TSLP: A key cytokine in inflammation and its therapeutic intervention

• Gene Information: Thymic stromal lymphopoietin (TSLP) is a protein-coding gene located on chromosome 5q22.1. It encodes a hemopoietic cytokine that is a member of the interleukin 7-like cytokine family.
• Protein Expression: TSLP is primarily expressed by activated epithelial cells (lung, gut), skin keratinocytes, and fibroblasts. Two main isoforms exist: the short form (sfTSLP) is constitutively expressed and plays a homeostatic role, while the long form (lfTSLP) is induced during inflammation.
• Signaling Pathway: TSLP exerts its effects by binding to a high-affinity heterodimeric receptor complex composed of the TSLP receptor chain (TSLPR) and the IL-7 receptor alpha chain (IL-7Rα).
• Therapeutic Inhibition: By blocking TSLP binding to its receptor, tezepelumab inhibits downstream inflammation and improves clinical outcomes, including reduced serum IgE levels, decreased airway eosinophils, reduced mucus production, and lowered cytokine secretion.

TSLP

• Exons 1–5 of the mouse Tslp gene, which encode the entire protein (from ATG to stop codon), are replaced with the corresponding human sequences.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human TSLP expression to be driven by the native mouse Tslp promoter, while endogenous mouse Tslp transcription and translation are abolished.

TSLPR

• A chimeric CDS encoding the human TSLPR extracellular and transmembrane domains fused to the mouse TSLPR cytoplasmic domain, followed by the mouse 3′ UTR and stop codon, is inserted immediately downstream of the mouse Tslpr signal peptide to replace part of exon 2 of the endogenous Tslpr gene.
• Expression of the chimeric TSLPR protein is driven by the native mouse Tslpr promoter, while endogenous mouse Tslpr transcription and translation are disrupted.

• Human TSLP mRNA is specifically and correctly expressed in B-hTSLP/hTSLPR mice.

Strain specific analysis of TSLP and TSLPR gene expression in wild type C57BL/6 mice and homozygous B-hTSLP/hTSLPR mice by RT-PCR. Mouse mRNA was prepared from ear grinding supernatant of wild-type C57BL/6 mice (+/+;+/+) and homozygous B-hTSLP/hTSLPR mice (H/H;H/H) stimulated with calcipotriol (MC903). Mouse Tslp mRNA was only detectable in wild type C57BL/6 mice (+/+;+/+). Human TSLP mRNA was only detectable in homozygous B-hTSLP/hTSLPR mice but not in wild type mice. Mouse mRNA was prepared from splenocytes of wild type C57BL/6 mice and homozygous B-hTSLP/hTSLPR mice. Mouse Tslpr mRNA was only detectable in wild type C57BL/6 mice. Human TSLPR mRNA was only detectable in homozygous B-hTSLP/hTSLPR mice but not in wild type mice.

• Mouse TSLP was detected exclusively in wild-type C57BL/6 mice
• Human TSLP was detected in homozygous B-hTSLP/hTSLPR mice, but not in wild-type mice.

Strain specific TSLP expression analysis in homozygous B-hTSLP/hTSLPR mice by ELISA. Calcipotriol (MC903) was dissolved in ethanol and topically applied on ears of either wild type C57BL/6 mice (+/+;+/+) or homozygous B-hTSLP/hTSLPR mice (H/H;H/H; n=3) for 7 days. Ear grinding supernatant from the two mice was analyzed  by ELISA. Mouse TSLP was detectable in wild type C57BL/6 mice. Human TSLP was exclusively detectable in homozygous B-hTSLP/hTSLPR mice but not in wild type mice. ND: not detectable.

• Mouse TSLPR was detected macrophages populations in wild-type C57BL/6 mice.
• Human TSLPR was detected on macrophages populations in B-hTSLP/hTSLPR mice, but not in wild-type C57BL/6 mice.

Strain specific TSLPR expression analysis in heterozygous B-hTSLP/hTSLPR mice by flow cytometry. Macrophages were collected from wild-type C57BL/6 mice (+/+;+/+) and heterozygous B-hTSLP/hTSLPR mice (H/+;H/+), and analyzed by flow cytometry with species-specific anti-TSLPR antibody (anti-mouse TSLPR (TSLP-R) Antibody: Biolegend,151805; anti-human TSLPR (TSLP-R) Antibody: Biolegend, 322805). Mouse TSLPR was detectable in wild-type C57BL/6 mice and heterozygous B-hTSLP/hTSLPR mice. Human TSLPR was detectable in heterozygous B-hTSLP/hTSLPR mice but not in wild-type mice.

• Mouse TSLPR was detected dendritic cells populations in wild-type C57BL/6 mice.
• Human TSLPR was detected on dendritic cells populations in B-hTSLP/hTSLPR mice, but not in wild-type C57BL/6 mice.

Strain specific TSLPR expression analysis in heterozygous B-hTSLP/hTSLPR mice by flow cytometry. Dendritic cells were induced from bone marrow of wild-type C57BL/6 mice (+/+;+/+) and heterozygous B-hTSLP/hTSLPR mice (H/+;H/+), and stimulated with LPS. Protein expression was analyzed by flow cytometry with species-specific anti-TSLPR antibodies (anti-mouse TSLPR (TSLP-R) Antibody: Biolegend,151805; anti-human TSLPR (TSLP-R) Antibody: Biolegend, 322805). Mouse TSLPR was detectable in wild-type C57BL/6 mice and heterozygous B-hTSLP/hTSLPR mice. Human TSLPR was exclusively detectable in heterozygous B-hTSLP/hTSLPR mice but not in wild-type mice.

• Human TSLPR was detectable on total splenocytes, CD11b+ cells (monocytes and neutrophils) and CD11c+ cells (DCs) of homozygous B-hTSLP/hTSLPR mice but not on those cells of wild-type C57BL/6 mice.

Human TSLPR expression analysis in homozygous B-hTSLP/hTSLPR mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hTSLP/hTSLPR mice, and analyzed by flow cytometry with species-specific anti-TSLPR antibody. Human TSLPR was detectable on total splenocytes, CD11b+ cells (monocytes and neutrophils) and CD11c+ cells (DCs) of homozygous B-hTSLP/hTSLPR mice but not on those cells of wild-type C57BL/6 mice.

• Human TSLPR was detectable on cDC2, mo-DC, cDC1 and pre-DC of homozygous B-hTSLP/hTSLPR mice but not on those cells of wild-type C57BL/6 mice.

Human TSLPR expression analysis in homozygous B-hTSLP/hTSLPR mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hTSLP/hTSLPR mice, and analyzed by flow cytometry with species-specific anti-TSLPR antibody. Human TSLPR was detectable on cDC2, mo-DC, cDC1 and pre-DC of homozygous B-hTSLP/hTSLPR mice but not on those cells of wild-type C57BL/6 mice.

• Human TSLPR was detectable on T cells and ILC2 cells of B-hTSLP/hTSLPR mice but not on those cells of wild-type C57BL/6 mice.

Human TSLPR expression analysis on T cells and ILC2 of homozygous B-hTSLP/hTSLPR mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+;+/+) and homozygous B-hTSLP/hTSLPR mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-TSLPR antibody (anti-mouse TSLPR (TSLP-R) Antibody: Biolegend,151805; anti-human TSLPR (TSLP-R) Antibody: Biolegend, 322805). Mouse TSLPR was detectable on T cells and ILC2 of wild-type C57BL/6 mice but not in homozygous B-hTSLP/hTSLPR mice. Human TSLPR was exclusively detectable on the two cells of homozygous B-hTSLP/hTSLPR mice.

• In B-hTSLP/hTSLPR mice: Mouse TARC was induced by human TSLP, but not by mouse TSLP.
• In wild-type C57BL/6 mice: Mouse TARC was induced by mouse TSLP, but not by human TSLP.
• These results demonstrate species-specific TSLP–TSLPR signaling and confirm functional activation of dendritic cells by human TSLP in B-hTSLP/hTSLPR mice.

Mouse TARC was induced with human TSLP and mouse TSLP in homozygous B-hTSLP/hTSLPR mice analyzed by ELISA. Dendritic cells were respectively induced with FLT3L from bone marrow of homozygous B-hTSLP/hTSLPR mice (H/H;H/H) and wild-type C57BL/6 mice (+/+;+/+), and stimulated with human TSLP or mouse TSLP in vitro. Concentration of mouse TARC secreted from DCs was assayed by ELISA. Mouse TARC was successfully induced with human TSLP, but not mouse TSLP in homozygous B-hTSLP/hTSLPR mice. Meanwhile mouse TARC was successfully induced with mouse TSLP, but not human TSLP in wild-type C57BL/6 mice. Results indicated that TSLP and TSLPR are not cross-reactive between mouse and human. Introduction of human TSLP and TSLPR genes in place of its mouse counterpart does not change the stimulation effect of TSLP on dendritic cells.

• The frequencies of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hTSLP/hTSLPR mice were similar to those in C57BL/6 mice.
• Humanization of TSLP and TSLPR does not affect normal immune cell development or splenic distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, bone marrow, and thymus were isolated from C57BL/6 and B-hTSLP/hTSLPR mice mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hTSLP/hTSLPR mice were comparable to those in C57BL/6 mice
• Humanization of TSLP and TSLPR does not affect normal T cell development, differentiation, or splenic distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and thymus were isolated from C57BL/6 and B-hTSLP/hTSLPR mice (female, 8-week-old, n = 3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Body weight growth curve of B-hTSLP/hTSLPR mice after birth. Newborn pups (30 males and 30 females, respectively) were obtained at weaning (Week 3; birth date +/- 3 days). Body weight was measured once every week (on the same day each week) for 9 weeks.

• Humanization of TSLP and TSLPR does not change blood cell composition and morphology

Complete blood count (CBC). Blood from female C57BL/6 and B-hTSLP/hTSLPR mice (n=8, 10-11-week-old) was collected and analyzed for CBC. The measurements of B-hTSLP/hTSLPR mice were similar to that in C57BL/6 mice, indicating that introduction of hTSLP and hTSLPR in place of its mouse counterpart does not change blood cell composition and morphology. Values are expressed as mean ± SEM.

• Humanization of TSLP and TSLPR does not change the health of related organs.

Blood chemistry tests of B-hTSLP/hTSLPR mice. Serum from the C57BL/6 and B-hTSLP/hTSLPR mice (n=8, 10-11-week-old) was collected and analyzed for levels of indicators. The measurements of B-hTSLP/hTSLPR mice were similar to that in C57BL/6 mice, indicating that introduction of hTSLP and hTSLPR in place of its mouse counterpart does not change the health of related organs. Values are expressed as mean ± SEM.

Experimental schedule for the induction of asthma model and in vivo efficacy of anti-human TSLP antibody in B-hTSLP/hTSLPR mice. B-hTSLP/hTSLPR mice (male, 7-week-old, n=6) were immunized with OVA etc. inducer to induce asthma. The anti-human TSLP antibody tezepelumab analog (in-house) was administered by intraperitoneal injection.

• CD45⁺ cells, eosinophils, and neutrophils were significantly reduced in the anti–human TSLP antibody–treated group (G3,G4) compared with the isotype control group (G2).

Analysis of inflammatory cells in BALF by FACS. Mouse asthma model was induced in B-hTSLP/hTSLPR mice and treated with anti-human TSLP antibody (Tezepelumab analog, synthesized in house). BALF was collected at the end of the experiment to detect infiltrated inflammatory cells in lung tissue. The results showed that CD45+ cells, eosinophils and neutrophils in the modeling group treated with isotype antibody (G2) was significantly higher than that in the non-modeling group (G1), while these cells in the groups (G3, G4) treated with anti-human TSLP antibody decreased significantly when compared with the G2 group. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. 

• Tezepelumab analog treatment reduced OVA-specific IgE and TARC levels compared with untreated controls.

Total IgE, OVA specific IgE in serum and TARC in BALF were  significantly reduced in the mouse asthma model treated with anti-human TSLP antibody. Serum was collected at the study endpoint. IgE and TARC levels were analyzed by ELISA. The results showed that the levels of total IgE, OVA specific IgE and TARC in mice treated with Tezepelumab analog (in house) was lower than that in untreated mice. Values are expressed as mean ± SEM. TARC: thymic and activating regulatory chemokine, also known as CCL17 (C-C motif chemokine ligand 17). *P < 0.05, **P < 0.01, ***P < 0.001. 

• Tezepelumab analog significantly reduced inflammatory infiltration and mucus secretion in lung tissue compared with untreated controls (G2).
• B-hTSLP/hTSLPR mice provide a robust in vivo preclinical model for evaluating anti–human TSLP antibodies.

H&E staining of asthma-like model in B-hTSLP/hTSLPR mice. Lung tissues were collected at the study endpoint and analyzed with H&E staining. The results showed that when compared to the non-modeling group (G1), there was a significant increase in inflammatory cell infiltration and mucus secretion in the lung tissue of the modeling mice that were treated with isotype antibody (G2). But when compared with the isotype antibody treated group (G2), there is a significant reduction in inflammatory cell infiltration and mucus secretion in the lung tissue of the groups treated with Tezepelumab analog (in house) (G3,G4). The results indicate that B-hTSLP/hTSLPR mice can successfully establish a mouse asthma-like model and provide a powerful preclinical model for in vivo evaluation of anti-human TSLP antibodies. Bold arrow: mucus; Narrow arrow: inflammatory cells; Triangle: eosinophils. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. 

• Tezepelumab analog significantly reduced lung mucus secretion compared with untreated controls (G1).

Periodic acid-Schiff (PAS) staining of asthma-like model in B-hTSLP/hTSLPR mice. Lung tissues were collected at the study endpoint and analyzed with PAS staining. The results showed that when compared to the non-modeling group (G1), there was a significant increase in mucus secretion in the lung tissue of the modeling mice that were treated with isotype antibody (G2). But when compared with the isotype antibody treated group (G2), there is a significant reduction in mucus secretion in the lung tissue of the groups treated with Tezepelumab analog (in house) (G3, G4). The results indicate that B-hTSLP/hTSLPR mice can successfully establish a mouse asthma-like model and provide a powerful preclinical model for in vivo evaluation of anti-human TSLP antibodies. Arrow: goblet cells; Triangle: mucus. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Experimental schedule for the induction of atopic dermatitis (AD) skin lesions and in vivo efficacy of anti-human TSLP antibody in B-hTSLP/hTSLPR mice. OXA was applied to the ear skin of mice on day 0, followed by nine challenges to the same site from days 7 to 25. The anti-human TSLP antibody tezepelumab analog (in-house) was administered by intraperitoneal injection. OXA, oxazolone.

• Tezepelumab significantly reduced ear thickness and serum total IgE compared with controls.

Efficacy of anti-human TSLP antibody in B-hTSLP/hTSLPR mice. Mice in each group were treated with anti-hTSLP antibody Tezepelumab analog (in house). (A) Statistical analysis of ear thickness in each group. Epidermis of ear began to desquamate from day 18. So the ear thicknesses were decreased from day 18 as shown in figure. (B) Body weight changes during the treatment. (C) Total IgE levels in serum. Serum was collected on day 26 and total IgE levels were measured by ELISA. (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001.

• Tezepelumab analog significantly reduced ear thickness and eosinophil infiltration scores compared with the isotype control.
• B-hTSLP/hTSLPR mice provide a robust in vivo preclinical model for evaluating anti–human TSLP antibodies.

Effects of anti-human TSLP antibody on ear skin of the AD mouse model. (A) Hematoxylin and eosin (H&E) staining. (B) Thickness of ear epidermal skin. (C) Score of eosinophils infiltrated in ear epidermal skin. (D) Total score of ear epidermal skin. Ear thickness and infiltration scores of eosinophils in ear skin of the groups treated with dexamethasone or tezepelumab analog(in house) were decreased significantly compared to that in the isotype control, demonstrating that the B-hTSLP/hTSLPR mice provide a powerful preclinical model for in vivo evaluation of anti-human TSLP antibodies. Infiltration score of eosinophils: 1=slight; 2=mild; 3=moderate; 4=severe. The content of the pathology total score evaluation includes the following aspects: epidermal hyperplasia in skin, erosion/crusting, hyperkeratosis and parakeratosis; inflammatory cell infiltration in dermis and subcutaneous.