Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Abstract

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

Keywords: adoptive cell therapy; allergic asthma; chimeric antigen receptor; ovalbumin mouse model; regulatory T cells.

Figure 1

Regulatory T cells (Tregs) engineered with a carcinoembryonic antigen (CEA)-specific chimeric antigen receptor (CAR) specifically redirect their suppressive capacity. (A) Modular composition of the CEA-specific CAR with the anti-CEA scFv binding domain and the combined CD28–CD3ζ signaling domains. (B) CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T effector cells (Teffs) with anti-CEA CAR were isolated from the spleens of the anti-CEA CAR transgenic mice. Tregs isolated from CEA transgenic mice or wild-type (wt) C57BL/6 mice stained positive for CD4, CD25, and FoxP3. (C) CAR Tregs suppressed the amplification of PKH26-labeled CAR Teffs that were stimulated by the agonistic anti-CD3/anti-CD28 antibodies directed against the TCR/CD28 or by the BW2064/26 antibody (anti-CAR) directed against the CAR. Samples were measured in triplicates. Statistical analyses were performed by the one-way ANOVA test. ***p < 0.001. (D) Latency-associated peptide (LAP) (TGF-β1) is increased on the surface of CAR engineered CD4⁺CD25⁺FoxP3⁺ Treg cells upon specific stimulation. The assay was performed in triplicates. Statistical analyses were performed by the Student’s t-test (*p < 0.05, **p < 0.01). (E) CAR Tregs were stimulated by the BW2064/36 monoclonal antibodies (mAb) (anti-CAR) as surrogate antigen or by an IgG₁ isotype matched control mAb. IL-10 released into the supernatant was recorded by a bead-based immunoassay. Data are representative for two independent experiments.

Figure 2

Chimeric antigen receptor (CAR) regulatory T cells (Tregs) home to the lung and spleen. (A) Lung slices from the carcinoembryonic antigen transgenic (CEAtg) mouse were stained for CEA (microscope magnifications 5× and 20×, respectively). CEA is expressed in a polarized fashion on the luminal site by the alveolar epithelia. (B) Gaussia Luciferase (Gluc)-labeled CAR Tregs and Gluc-labeled non-modified wild-type (wt) Tregs were recorded by bioluminescent imaging in the lungs, spleen, stomach, and kidney in the same mouse 36 h after Treg transfer to the ovalbumin-sensitized mice. One representative mouse out of three from each group is shown. Statistical analyses were performed using the Student’s t-test. *p < 0.05, **p < 0.01; n.d., not detectable.

Figure 3

Adoptive transfer of chimeric antigen receptor (CAR) regulatory T cells (Tregs) reduces airway hyper-reactivity, inflammation, mucus production, and eosinophilia in mice with induced experimental asthma. (A) Schematic outline of the experimental protocol. (B) Lung resistance (R) after exposure to increasing concentrations of methacholine (MeCh) was recorded as described in Section “Materials and Methods.” Data from n = 9–11 mice from three independent experiments are shown. Statistical analyses were performed with the two-way ANOVA test. (C,D) Stained whole lung sections were quantified for (C) hematoxylin–eosin (H&E) or (D) PAS signals (number of pixels), n = 4–6 (H&E) and n = 6–9 (PAS) each from 3 independent experiments; scale bar = 100 μm. (E) Absolute numbers of cells in the bronchoalveolar lavage fluid (BALF); n = 5–7 in one of four independent experiments. Alum, mice treated with alum adjuvant only; ovalbumin (OVA), mice treated with OVA in alum adjuvant; CAR Tregs, mice treated with OVA in alum adjuvant and subsequent one dose of CAR Treg cells; wild-type (wt) Tregs, mice treated with OVA in alum adjuvant and subsequent one dose of unmodified wt Treg cells. Statistical analyses were performed by the one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant, n.d., not detectable.

Figure 4

Chimeric antigen receptor (CAR) regulatory T cells (Tregs) reduce antigen-specific T helper-2 cytokine production by lung cells and splenocytes. IL-5, IL-13, and IL-10 levels were measured by bead-based assay in lung, spleen (SPL), and tracheobronchial lymph node (LN) cell culture supernatants after in vitro restimulation with 200 µg/ml ovalbumin (OVA). Data represent three independent experiments including pooled samples from more than 6 mice per group. Statistical analyses were performed by the one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

IL-5 and allergen-specific IgE levels are diminished upon chimeric antigen receptor (CAR) regulatory T cell (Treg) treatment of carcinoembryonic antigen transgenic (CEAtg) mice. (A) IL-5 levels in sera from CEAtg mice were determined at different times of the experimental protocol (Figure 3A), n = 3 mice per group per time point; statistical analysis was performed using the two-way ANOVA test. (B) The amount of ovalbumin (OVA)-specific IgE (n = 6–8 mice), IgG₁ (n = 6–9), and IgG_2a (n = 6–17) in sera of CEAtg mice. Data are pooled from two to three independent experiments. (C) Total number of bronchoalveolar lavage fluid (BALF) cells in wild-type (wt) C57BL/6 mice without CEA expression, treated as described in Section “Materials and Methods.” (D) OVA-specific IgE at day 12 in sera of C57BL/6 mice was recorded by ELISA. Statistical analysis was performed by the one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Chimeric antigen receptor (CAR) regulatory T cells (Tregs) accumulate in the lung, spleen (SPL), and tracheobronchial lymph nodes (LNs) after challenge with antigen. (A) Lung and SPL cells were isolated at day 13 from carcinoembryonic antigen transgenic mice treated with CAR Tregs or non-modified [wild-type (wt)] Tregs and fluorescently stained for nuclei (DAPI), and CD4, FoxP3, and CAR expression. One representative mouse out of three per group is shown. (B) Detection of CAR Tregs in the SPL and tracheobronchial LNs by RT-PCR at the end of experimental protocol (day 13). The specific RT-PCR CAR fragment is 565 bp in size. As expected, tissues from mice which received non-modified Tregs (SPL ctrl) did not show a CAR-derived signal. Bronchoalveolar lavage fluid (BALF) did not contain detectable CAR Treg cells. Lad, ladder of DNA fragments of different sizes; H₂O ctrl, RT-PCR in the absence of RNA; Pos ctrl, RT-PCR with RNA from purified CAR T cells; m, mouse; s, sample consisting of cells that are pooled from five to six mice.