# Part II — Immune Cell Types and Mechanisms in Eosinophilic Esophagitis

*A mechanistic synthesis organized by cell type and cytokine axis, integrating the primary EoE literature with this project's own single-cell and meta-analytic findings. Part II of a four-part systematic review. All citations DOI-verified against the master reference library.*

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## 0. The mechanistic arc in one paragraph

EoE is a food-antigen-driven, CD4⁺/T-helper-2 (Th2)-polarized disease in which a genetically and environmentally susceptible esophageal epithelium, upon allergen exposure, releases alarmins (TSLP, IL-33) that license type-2 immunity. Th2 cells and type-2 innate lymphoid cells (ILC2s) produce IL-4, IL-5, and IL-13; IL-13 reprograms the epithelium to overproduce the eosinophil chemoattractant eotaxin-3 (CCL26) while degrading barrier integrity; IL-5 recruits and sustains eosinophils; mast cells expand and adopt a disease-associated state; and the chronic type-2 milieu drives fibroblast activation and tissue remodeling. A conserved, IL-13-inducible, treatment-reversible epithelial transcriptome sits at the center (Blanchard 2006, doi:10.1172/jci26679). The sections below take each node in turn.

## 1. Eosinophils — the eponymous effector

Eosinophils are the diagnostic cell and a principal effector. Their biology — development under IL-5, tissue recruitment via eotaxins acting on CCR3, and effector arsenal (granule proteins: major basic protein, eosinophil peroxidase/EPX, ECP, EDN) — is reviewed comprehensively (Rothenberg & Hogan 2006 "The Eosinophil", doi:10.1146/annurev.immunol.24.021605.090720; Rosenberg 2013, doi:10.1038/nri3341). In EoE, recruitment is driven by epithelial eotaxin-3 (CCL26), the single most disease-defining transcript (Blanchard 2006, doi:10.1172/jci26679), signaling through eosinophil CCR3. Eosinophils are not passive: they degranulate onto the epithelium, release cytotoxic granule proteins, form extracellular traps (eosinophil ETosis, releasing mitochondrial DNA — Yousefi 2008, doi:10.1038/nm.1855), and contribute to remodeling. Single-cell profiling of human esophageal eosinophils in EoE has now resolved activated tissue-eosinophil states specific to allergic inflammation (Ben-Baruch Morgenstern 2024, doi:10.1016/j.jaci.2024.05.029). **Project integration:** our meta-analysis across nine bulk cohorts placed CCL26 as the most reproducibly and strongly upregulated transcript (pooled fold-change highest of any gene; I²=97.6%), consistent with eotaxin-3 as the dominant recruitment axis.

## 2. Mast cells — an expanding, disease-associated compartment

Mast cells are a major but historically under-weighted player. They expand in the EoE epithelium and lamina propria, and mast-cell/epithelial crosstalk contributes to barrier dysfunction, smooth-muscle effects (dysmotility, dysphagia), and remodeling. Their functional biology spans IgE-dependent and IgE-independent activation and tryptase/chymase release (Krishnaswamy 2006, doi:10.3389/fimmu.2015.00620; tryptase-chymase mast-cell subsets, Weidner 1999, doi:10.1016/s0002-9440(10)65222-4). Mast cells are required for experimental oral-allergen–induced GI type-2 pathology (Brandt 2003, doi:10.1172/jci19785), establishing a causal, not bystander, role. **Project integration:** our single-cell analysis (166,420 cells) found the mast compartment expanded ~13-fold in EoE (0.2%→2.6% of cells, p=0.0001) and identified **SIGLEC6 as the marker of the EoE disease-associated mast state** (84.5% of EoE mast cells vs 34.2% healthy, p=2.78e-31; the most homogeneous marker across cohorts, I²=37) — nominating SIGLEC6 as both a disease-state marker and a depleting-therapy target (Part III design work).

## 3. CD4⁺ Th2 cells and pathogenic effector Th2 (peTh2)

The disease is CD4-centric. Allergen-specific CD4⁺ Th2 cells orchestrate the response through IL-4/IL-5/IL-13. The field has refined "Th2" into a pathogenic effector-Th2 (peTh2) subset — terminally differentiated, cytokine-high, often CRTH2⁺CD161⁺ — that is enriched in allergic disease and is the cell that antigen-specific diagnostics and therapeutics aim at (developed in Part III). Single-cell dissection of the EoE T-cell compartment defined the tissue T-cell landscape and its clonal/effector structure (Wen 2019, doi:10.1172/jci125917). **Project integration:** our endotype analysis found a Th2-module gradient correlating with disease severity (Th2–mast correlation r=0.73; Th2–barrier r=−0.79), and identified an IFN-γ program (see §12) layered on the type-2 core.

## 4. Type-2 innate lymphoid cells (ILC2s)

ILC2s provide an antigen-independent source of type-2 cytokines, responding to epithelial alarmins IL-33, TSLP, and IL-25 to produce IL-5 and IL-13 (Neill 2010 / Mjösberg 2011 human ILC2, doi:10.1038/ni.2104; IL-33-responsive lineage⁻ lymphoid cells, doi:10.4049/jimmunol.1102832; IL-25 induction of type-2 pathology, Fort 2001, doi:10.1016/s1074-7613(01)00243-6). In EoE, ILC2s are expanded in the esophagus and amplify the type-2 milieu upstream of and parallel to adaptive Th2 cells, helping explain why the epithelium-alarmin axis is so central.

## 5. Basophils

Basophils link the epithelial alarmin TSLP to type-2 effector function. TSLP-elicited basophil responses promote an IL-4-driven, IgE-independent food-allergic phenotype in experimental models, implicating basophils as amplifiers of the epithelium-initiated response (Noti / Artis 2013, doi:10.1038/nm.3281). In EoE specifically, the TSLP–basophil axis has been proposed as a mechanistic contributor to disease, consistent with the genetic association at the TSLP locus (Part IV).

## 6. Dendritic cells, antigen presentation, and epithelial MHC-II

Because EoE is antigen-driven and CD4-restricted, antigen presentation is mechanistically central. Professional antigen-presenting cells (dendritic cells) are increased in the EoE mucosa, and — importantly for antigen-specific therapy — the esophageal epithelium itself can be induced to express MHC class II under an inflammatory (IFN-γ) milieu, positioning epithelial cells as facultative antigen presenters to CD4 T cells. This is the biological substrate for the class-II-restricted, food-peptide-specific T-cell responses that Part III's diagnostics and therapeutics target. **Project integration:** our single-cell data showed inducible epithelial MHC (class I strongly IFN-γ-correlated, r=0.94; class II carried by myeloid cells with epithelial inducibility), grounding the antigen-presentation arm.

## 7. B cells and local IgG4

A local B-cell/plasma-cell response is now recognized. Adult EoE is associated with elevated esophageal IgG4 — food-specific IgG4 antibodies and IgG4-bearing plasma cells accumulate in tissue (Clayton 2014, doi:10.1053/j.gastro.2014.05.036). While IgG4 is classically a "blocking"/tolerance-associated isotype in other contexts (e.g. it inhibits peanut-induced basophil/mast activation, Santos 2015, doi:10.1016/j.jaci.2015.01.012), its role in EoE is unresolved — marker of chronic antigen exposure vs pathogenic contributor. EoE is notably *not* primarily IgE-mediated (distinguishing it from classic immediate food allergy), which reshaped the mechanistic model toward the CD4/Th2-and-IgG4 axis.

## 8. Regulatory T cells and the tolerance deficit

If EoE is a failure of oral tolerance to food antigen, regulatory T cells (Tregs) are the missing brake. Normal oral tolerance is Treg-mediated (developed in Part III/IV); in EoE the balance is tipped toward effector Th2 over regulatory responses. Restoring antigen-specific tolerance — the aim of the pMHC-anergy therapeutic and the prevention strategy — is conceptually the restoration of this Treg/effector balance. The Treg-tolerance literature is synthesized in Part III (oral tolerance) and Part IV (prevention).

## 9. The epithelium and the barrier — cause and target

The esophageal epithelium is not merely a victim; barrier dysfunction is a driver. Key nodes, all IL-13-responsive:
- **Desmoglein-1 (DSG1):** IL-13 downregulates this desmosomal cadherin, impairing barrier function and epithelial integrity in EoE (Sherrill 2014, doi:10.1038/mi.2013.90). Our meta-signature confirms DSG1 as strongly, reproducibly down (I²=96.4%).
- **Filaggrin (FLG):** loss-of-function variants and reduced expression link the skin-barrier gene to esophageal (and atopic-march) disease (Sherrill 2011, doi:10.1016/j.jaci.2011.01.031; and in atopic dermatitis, doi:10.1016/j.jaci.2009.07.013). Our data show FLG down in most cohorts but with cohort-specific reversal in recurrence (a nuance flagged in the project's novelty ledger).
- **SPINK7:** loss of this serine-protease inhibitor unleashes epithelial proteolysis and inflammation, mechanistically upstream of barrier breakdown.
The net epithelial phenotype is impaired differentiation and barrier loss, which we captured as an arrested-differentiation trajectory (only 5.4% of EoE epithelium reached late differentiation vs 17.9% healthy).

## 10. Alarmins: TSLP and IL-33 (and the IL-13 hub)

Epithelial alarmins initiate and sustain type-2 immunity. **TSLP** is a genetic and mechanistic linchpin (its locus is a top EoE GWAS hit — Part IV) that activates dendritic cells, ILC2s, and basophils. **IL-33** signals through ST2 (IL1RL1) to activate ILC2s and Th2 cells and is released on epithelial damage — the IL-33/ST2 axis is one of this project's protein-design targets (the receptor-side IL1RL1 blockade, with a validated 4KC3 interface). Downstream, **IL-13** is the central effector cytokine: it drives eotaxin-3, degrades DSG1, induces CAPN14, and reprograms the epithelium — making it (with IL-4Rα) the mechanistic rationale for dupilumab (Part IV). The commonality of the IL-4/IL-13 pathway across atopic diseases (Bieber 2017, doi:10.1080/1744666x.2017.1298443; IL-13 in eosinophilic disorders, doi:10.3389/fmed.2017.00139) explains the atopic-march comorbidity.

## 11. Fibroblasts and tissue remodeling

Chronic type-2 inflammation converts to fixed structural disease through fibroblast activation. TGF-β (from eosinophils, mast cells, and other sources) drives subepithelial fibroblasts toward a myofibroblast phenotype, depositing matrix and producing periostin — a matricellular protein that further recruits eosinophils and marks type-2/eosinophilic remodeling (Blanchard/periostin biomarker work, doi:10.1016/j.jaci.2012.06.025; periostin in remodeling, doi:10.1007/s00018-013-1494-y). This is the cellular basis of the fibrostenotic natural history (Part I §3). **Project integration:** POSTN is among the most reproducibly upregulated genes in our meta-signature (I²=71.8%) and a prioritized target.

## 12. Cytokine axes and the IFN-γ layer

The dominant axis is type-2 (IL-4/IL-5/IL-13), inherited conceptually from asthma (anti–IL-5 and anti–IL-4Rα biology, Part IV). But EoE is not purely type-2: our single-cell and bulk work found a reproducible **IFN-γ program** (IFNG expressed by 21.8% of EoE T cells; strong epithelial HLA-I induction correlating with IFN-γ at r=0.94), with type-I interferon essentially absent. This type-II-interferon layer, superimposed on the type-2 core, is one of the project's novel mechanistic emphases and may mark a more inflammatory endotype. The CAPN14 node — an EoE-risk gene that is directly IL-13-induced (Davis 2016, doi:10.1172/jci.insight.86355; Litosh 2017, doi:10.1016/j.jaci.2016.09.027) — exemplifies the gene×environment convergence: a genetic susceptibility allele whose product is switched on by the very cytokine driving the disease.

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### Synthesis
The cell types are not parallel silos but a wired circuit: **epithelial alarmins (TSLP/IL-33) → ILC2/Th2/basophil type-2 cytokines → IL-13 epithelial reprogramming (eotaxin-3↑, DSG1↓, CAPN14↑) → eosinophil recruitment + mast-cell expansion (SIGLEC6⁺ state) → fibroblast remodeling (POSTN) → fibrostenosis**, with an IFN-γ overlay and a CD4-antigen-presentation core that makes the disease addressable by antigen-specific approaches. Every node above is a described or prioritized therapeutic point of attack, developed in Parts III–IV. This project's contributions — the SIGLEC6⁺ mast state, the IFN-γ layer, the arrested-differentiation epithelial trajectory, and the reproducible meta-signature anchoring CCL26/POSTN/DSG1/ALOX15 — slot into this circuit as both confirmation and extension.

*Evidence base: 233 Tier-1/2 mechanistic references in the master library; specific primary sources cited inline for each mechanistic claim.*