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Supplemental material, sj-docx-1-dhj-10.1177_20552076231152989 for Driving time-based identification of gaps in specialised care coverage: An example of neuroinflammatory diseases in Germany by Lars Masanneck, Saskia Räuber, Christina B Schroeter, Sophie Lehnerer, Tjalf Ziemssen, Tobias Ruck, Sven G. Meuth and Marc Pawlitzki in Digital Health

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPβ in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron–microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo–like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.

Additional file 2: Figure S2. Titration curves of oral cladribine treatment in mice. In preliminary experiments, we analyzed the plasma [ng/ml] and brain concentrations [ng/g] of oral cladribine treatment with different doses (3.25, 5 and 10 mg/kg bodyweight) over a time period of 2.5 h. Every 30 min blood was drawn and a proportion of animals was killed to obtain brain tissue for titration analyses. Curves show the maximal concentration of cladribine in blood and brain tissue (Cmax [ng/ml or ng/g]), the time to maximal concentration (tmax [hours]) and the area under the curve (AUC [ng*h/ml or ng*h/g]).

Additional file 3: Figure S3. Gating strategy for flow cytometry. Single immune cells from the periphery (A, here exemplary shown with spleen tissue) and the central nervous system (CNS; B, here exemplary shown with spinal cord tissue) were simultaneously analyzed by flow cytometry. Total leukocytes were identified by forward scatter (FSC) and sideward scatter (SSC) and cell-doublets were removed by FSC width and FSC height gating. From these cells, we identified leukocytes subsets based on their surface marker expression: CD45R+ B cells; CD3+CD4+ T-helper cells, CD3+CD8+ cytotoxic T cells and CD4+CD8+ double positive T cells; CD3−NK1.1+ natural killer (NK) cells; CD11b+CD11c− monocytes and macrophages (M/M) and CD11b+CD11c+ dendritic cells. Regarding the CNS tissue, prior to discrimination of leukocyte subsets, we differentiated microglia cells (CD45med) from infiltrated leukocytes (CD45high).

Additional file 7: Figure S7. Proliferation assay of splenocytes from naïve wild-type mice, with and without cladribine treatment in vitro. A: Proliferated cells in % after cladribine treatment (with 0.1 or 1 μM cladribine) versus vehicle-treated cells. We compared the unstimulated to the stimulated setting (stimulated with 1 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 3 days) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). B: Mean fluorescence intensities (MFIs) of migration and activation markers in all viable stimulated cells upon vehicle- compared to cladribine-treatment (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 1: Figure S1. Experimental scheme. Active MOG EAE was induced as previously described by immunization (day 0) of C57BL/6J mice with MOG35–55 peptide, followed by pertussis toxin (PTX) injections (day 0 and day 2) (15). Mice were divided into two experimental groups: Group 1 received cladribine via oral gavage (10 mg/kg from day 5 to day 9), while group 2 received only the vehicle for the same period of time. On day 10 post-EAE induction, focal EAE lesions were generated by stereotactic injection of proinflammatory cytokines (interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) into the auditory cortex to induce cortical grey matter lesions. Experimental read-out (electrophysiological recordings, flow cytometric immunophenotyping, histology) was performed either on dmax (defining the day of maximal clinical deterioration) or on day 27 post-EAE induction (to assess the chronical EAE state).

Additional file 6: Figure S6. Migration assay of leukocytes in organ cultures of spleens from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells upon cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in spleens (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 2. Suppl. Table 2. Clinical and demographic characteristics of included MC.

Additional file 5: Figure S5. Migration assay of leukocytes in organ cultures of cervical LNs from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells after cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in LNs (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.0001 = ****.

Additional file 4: Figure S4. Gating strategy for sorting of macrophages. CD11b+ cells were isolated from murine CNS and spinal cord via magnetic beads. Subsequently, CD11b+ cells were simultaneously analyzed and sorted by flow cytometry. Total CD11b+ cells were identified by forward scatter (FSC) and sideward scatter (SSC) (A), and cell-doublets were removed by SSC width and SSC height gating (B). From these cells, we discriminated macrophages based on their surface marker expression of CD45highCD11bhigh (C). Macrophages were sorted for downstream experiments (D). Plots (E)-(F) show the distribution of microglia (CD45intermCD11bhigh) in comparison to macrophages.

Additional file 3. Suppl. Table 3. Trigger factors.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.