Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood–brain barrier. The recent identification and characterization of a small population of regulatory T (Treg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident Treg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Recordings were processed and analyzed using Multi Channel Experimenter software (Multi Channel Systems).

Supplementary Resource 2. Code for single-cell RNA-seq analysis for wildtype vs
αCamKII IL-2 mice. Code is provided in .pdf and .Rmd formats.

Supplementary Resource 3. Code for single-cell RNA-seq analysis for PHP.GFAP-GFPvs PHP.GFAP-IL-2-treated mice.
Code is provided in .pdf and .Rmd formats.
Representative video of a conventional CD4 T cell and a Treg, in the mid-brain region.
Supplementary Video 2. Brain Treg cells in αCamKII IL-2 brain. 3D surface rendering of an αCamKII IL-2 perfused brain, stained for CD4 (green), Foxp3 (red), CD31 (white) and DAPI (blue). Representative video of a CD4 T cell cluster, consisting of two conventional CD4 T cell and four Treg cells, in the mid-brain region.
Supplementary Video 3. CD4 T cells in control brain. 3D surface rendering of a PHP.GFAP-GFP-treated perfused brain, stained for CD4 (green), Foxp3 (red), CD31 (white) and DAPI (blue). Representative video of a conventional CD4 T cell and a Treg, within the mid-brain.

Supplementary Figure 1: Confocal identification of brain Treg cells in αCamKII IL-2
mice. Healthy perfused mouse brains from wildtype and αCamKII IL-2 mice were compared by immunofluorescent confocal imaging. CD4 (green), Foxp3 (red), CD31 (vasculature,white) and DAPI (blue). Single and combined channel representative images of CD4 T cells in the mid-brain, with close-up imaging of identified CD4 T cells. A representative picture of three individual mouse samples is shown (n = 3). Scale bar, 10 µm. representative picture is shown (n = 3, 1). Scale bar coronal section, 100 µm. Scale bar regional image, 100 µm. Scale bar insets, 10 µm.    Control -brain

Supplementary Figure 4: Treg cells in brain regions in αCamKII
Control -blood g Supplementary Figure 6: Synthetic expansion of brain regulatory T cells preserves the transcriptional profile. Brain CD11b + cells, brain CD45 + CD4 + cells and blood CD45 + CD4 + positive cells were sorted from wildtype or αCamKII IL-2 mice for 10X single-cell sequencing (n=2). Data processing, filtering and reclustering (as in Supplementary Fig. 5) identified 6332 cells classified as T cells. a, Differential gene expression displayed for brain Treg cells from wildtype vs αCamKII IL-2 mice, b blood Treg cells from wildtype vs αCamKII IL-2 mice, c brain conventional T cells from wildtype vs αCamKII IL-2 mice, d blood conventional T cells from wildtype vs αCamKII IL-2 mice, or e blood vs brain Treg cells. Vertical lines mark fold changes 0.4 and -0.4 and horizontal lines mark the adjusted P value of 0.05. Selected significant changes are annotated. Statistical analyses were performed using Wilcoxon rank sum test to perform differential expression analysis. The P value adjusted is based on Bonferroni correction using all features in the dataset. Statistical analyses were performed using Wilcoxon rank sum test to perform differential expression analysis. The P value adjusted is based on Bonferroni correction using all features in the dataset (d). Control αCamKII IL-2 Supplementary Figure 8: Progressive neurological damage following traumatic brain injury. Controlled cortical impact was performed to induce moderate TBI, with examination on days 1, 2, 3 and 7 post-TBI (n = 3, 3, 1, 3). a, Macroscopic damage to the surface of the brain at the injury site, representative photo. Scale bar, 0.5 cm. b, Representative immunofluorescence staining of the cortical tissue after controlled cortical impact. GFAP (astrocytes), Iba1 (microglia), DAPI (nuclei). Scale bars, 500 µm. c, Measure of total integrated GFAP intensity in the cortical area adjacent to the impact site at 1, 2, 3 and 7 days post-TBI. Mean ± s.e.m. Figure 9: Normal peripheral influx following traumatic brain injury in αCamKII IL-2 mice. Control (wildtype) littermates and αCamKII IL-2 mice were tamoxifentreated at 6 weeks, and controlled cortical impacts to induce moderate TBI were given at 12 weeks. Mice were examined at 15 days post-TBI (n=4,4). TBI-induced perfused brains from littermate control and αCamKII IL-2 mice were compared by high-dimensional flow cytometry. Statistical analyses were performed using a non-parametric Mann-Whitney U-test. 1 -Naive Representative gating strategy used to quantify Treg cell numbers in the brain using flow cytometry.  CD4  496  372  1676  88  152  254  1160  298  Tregs  328  164  1114  68  474  284  1056  332  CD8  2276  786  3272  470  1254  700  3350  1032  TCRγδ  518  746  3008  64  1634  456  2164 Areg  22,2  0  22,2  30  27,3  50  10,2  31,8  50  60  IL10  5,56  22,2  0  60  18,2  33,3  18,4  50  50  50  IL17  0  11,1  0  10  0  0  2,