Panel A shows construct schematics for a luciferase reporter and CAIleR 8 with Thy1.1, P2A, signal peptide, scFv8, CD28 hinge, transmembrane and intracellular domains, and CD3 zeta, alongside an experimental timeline from day 0 to day 14 covering murine T cell isolation, retroviral transduction, and intravenous injection. Panel B shows a BALB/c mouse timeline with intravenous injection at day 0 and intranasal allergen instillations from day 0 to day 11 with sacrifice at day 14. Panel C shows flow cytometry plots confirming CAIleR 8 transduction at 80.5 percent and Th1.1 expression on polyclonal T cells at 84 percent. Panel D shows a scatter dot plot of absorbance at 450 nanometers for Bet v1 IgG1, where BPE CAIleR 8 reaches approximately 1.5 compared to PBS CAIleR 8 near 0.5 with significant differences. Panel E shows bar graphs of CD3 positive Thy1.1 positive percent across lung, mediastinal lymph node, cervical lymph node, and spleen with no significant differences between groups. Panel F shows bar graphs of proliferation percent where BPE CAIleR 8 reaches approximately 4 percent in lung and cervical lymph node, significantly higher than PBS CAIleR 8 and BPE polyclonal. Panel G shows the CAR A2 and CAIleR 8 construct schematics and an in vivo experimental timeline from day 0 to day 15 with intranasal allergen instillations and serum collection. Panel H shows absorbance at 450 nanometers for Bet v1 IgG1 reaching approximately 1.5 for BPE compared to near 0 for PBS. Panel I shows transduction efficiency reaching approximately 65 percent for both CAR A2 and CAIleR 8. Panel J shows flow cytometry plots of CD69 versus CD25 across dendritic cell, Bet v1, serum PBS, and serum BPE conditions, where CAIleR 8 with dendritic cells, Bet v1, and serum BPE reaches approximately 30 percent CD25 positive CD69 positive, confirmed by bar plots showing significant differences. Panel K shows a schematic of serum collection and autologous coculture from birch allergic donors with lentiviral transduction of CAR and CAIleR T cells. Panel L shows absorbance at 450 nanometers reaching approximately 3.5 for BPE BALB/c and approximately 2 for allergic human donors. Panel M shows a bar graph of CD25 positive CD71 positive percent where CAIleR 8 with K562 CD64, mAb 10, and human allergic donor serum reaches approximately 65 percent.
CAlleR 8 murine T cell activation and proliferation in birch-sensitized mice. (A) Experimental design of murine CAlleR 8 and polyclonal Teff generation. (B) Experimental design of the allergic airway inflammation mouse model with adoptive transfer of transduced Teff at day 10. (C) Transduction efficiency of Tconv after 7 days of expansion. (D) Bet v1 IgG1 detection in murine sera by ELISA (n = 6). (E) Cumulative data of the percentage of transduced (Thy1.1+) Tconv detected among the CD3+ cells in the different collected organs (lung, mLN, cLN, and spleen) (n = 5–6). (F) Cumulative data of the percentage of Thy1.1+CD4+ cells that proliferated (gated on CFSE−) in the different organs in mice exposed to PBS or BPE (n = 6). (G) Experimental design of CAR A2 and CAlleR 8 Teff production and coculture with BMDCs in the presence of Bet v1 and serum from mice exposed to either PBS or BPE. (H) Bet v1–specific IgG1 detection in the serum of mice exposed to PBS (n = 2) or BPE (n = 3) by ELISA. (I) Cumulative data of transduction efficiency (Thy1.1+) of CAR A2 and CAlleR 8 Teff after 7 days of expansion (n = 3). (J) Representative flow cytometry plots and cumulative data showing the expression of CD25 and CD69 in CAR A2 and CAlleR 8 Teff after 24-h coculture with Bet v1 (0.5 μg/ml), mouse serum (10%), and/or BMDCs (BMDC:Teff ratio of 1:5) (for each condition and for each serum, three biologically independent samples of Teff were tested). (K) Schematic representation of the experimental design. (L) Anti-Bet v1 IgG detection in the serum (1:500 dilution) of birch pollen–sensitized mice (n = 6) and allergic donors (n = 3). (M) Cumulative data showing the percentage of untransduced, CD19 CAR, and CAlleR 8 T cells expressing CD25 and CD71 after 48-h coculture with Bet v1 (0.5 μg/ml), K562 expressing CD64 (1:5 ratio), and 20% of autologous serum from allergic donors or mAb 10 (1 μg/ml) (n = 3 allergic donors). Data in E, F, H, I, L, and M are represented by the mean ± SEM and by median ± interquartile range in D and J. P values in D were calculated with the Kruskal–Wallis test with Dunn’s multiple comparison test, in E, F, and M with two-way ANOVA and Tukey’s multiple comparison test, in J with the Mann–Whitney U test, and in L with an unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. cLN, cervical lymph node; Tconv, conventional T cell.