{
  "version": 3,
  "created_at": "2026-07-08T00:28:25.434Z",
  "task_summary": "Phase 4: Prospective preclinical pMHC-TCR therapy framework — 8-allergen MHC-II epitope mapping, patient HLA stratification, T-cell assay design, mechanism specification, and translational workflow roadmap.",
  "agents": [],
  "phases": [
    {
      "name": "Plan",
      "delegations": [
        {
          "steps": [
            {
              "title": "Task 1a: Fetch 8-allergen sequences from UniProt",
              "description": "Query UniProt for canonical sequences of 8 common EoE food triggers: milk (β-lactoglobulin P24627, α/β-casein P02662/P02663), wheat gliadin (P04730), egg ovomucoid (P01005), soy glycinin (P04347) and β-conglycinin, peanut Ara h 2 (P43238), fish parvalbumin (P02624), shellfish tropomyosin (P09495), tree nuts (almond 2S albumin, cashew 2S albumin, walnut seed protein). Output: `allergen_sequences.fasta` with full-length and major epitope-bearing domains annotated."
            },
            {
              "title": "Task 1b: MHC-II epitope prediction across allele panel",
              "description": "Perform full HLA-II binding prediction (mhcnuggets, 15-mer cores) for all 8 allergens across representative population-dominant alleles: HLA-DRB1*01:01, *03:01, *04:01, *07:01, *15:01 (covers ~50% European ancestry), plus DQA1/DQB1 pairs (DQA1*01:01/DQB1*05:01, DQA1*05:01/DQB1*02:01) and DPA1/DPB1 heterodimers. Classify binders: strong (IC50 <500nM), moderate (500–5000nM). Output: `allergen_epitope_panel.csv` (allergen, epitope_seq, start_pos, allele, IC50_nM, rank, strong_Y/N)."
            },
            {
              "title": "Task 1c: Allergen-epitope-HLA matrix & visualization",
              "description": "Consolidate predictions: (1) per-allergen heatmap (epitopes × alleles, IC50 colored), (2) cumulative strong-binder load by allergen and by allele (bar plots, population-frequency weighted), (3) epitope uniqueness (shared vs allergen-private), (4) cross-reactivity matrix (epitope sequence similarity, BLOSUM62). Outputs: `allergen_epitope_heatmap.png`, `epitope_load_by_allergen.csv/png`, `epitope_uniqueness.csv`, `cross_reactivity_matrix.csv`, consolidated `allergen_pmhc_dossier.md`."
            },
            {
              "title": "Task 2: Patient HLA stratification & recruitment design",
              "description": "Integrate population allele frequencies (NMDP, 1000 Genomes, European ancestry n≥1000): (1) rank HLA alleles by frequency (common: >5%, rare: <2%), (2) compute per-patient epitope burden (sum of strong-binder epitopes for each HLA allele pair, weighted by population), (3) stratify patients into Tier-High (top-quartile epitope burden, common HLA) vs Tier-Low (bottom-quartile, rare HLA), (4) calculate statistical power (n/group for epitope-specific T-cell response detection). Outputs: `hla_allele_frequencies.csv`, `patient_epitope_burden_tiers.csv`, `recruitment_stratification_rationale.md`."
            },
            {
              "title": "Task 3: T-cell reactivity assay protocol specification",
              "description": "Design IND-enabling protocol: (1) T-cell isolation from fresh EoE esophageal biopsies (enzymatic digestion, magnetic bead enrichment) or PBMC, (2) allergen-specific in vitro stimulation (dominant epitopes as peptide pools, autologous APC loading), (3) IL-2 expansion (14d, 100–200 IU/mL), (4) T-cell cloning (limiting dilution or single-cell sorting), (5) readouts—proliferation (3H-thymidine incorporation 0–72h), cytokine production (IFNγ, IL-5, IL-13 by ELISA/Luminex/ELIspot at 24–48h), activation phenotype (flow: CD25+/HLA-DR+ CD4+ T cells, tetramer+), TCR clonality (CDR3 sequencing). Output: `t_cell_assay_protocol.md` (step-by-step SOP, troubleshooting, data interpretation criteria)."
            },
            {
              "title": "Task 4: pMHC-TCR mechanism specification & literature synthesis",
              "description": "Literature review (OpenAlex, PubMed): (1) pMHC-multimer-based T-cell depletion mechanisms (adoptive transfer of pMHC-loaded DC, TCR-ligation-induced deletion, Fas/FasL signaling), (2) costimulatory blockade (anti-CD28, anti-ICOS), (3) anergy induction models (IL-10/TGF-β, regulatory T-cell expansion), (4) humanized mouse models (HLA transgenic, human immune reconstitution), (5) human ex vivo validation (esophageal organoid co-culture). Integrate findings into mechanism-of-action roadmap. Outputs: `pmhc_tcr_mechanism_review.md` (mechanism modules with citations, decision trees for approach selection), `mechanism_decision_matrix.csv`."
            },
            {
              "title": "Task 5: Preclinical translational workflow roadmap",
              "description": "Synthesize Tasks 1–4 into Phase-1–4 study plan: **Phase 4.1 (Patient Characterization):** HLA genotyping (n=40 EoE, stratified by epitope burden), esophageal biopsy T-cell isolation & polyclonal expansion (14d IL-2), baseline immune profiling (flow, TCR clonality, Luminex multiplex cytokines). **Phase 4.2 (Epitope Mapping):** Allergen-peptide pool stimulation (dose-response: 0.1–10 μg/mL), epitope-specific T-cell cloning, single-clone TCR sequencing. **Phase 4.3 (Mechanism Validation):** pMHC-multimer therapy in vitro (T-cell lines + pMHC-loaded APC, readouts: apoptosis/anergy/proliferation suppression at day 5–10), humanized mouse model (HLA-transgenic recipient, human T-cell transfer, OVA or food-peptide challenge). **Phase 4.4 (IND Enabling):** PK/PD of pMHC-therapeutic in mice, human ex vivo esophageal culture (patient-derived epithelium + T cells + pMHC-therapy), safety & tolerability readouts. Outputs: `preclinical_workflow_roadmap.md` (Gantt chart, success criteria, n/power, timelines), `iind_enabling_checklist.csv`."
            },
            {
              "title": "Consolidate Phase 4 package & final report",
              "description": "Synthesize all Task outputs into master deliverable: (1) Phase-4-executive summary (pMHC-therapy rationale, patient stratification, mechanism, translational plan), (2) allergen-epitope compendium with clinical application notes, (3) T-cell assay SOP + validation data template, (4) mechanism roadmap + literature citations, (5) preclinical study protocol with regulatory/GLP alignment guidance. Output: `eoe_phase4_pmhc_therapy_package.md`, consolidated artifact bundle `phase4_deliverables.tar.gz`."
            }
          ]
        }
      ],
      "id": "phase-0"
    }
  ],
  "desired_outputs": [
    "allergen_sequences.fasta (8 allergens, major domains annotated)",
    "allergen_epitope_panel.csv (complete IC50 predictions across HLA-II alleles)",
    "allergen_epitope_heatmap.png, epitope_load_by_allergen.png (visualizations)",
    "hla_allele_frequencies.csv, patient_epitope_burden_tiers.csv (recruitment stratification)",
    "t_cell_assay_protocol.md (IND-ready SOP)",
    "pmhc_tcr_mechanism_review.md (literature synthesis with mechanism decision tree)",
    "preclinical_workflow_roadmap.md (phase 1–4 study plan with timelines & success criteria)",
    "eoe_phase4_pmhc_therapy_package.md (consolidated Phase 4 report)",
    "phase4_deliverables.tar.gz (complete deliverable bundle)"
  ],
  "feasibility": {
    "confidence": "high",
    "rationale": "All component data and tools are in scope: UniProt sequence retrieval is stable; mhcnuggets (HLA-II) and mhcflurry (HLA-I) are already validated in prior phases; literature synthesis is feasible via OpenAlex/PubMed. Computational tasks are GPU-independent (epitope prediction runs on CPU). Clinical protocol design is standard translational template. Only limitation: in vivo model validation requires prospective patient cohorts (scoped as Phase 4.3 spec, not execution)."
  }
}