Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells engraft and deplete DC1 in immunodeficient mice. RAG2 −/− mice were iv injected with 1 x 10 6 XCR1, control, or mock transduced (A, B, G–J, P) CD8 + , ( C-F, O ) CD3 + (+ 10 μg IL-2), or (K–N, Q) CD4 + T cells on day 0 (n = 3/group). On day 7 or 12, spleen and ILN were collected and analyzed via flow cytometric analysis for CAR-T and DC subsets. Shown (A) are representational dot plots of the following: row 1, donor T cells (TCRβ + CD8 + ) assessed for SSC-A (y-axis) and mCherry (x-axis); row 2, recipient CD45 + cells for MHCII (y-axis) and CD11c (x-axis). Shown (B) are representational dot plots of recipient DCs (MHCII + CD11c + ) for CD11b (y-axis) and XCR1 (x-axis), and DCs for BST2 (y-axis) and XCR1 (x-axis) from the spleen of mice treated with CD8 + CAR-T cells. Graphed are the percent CAR + (mCherry + ) of the (C) TCRβ + (G) , CD8 + , and (K) CD4 + T cells and (D, H, L) the percent DCs (MHCII + CD11c + ) of total CD45 + from the ILN and spleen of mice treated with (C, D) CD3 + (G, H) , CD8 + , and (K, L) CD4 + CAR-T cells. The percent DC1 (XCR1 + CD11b − ), DC2 (XCR1 − CD11b + ), and pDC (XCR1 − BST2 + ) of total DC are shown for the spleen (E, I, M) and the ILN (F, J, N) for mice treated with (E, F) CD3 + , (I, J) CD8 + , and (M, N) CD4 + CAR-T cells. Graphed (O) is the Percent change in body weight from day 0 to the end of the experiment on day 12 of mice treated with CD3 + CAR-T cells. On day 7, a quarter of the total splenocytes were restimulated with 10 μg/mL LPS in T cell media for 24 h, and then ng/mL IL-12 was determined via MSD. Shown (P, Q) are ng/mL of IL-12 from (P) CD8 + or (O) CD4 + CAR-T cell-treated mice. Statistical significance was analyzed by use of one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05. Error bars represent SEM. Data (A–J) are representative of three independent similar experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Control

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells systemically deplete DC1 when engrafted with encephalitogenic T cells in immunodeficient mice. RAG2 −/− mice (n = 4/group) were iv injected with 1 x 10 6 XCR1, control, or mock transduced CD45.1 + CD8 + CAR-T alongside 1 x 10 6 encephalitogenic CD4 + CD45.2 + T cells and 10 µg of IL-2 IP on day 0. On day 6, ILN, CLN, spleen, lung, and liver were collected and analyzed via flow cytometric analysis for CAR-T cells and DC subsets. Shown (A) the percent CAR + (mCherry + ) CD45.1 + of viable and (B) the percent Ki67 + of donor CD45.1 + cells and (C) the percent DC of total CD45.2 + . Shown (D) are representational dot plots of DC (MHCII + CD11c + ) for XCR1 (y-axis) and CD11b (x-axis) for each organ. Graphed are (E) the percent DC1 (XCR1 + CD11b − ), (F) DC2 (XCR1 − CD11b + ), and (G) pDC (XCR1 − BST2 + ) of total DCs. Statistical significance was analyzed by use of one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Control

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells suppressed the onset of CD4 + T cell-induced passive EAE. (A-G) EAE was induced in RAG2 −/− mice by iv injection with 2 × 10 6 CD45.2 + CD4 + encephalitogenic T cells followed by PTx on days 0 and 2. In addition, mice were treated with 2 × 10 6 XCR1, control, or mock CD45.1 + CD8 + CAR-T (n = 8/group) and 10 µg of IL-2. Shown (A) are the mean clinical EAE scores and (B) the mean percent change in body weight from day 0 through the end of the experiment on day 24 for each group. Shown (C) is the initial day of EAE disease onset from day 0 through day 24 for XCR1 and control CAR-T treatment groups. Spleen, ILN, and CNS were harvested from five mice from each group on day 24 and analyzed via flow cytometry for presence of CAR-T cells and DC subset. Shown (D) are representative dot plots of the following: first row, CD45.1 + donor cells assessed for mCherry; second row, CD45.2 + cells assessed for MHCII (y-axis) and CD11c (x-axis); and third row, DC (CD11c + MHCII + ) assessed for SSC-A (y-axis) and XCR1 (X-axis) from the spleen. The percent (E) CAR + (mCherry + ) T cells, (F) percent DC (CD11c + MHCII + ) of the CD45.2 + cells, and (G) percent DC1 (XCR1 + ) of the total CD45.2 + DC are shown for each organ on day 24. Statistical significance was analyzed by use of (A) Friedman test followed by Dunn’s multiple comparisons test, (B) two-way repeated measures ANOVA followed by Šídák multiple comparison test, (C) Gehan–Breslow–Wilcoxon test, (E–G) one-way ANOVA Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM. Data are representative of two independent experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: IV Injection, Control, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 CAR-T cells suppress the onset of CD3 + T cell-induced EAE. RAG2 −/− mice were iv injected with 1 x 10 6 purified CD3 + encephalitogenic T cells, to induce EAE, on day 0. Subsequently, RAG2 −/− mice were injected IP with 100 ng PTx on days 0 and 2. In addition, mice were iv treated with 1 x 10 6 XCR1 or control CD8 + CAR T cells (n = 6/group) on day 0. Shown (A) are the daily mean clinical EAE scores and (B) the mean percent change in body weight and (C) the initial day of EAE onset from day 0 through day 14 for XCR1 and control CAR-T cell-treated mice. The CNS was harvest on day 19 and analyzed via flow cytometry for the presence of CAR-T cells and different DC subsets. Shown (D) are representational dot plots; first column, CAR-T cells (CD45.1 + CD8 + ) assessed for CD8 (y-axis) and mCherry (x-axis); second column, total DCs (MHCII + ) assessed for SSC-A (y-axis) and CD11c (x-axis); third column, DCs (MHCII + CD11c + ) assessed for XCR1 (y-axis) and CD11b (X-axis); fourth column, DCs assessed for BST2 (y-axis) and CD11b (X-axis). The percent CAR + T cells of CD45.1 + donor cells (E) and the percent DCs of total CD45.2 + F4/80 − MHCII + cells (F) for CAR-treated mice. Shown (G) are the percent DC1, DC2, and pDC, of total DCs. Statistical significance was analyzed by use of (A) Wilcoxon matched pairs test, (B) two-way repeated measures ANOVA, (C) Gehan–Breslow–Wilcoxon test and (D-G) unpaired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Injection, Purification, Control, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: CAR-T cells and CAR-Tregs targeting conventional type-1 dendritic cell suppress experimental autoimmune encephalomyelitis

doi: 10.3389/fimmu.2023.1235222

Figure Lengend Snippet: XCR1 and MOG CAR-Tregs are functional and suppress EAE. (A, B) 50,000 XCR1, control, or MOG CD4 + CAR-Treg were cocultured with 5 x 10 5 irradiated RAW, RAW–XCR1, HEK, or HEK–MOG overnight in complete media with 3 ng/mL IL-2. Shown (A) are representational histograms of CD69 (x-axis) expression of XCR1 (top) or MOG (bottom) CAR-Treg cocultured with respective cell lines, mouse T-activator beads “CD3/CD28” at a 10:1 ratio, or alone. Graphed (B) are the percent CD69 + of XCR1 (left), MOG (middle), or control (right) CAR-Treg following cocultured with respective cell lines. (C, D) 25,000 CTV-stained OTII responder T cells were cocultured with 50,000 irradiated (30 Gy) BMDC and 50,000 RAW, RAW–XCR1, or RAW–MOG cells in complete media with 64 nM OVA323-339. XCR1 or MOG CAR-Treg were titrated and added at designated ratios. Shown (C) are representative histograms of CTV dilution of responder OTII T cells under each condition. Graphed (D) is the percent suppression of responder OTII T cells at designated ratios with XCR1 or MOG CAR-Tregs cocultured with RAW–XCR1 (left) and RAW–MOG (right). (E) RAG2 −/− (n = 4–5/group) were iv injected with 1 x 10 6 purified CD4 + encephalitogenic T cells and 5 × 10 5 XCR1, control, MOG, or mock CAR-Treg on day 0 and IP with 120 ng PTx on days 0 and 2. Shown (E) are the daily mean clinical EAE scores from day 0 through day 20. (F) C57BL/6 mice (10/group) were injected with MOG 35–55 in CFA followed by 120 ng PTx IP on days 0 and 2. 5 × 10 5 XCR1, control, MOG CAR-Treg, or PBS were injected on day 1. Shown (F) are the mean clinical EAE scores from day 0 through day 15. (G) C57BL/6 mice (5–10/group) were injected with 2 × 10 6 purified CD4 + encephalitogenic T cells followed by 120 ng PTx IP on days 0 and 2. Then, 2.5 × 10 5 XCR1, control, or mock CAR-Treg were iv injected on day 13. Shown (G) are the mean clinical EAE scores from day 0 through day 40. Statistical significance was analyzed by use of (B, D) unpaired two-tailed t test and (E, G) Friedman test followed by Dunn’s multiple comparisons test for day 8 through end of experiment. *p < 0.05, ****p < 0.0001 Error bars represent SEM. Data (A-D) are representative of three independent experiments.

Article Snippet: Female C57BL/6J “CD45.2” (Stock# 000664), B6.SJL- Ptprc a Pepc b /BoyJ “CD45.1” (Stock# 002014), B6.Cg-Foxp3tm2Tch/J “FIG” (Stock# 006772), and B6.Cg-Tg(TCRaTCRb)425Cbn/J “OTII” (Stock# 004194) were purchased from Jackson Laboratories; C57BL/6NCrl “CD45.2” and B6.SJL- Ptprc a Pepc b /BoyCrCrl “CD45.1” were purchased from Charles River Laboratories; B6.129S6- Rag2 tm1Fwa “RAG2 −/− “ were purchased from Taconic; and XCR1-DTR-cre mice were designed and generated by Horizon Discovery using CRISPR-Cas9 (now Inotiv Co, St. Louis, MO) and colony maintained at Charles River Laboratory.

Techniques: Functional Assay, Control, Irradiation, Expressing, Staining, Injection, Purification, Two Tailed Test