one-way ANOVA with Tukey’s\nmultiple comparisons tests. + versus naïve mice, # versus H74DS3M8-treated EAE mice, and * denotes direct\ncomparison of groups ++++p<0.0001, ####p<0.0001, ****p<0.0001). H74 = H74DS3M8. Data are means ± SD.\n-------------------------------------------------\nReviewer #3 (Remarks to the Author):\nGeneral comments:\nI've reviewed the manuscript titled \"Radiolabeled dendrimer non-invasively tracks innate immune activation in\nmultiple sclerosis mice after immunomodulatory therapy.\" This is an interesting study exploring the use of a novel\nPET tracer (18F-FMD) for detecting activated myeloid cells in an experimental autoimmune encephalomyelitis\n(EAE) mouse model of multiple sclerosis.\nThe study demonstrates that 18F-FMD can detect neuroinflammation even before clinical symptoms appear in\nthe EAE model. Multiple validation methods (PET imaging, ex vivo gamma counting, autoradiography, flow\ncytometry) strengthen the findings.\nThe comparison of two different therapies (H74DS3M8 and fingolimod) provides insights into different\nmechanisms of action.\nThe selectivity of the tracer for activated myeloid cells is well-documented.\nHowever, because of the reasons below, I would recommend rejection of this paper:\nComment 1:\nMethodological concerns: The study lacks appropriate controls for the specificity of 18F-FMD uptake. While flow\ncytometry with Cy5-HD is used as a surrogate, direct validation that 18F-FMD and Cy5-HD target identical cell\npopulations was not adequately demonstrated. The authors should have included additional control experiments\nto rule out non-specific binding.\nResponse: Thank you for this important comment. We apologize for not clearly conveying the rationale for using\nCy5-HD as a surrogate for ¹⁸F-FMD. We have now added text to the discussion section clarifying that neither\n¹⁸F-FMD nor Cy5-HD binds to specific cell surface markers on activated myeloid cells. Instead, their uptake is\ndriven by fluid-phase endocytosis, a well-established biophysical mechanism by which generation 4 hydroxyl\ndendrimers are selectively internalized by activated myeloid cells due to their heightened endocytic activity. This\nprocess doesn't require specific receptor binding. Prior studies have demonstrated that payload conjugation\n(such as Cy5 or ¹⁸F) does not alter dendrimer structure, surface charge, or uptake behavior (Lesniak et al., Mol\nPharm 2013; Mishra et al., ACS Nano 2014; Zhang et al., J Neuroinflammation 2016).\nWe have added the following text to the discussion section:\n“To investigate the immune cells that endocytose 18F-FMD, we used Cy5-HD as a fluorescent surrogate\nfor the PET tracer. Cy5-HD mirrors the structure and function of 18F-FMD. Both compounds lack affinity for\nspecific cell surface markers and are internalized via fluid-phase endocytosis, a non-receptor-mediated\nmechanism that is increased in activated myeloid cells during neuroinflammation. This uptake mechanism\nensures that tracer localization reflects active cellular processes rather than passive diffusion or nonspecific\nretention. Once internalized, endocytosed dendrimers are trafficked to lysosomes and stably retained, enabling\nprolonged intracellular signal55–57. Importantly, the molecular weights of 18F-FMD (14.462 kDa) and Cy5-HD\n(15.084 kDa) are nearly identical, with the shared dendrimer scaffold (14.277 kDa) comprising the majority of\nthe mass. Each construct carries only a single 18F or Cy5 payload, which does not alter the core structure,\nsurface charge, hydrophilicity, or biodistribution of the dendrimer43,58–60. This molecular consistency supports the\nuse of Cy5-HD as a reliable surrogate for 18F-FMD in mechanistic and cell-type validation studies.”\nComment 2:\nInsufficient statistical power: The sample sizes in several experimental groups (n=4-7 per group) appear\nunderpowered for the conclusions being drawn, particularly in the treatment response studies. This raises\nquestions about the reproducibility of the findings.\nResponse: Thank you for this important comment. We fully agree that statistical power and reproducibility are\ncritical, particularly in treatment response studies. We also want to clarify that both PET imaging with 18F-FMD\nand treatment studies presented in this manuscript were independently reproduced by two different graduate\nstudents at separate times. All datasets were analyzed by two blinded people for enhanced rigor. In both cases,\nthe results were statistically significant and highly consistent, supporting the reproducibility and robustness of\nour findings.\nTo further address concerns about statistical power, we have performed additional flow cytometry studies and\nincorporated data from mice that were initially excluded due to suboptimal perfusion. Importantly, hydroxyl\ndendrimers such as 18F-FMD and Cy5-HD are not retained in blood for long, and the data from these mice were\nnot significantly different from the rest of the group. Including them helped us increase the numbers to n≥5\nanimals per group across all treatment arms.\nThis sample size was determined based on power analyses, the reproducibility of data, and in compliance with\nour APLAC (Administrative Panel on Laboratory Animal Care) guidelines, which mandate the use of the minimum\nnumber of animals necessary to obtain statistically valid and biologically meaningful outcomes.\nWe have revised all relevant figures and figure legends to clearly indicate the final sample sizes used in each\nexperiment.\n“FIGURE 6. Flow cytometry reveals increased numbers of immune cells in spinal cord and blood from\nEAE mice and that immune expansion and infiltration is significantly reduced following treatment with\nH74DS3M8 or FTY720. a EAE was induced in C57BL/6J mice, and a group of them was treated with either\nH74DS3M8, FTY720, or vehicle. Blood was collected and spinal cords were harvested for single-cell flow\ncytometry at different disease states. Summarized proportion of infiltrating macrophages, resting microglia,\nmonocytes/dendritic cells, neutrophils, T cells, and B cells in the b spinal cord and c blood of naïve (n=5-11),\npre-EAE (n=5-9), EAE with vehicle (n=5-16), EAE with H74DS3M8 (n=5-9), and EAE with FTY720 mice (n=5).\nH74 = H74DS3M8. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple\ncomparisons tests. + versus naïve mice, # versus H74DS3M8-treated EAE mice, and * denotes direct\ncomparison of groups (+p<0.05, ++p<0.01, +++p<0.001, ++++p<0.0001, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001,\n*p<0.05, **p<0.01, ****p<0.0001). Data are means ± SD.”\n“FIGURE 7. Cy5-labeled hydroxyl dendrimers are specifically endocytosed by activated myeloid cells in\nEAE mice and extent of uptake reflects PET imaging findings using 18F-FMD. Percentage of Cy5-HD+ cells\nin a spinal cord and b blood of naïve (n=11), pre-EAE (n=9), EAE with vehicle (n=5-16), EAE with H74DS3M8\n(n=5-8), and EAE with FTY720 mice (n=5). H74 = H74DS3M8. Statistical analyses were performed using one-\nway ANOVA with Tukey’s multiple comparisons tests. + versus naïve mice, # versus H74DS3M8-treated EAE\nmice, and * denotes direct comparison of groups (+p<0.05, ++p<0.01, ++++p<0.0001, #p<0.05, ####p<0.0001,\n**p<0.01, ****p<0.0001). Data are means ± SD.”\nComment 3:\nLimited translational relevance: While the EAE mouse model is well-established, it primarily recapitulates aspects\nof relapsing-remitting MS. The manuscript claims relevance for progressive forms of MS but provides insufficient\nevidence supporting this assertion. The direct applicability to human MS patients remains speculative.\nResponse: Thank you for this comment. Please refer to our response to Reviewer 2, comment 1 for full detail\non the limitations of the EAE model and the translational relevance of our tracer.\nWe agree that the MOG EAE model does not recapitulate progressive MS, and we have removed all mentions\n35-55\nsuggesting that it models the progressive form of the disease. In this study, our goal was not to replicate the full\nclinical spectrum of MS, but rather to evaluate the cellular specificity and uptake mechanism of our PET tracer\nin vivo. We demonstrated that ¹⁸F-FMD is taken up by activated myeloid cells—a key immune population\nimplicated across multiple stages of MS. Importantly, since we have translated this tracer for clinical use, we are\ncurrently evaluating its performance in patients with progressive MS (clinical trial NCT05395624). Preliminary\ndata from the clinical trial have recently been presented at the Society of Nuclear Medicine and Molecular\nImaging 2025 (Moradi et al., J Nuc Med 2024, reference 77). Additional human data will be reported in detail in\nfuture publications.\n[77] Moradi, F. et al. [18F]Flurimedrimer, a Novel Nanomedicine Radiotracer, Selectively Targeting Activated\nMicroglia in Human Brain. J. Nucl. Med. 66, 251092 (2025).\nComment 4:\nConfounding factors not addressed: The potential impact of BBB disruption on tracer uptake (common in EAE\nmodels) is not sufficiently addressed. The authors do not adequately demonstrate that 18F-FMD signal is\nspecifically related to activated myeloid cells rather than passive accumulation due to increased vascular\npermeability.\nResponse: We thank the reviewer for raising this important point. We agree that the EAE model used in our\nstudy is characterized by active inflammation and associated BBB dysfunction. We have added this as a\nlimitation in our discussion section. The selective uptake and retention of ¹⁸F-FMD is by a distinct biophysical\nmechanism: fluid-phase endocytosis by activated myeloid cells. Hydroxyl dendrimers such as 18F-FMD are\ninternalized through non-receptor-mediated endocytosis, a process that is markedly upregulated in activated\nmicroglia and macrophages during neuroinflammation. Importantly, this tracer is not retained intracellularly in\nresting or non-activated immune cells. In the absence of high endocytic activity, dendrimers do not exhibit\nsustained accumulation. Therefore, even in the context of BBB permeability, non-specific leakage alone does\nnot account for the tracer signal observed in our study. We have added additional data to Figure 7 and\nSupplementary Figure 7 in the manuscript (please see the figures in our response to Reviewer 1, comment 1) to\nshow the selective uptake by activated microglia and infiltrating macrophages but not by resting microglia.\nFurthermore, our ex vivo autoradiography data (Figure 3b) demonstrate that 18F-FMD signal spatially colocalize\nwith regions of increased immune cell infiltration as confirmed by H&E and CD68 (a marker of activated myeloid\ncells) IHC staining. Regions without activated immune cells–despite being within the CNS and theoretically\nexposed to systemic tracer–do not show appreciable uptake. This strongly supports the conclusion that the PET\nsignal reflects specific uptake by activated myeloid cells rather than non-specific accumulation due to vascular\npermeability alone.\nCollectively, these findings are consistent with prior preclinical studies demonstrating selective uptake and\nlysosomal retention of hydroxyl dendrimers in activated, but not resting, myeloid cells (Thomas et al.,\nBiomacromolecules 2009; 10(12):3207-14, Alnasser et al., Molecules 2018; 23(5):1025, and Albertazzi et al.,\nMolecular Pharmaceutics 2010; 7 (3), 680-688). We have clarified this mechanism in the revised discussion\nsection:\n“To investigate the immune cells that endocytose 18F-FMD, we used Cy5-HD as a fluorescent surrogate for the\nPET tracer. Cy5-HD mirrors the structure and function of 18F-FMD. Both compounds lack affinity for specific cell\nsurface markers and are internalized via fluid-phase endocytosis, a non-receptor-mediated mechanism that is\nincreased in activated myeloid cells during neuroinflammation. This uptake mechanism ensures that tracer\nlocalization reflects active cellular processes rather than passive diffusion or nonspecific retention. Once\ninternalized, endocytosed dendrimers are trafficked to lysosomes and stably retained, enabling prolonged\nintracellular signal55–57. Importantly, the molecular weights of 18F-FMD (14.462 kDa) and Cy5-HD (15.084 kDa)\nare nearly identical, with the shared dendrimer scaffold (14.277 kDa) comprising the majority of the mass. Each\nconstruct carries only a single 18F or Cy5 payload, which does not alter the core structure, surface charge,\nhydrophilicity, or biodistribution of the dendrimer43,58–60. This molecular consistency supports the use of Cy5-HD\nas a reliable surrogate for 18F-FMD in mechanistic and cell-type validation studies.”\nWe have also added a paragraph in the discussion section to discuss BBB disruption in this EAE model and how\nEAE models can be useful for studying MS-like neuroinflammation.\n“In addition, EAE models are associated with BBB disruption, which may enable some degree of tracer access\nto CNS parenchyma. However, since the selective uptake and retention of 18F-FMD is driven by fluid-phase\nendocytosis by activated myeloid cells, the tracer is not retained in resting or non-activated immune cells. In the\nabsence of high endocytic activity, hydroxyl dendrimers do not exhibit sustained accumulation. Therefore, even\nin the context of increased BBB permeability, nonspecific leakage alone does not account for the tracer signal\nobserved in our study.\nDespite these limitations, EAE models remain a powerful and well-characterized preclinical platform for\nstudying MS-like neuroinflammation. In our study, we leveraged the EAE model to evaluate the in vivo\nperformance of 18F-FMD and its ability to selectively monitor activated myeloid cells. While this model does not\ncapture the full spectrum of disease progression seen in clinical MS, it provides a robust system for assessing\nsystemic tracer distribution and uptake by activated myeloid populations within the CNS and throughout the\nbody. These preclinical findings serve as a foundation for translation to human studies.”\nComment 5:\nIncomplete characterization of H74DS3M8: The novel CSF1R dendranib is inadequately characterized\npharmacologically. Basic properties such as plasma half-life, brain penetrance, and selectivity profiling are\nmissing, making it difficult to interpret the therapeutic response data.\nResponse: Thank you for raising this important point and great suggestion of including pharmacokinetics of\nH74DS3M8. We have performed additional studies to further characterize the pharmacological profile of\nH74DS3M8. These new data have been incorporated into the revised manuscript, with corresponding details\nadded to the methods section and Supplementary Figures 1 and 2 and Supplementary Tables 3 and 4.\nTable S3\nKinase\nCompound CSF1R ABL1 c-Kit c-Src EGFR PDGFRβ\nH74DS3M8 0.273 0.096 0.466 <3.81E-3 427 15.250\nSUPPLEMENTARY TABLE 3: Kinase inhibition profile of H74DS3M8. IC values (nM) determined in a kinase\n50\npanel assay showed that H74DS3M8 has sub-nanomolar potency against CSF1R, ABL1, c-Kit, and c-Src, with\nsignificantly reduced off-target activity against EGFR and PDGFRβ. n=3-4 per group.\nKinase profiling of H74DS3M8 demonstrates sub-nanomolar potency against CSF1R (IC = 0.273 nM), ABL1\n50\n(0.096 nM), c-Kit (0.466 nM), and c-Src (<3.81E-3 nM), with minimal off-target against kinases such as EGFR\n(427 nM) and PDGFRβ (15.25 nM), indicating strong target selectivity (Supplementary Table 3).\nSUPPLEMENTARY FIGURE 2. Noncompartmental pharmacokinetic (PK) analysis of H74DS3M8 using\nsparse sampling. Mean plasma concentration-time profiles of H74DS3M8 following a intraperitoneal (i.p.) and\nb intravenous (i.v.) administration. Concentrations are plotted on a logarithmic scale. Dotted lines represent the\nlog-linear fit to the terminal phase used for noncompartmental analysis. Terminal half-lives (HL_Lambda_z) were\nestimated to be .44 hours for i.p. and 3.15 hours for i.v. administration, based on 3 and 4 terminal-phase data\npoints, respectively. Goodness of fit for the regression is reported as R2 and adjusted R2 (R2 ). Adjusted R2\nadj\naccounts for the number of data points and model parameters. Data are means ± SD. PK parameters were\nderived from composite profiles generated using sparsely sampled animals per time point.\nTable S4\nDose T Cmax Tmax AUClast CL Bioavailability\nCompound Route 1/2\n(mg/kg) (h) (ng/mL) (h) (h*ng/mL) (mL/h/kg) (F)\ni.p. 60 3.44 17,100 0.5 259,000 231 0.15\nH74DS3M8\ni.v. 60 3.15 1,060,000 0.5 1,680,000 35.7 1.00\nSUPPLEMENTARY TABLE 4: Pharmacokinetic parameters of H74DS3M8 following single-dose\nintraperitoneal (i.p.) and intravenous (i.v.) administration in mice. Noncompartmental analysis was used to\ndetermine key pharmacokinetic parameters after administration of a single 60 mg/kg dose of H74DS3M8 via i.p.\nor i.v. route. Parameters include terminal half-life (T ), maximum plasma concentration (Cmax), time to\n1/2\nmaximum concentration (Tmax), area under the curve (AUClast), systemic clearance (CL), and absolute\nbioavailability (F). n=3 per group.\nH74DS3M8 exhibited favorable pharmacokinetic properties following both intraperitoneal (i.p.) and intravenous\n(i.v.) administration in mice. After a single 60 mg/kg dose, the compound showed a terminal half-life of 3.44 hours\n(i.p.) and 3.15 hours (i.v.). Systemic exposure was substantially higher after i.v. dosing, with a Cmax of 1,060,000\nng/mL and AUClast of 1,680,000 h*ng/mL, compared to 17,100 ng/mL and 259,000 h*ng/mL via the i.p. route.\nThe estimated absolute bioavailability of 15% after i.p. administration suggests limited systemic absorption.\nClearance was 231 mL/h/kg (i.p.) and 35.7 mL/h/kg (i.v.), consistent with rapid elimination following non-i.v.\ndosing. These data support the suitability of H74DS3M8 for systemic delivery and therapeutic use.\nWe also evaluated the compound’s functional potency in vitro using a cell proliferation assay in BV2 cells, where\nH74DS3M8 demonstrated dose-dependent growth inhibition with an IC of 343 nM at 52 hours (Supplementary\ntargeting proliferative myeloid populations.\nSUPPLEMENTARY FIGURE 1. H74DS3M8 inhibits BV2 cell proliferation in a dose-dependent manner. a\nRepresentative phase-contrast microscopy images (10x magnification) shows reduced cell density with\nincreasing concentrations of H74DS3M8 (vehicle, 370 nM, and 3 µM) after 52 hours of treatment. B Live-cell\nimaging using the IncuCyte system demonstrates dose-dependent suppression of cell confluence over time\nacross a range of H74DS3M8 concentrations (0.005-10 µM). c Quantification of growth inhibition at 52 hours\nshows an IC50 of 343 nM for H74DS3M8. H74 = H74DS3M8. Data are means ± SD.\nWith respect to CNS penetrance, while direct quantification of CNS drug levels is difficult, several lines of\nevidence support CNS delivery of H74DS3M8 in vivo. The compound is based on the same hydroxyl dendrimer\nscaffold (and shares the same biophysical delivery) as our 18F-FMD PET tracer (14.462 kDa vs. 20.320 kDa for\nH74DS3M8), which successfully crossed the BBB and selectively accumulated in regions of neuroinflammation,\nas shown in our imaging data in Figures 2-5. Importantly, H74DS3M8 was administered at a substantially higher\nmass dose (micromolar vs. nanomolar), increasing the likelihood of CNS uptake.\nFurthermore, the EAE model used in this study is characterized by BBB disruption, which enhances CNS\npenetration of systemically administered agents. This mirrors well-documented BBB dysfunction observed in\nhuman MS, particularly during active lesions. Numerous studies using gadolinium-enhanced MRI, histology,\nand molecular markers have confirmed BBB leakage in MS patients during both relapsing and progressive\nphases (Lassmann et al., Nat Rev Neurol 2012; Minagar & Alexander, Neurochem Res 2003). Activated myeloid\ncells and reactive endothelial changes further promote permeability in both the EAE model and human disease.\nGiven the shared dendrimer architecture, the higher dosing of H74DS3M8, and the presence of BBB dysfunction\nin EAE and human MS, we believe it is reasonable to conclude that H74DS3M8 reaches the CNS where it can\nthen have its desired effect on its target myeloid cell populations. Together, these factors support the\ninterpretation that the observed therapeutic effects are due to on-target modulation of CNS immune activity.\nWe have added the following text to the results section:\n“H74DS3M8 is a hydroxyl dendrimer conjugated with eight novel dasatinib payloads, engineered to selectively\ntarget activated myeloid cells via fluid-phase endocytosis and inhibit CSF1R with high potency. Kinase profiling\nrevealed sub-nanomolar potency against CSF1R, ABL1, c-Kit, and c-Src, with minimal off-target activity against\nEGFR and PDGFRβ (Supplementary Table 3). In cell-based assays, H74DS3M8 inhibited proliferation in BV2\nmicroglial cells in a dose-dependent manner, with an IC of 343 nM at 52 hours (Supplementary Fig. 1).\nmechanism of action of H74DS3M8 from other CSF1R inhibitors whose primary mechanism is through microglial\nkilling or depletion. Pharmacokinetic studies demonstrated sustained systemic exposure following both\nintraperitoneal (i.p.) and intravenous administration, supporting its suitability for in vivo applications\n(Supplementary Table 4). Noncompartmental analysis of sparse-sampling data revealed log-linear elimination\nkinetics with terminal plasma half-lives (HL_Lambda_z) of 3.44 hours (i.p.) and 3.15 hours (i.v.), respectively\n(Supplementary Fig. 2), consistent with a clearance profile appropriate for therapeutic targeting of CNS\ninflammation.“\nComment 6:\nOverlapping author affiliations with Ashvattha Therapeutics: Several authors are affiliated with the company\ndeveloping both 18F-FMD and H74DS3M8, creating a potential conflict of interest that may influence the\ninterpretation of results.\nResponse: Thank you for your comment. This study was sponsored by Ashvattha Therapeutics; however, all\nexperiments, data analysis, and interpretation were independently led and conducted by our research team at\nStanford. We have fully disclosed all author affiliations and potential conflicts of interest in the manuscript to\nensure transparency. While the study design and analyses were discussed with Ashvattha, they did not influence\nthe interpretation and conclusions of the study.\nComment 7:\nI would suggest the authors address these concerns with additional experiments and resubmit a more\ncomprehensive manuscript with expanded controls and improved statistical rigor.\nResponse: Thank you for raising these very helpful comments. We have addressed all concerns by performing\nadditional experiments to strengthen the manuscript. Specifically, we expanded the control groups where\nappropriate, increased the sample size to at least n=5 in all treatment response studies to improve statistical\nrigor, and revised the relevant text and figures accordingly. We believe these revisions significantly enhance the\nrobustness, clarity, and interpretability of our findings.\nRadiolabeled dendrimer non-invasively tracks innate immune activation in multiple sclerosis\nmice after immunomodulatory therapy\nResponse to Reviewers’ Comments:\nReviewer #1 (Remarks to the Author):\nGeneral comments:\nI greatly appreciate that the authors have effectively addressed the issues I raised in my previous review,\nperforming new analyses and updating their figures and results accordingly.\nHowever, a couple of important issues remain to be addressed:\nComment 1:\nRegarding the response to comment 2: The authors state that it is not possible to include post-mortem data from\nprogressive MS patients due to the biophysical mechanisms of PET tracer uptake. In this case, it is essential to\nclearly state in the limitations section of the manuscript that, as it currently stands, the presented results do not\ndemonstrate the utility of this novel tracer in the context of progressive MS. Any such utility can only be\nhypothesized at this stage.\nResponse: Thank you so much for this very helpful comment. We agree that this is an important limitation and\nhave added a paragraph to the discussion section to address this point.\nThe following text has been added to the discussion section:\n“Another important limitation is that, due to the biophysical mechanism of uptake of our PET tracer via fluid-\nphase endocytosis by activated myeloid cells, it is not possible to evaluate 18F-FMD specificity or uptake patterns\nin vitro using post-mortem tissue sections. Both the Cy5- and 18F-labeled dendrimer conjugates require living,\nmetabolically active cells for internalization, and endocytic activity ceases upon cell death. Consequently,\nanalyses of these agents in fresh-frozen or fixed MS tissues are not possible.”\nComment 2:\nRegarding the response to comment 3: I appreciate that the authors have included a reference to the revised\nimplication—that this novel tracer could enable a more accurate or earlier diagnosis of MS is not supported by\nthe presented data and should be removed from all sections of the manuscript. For instance, statements such\nas “These preclinical results highlight the potential utility of 18F-FMD in a clinical setting, particularly as the field\nmoves toward earlier diagnosis of MS,” or “However, diagnostic uncertainty remains a challenge in cases with\nambiguous radiographic or clinical presentations,” imply that the novel PET tracer may help resolve diagnostic\nuncertainty. However, this implication is not supported by the presented data, nor is it plausible even as a\ntheoretical hypothesis based on the current evidence. Therefore, any reference of the new tracer having a\npotential to allow an earlier or more accurate diagnosis of MS have to be removed from the manuscript.\nInstead, the authors could emphasize that while conventional MRI is widely and effectively used both to diagnose\nMS and to monitor treatment efficacy, it lacks specificity for innate immune cells—cells known to play a key role\nin the neurodegenerative aspects of MS pathology. Furthermore, as new treatments are being developed that\nspecifically target innate immune cells, there is a growing need for advanced imaging techniques capable of\nevaluating the efficacy of these therapies. In this context, the novel tracer may fill an important gap, representing\na promising method to specifically image pro-inflammatory innate immune cell activity in the brains of people\nwith MS.\nResponse: We thank the reviewer for this important clarification. We have carefully removed all statements\nsuggesting that this tracer may contribute to earlier or more accurate MS diagnosis. The revised text now\nemphasizes the established role of conventional MRI for diagnosis and monitoring, while highlighting that its\nlimitation lies in the lack of specificity for innate immune cells. We further revised the discussion to underscore\nthat 18F-FMD PET is positioned not as a diagnostic tool, but rather as a complementary approach with potential\nutility in evaluating therapeutic responses and monitoring innate immune dynamics, particularly as new\ntreatments targeting these cell populations are being developed.\nThe following text has been modified in the discussion section:\n“Conventional MRI remains the cornerstone of MS care, serving as the primary tool for both diagnosis and\nlongitudinal monitoring of treatment efficacy. The revised McDonald criteria, introduced at the 40th Congress of\nthe European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) in 2024, have further\nexpanded the role of clinical and imaging parameters, reinforcing the central role of structural MRI in facilitating\nmore timely diagnoses61. However, while MRI provides critical structural information, it lacks specificity for innate\nimmune cells–cell populations increasingly recognized as key drivers of the neurodegenerative aspects of MS\npathology. As new therapeutic strategies emerge that target these cells directly, there is a growing need for\nadvanced imaging approaches capable of capturing their dynamics and treatment responses in vivo. In this\ncontext, 18F-FMD PET has the potential to offer a complementary approach by enabling cell-specific, real-time\nvisualization of innate immune activation. Herein, we demonstrated that 18F-FMD PET sensitively detected\nreductions in CNS inflammatory activity in EAE mice following treatment with either FTY720, an FDA-approved\nS1P receptor modulator, or H74DS3M8, a novel CSF1R-targeting dendranib. These reductions were\ncorroborated by flow cytometry and ex vivo analyses, underscoring the tracer’s ability to track immune\nmodulation with high specificity. Together, these results support 18F-FMD PET as a promising biomarker for\nmonitoring innate immune dynamics and therapeutic efficacy, complementing structural MRI and potentially\nguiding the development and evaluation of emerging MS therapies.”\nReviewer #2 (Remarks to the Author):\nGeneral comments:\nThe authors have sufficiently addressed my comments. The additions / modifications made, significantly\nenhanced the manuscript. I have no additional comments.\nResponse: Thank you! We appreciate your thoughtful review and positive feedback.\nReviewer #3 (Remarks to the Author):\nGeneral comments:\nThis manuscript presents a technically rigorous evaluation of ¹⁸F-FMD as a PET tracer for detecting activated\nmyeloid cells in an EAE mouse model of multiple sclerosis. The authors employ multiple complementary\nmethodologies and demonstrate impressive technical expertise in radiotracer development, PET imaging, and\nimmunological characterization. The work addresses an important clinical need for biomarkers that can detect\nneuroinflammation and monitor therapeutic responses in MS.\nHowever, several fundamental limitations raise questions about the translational relevance of these findings:\nComment 1:\nModel Limitations: The EAE model involves artificial immune activation using Complete Freund's Adjuvant and\npertussis toxin, creating what the authors acknowledge as \"strong peripheral innate immune activation\" that\n\"alters CNS-resident immune cell behavior.\" This artificial immune environment may not adequately represent\nthe pathophysiology of human MS, where disease initiation occurs through different mechanisms.\nResponse: Thank you for raising this critical point. We agree that the EAE model presented in this manuscript\ninvolves artificial immune activation, and as such cannot fully recapitulate the pathophysiology of human MS.\nWe performed these EAE studies specifically to evaluate the performance of our tracer in a preclinical MS model\nand to obtain proof-of-concept data showing that 18F-FMD can detect innate immune activation in mice with MS-\nlike symptoms and monitor therapeutic responses to two different therapies. These data strengthen the feasibility\nand safety rationale for translation by demonstrating selective targeting of activated myeloid cells, favorable\npharmacokinetics, and the ability to measure therapeutic modulation in vivo.\nOur major objective was to move into human studies as quickly as possible. Excitingly, this tracer is currently\nbeing evaluated in a phase 1/2 clinical trial (NCT05395624). Our goal is to image as many MS patients as\npossible, including individuals across the spectrum of MS subtypes, to rigorously establish clinical utility. We also\nrecognize that all animal models fall short in fully capturing human disease complexity; thus, our preclinical data\nare not intended to be directly translationally definitive but rather serve as an essential bridge enabling us to\nadvance safely and rationally into studies in people.\nThe following texts have been modified in the discussion section:\n“The induction of EAE via MOG peptide and Complete Freund’s adjuvant (CFA) is an artificial process that\n35-55\ndoes not naturally occur in human MS. CFA triggers strong peripheral innate immune activation to facilitate\nantigen presentation, which in turn alters CNS-resident immune cell behavior80. This preclinical immune milieu\nmay therefore not adequately represent the pathophysiology of human MS, underscoring the need for clinical\nstudies to evaluate the performance of 18F-FMD.”\n“This trial aims to validate the sensitivity, specificity, and uptake patterns of 18F-FMD in patients, and to establish\nwhether they mirror those observed in preclinical models. In addition, the study will evaluate the tracer’s utility\nfor monitoring neuroinflammatory activity, assessing treatment response, and enabling patient stratification in\nclinical settings.”\nComment 2:\nBlood-Brain Barrier Considerations: The authors acknowledge that EAE involves BBB disruption, which may\nfacilitate tracer access to CNS parenchyma. While they argue that selective uptake depends on endocytic activity\nrather than passive leakage, this mechanism has not been validated under conditions of intact BBB function.\nHuman MS exhibits more heterogeneous and potentially less extensive BBB compromise, raising questions\nabout tracer performance in clinical settings.\nResponse: We thank the reviewer for this important comment. We agree that this is a major consideration when\ninterpreting our results. The EAE model involves BBB disruption, and translation into the clinical context will\nrequire consideration of the heterogeneity and varying extent of BBB compromise across different stages of MS.\nWhile our tracer uptake depends on endocytic activity rather than passive leakage, we acknowledge that BBB\nintegrity may influence performance under conditions of more limited or heterogeneous barrier disruption.\nAccordingly, we have revised the limitation paragraph in our discussion to explicitly highlight BBB heterogeneity\nin human MS:\n“Furthermore, BBB disruption in human MS is more heterogenous and typically less pronounced than in EAE,\ncomplicating direct extrapolation from animal models. Definitive evaluation of tracer uptake patterns will therefore\nrequire clinical validation, where the interplay of BBB integrity, immune activation, and disease heterogeneity\ncan be directly assessed.”\nAt this time, we cannot fully know how our approach will perform in the setting of intact or partially compromised\nBBB until clinical studies are completed. We have therefore added a sentence to the limitations paragraph of the\nmanuscript to alert readers to this caveat. We consider this a key issue for evaluation and agree that human\nstudies will ultimately be required to address it directly. Importantly, we are actively investigating this question in\nour ongoing clinical trial (NCT05395624; preliminary data published in Moradi, F. et al. J. Nucl. Med. 66, 251092\n[2025]; reference 77). Our goal is to understand how 18F-FMD performs across the full spectrum of MS subtypes\n(including RRMS, PPMS, SPMS, and PRMS) to establish its clinical utility in patients, where BBB compromise\nis heterogenous and variable over time.\n[77] Moradi, F. et al. [18F]Flurimedrimer, a Novel Nanomedicine Radiotracer, Selectively Targeting Activated\nMicroglia in Human Brain. J. Nucl. Med. 66, 251092 (2025).\nComment 3:\nSpecies Translation Gap: The substantial differences between murine and human immune systems, particularly\nregarding microglial activation patterns and myeloid cell responses, create uncertainty about cross-species\napplicability. The behavior of the 14.5kDa dendrimer may differ significantly in human physiology.\nResponse: Thank you for raising this critical consideration. We agree that cross-species differences in microglial\nand myeloid biology are a major consideration. Importantly, the hydroxyl-terminated PAMAM dendrimer platform\n(the ~14.5 kDa scaffold used in 18F-FMD) has been evaluated in humans as OP-101 (dendrimer-N-\nacetylcysteine). In a multicenter, randomized, double-blind phase 2a trial in adults with severe COVID-19, single-\ndose intravenous OP-101 was well tolerated, reduced systemic inflammation, and lowered neurological injury\nmarkers (NfL and GFAP) versus placebo – consistent with selective delivery to activated macrophage/microglia-\nlineage cells in vivo (Gusdon et al. Sci Transl Med. 2022 Jul 20;14(654):eabo2652). These data provide direct\nhuman evidence that this hydroxyl dendrimer platform behaves safely and with the intended immune-cell-\ntargeting pharmacology in an inflammatory clinical setting.\nIn addition, the in vivo pharmacology of hydroxyl dendrimers has been extensively validated in more than 30\nanimal models of neuroinflammation, including non-human primate and canine models (Kannan 2012; Kannan\n2014; Mishra 2014; Nance 2015; Guo 2016; Nance 2016; Zhang 2016; Nance 2017; Niño 2018; Turk 2018;\nLiaw 2020; Sharma 2020; Cabeza 2021; Henningfield 2024). Across these studies, hydroxyl dendrimers consistently\ncross the BBB in the presence of neuroinflammation, distribute rapidly after systemic administration, and are\nselectively internalized by activated macrophages and microglia in proportion to disease severity. Notably,\nHenningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) showed that Cy5-labeled hydroxyl dendrimers\npreferentially accumulated in plaque-associated microglia in 5xFAD mice. The 5xFAD model is characterized by\na comparatively intact BBB, with only localized and age-dependent permeability changes, underscoring that\ndendrimer uptake in this setting reflects genuine cellular targeting rather than nonspecific paracellular leakage.\nFinally, large-animal studies reported by other groups provide additional confidence in translational applicability.\nIn non-human primates with ischemic optic neuropathy and in canines subjected to hypothermic circulatory arrest,\nhydroxyl dendrimers selectively trafficked to injured CNS tissue and glia while showing negligible uptake in\nuninvolved regions (Guo et al. PLoS One. 2016 Apr 29;11(4):e0154437; Mishra et al. ACS Nano. 2014 Mar\n25;8(3):2134-47). These findings suggest preservation of the dendrimer’s CNS trafficking and targeting\nmechanisms across species.\nTaken together, the convergence of direct human trial data with OP-101, consistent findings across small- and\nlarge-animal neuroinflammatory models, and mechanistic studies showing selective uptake by activated\nmicroglia and macrophages provide strong evidence that the pharmacology of hydroxyl dendrimers is conserved\nacross species. The aim of our current study is to establish in vivo tracer specificity and pharmacology in a well-\ncharacterized preclinical MS model as a bridge to clinical imaging. Building on the human OP-101 results and\nlarge-animal evidence above, we are now evaluating 18F-FMD across all clinical MS subtypes to directly quantify\ntracer performance in human physiology.\nComment 4:\nLimited Disease Modeling: Testing in only one MS model (MOG35-55 EAE) provides insufficient evidence for\nbroader applicability. Human MS encompasses diverse subtypes with varying inflammatory profiles that may not\nbe adequately represented by this single model.\nResponse: We thank the reviewer for this comment. We acknowledge that there are multiple preclinical models\nof MS, each of which captures different aspects of disease biology. However, the goal of this study was not to\ntest tracer performance across a wide range of models, but rather to understand how our tracer performs in vivo\nin a model that develops both neuroinflammation and lesions. Using the MOG EAE model allowed us to\n35-55\nassess the tracer’s uptake, biodistribution, and specificity in the context of acute neuroinflammation, thereby\nproviding essential information about the sensitivity and specificity of this imaging approach in vivo.\nSince no single mouse model can fully recapitulate the pathophysiology or heterogeneity of human MS, our\nultimate goal was to move into clinical studies which are currently ongoing (NCT05395624), where the tracer is\nbeing evaluated directly in patients across the full spectrum of MS subtypes. As such, the EAE experiments were\ndesigned as a necessary preclinical step to establish feasibility and inform translation, rather than to exhaustively\nmodel the diversity of MS disease presentations in animals.\nComment 5:\nEthical Considerations: The extensive use of animals (160 mice) subjected to neurological disease with paralysis\nas endpoints appears disproportionate given the uncertain translational value. The authors have not\ndemonstrated that alternative research approaches (human tissue studies, computational modeling, existing\nclinical data) were adequately explored before conducting this extensive animal study.\nResponse: We appreciate this important point and agree that ethical considerations are central in animal\nresearch. Alternative approaches, however, are not feasible for the aims of this study. The goal of our work was\nto assess the uptake, biodistribution, and specificity of 18F-FMD in vivo. At present, there are no available\ncomputational models or in vitro techniques capable of recapitulating the complex in vivo behavior of radiotracers,\nincluding their stability, kinetics, biodistribution, specificity, and non-specific binding. While computational\nmodeling has shown promise in drug discovery, predictive modeling for PET tracer performance remains limited,\nand we are actively exploring this area in our lab.\nIn addition, the biophysical mechanism of uptake for 18F-FMD (via fluid-phase endocytosis by activated myeloid\ncells) cannot be meaningfully reproduced in tissue sections or static in vitro systems as these cannot replicate\nthe dynamic cell-environment interactions present in living organisms. New PET radiotracers must undergo\nevaluation in rodents and/or large animas (e.g., non-human primates) as a critical step before human studies.\nWe would also like to emphasize that the number of mice used was carefully justified. All experiments were\ndesigned a priori to achieve appropriate statistical power; we used the minimum number of animals necessary\nto rigorously test our hypotheses across multiple treatment arms and confirmatory analyses. Although 160 mice\nwere used in total, they were essential to establish the feasibility and safety rationale for moving into humans.\nFinally, we note that this tracer is currently being evaluated in a phase 1/2 clinical trial (NCT05395624). These\nanimal studies were not conducted in isolation but rather as an ethically justified bridge, designed to provide the\nproof-of-concept data necessary to support safe, rational translation into people. Our goal is to image as many\nMS patients as possible to rigorously establish clinical utility.\nComment 6:\nHow do the authors address potential partial volume effects from renal clearance affecting spinal cord\nquantification in such small anatomical structures?\nResponse: We thank the reviewer for raising this important point and apologize for not explaining this more\nclearly in the discussion section. To clarify, we addressed the potential issue for partial volume effects from renal\nclearance impacting spinal quantification through two different ex vivo approaches: gamma counting and\nautoradiography. To further explain these processes, spinal cords and kidneys were carefully dissected out of\neach mouse following cardiac perfusion to remove any potential contributions of tracer signal in blood. For\ngamma counting, the tissues were weighed wet, transferred into separate gamma counting tubes, and measured\nusing a gamma counter to obtain absolute quantitation of tracer concentration in individual organs, normalized\nto weight. This technique allows us to determine absolute tracer concentrations in each tissue without any\npotential for spillover or partial volume effects that can occur in PET imaging. To further understand the tracer\nuptake pattern (with higher resolution) in spinal cords, we embedded them in OCT, cryosectioned into thin (60\nµm) slices, and exposed on digital phosphorous films to obtain high-resolution spatial distribution data. These\nex vivo analyses, performed independently of the in vivo PET data, confirmed that tracer uptake within the spinal\ncord reflected true biological signal and was not artifactual due to renal clearance or blood pool signal or any\nother partial volume effects. Together, these complementary methods strengthen the robustness of our\nquantification and provide confidence in the specificity of our findings.\nThe following text has been modified in the discussion section:\n“Given the renal clearance of 18F-FMD and the small size of EAE mice, there is a possibility of partial volume\neffects from adjacent kidneys affecting signal quantification in the lumbar spinal cord. Partial volume corrections\nusing post-reconstruction- and reconstruction-based methods could help circumvent this limitation73. However,\nthese correction techniques are challenging to implement effectively in mouse models due to the extremely small\nsize and thin geometry of the spinal cord, which often falls below the spatial resolution of small-animal PET\nscanners74,75. This leads to significant spillover and underestimation of true activity even after correction,\nparticularly when adjacent high-uptake organs like kidneys are involved. Instead, we mitigated this issue by\ndissecting the kidneys and spinal cord out of each mouse for gamma counting and performed autoradiography\non thin spinal cord sections. These techniques eliminate contributions from circulating tracer and provide higher\nsensitivity and spatial resolution respectively, without any possibility for spillover from adjacent tissues.\nUsing\nthese methods, we observed a dramatic elevation in spinal cord signal in EAE mice compared to naïve controls,\ncorroborating in vivo PET findings. Importantly, this limitation is less relevant in larger species, such as non-\nhuman primates or humans, where the increased anatomical separation between kidneys and spinal cord\nreduces the likelihood of signal interference, allowing for more accurate delineation of regions of interest.”\nComment 7:\nCould the authors provide additional validation using models with intact blood-brain barriers to confirm specificity\nmechanisms?\nResponse: Thank you so much for this comment. We fully agree that confirming the specificity of the mechanism\nof tracer uptake is important. As mentioned in our manuscript, 18F-FMD does not accumulate in healthy CNS\ntissues, but only accumulates in regions containing neuroinflammation, where it is selectively endocytosed by\nactivated myeloid cells. We understand your concern regarding whether the uptake of our tracer depends\nprimarily on BBB disruption, and that it would be ideal to test in a model with an intact BBB. As you know,\nmicroglial activation is closely linked to BBB function. Once there are activated myeloid cells present in the CNS,\nthey release cytokines, chemokines, and reactive oxygen species (among other mediators) that modulate\nendothelial cells and pericytes. These chemical signals can subtly (or overtly) alter tight junction integrity,\nendothelial pathways, transporter activity, and thus passage of PET tracers into and out of the CNS (Yoon et al.\nNat Commun 16, 699 (2025); Mayer and Fischer, Front Cell Neurosci. 2024 Mar 13;18:1360195; da Fonseca et\nal. Front Cell Neurosci. 2014 Nov 3;8:362). It is thus challenging to identify a suitable model that contains our\ntarget of interest (i.e., activated myeloid cells) in absence of BBB dysfunction.\nWe agree that EAE mice do indeed exhibit significant BBB compromise, and therefore you raise a valid question\nabout the specificity of the mechanism of uptake of our tracer. To address your concern, we have added\nadditional empirical data below validating the uptake of 18F-FMD in models with negligible BBB dysfunction.\nEvidence of elevated 18F-FMD uptake in the cortex and hippocampus of 5xFAD mice at an early stage of\ndisease, known to have largely intact BBB (cited data; open access)\nWe evaluated 18F-FMD in the 5xFAD Alzheimer’s disease mouse model, which is known to have a largely intact\nBBB before 4 months of age and only modestly altered between 4-8 months (Zhukov et al., Front Aging Neurosci.\n5xFAD mice (Figure 1 and Table 1), which is an early stage of disease, 18F-FMD uptake was threefold higher in\ncortex and hippocampus (regions with known AD pathology and microglial activation) compared to wild-type\ncontrols, with higher sensitivity than TSPO-PET using 18F-GE180 (a small molecule that crosses the intact rodent\nBBB and binds to the translocator protein 18kDa, a widely explored PET imaging biomarker of\nneuroinflammation). This data provides strong evidence that the predominate mechanism of 18F-FMD uptake is\nvia endocytosis of activated microglia rather than passive diffusion across a compromised BBB alone.\nFigure 1. Coronal view of representative 3.75-month-old 5xFAD transgenic and wild-type mouse brain PET\nimages (50-60 min p.i. summed) using either 18F-FMD or 18F-GE180, co-registered with CT images. Carlson et\nal., Alzheimer's Dement. 2022, 18: e060700.\nTable 1. Tracer signal in 5xFAD mice normalized to wild-type mice signal (equivalent to signal-to-background\nratios) in brain regions known to contain amyloid pathology and microglial activation in 5xFAD mice imaged with\neither 18F-FMD or 18F-GE180 at 3.75 months old.\n18F-FMD (n=4 WT, n=4 5xFAD) 18F-GE180 (n=4 WT, n=5 5xFAD)\nCortex 3.18 1.21\nHippocampus 3.05 1.24\nWhole Brain 3.14 1.21\nComplementary evidence from fluorescent dendrimer analog of 18F-FMD (cited data; open access)\nHenningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) confirmed the specificity of the fourth-\ngeneration polyamidoamine (PAMAM) hydroxyl dendrimer (same scaffold as 18F-FMD), showing that this Cy5-\nlabeled dendrimer was regionally and selectively phagocytosed by plaque-associated microglia (PAMs) in 7-\nmonth-old 5xFAD brains (Figure 1c). Their work corroborated our in vivo PET imaging data in 5xFAD mice and\nshowed robust Cy5 signal around dense-core plaques (Amylo-Glo-positive) and co-localization to Iba1+ microglia\nand CD68+ microglial lysosomes in hippocampus and samatosensory cortex. Wild-type mice, by contrast,\nexhibited negligible Cy5 signal. Additionally, staining for astrocytes, neurons, and oligodendrocytes showed no\nCy5 signal associated with those cells in the subiculum or somatosensory cortex (Fig. 1m-q).\nFigure 1c from Henningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) demonstrates co-localization\nof Cy5-labeled hydroxyl dendrimers (red) with microglia (Iba1, green) and dense core plaques (Amylo-Glo, blue).\nFigure 1m-q from Henningfield et al. showing G4 hydroxyl dendrimers are selectively endocytosed by plaque-\nassociated microglia in the subiculum (p) and somatosensory cortex (q) regions of 5xFAD brain with no uptake\nby astrocytes (m, m1), neurons (n), oligodendrocytes (o, o1).\nAdditional validation across models with limited BBB disruption (from Carlson et al., with permission)\nWe have also previously published a study assessing 18F-FMD in an LPS model induced by intraperitoneal\ninjection of LPS (rather than intracerebroventricularly; Carlson et al., Mol Imaging Biol. 2023;25:1063-1072),\nwhere BBB alterations are modest compared to the EAE model used in our current study. In LPS mice, we\ndemonstrated a correlation between whole-brain 18F-FMD uptake and murine sepsis score – with increasing\nscore relating to elevated levels of activated microglia (Figure 4c).\nFigure 4c shows strong correlation between 18F-FMD whole brain signal and severity of sepsis symptoms using\nmurine sepsis score (MSS). Adapted from Carlson et al., “Development and Initial Assessment of [18F]OP-801:\na Novel Hydroxyl Dendrimer PET Tracer for Preclinical Imaging of Innate Immune Activation in the Whole Body\nand Brain,” Molecular Imaging and Biology, 25, 1063-1072 (2023), Springer Nature. Permission obtained via\nRightsLink.\nMoreover, the mechanism of uptake and biodistribution of these hydroxyl dendrimers has been characterized\nextensively in over 30 animal models of neuroinflammation across species (including rodent, canine, and primate\nmodels) by other groups. In these studies, G4 dendrimers consistently accumulated at sites of injury or\nneuroinflammation but not in healthy brain tissue (Kannan 2012; Kannan 2014; Mishra 2014; Nance 2015; Guo\n2016; Nance 2016; Zhang 2016; Nance 2017; Niño 2018; Turk 2018; Henningfield 2020; Liaw 2020; Sharma\n2020; Cabeza 2021).\nTaken together, these data (in particular, the 5xFAD mouse data collected by our lab using 18F-FMD in addition\nto 5xFAD data collected and published by a separate lab using the fluorescently labeled version of this same\ndendrimer), along with extensive G4 dendrimer biodistribution studies in multiple species, provide strong\nevidence that 18F-FMD can cross the BBB both under conditions of overt disruption (e.g., EAE) and, importantly,\nin models with minimal impairment (e.g., early-stage 5xFAD and i.p. LPS). Crucially, tracer retention occurs only\nin regions containing activated myeloid cells, thus indicating that the predominate mechanism of uptake and\nretention is endocytosis by activated myeloid cells.\nThe following text has been modified in the introduction section to more clearly articulate the mechanism of\nuptake of our tracer:\n“Fourth-generation hydroxyl dendrimers have been demonstrated to penetrant regions of a dysfunctional–but\nnot healthy, intact–blood-brain barrier (BBB), where they are quickly and selectively taken up by activated\nmyeloid cells via fluid phase endocytosis, and rapidly cleared from tissue that does not contain these cells27–34.”\nMechanistic evidence of selective endocytosis of generation 4 hydroxyl dendrimers by activated myeloid\ncells\nWe used a CSF1R inhibitor dendranib to selectively inhibit the activation and proliferation of myeloid cells in EAE\nmice (to investigate the specificity of our tracer uptake). In vivo PET imaging, complemented by ex vivo gamma\ncounting and autoradiography, demonstrated significantly reduced 18F-FMD uptake in CNS regions with known\nEAE pathology, including the medulla, cerebellum, pons, and both the cervical/thoracic and lumbar spinal cord\n(Figures 4 and 5 in the manuscript). Flow cytometry with a fluorescent analog of 18F-FMD (Cy5-labeled G4\nhydroxyl dendrimer; Cy5-HD) further confirmed selective endocytosis of hydroxyl dendrimers by activated\nmicroglia and infiltrating myeloid cells. Notably, CSF1R dendranib treatment led to a marked reduction in Cy5-\nHD+ activated microglia (46-fold) and infiltrating macrophages (19-fold).\nGiven the breath of preclinical validation already generated, and as the reviewer noted in comment 5, we believe\nthat additional rodent models would add limited translational value. Instead, our priority is to complete our current\nhuman studies. In our ongoing clinical trial (NCT05395624; preliminary data published in Moradi, F. et al. J. Nucl.\nMed., 2025, 66, 251092; reference 77), our goal is to understand how 18F-FMD performs across the full spectrum\nof MS subtypes (including RRMS, PPMS, SPMS, and PRMS) to establish its clinical utility in patients, where\nBBB compromise is heterogeneous and variable over time.\nComment 8:\nWhat evidence supports the assumption that fluid-phase endocytosis rates in activated murine myeloid cells\naccurately reflect those in human cells?\nResponse: We appreciate this thoughtful comment. While cross-species differences in endocytic activity are\nindeed important to consider, there is direct evidence that fluid-phase endocytosis of hydroxyl dendrimers is\nconserved across rodent and human cells and tissues.\nFirst, Kambhampati et al. (Eur J Pharm Biopharm. 2015 Sep;95(Pt B):239-49) performed a side-by-side in vitro\ncharacterization of hydroxyl dendrimer uptake and release kinetics in murine BV2 microglial cells and in human\nretinal pigment epithelial (RPE) cells. The study demonstrated highly consistent patterns of dendrimer\ninternalization, trafficking, and intracellular retention between the murine and human cell systems, supporting\nconserved uptake mechanisms across species at the cellular level.\nSecond, Kambhampati et al. (J Control Release. 2021 Jul 10;335:527-540) provided in vivo confirmation in both\nrodent and human tissues. In a rat model of choroidal neovascularization (CNV), hydroxyl dendrimers selectively\naccumulated in activated myeloid and glial cells. Importantly, the same paper also demonstrated dendrimer\nuptake in post-mortem human diabetic eyes, localizing to activated myeloid and glial populations. This direct\ncomparison across rat and human tissue provides strong evidence that the endocytic targeting behavior of\nhydroxyl dendrimers is conserved across species, including in relevant human disease contexts.\nCollectively, these studies help bridge the translational gap by showing that the fluid-phase endocytosis of\nhydroxyl dendrimers by activated myeloid cells is not unique to murine systems but is also observed in human\ncells. This supports the rationale for extrapolating from murine models to human applications in the context of\n18F-FMD pharmacology."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I greatly appreciate that the authors have effectively addressed the issues I raised in my previous review, performing new analyses and updating their figures and results accordingly.\nHowever, a couple of important issues remain to be addressed:\n1) Regarding the response to comment 2:\nThe authors state that it is not possible to include post-mortem data from progressive MS patients due to the biophysical mechanisms of PET tracer uptake. In this case, it is essential to clearly state in the limitations section of the manuscript that, as it currently stands, the presented results do not demonstrate the utility of this novel tracer in the context of progressive MS. Any such utility can only be hypothesized at this stage.\n2) Regarding the response to comment 3:\nI appreciate that the authors have included a reference to the revised 2024 McDonald criteria; however, I believe the main point of my comment was missed. The claim—or even the implication—that this novel tracer could enable a more accurate or earlier diagnosis of MS is not supported by the presented data and should be removed from all sections of the manuscript. For instance, statements such as “These preclinical results highlight the potential utility of 18F-FMD in a clinical setting, particularly as the field moves toward earlier diagnosis of MS,” or “However, diagnostic uncertainty remains a challenge in cases with ambiguous radiographic or clinical presentations,” imply that the novel PET tracer may help resolve diagnostic uncertainty. However, this implication is not supported by the presented data, nor is it plausible even as a theoretical hypothesis based on the current evidence. Therefore, any reference of the new tracer having a potential to allow an earlier or more accurate diagnosis of MS have to be removed from the manuscript.\nInstead, the authors could emphasize that while conventional MRI is widely and effectively used both to diagnose MS and to monitor treatment efficacy, it lacks specificity for innate immune cells—cells known to play a key role in the neurodegenerative aspects of MS pathology. Furthermore, as new treatments are being developed that specifically target innate immune cells, there is a growing need for advanced imaging techniques capable of evaluating the efficacy of these therapies. In this context, the novel tracer may fill an important gap, representing a promising method to specifically image pro-inflammatory innate immune cell activity in the brains of people with MS.", "reviewer_2": "The authors have suffieciently addressed my comments. The additions / modifications made, significantly enhanced the manuscript. I have no additional comments.", "reviewer_3": "This manuscript presents a technically rigorous evaluation of ¹⁸F-FMD as a PET tracer for detecting activated myeloid cells in an EAE mouse model of multiple sclerosis. The authors employ multiple complementary methodologies and demonstrate impressive technical expertise in radiotracer development, PET imaging, and immunological characterization. The work addresses an important clinical need for biomarkers that can detect neuroinflammation and monitor therapeutic responses in MS.\nHowever, several fundamental limitations raise questions about the translational relevance of these findings:\nModel Limitations: The EAE model involves artificial immune activation using Complete Freund's Adjuvant and pertussis toxin, creating what the authors acknowledge as \"strong peripheral innate immune activation\" that \"alters CNS-resident immune cell behavior.\" This artificial immune environment may not adequately represent the pathophysiology of human MS, where disease initiation occurs through different mechanisms.\nBlood-Brain Barrier Considerations: The authors acknowledge that EAE involves BBB disruption, which may facilitate tracer access to CNS parenchyma. While they argue that selective uptake depends on endocytic activity rather than passive leakage, this mechanism has not been validated under conditions of intact BBB function. Human MS exhibits more heterogeneous and potentially less extensive BBB compromise, raising questions about tracer performance in clinical settings.\nSpecies Translation Gap: The substantial differences between murine and human immune systems, particularly regarding microglial activation patterns and myeloid cell responses, create uncertainty about cross-species applicability. The behavior of the 14.5kDa dendrimer may differ significantly in human physiology.\nLimited Disease Modeling: Testing in only one MS model (MOG35-55 EAE) provides insufficient evidence for broader applicability. Human MS encompasses diverse subtypes with varying inflammatory profiles that may not be adequately represented by this single model.\nEthical Considerations: The extensive use of animals (160 mice) subjected to neurological disease with paralysis as endpoints appears disproportionate given the uncertain translational value. The authors have not demonstrated that alternative research approaches (human tissue studies, computational modeling, existing clinical data) were adequately explored before conducting this extensive animal study.\nHow do the authors address potential partial volume effects from renal clearance affecting spinal cord quantification in such small anatomical structures?\nCould the authors provide additional validation using models with intact blood-brain barriers to confirm specificity mechanisms?\nWhat evidence supports the assumption that fluid-phase endocytosis rates in activated murine myeloid cells accurately reflect those in human cells?"}, "author_response": "Radiolabeled dendrimer non-invasively tracks innate immune activation in multiple sclerosis\nmice after immunomodulatory therapy\nResponse to Reviewers’ Comments:\nReviewer #1 (Remarks to the Author):\nGeneral comments:\nI greatly appreciate that the authors have effectively addressed the issues I raised in my previous review,\nperforming new analyses and updating their figures and results accordingly.\nHowever, a couple of important issues remain to be addressed:\nComment 1:\nRegarding the response to comment 2: The authors state that it is not possible to include post-mortem data from\nprogressive MS patients due to the biophysical mechanisms of PET tracer uptake. In this case, it is essential to\nclearly state in the limitations section of the manuscript that, as it currently stands, the presented results do not\ndemonstrate the utility of this novel tracer in the context of progressive MS. Any such utility can only be\nhypothesized at this stage.\nResponse: Thank you so much for this very helpful comment. We agree that this is an important limitation and\nhave added a paragraph to the discussion section to address this point.\nThe following text has been added to the discussion section:\n“Another important limitation is that, due to the biophysical mechanism of uptake of our PET tracer via fluid-\nphase endocytosis by activated myeloid cells, it is not possible to evaluate 18F-FMD specificity or uptake patterns\nin vitro using post-mortem tissue sections. Both the Cy5- and 18F-labeled dendrimer conjugates require living,\nmetabolically active cells for internalization, and endocytic activity ceases upon cell death. Consequently,\nanalyses of these agents in fresh-frozen or fixed MS tissues are not possible.”\nComment 2:\nRegarding the response to comment 3: I appreciate that the authors have included a reference to the revised\nimplication—that this novel tracer could enable a more accurate or earlier diagnosis of MS is not supported by\nthe presented data and should be removed from all sections of the manuscript. For instance, statements such\nas “These preclinical results highlight the potential utility of 18F-FMD in a clinical setting, particularly as the field\nmoves toward earlier diagnosis of MS,” or “However, diagnostic uncertainty remains a challenge in cases with\nambiguous radiographic or clinical presentations,” imply that the novel PET tracer may help resolve diagnostic\nuncertainty. However, this implication is not supported by the presented data, nor is it plausible even as a\ntheoretical hypothesis based on the current evidence. Therefore, any reference of the new tracer having a\npotential to allow an earlier or more accurate diagnosis of MS have to be removed from the manuscript.\nInstead, the authors could emphasize that while conventional MRI is widely and effectively used both to diagnose\nMS and to monitor treatment efficacy, it lacks specificity for innate immune cells—cells known to play a key role\nin the neurodegenerative aspects of MS pathology. Furthermore, as new treatments are being developed that\nspecifically target innate immune cells, there is a growing need for advanced imaging techniques capable of\nevaluating the efficacy of these therapies. In this context, the novel tracer may fill an important gap, representing\na promising method to specifically image pro-inflammatory innate immune cell activity in the brains of people\nwith MS.\nResponse: We thank the reviewer for this important clarification. We have carefully removed all statements\nsuggesting that this tracer may contribute to earlier or more accurate MS diagnosis. The revised text now\nemphasizes the established role of conventional MRI for diagnosis and monitoring, while highlighting that its\nlimitation lies in the lack of specificity for innate immune cells. We further revised the discussion to underscore\nthat 18F-FMD PET is positioned not as a diagnostic tool, but rather as a complementary approach with potential\nutility in evaluating therapeutic responses and monitoring innate immune dynamics, particularly as new\ntreatments targeting these cell populations are being developed.\nThe following text has been modified in the discussion section:\n“Conventional MRI remains the cornerstone of MS care, serving as the primary tool for both diagnosis and\nlongitudinal monitoring of treatment efficacy. The revised McDonald criteria, introduced at the 40th Congress of\nthe European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) in 2024, have further\nexpanded the role of clinical and imaging parameters, reinforcing the central role of structural MRI in facilitating\nmore timely diagnoses61. However, while MRI provides critical structural information, it lacks specificity for innate\nimmune cells–cell populations increasingly recognized as key drivers of the neurodegenerative aspects of MS\npathology. As new therapeutic strategies emerge that target these cells directly, there is a growing need for\nadvanced imaging approaches capable of capturing their dynamics and treatment responses in vivo. In this\ncontext, 18F-FMD PET has the potential to offer a complementary approach by enabling cell-specific, real-time\nvisualization of innate immune activation. Herein, we demonstrated that 18F-FMD PET sensitively detected\nreductions in CNS inflammatory activity in EAE mice following treatment with either FTY720, an FDA-approved\nS1P receptor modulator, or H74DS3M8, a novel CSF1R-targeting dendranib. These reductions were\ncorroborated by flow cytometry and ex vivo analyses, underscoring the tracer’s ability to track immune\nmodulation with high specificity. Together, these results support 18F-FMD PET as a promising biomarker for\nmonitoring innate immune dynamics and therapeutic efficacy, complementing structural MRI and potentially\nguiding the development and evaluation of emerging MS therapies.”\nReviewer #2 (Remarks to the Author):\nGeneral comments:\nThe authors have sufficiently addressed my comments. The additions / modifications made, significantly\nenhanced the manuscript. I have no additional comments.\nResponse: Thank you! We appreciate your thoughtful review and positive feedback.\nReviewer #3 (Remarks to the Author):\nGeneral comments:\nThis manuscript presents a technically rigorous evaluation of ¹⁸F-FMD as a PET tracer for detecting activated\nmyeloid cells in an EAE mouse model of multiple sclerosis. The authors employ multiple complementary\nmethodologies and demonstrate impressive technical expertise in radiotracer development, PET imaging, and\nimmunological characterization. The work addresses an important clinical need for biomarkers that can detect\nneuroinflammation and monitor therapeutic responses in MS.\nHowever, several fundamental limitations raise questions about the translational relevance of these findings:\nComment 1:\nModel Limitations: The EAE model involves artificial immune activation using Complete Freund's Adjuvant and\npertussis toxin, creating what the authors acknowledge as \"strong peripheral innate immune activation\" that\n\"alters CNS-resident immune cell behavior.\" This artificial immune environment may not adequately represent\nthe pathophysiology of human MS, where disease initiation occurs through different mechanisms.\nResponse: Thank you for raising this critical point. We agree that the EAE model presented in this manuscript\ninvolves artificial immune activation, and as such cannot fully recapitulate the pathophysiology of human MS.\nWe performed these EAE studies specifically to evaluate the performance of our tracer in a preclinical MS model\nand to obtain proof-of-concept data showing that 18F-FMD can detect innate immune activation in mice with MS-\nlike symptoms and monitor therapeutic responses to two different therapies. These data strengthen the feasibility\nand safety rationale for translation by demonstrating selective targeting of activated myeloid cells, favorable\npharmacokinetics, and the ability to measure therapeutic modulation in vivo.\nOur major objective was to move into human studies as quickly as possible. Excitingly, this tracer is currently\nbeing evaluated in a phase 1/2 clinical trial (NCT05395624). Our goal is to image as many MS patients as\npossible, including individuals across the spectrum of MS subtypes, to rigorously establish clinical utility. We also\nrecognize that all animal models fall short in fully capturing human disease complexity; thus, our preclinical data\nare not intended to be directly translationally definitive but rather serve as an essential bridge enabling us to\nadvance safely and rationally into studies in people.\nThe following texts have been modified in the discussion section:\n“The induction of EAE via MOG peptide and Complete Freund’s adjuvant (CFA) is an artificial process that\n35-55\ndoes not naturally occur in human MS. CFA triggers strong peripheral innate immune activation to facilitate\nantigen presentation, which in turn alters CNS-resident immune cell behavior80. This preclinical immune milieu\nmay therefore not adequately represent the pathophysiology of human MS, underscoring the need for clinical\nstudies to evaluate the performance of 18F-FMD.”\n“This trial aims to validate the sensitivity, specificity, and uptake patterns of 18F-FMD in patients, and to establish\nwhether they mirror those observed in preclinical models. In addition, the study will evaluate the tracer’s utility\nfor monitoring neuroinflammatory activity, assessing treatment response, and enabling patient stratification in\nclinical settings.”\nComment 2:\nBlood-Brain Barrier Considerations: The authors acknowledge that EAE involves BBB disruption, which may\nfacilitate tracer access to CNS parenchyma. While they argue that selective uptake depends on endocytic activity\nrather than passive leakage, this mechanism has not been validated under conditions of intact BBB function.\nHuman MS exhibits more heterogeneous and potentially less extensive BBB compromise, raising questions\nabout tracer performance in clinical settings.\nResponse: We thank the reviewer for this important comment. We agree that this is a major consideration when\ninterpreting our results. The EAE model involves BBB disruption, and translation into the clinical context will\nrequire consideration of the heterogeneity and varying extent of BBB compromise across different stages of MS.\nWhile our tracer uptake depends on endocytic activity rather than passive leakage, we acknowledge that BBB\nintegrity may influence performance under conditions of more limited or heterogeneous barrier disruption.\nAccordingly, we have revised the limitation paragraph in our discussion to explicitly highlight BBB heterogeneity\nin human MS:\n“Furthermore, BBB disruption in human MS is more heterogenous and typically less pronounced than in EAE,\ncomplicating direct extrapolation from animal models. Definitive evaluation of tracer uptake patterns will therefore\nrequire clinical validation, where the interplay of BBB integrity, immune activation, and disease heterogeneity\ncan be directly assessed.”\nAt this time, we cannot fully know how our approach will perform in the setting of intact or partially compromised\nBBB until clinical studies are completed. We have therefore added a sentence to the limitations paragraph of the\nmanuscript to alert readers to this caveat. We consider this a key issue for evaluation and agree that human\nstudies will ultimately be required to address it directly. Importantly, we are actively investigating this question in\nour ongoing clinical trial (NCT05395624; preliminary data published in Moradi, F. et al. J. Nucl. Med. 66, 251092\n[2025]; reference 77). Our goal is to understand how 18F-FMD performs across the full spectrum of MS subtypes\n(including RRMS, PPMS, SPMS, and PRMS) to establish its clinical utility in patients, where BBB compromise\nis heterogenous and variable over time.\n[77] Moradi, F. et al. [18F]Flurimedrimer, a Novel Nanomedicine Radiotracer, Selectively Targeting Activated\nMicroglia in Human Brain. J. Nucl. Med. 66, 251092 (2025).\nComment 3:\nSpecies Translation Gap: The substantial differences between murine and human immune systems, particularly\nregarding microglial activation patterns and myeloid cell responses, create uncertainty about cross-species\napplicability. The behavior of the 14.5kDa dendrimer may differ significantly in human physiology.\nResponse: Thank you for raising this critical consideration. We agree that cross-species differences in microglial\nand myeloid biology are a major consideration. Importantly, the hydroxyl-terminated PAMAM dendrimer platform\n(the ~14.5 kDa scaffold used in 18F-FMD) has been evaluated in humans as OP-101 (dendrimer-N-\nacetylcysteine). In a multicenter, randomized, double-blind phase 2a trial in adults with severe COVID-19, single-\ndose intravenous OP-101 was well tolerated, reduced systemic inflammation, and lowered neurological injury\nmarkers (NfL and GFAP) versus placebo – consistent with selective delivery to activated macrophage/microglia-\nlineage cells in vivo (Gusdon et al. Sci Transl Med. 2022 Jul 20;14(654):eabo2652). These data provide direct\nhuman evidence that this hydroxyl dendrimer platform behaves safely and with the intended immune-cell-\ntargeting pharmacology in an inflammatory clinical setting.\nIn addition, the in vivo pharmacology of hydroxyl dendrimers has been extensively validated in more than 30\nanimal models of neuroinflammation, including non-human primate and canine models (Kannan 2012; Kannan\n2014; Mishra 2014; Nance 2015; Guo 2016; Nance 2016; Zhang 2016; Nance 2017; Niño 2018; Turk 2018;\nLiaw 2020; Sharma 2020; Cabeza 2021; Henningfield 2024). Across these studies, hydroxyl dendrimers consistently\ncross the BBB in the presence of neuroinflammation, distribute rapidly after systemic administration, and are\nselectively internalized by activated macrophages and microglia in proportion to disease severity. Notably,\nHenningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) showed that Cy5-labeled hydroxyl dendrimers\npreferentially accumulated in plaque-associated microglia in 5xFAD mice. The 5xFAD model is characterized by\na comparatively intact BBB, with only localized and age-dependent permeability changes, underscoring that\ndendrimer uptake in this setting reflects genuine cellular targeting rather than nonspecific paracellular leakage.\nFinally, large-animal studies reported by other groups provide additional confidence in translational applicability.\nIn non-human primates with ischemic optic neuropathy and in canines subjected to hypothermic circulatory arrest,\nhydroxyl dendrimers selectively trafficked to injured CNS tissue and glia while showing negligible uptake in\nuninvolved regions (Guo et al. PLoS One. 2016 Apr 29;11(4):e0154437; Mishra et al. ACS Nano. 2014 Mar\n25;8(3):2134-47). These findings suggest preservation of the dendrimer’s CNS trafficking and targeting\nmechanisms across species.\nTaken together, the convergence of direct human trial data with OP-101, consistent findings across small- and\nlarge-animal neuroinflammatory models, and mechanistic studies showing selective uptake by activated\nmicroglia and macrophages provide strong evidence that the pharmacology of hydroxyl dendrimers is conserved\nacross species. The aim of our current study is to establish in vivo tracer specificity and pharmacology in a well-\ncharacterized preclinical MS model as a bridge to clinical imaging. Building on the human OP-101 results and\nlarge-animal evidence above, we are now evaluating 18F-FMD across all clinical MS subtypes to directly quantify\ntracer performance in human physiology.\nComment 4:\nLimited Disease Modeling: Testing in only one MS model (MOG35-55 EAE) provides insufficient evidence for\nbroader applicability. Human MS encompasses diverse subtypes with varying inflammatory profiles that may not\nbe adequately represented by this single model.\nResponse: We thank the reviewer for this comment. We acknowledge that there are multiple preclinical models\nof MS, each of which captures different aspects of disease biology. However, the goal of this study was not to\ntest tracer performance across a wide range of models, but rather to understand how our tracer performs in vivo\nin a model that develops both neuroinflammation and lesions. Using the MOG EAE model allowed us to\n35-55\nassess the tracer’s uptake, biodistribution, and specificity in the context of acute neuroinflammation, thereby\nproviding essential information about the sensitivity and specificity of this imaging approach in vivo.\nSince no single mouse model can fully recapitulate the pathophysiology or heterogeneity of human MS, our\nultimate goal was to move into clinical studies which are currently ongoing (NCT05395624), where the tracer is\nbeing evaluated directly in patients across the full spectrum of MS subtypes. As such, the EAE experiments were\ndesigned as a necessary preclinical step to establish feasibility and inform translation, rather than to exhaustively\nmodel the diversity of MS disease presentations in animals.\nComment 5:\nEthical Considerations: The extensive use of animals (160 mice) subjected to neurological disease with paralysis\nas endpoints appears disproportionate given the uncertain translational value. The authors have not\ndemonstrated that alternative research approaches (human tissue studies, computational modeling, existing\nclinical data) were adequately explored before conducting this extensive animal study.\nResponse: We appreciate this important point and agree that ethical considerations are central in animal\nresearch. Alternative approaches, however, are not feasible for the aims of this study. The goal of our work was\nto assess the uptake, biodistribution, and specificity of 18F-FMD in vivo. At present, there are no available\ncomputational models or in vitro techniques capable of recapitulating the complex in vivo behavior of radiotracers,\nincluding their stability, kinetics, biodistribution, specificity, and non-specific binding. While computational\nmodeling has shown promise in drug discovery, predictive modeling for PET tracer performance remains limited,\nand we are actively exploring this area in our lab.\nIn addition, the biophysical mechanism of uptake for 18F-FMD (via fluid-phase endocytosis by activated myeloid\ncells) cannot be meaningfully reproduced in tissue sections or static in vitro systems as these cannot replicate\nthe dynamic cell-environment interactions present in living organisms. New PET radiotracers must undergo\nevaluation in rodents and/or large animas (e.g., non-human primates) as a critical step before human studies.\nWe would also like to emphasize that the number of mice used was carefully justified. All experiments were\ndesigned a priori to achieve appropriate statistical power; we used the minimum number of animals necessary\nto rigorously test our hypotheses across multiple treatment arms and confirmatory analyses. Although 160 mice\nwere used in total, they were essential to establish the feasibility and safety rationale for moving into humans.\nFinally, we note that this tracer is currently being evaluated in a phase 1/2 clinical trial (NCT05395624). These\nanimal studies were not conducted in isolation but rather as an ethically justified bridge, designed to provide the\nproof-of-concept data necessary to support safe, rational translation into people. Our goal is to image as many\nMS patients as possible to rigorously establish clinical utility.\nComment 6:\nHow do the authors address potential partial volume effects from renal clearance affecting spinal cord\nquantification in such small anatomical structures?\nResponse: We thank the reviewer for raising this important point and apologize for not explaining this more\nclearly in the discussion section. To clarify, we addressed the potential issue for partial volume effects from renal\nclearance impacting spinal quantification through two different ex vivo approaches: gamma counting and\nautoradiography. To further explain these processes, spinal cords and kidneys were carefully dissected out of\neach mouse following cardiac perfusion to remove any potential contributions of tracer signal in blood. For\ngamma counting, the tissues were weighed wet, transferred into separate gamma counting tubes, and measured\nusing a gamma counter to obtain absolute quantitation of tracer concentration in individual organs, normalized\nto weight. This technique allows us to determine absolute tracer concentrations in each tissue without any\npotential for spillover or partial volume effects that can occur in PET imaging. To further understand the tracer\nuptake pattern (with higher resolution) in spinal cords, we embedded them in OCT, cryosectioned into thin (60\nµm) slices, and exposed on digital phosphorous films to obtain high-resolution spatial distribution data. These\nex vivo analyses, performed independently of the in vivo PET data, confirmed that tracer uptake within the spinal\ncord reflected true biological signal and was not artifactual due to renal clearance or blood pool signal or any\nother partial volume effects. Together, these complementary methods strengthen the robustness of our\nquantification and provide confidence in the specificity of our findings.\nThe following text has been modified in the discussion section:\n“Given the renal clearance of 18F-FMD and the small size of EAE mice, there is a possibility of partial volume\neffects from adjacent kidneys affecting signal quantification in the lumbar spinal cord. Partial volume corrections\nusing post-reconstruction- and reconstruction-based methods could help circumvent this limitation73. However,\nthese correction techniques are challenging to implement effectively in mouse models due to the extremely small\nsize and thin geometry of the spinal cord, which often falls below the spatial resolution of small-animal PET\nscanners74,75. This leads to significant spillover and underestimation of true activity even after correction,\nparticularly when adjacent high-uptake organs like kidneys are involved. Instead, we mitigated this issue by\ndissecting the kidneys and spinal cord out of each mouse for gamma counting and performed autoradiography\non thin spinal cord sections. These techniques eliminate contributions from circulating tracer and provide higher\nsensitivity and spatial resolution respectively, without any possibility for spillover from adjacent tissues.\nUsing\nthese methods, we observed a dramatic elevation in spinal cord signal in EAE mice compared to naïve controls,\ncorroborating in vivo PET findings. Importantly, this limitation is less relevant in larger species, such as non-\nhuman primates or humans, where the increased anatomical separation between kidneys and spinal cord\nreduces the likelihood of signal interference, allowing for more accurate delineation of regions of interest.”\nComment 7:\nCould the authors provide additional validation using models with intact blood-brain barriers to confirm specificity\nmechanisms?\nResponse: Thank you so much for this comment. We fully agree that confirming the specificity of the mechanism\nof tracer uptake is important. As mentioned in our manuscript, 18F-FMD does not accumulate in healthy CNS\ntissues, but only accumulates in regions containing neuroinflammation, where it is selectively endocytosed by\nactivated myeloid cells. We understand your concern regarding whether the uptake of our tracer depends\nprimarily on BBB disruption, and that it would be ideal to test in a model with an intact BBB. As you know,\nmicroglial activation is closely linked to BBB function. Once there are activated myeloid cells present in the CNS,\nthey release cytokines, chemokines, and reactive oxygen species (among other mediators) that modulate\nendothelial cells and pericytes. These chemical signals can subtly (or overtly) alter tight junction integrity,\nendothelial pathways, transporter activity, and thus passage of PET tracers into and out of the CNS (Yoon et al.\nNat Commun 16, 699 (2025); Mayer and Fischer, Front Cell Neurosci. 2024 Mar 13;18:1360195; da Fonseca et\nal. Front Cell Neurosci. 2014 Nov 3;8:362). It is thus challenging to identify a suitable model that contains our\ntarget of interest (i.e., activated myeloid cells) in absence of BBB dysfunction.\nWe agree that EAE mice do indeed exhibit significant BBB compromise, and therefore you raise a valid question\nabout the specificity of the mechanism of uptake of our tracer. To address your concern, we have added\nadditional empirical data below validating the uptake of 18F-FMD in models with negligible BBB dysfunction.\nEvidence of elevated 18F-FMD uptake in the cortex and hippocampus of 5xFAD mice at an early stage of\ndisease, known to have largely intact BBB (cited data; open access)\nWe evaluated 18F-FMD in the 5xFAD Alzheimer’s disease mouse model, which is known to have a largely intact\nBBB before 4 months of age and only modestly altered between 4-8 months (Zhukov et al., Front Aging Neurosci.\n5xFAD mice (Figure 1 and Table 1), which is an early stage of disease, 18F-FMD uptake was threefold higher in\ncortex and hippocampus (regions with known AD pathology and microglial activation) compared to wild-type\ncontrols, with higher sensitivity than TSPO-PET using 18F-GE180 (a small molecule that crosses the intact rodent\nBBB and binds to the translocator protein 18kDa, a widely explored PET imaging biomarker of\nneuroinflammation). This data provides strong evidence that the predominate mechanism of 18F-FMD uptake is\nvia endocytosis of activated microglia rather than passive diffusion across a compromised BBB alone.\nFigure 1. Coronal view of representative 3.75-month-old 5xFAD transgenic and wild-type mouse brain PET\nimages (50-60 min p.i. summed) using either 18F-FMD or 18F-GE180, co-registered with CT images. Carlson et\nal., Alzheimer's Dement. 2022, 18: e060700.\nTable 1. Tracer signal in 5xFAD mice normalized to wild-type mice signal (equivalent to signal-to-background\nratios) in brain regions known to contain amyloid pathology and microglial activation in 5xFAD mice imaged with\neither 18F-FMD or 18F-GE180 at 3.75 months old.\n18F-FMD (n=4 WT, n=4 5xFAD) 18F-GE180 (n=4 WT, n=5 5xFAD)\nCortex 3.18 1.21\nHippocampus 3.05 1.24\nWhole Brain 3.14 1.21\nComplementary evidence from fluorescent dendrimer analog of 18F-FMD (cited data; open access)\nHenningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) confirmed the specificity of the fourth-\ngeneration polyamidoamine (PAMAM) hydroxyl dendrimer (same scaffold as 18F-FMD), showing that this Cy5-\nlabeled dendrimer was regionally and selectively phagocytosed by plaque-associated microglia (PAMs) in 7-\nmonth-old 5xFAD brains (Figure 1c). Their work corroborated our in vivo PET imaging data in 5xFAD mice and\nshowed robust Cy5 signal around dense-core plaques (Amylo-Glo-positive) and co-localization to Iba1+ microglia\nand CD68+ microglial lysosomes in hippocampus and samatosensory cortex. Wild-type mice, by contrast,\nexhibited negligible Cy5 signal. Additionally, staining for astrocytes, neurons, and oligodendrocytes showed no\nCy5 signal associated with those cells in the subiculum or somatosensory cortex (Fig. 1m-q).\nFigure 1c from Henningfield et al. (Alzheimers Res Ther. 2024 May 6;16(1):101) demonstrates co-localization\nof Cy5-labeled hydroxyl dendrimers (red) with microglia (Iba1, green) and dense core plaques (Amylo-Glo, blue).\nFigure 1m-q from Henningfield et al. showing G4 hydroxyl dendrimers are selectively endocytosed by plaque-\nassociated microglia in the subiculum (p) and somatosensory cortex (q) regions of 5xFAD brain with no uptake\nby astrocytes (m, m1), neurons (n), oligodendrocytes (o, o1).\nAdditional validation across models with limited BBB disruption (from Carlson et al., with permission)\nWe have also previously published a study assessing 18F-FMD in an LPS model induced by intraperitoneal\ninjection of LPS (rather than intracerebroventricularly; Carlson et al., Mol Imaging Biol. 2023;25:1063-1072),\nwhere BBB alterations are modest compared to the EAE model used in our current study. In LPS mice, we\ndemonstrated a correlation between whole-brain 18F-FMD uptake and murine sepsis score – with increasing\nscore relating to elevated levels of activated microglia (Figure 4c).\nFigure 4c shows strong correlation between 18F-FMD whole brain signal and severity of sepsis symptoms using\nmurine sepsis score (MSS). Adapted from Carlson et al., “Development and Initial Assessment of [18F]OP-801:\na Novel Hydroxyl Dendrimer PET Tracer for Preclinical Imaging of Innate Immune Activation in the Whole Body\nand Brain,” Molecular Imaging and Biology, 25, 1063-1072 (2023), Springer Nature. Permission obtained via\nRightsLink.\nMoreover, the mechanism of uptake and biodistribution of these hydroxyl dendrimers has been characterized\nextensively in over 30 animal models of neuroinflammation across species (including rodent, canine, and primate\nmodels) by other groups. In these studies, G4 dendrimers consistently accumulated at sites of injury or\nneuroinflammation but not in healthy brain tissue (Kannan 2012; Kannan 2014; Mishra 2014; Nance 2015; Guo\n2016; Nance 2016; Zhang 2016; Nance 2017; Niño 2018; Turk 2018; Henningfield 2020; Liaw 2020; Sharma\n2020; Cabeza 2021).\nTaken together, these data (in particular, the 5xFAD mouse data collected by our lab using 18F-FMD in addition\nto 5xFAD data collected and published by a separate lab using the fluorescently labeled version of this same\ndendrimer), along with extensive G4 dendrimer biodistribution studies in multiple species, provide strong\nevidence that 18F-FMD can cross the BBB both under conditions of overt disruption (e.g., EAE) and, importantly,\nin models with minimal impairment (e.g., early-stage 5xFAD and i.p. LPS). Crucially, tracer retention occurs only\nin regions containing activated myeloid cells, thus indicating that the predominate mechanism of uptake and\nretention is endocytosis by activated myeloid cells.\nThe following text has been modified in the introduction section to more clearly articulate the mechanism of\nuptake of our tracer:\n“Fourth-generation hydroxyl dendrimers have been demonstrated to penetrant regions of a dysfunctional–but\nnot healthy, intact–blood-brain barrier (BBB), where they are quickly and selectively taken up by activated\nmyeloid cells via fluid phase endocytosis, and rapidly cleared from tissue that does not contain these cells27–34.”\nMechanistic evidence of selective endocytosis of generation 4 hydroxyl dendrimers by activated myeloid\ncells\nWe used a CSF1R inhibitor dendranib to selectively inhibit the activation and proliferation of myeloid cells in EAE\nmice (to investigate the specificity of our tracer uptake). In vivo PET imaging, complemented by ex vivo gamma\ncounting and autoradiography, demonstrated significantly reduced 18F-FMD uptake in CNS regions with known\nEAE pathology, including the medulla, cerebellum, pons, and both the cervical/thoracic and lumbar spinal cord\n(Figures 4 and 5 in the manuscript). Flow cytometry with a fluorescent analog of 18F-FMD (Cy5-labeled G4\nhydroxyl dendrimer; Cy5-HD) further confirmed selective endocytosis of hydroxyl dendrimers by activated\nmicroglia and infiltrating myeloid cells. Notably, CSF1R dendranib treatment led to a marked reduction in Cy5-\nHD+ activated microglia (46-fold) and infiltrating macrophages (19-fold).\nGiven the breath of preclinical validation already generated, and as the reviewer noted in comment 5, we believe\nthat additional rodent models would add limited translational value. Instead, our priority is to complete our current\nhuman studies. In our ongoing clinical trial (NCT05395624; preliminary data published in Moradi, F. et al. J. Nucl.\nMed., 2025, 66, 251092; reference 77), our goal is to understand how 18F-FMD performs across the full spectrum\nof MS subtypes (including RRMS, PPMS, SPMS, and PRMS) to establish its clinical utility in patients, where\nBBB compromise is heterogeneous and variable over time.\nComment 8:\nWhat evidence supports the assumption that fluid-phase endocytosis rates in activated murine myeloid cells\naccurately reflect those in human cells?\nResponse: We appreciate this thoughtful comment. While cross-species differences in endocytic activity are\nindeed important to consider, there is direct evidence that fluid-phase endocytosis of hydroxyl dendrimers is\nconserved across rodent and human cells and tissues.\nFirst, Kambhampati et al. (Eur J Pharm Biopharm. 2015 Sep;95(Pt B):239-49) performed a side-by-side in vitro\ncharacterization of hydroxyl dendrimer uptake and release kinetics in murine BV2 microglial cells and in human\nretinal pigment epithelial (RPE) cells. The study demonstrated highly consistent patterns of dendrimer\ninternalization, trafficking, and intracellular retention between the murine and human cell systems, supporting\nconserved uptake mechanisms across species at the cellular level.\nSecond, Kambhampati et al. (J Control Release. 2021 Jul 10;335:527-540) provided in vivo confirmation in both\nrodent and human tissues. In a rat model of choroidal neovascularization (CNV), hydroxyl dendrimers selectively\naccumulated in activated myeloid and glial cells. Importantly, the same paper also demonstrated dendrimer\nuptake in post-mortem human diabetic eyes, localizing to activated myeloid and glial populations. This direct\ncomparison across rat and human tissue provides strong evidence that the endocytic targeting behavior of\nhydroxyl dendrimers is conserved across species, including in relevant human disease contexts.\nCollectively, these studies help bridge the translational gap by showing that the fluid-phase endocytosis of\nhydroxyl dendrimers by activated myeloid cells is not unique to murine systems but is also observed in human\ncells. This supports the rationale for extrapolating from murine models to human applications in the context of\n18F-FMD pharmacology."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all of my previous comments, and I have no further revisions to suggest for this very nice manuscript.", "reviewer_3": "The authors responded to my comments very well.\nMy previous translational concerns, while not completely resolved, are substantially mitigated by the new evidence provided.\nThank you."}, "author_response": ""}]

Decision letter