Fig 2: Transduction of neural cells upon IC GT and brain myeloid cell reconstitution upon BMT in SD mice(A) Schematic of LV.hexA+ LV.hexBinjection and immunofluorescence (IF) confocal image showing clearance of GM2 storage and reduction in CD68+ cells in a region of SD brain tissue (box) close to the LV injection site. Top panel: GM2, red. Bottom panel: CD68, red; IBA1, blue. Nuclei are counterstained with Hoechst (white). Scale bar, 25 μm. (B and C) Percentages of donor-derived GFP+ cells in the peripheral blood (PB; (B) and bone marrow (BM; (C) of BMT- and IC GT + BMT-treated SD and WT mice evaluated 30 days (PB) and 60 days (BM) after BMT. The percentage of GFP+ cells in the BM of tgGFP mice (donors) is shown for comparison. (D) Cellular composition of the BM analyzed 60 days after transplantation into BMT- and IC GT + BMT-treated SD mice and age-matched UT controls (WT, SD, and tgGFP mice). CD19, marker for B cells; CD11b, marker for monocytes, neutrophils, and natural killer (NK) cells; CD3, marker of mature T cells. Data represent the mean ± SEM; n = 3–13 mice/group. (E) IF image showing the distribution of GFP+ cells in a whole brain sagittal section of an IC GT + BMT-treated SD mouse at 120 days. Boxes highlight brain regions in (F) at higher magnification. (F) IF pictures showing GFP+ cells engrafted into the olfactory bulb (OB), hippocampus (Hip), thalamus (Thal), and pons/medulla (Pons) of IC GT + BMT-treated SD mice at 120 days and 240 days. (E) and (F) Direct GFP fluorescence, green; nuclei counterstained with Hoechst, gray. Scale bars, 3,000 μm (E) and 500 μm (F).

Fig 3: The effect of tumor size on the ability of FA-TLR7-1A to enhance anti-CD19 CAR T cell therapy of solid 4T1-mCD19 tumors. Analysis of tumor size and body weight change of mice in which treatments started when the average tumor sizes reached (A) 50 mm3, (B) 90 mm3, and (C) 130 mm3. (D) Comparison of the relative tumor sizes on day 26 after initiation of therapy. The number of mice per treatment group were as follows: for mice with 50mm3 tumors: “No treatment”, n=5; “3 nmol FA-TLR7-1A, 5W”, n=6; “CAR T+PBS”, n=6; “CAR T+3n mol FA-TLR7-1A, 5W”, n=9. For mice with either 90mm3 or 130mm3, n=5 for all the groups. Mean ± SEM. Statistical significance between multiple groups was determined using a 1-way ANOVA followed by a Tukey post hoc analysis. Statistical significance between two groups was determined using an unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant P ≥ 0.05).

Fig 4: The immune adhesion of Foe‐TEVs via enhanced subcapsular capturing and effector interacting. A) The distribution of Foe‐TEVs in lymphatic vessels and blood vessels presented by immunofluorescence staining in allograft. B) Confocal imaging of Foe‐TEVs distribution in subcapsular sinus of draining lymph node. C) The distribution of Foe‐TEVs under the capsule and in the corticomedullary junction shown by multiple immunofluorescence staining. D) The interaction between Foe‐TEVs and medullary macrophage in the medulla shown by multiple immunofluorescence staining. E) The interaction of Foe‐TEVs with T cells (CD3+), marginal zone B cells (CD19+), and follicular B cells (CD21+) in germinal centers. F) Flow cytometry analysis of adhesion of Foe‐TEVs to CD11b+CD11c+DCs, F4‐80+macrophages, CD45+CD3+T cells, and CD45+CD19+B cells. G) Quantification of leukocyte percentage with Dil+ TEVs 1 and 6 h after intravenous injection of Vector‐TEVs or Foe‐TEVs (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the Vector‐TEVs 6 h group; ### P < 0.001 compared to the Foe‐TEVs 1 h group. H) Confocal images showing tracks of Foe‐TEVs and Vector‐TEVs contacting with SCS macrophages under subcapsular sinus of lymph nodes. I) The instant velocity of individual Vector‐TEVs and Foe‐TEVs. J) The mean velocity of Vector‐TEVs and Foe‐TEVs under subcapsular sinus of lymph nodes (n = 300). K) The arrest coefficient of Vector‐TEVs and Foe‐TEVs (n = 300). ***P < 0.001. Values are presented as mean ± SEM. Statistical analysis was determined by two‐tailed Student's t‐test.

Fig 5: Engineering primary B cells and adoptive transfer of cells to WT mice.a Targeting antibody genes to the mouse heavy chain (HC) locus (H-targeting). Donor DNA encoding (1) a HC V-gene promoter, (2) the VRC01 ? chain (with mouse constant gene and P2A peptide), (3) the VRC01 HC variable (VDJ) region and donor splice site is inserted at a CRISPR-Cas9 cut site in JH4 for expression as a functional antigen receptor using endogenous downstream HC constant genes. Regions of homology (HR) flanking bnAb donor-DNA allow for its incorporation at the DNA break-site by homology-directed repair (HDR). b Targeting antibody genes to the mouse heavy and kappa loci (H + ? targeting). To engineer the IgH locus, donor DNA encoding (1) a HC V-gene promoter (2) VRC01 VDJ gene, and constant gene donor splice site is inserted as above. To engineer the Ig? locus, donor DNA encoding (1) a ? V-gene promoter, (2) VRC01 ? variable (VJ) region and constant gene donor splice site is likewise inserted into a CRISPR-Cas9 cut site in J?5, for expression of VRC01 H and ? chains from their endogenous loci spliced to cell-native constant genes. c Targeting efficiency. Successfully targeted B cells expressing VRC01 as cell surface antigen receptor were detected as live, single, KO11-,eOD-GT8-AF647+, and eOD-GT8-AF488+ cells by flow cytometry. eOD-GT8 double-positive cells are shown for LPS activated (mock), H + ?, and H-targeted B cell cultures. d LPS-activated donor cells acquire a memory phenotype in vivo after adoptive transfer. Non-engineered primary B cells were either directly transferred or cultured for 48 h in LPS before adoptive transfer into host mice. The fractions of donor (CD45.1+) cells that showed a memory cell (MC) phenotype after 14 d in vivo are shown for n = 6 mice in each group. Memory B cells are gated as live CD19+CD38+GL7-IgD-. e Engineered cells become germinal center independent (CD73-) memory in vivo. H-targeted cells were adoptively transferred into host mice. 14 days later, successfully engineered (GT8+) cells were analyzed by flow cytometry. Host naïve and memory B cell populations are compared for their expression of CD73, PD-L2, and CD80 memory markers. f Quantitation of B cells gated as in (e). The fraction of successfully targeted cells with the indicated cell surface memory cell markers are given for n = 4 engrafted mice. These are compared with unsuccessfully engineered cells in the targeted population (GT8-), mock engineered, or host cell controls. Data are presented as mean value ± SD in Figure d and f.