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.

IL7R

• Exons 1–6 of the mouse Il7r gene encoding the extracellular domain are replaced with the corresponding human IL7R exons 1–6.

• Both long and short human TSLP isoforms are specifically and correctly expressed in B-hTSLP/hTSLPR mice plus.

Long and short isoforms of human TSLP were detectable in B-hTSLP/hTSLPR mice plus by RT-PCR and sequencing. Ear tissues were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTSLP/hTSLPR mice plus (H/H). (A) Primers were designed to detect the long-form (lfTSLP) and short-form (sfTSLP) isoforms of human TSLP. (B) lfTSLP mRNA was detectable in B-hTSLP/hTSLPR mice plus, but not in wild-type mice. (C) sfTSLP mRNA was detectable in B-hTSLP/hTSLPR mice plus, but not in wild-type mice. Sequencing of the short-form PCR products confirmed that the amplified sequences were consistent with database reference sequences.

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

Strain-specific TSLP expression evaluated by ELISA in wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus. Calcipotriol (MC903), dissolved in ethanol, was topically applied to the ears of wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus for 7 days (male, 8-week-old, n = 3). Mouse and human TSLP levels in ear tissue homogenates were quantified by ELISA (mouse TSLP, BioLegend 434107; human TSLP, BioLegend 434207).

• Mouse TSLPR was detected on cDCs, pDCs, and non-DC populations in wild-type C57BL/6 mice, but not in TSLPR-humanized mice.
• Human TSLPR was detected on cDCs, pDCs, and non-DC populations in B-hTSLP/hTSLPR and B-hTSLP/hTSLPR mice plus, but not in wild-type C57BL/6 mice.

Mouse and human TSLPR expression analysis in splenocytes. Splenocytes were collected from wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus. TSLPR expression on cDCs, pDCs, and non-DCs was analyzed by flow cytometry using anti-mouse TSLPR antibody and anti-human TSLPR antibody.

• Mouse TSLPR was detected on cDCs, pDCs, and non-DC populations in wild-type C57BL/6 mice, but not in TSLPR-humanized mice.
• Human TSLPR was detected on cDCs, pDCs, and non-DC populations in B-hTSLP/hTSLPR and B-hTSLP/hTSLPR mice plus, but not in wild-type C57BL/6 mice.

Mouse and human TSLPR expression analysis in bone marrow. Bone marrow cells were collected from wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus. TSLPR expression on bone marrow cells was analyzed by flow cytometry using anti-mouse TSLPR antibody and anti-human TSLPR antibody.

• Human TSLPR was highly expressed on cDC1 in B-hTSLP/hTSLPR and B-hTSLP/hTSLPR mice plus.

Human TSLPR expression analysis in cDC1 from bone marrow. Bone marrow was collected from wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus. Human TSLPR expression on cDC1 was analyzed by flow cytometry using species-specific antibodies.

• The frequencies of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hTSLP/hTSLPR mice plus were similar to those in C57BL/6 mice.
• Humanization of TSLP, TSLPR, and IL7R 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, lymph nodes, and thymus were isolated from C57BL/6 and B-hTSLP/hTSLPR mice plus (female, 9-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 plus were comparable to those in C57BL/6 mice.
• Humanization of TSLP, TSLPR, and IL7R 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, lymph nodes, and thymus were isolated from C57BL/6 and B-hTSLP/hTSLPR mice plus (female, 9-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

In B-hTSLP/hTSLPR and B-hTSLP/hTSLPR mice plus:

• Mouse TARC was induced by human TSLP, but not by mouse TSLP.
• Mouse TARC levels were higher in B-hTSLP/hTSLPR mice plus compared with B-hTSLP/hTSLPR mice.

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 models.

Mouse TARC induction by human and mouse TSLP in wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice and B-hTSLP/hTSLPR mice plus. Dendritic cells were generated from the bone marrow of wild-type C57BL/6 mice, homozygous B-hTSLP/hTSLPR mice, and B-hTSLP/hTSLPR mice plus using FLT3L, and then stimulated in vitro with human TSLP or mouse TSLP. Mouse TARC levels secreted by dendritic cells were measured by ELISA.

• TSLPR and IL7R mRNA were detectable in both B-hTSLP/hTSLPR mice plus and wild-type C57BL/6JNidfc mice across multiple immune organs.

Strain specific analysis of TSLPR and IL7R expression in wild-type C57BL/6JNidfc mice and homozygous B-hTSLP/hTSLPR mice plus by RT-qPCR. RNA was isolated from multiple organs of wild-type C57BL/6JNidfc mice (+/+) (female, 8-week-old, n = 3) and homozygous B-hTSLP/hTSLPR mice plus (female, 8-week-old, n = 3), followed by cDNA synthesis via reverse transcription and quantitative real-time PCR using TSLPR- and IL7R-specific primers. Values are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA.  *P < 0.05, **P < 0.01, ***p < 0.001. Data were normalized to C57BL/6JNidfc liver.

• TSLPR and IL7R mRNA were detectable in both B-hTSLP/hTSLPR mice plus and wild-type C57BL/6JNidfc mice.
• TSLPR mRNA was highly expressed in testis.
• IL7R mRNA was highly expressed in various immune organs.

Strain-specific analysis of TSLPR and IL7R expression in wild-type C57BL/6JNidfc mice and homozygous B-hTSLP/hTSLPR mice plus by RT–qPCR. RNA was isolated from multiple organs of wild-type C57BL/6JNidfc mice (+/+) (male, 8-week-old, n = 3) and homozygous B-hTSLP/hTSLPR mice plus (male, 8-week-old, n = 3), followed by cDNA synthesis by reverse transcription and quantitative real-time PCR using TSLPR- and IL7R-specific primers. Values are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Data were normalized to C57BL/6JNidfc liver.

Growth curve of B-hTSLP/hTSLPR mice plus. Eight-week-old mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 12 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD. The growth curve follows a normal distribution, with approximately 68% of values falling within ± SD.

• Hematology analysis in B-hTSLP/hTSLPR mice plus.

Complete blood count (CBC) of B-hTSLP/hTSLPR mice plus. Values are expressed as mean ± SD.

• Blood biochemical analysis in B-hTSLP/hTSLPR mice plus.

Blood biochemical parameters of B-hTSLP/hTSLPR mice plus are shown. Values are expressed as mean ± SD.

• No abnormalities were observed.

Organs of female B-hTSLP/hTSLPR mice plus (12-week-old, n = 10).

• No abnormalities were observed.

Organs of male B-hTSLP/hTSLPR mice plus (12-week-old, n = 10).

• No abnormalities were observed.

Average weights of major organs in B-hTSLP/hTSLPR mice plus.

• No obvious abnormalities were observed in any organs examined (brain, heart, lung, liver, spleen, stomach, small intestine, colon, kidney, ovary, uterus ,testis, lymph node, thymus and bone marrow).

Histopathological analysis of organs in B-hTSLP/hTSLPR mice plus. Major organs from B-hTSLP/hTSLPR mice plus were collected at 12 weeks of age and analyzed by H&E staining (male, n = 10; female, n = 10).

Experimental schedule for the induction of asthma model and in vivo efficacy of anti-human TSLP antibody in B-hTSLP/hTSLPR mice plus. B-hTSLP/hTSLPR mice plus (female, 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 (n = 6).

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

OVA-specific IgE in serum and TARC in BALF were significantly reduced in the mouse asthma model treated with anti-TSLP antibody. Serum was collected at the study endpoint, and IgE and TARC levels were analyzed by ELISA. Values are expressed as mean ± SEM. TARC, thymus and activation-regulated chemokine, also known as CCL17 (C-C motif chemokine ligand 17). *P < 0.05, **P < 0.01, ***P < 0.001.

• CD45⁺ cells, eosinophils, and neutrophils were significantly reduced in the anti–human TSLP antibody–treated group (G2) compared with the isotype control group (G1).

Analysis of inflammatory cells in BALF by FACS. A mouse asthma model was induced in B-hTSLP/hTSLPR mice plus and treated with an anti-human TSLP antibody (Tezepelumab analog, synthesized in-house). BALF was collected at the end of the experiment to assess infiltrating inflammatory cells in lung tissue. Values are expressed as mean ± SEM. *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 (G1).
• B-hTSLP/hTSLPR mice plus provide a robust in vivo preclinical model for evaluating anti–human TSLP antibodies.

Hematoxylin and eosin (H&E) staining of an asthma model in B-hTSLP/hTSLPR mice plus. Lung tissues were collected at the study endpoint and analyzed by H&E staining. Black arrows: inflammatory cells, black triangles: eosinophils, asterisks: mucus. 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 an asthma model in B-hTSLP/hTSLPR mice plus. Lung tissues were collected at the study endpoint and analyzed by PAS staining. Arrows: goblet cells, triangles: 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 plus. 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 (n = 6). OXA, oxazolone.

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

Efficacy of anti-human TSLP antibody in B-hTSLP/hTSLPR mice plus. Mice in each group were treated with the anti-hTSLP antibody tezepelumab analog (in-house). (A) Statistical analysis of ear thickness in each group. Epidermal desquamation of the ear began on day 18; therefore, ear thickness decreased from day 18, as shown in the figure. (B) Body weight changes during treatment. (C) Total serum IgE levels, measured by ELISA using serum collected on day 26 (n = 6). Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

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

Effects of anti-human TSLP antibody on ear skin in an AD mouse model. (A) Hematoxylin and eosin (H&E) staining. (B) Ear epidermal thickness. (C) Eosinophil infiltration score in ear epidermal skin. (D) Total score of ear epidermal skin. Eosinophil infiltration was scored as follows: 1 = slight; 2 = mild; 3 = moderate; 4 = severe. The total pathology score included epidermal hyperplasia, erosion/crusting, hyperkeratosis, parakeratosis, and inflammatory cell infiltration in the dermis and subcutaneous tissue. *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 plus provide a robust in vivo preclinical model for evaluating anti–human TSLP antibodies.

Effects of anti-human TSLP antibody on ear skin in an AD mouse model. (A) Hematoxylin and eosin (H&E) staining. (B) Ear epidermal thickness. (C) Eosinophil infiltration score in ear epidermal skin. (D) Total score of ear epidermal skin. Eosinophil infiltration was scored as follows: 1 = slight; 2 = mild; 3 = moderate; 4 = severe. The total pathology score included epidermal hyperplasia, erosion/crusting, hyperkeratosis, parakeratosis, and inflammatory cell infiltration in the dermis and subcutaneous tissue. *P < 0.05, **P < 0.01, ***P < 0.001.