# EoE Allergen-Specific T-Cell Reactivity Assay Protocol
## Isolation, Expansion, Characterization & pMHC Therapy Sensitivity

**Version:** 1.0 (Phase 4 IND-Enabling)  
**Status:** Study Protocol  
**Last Revised:** Phase 4 Development  
**Regulatory Context:** IND Preclinical Section 2.3 (Pharmacology/Toxicology), Section 2.4.2 (Methods)

---

## 1. OBJECTIVE & RATIONALE

### Primary Objective
To establish a standardized assay for quantifying food allergen-specific CD4+ T-cell proliferation and cytokine production from eosinophilic esophagitis (EoE) patient samples, enabling ex vivo measurement of pMHC-TCR therapeutic efficacy.

### Secondary Objectives
1. Clone antigen-specific T-cell lines for TCR sequencing and mechanism validation
2. Assess polyfunctionality (multi-cytokine producers: IFNγ + IL-5 + IL-13)
3. Measure activation phenotypes (CD25, HLA-DR, PD-1, PD-L1)
4. Validate the predictive value of each patient's individualized epitope burden (does allergen-specific response track with personalized predicted burden?) — burden as a mechanistic covariate, not an eligibility criterion

### Rationale
Food-driven EoE is CD4+/Th2-mediated (Phase 2 findings: IFNγ 21.8% T cells, Th2 endotype r=0.73 with mast activation). Allergen-specific CD4+ T cells expanded from patient esophageal biopsies or peripheral blood mononuclear cells (PBMC) serve as an ex vivo disease model to test:
  - Epitope-specific reactivity (validating computational epitope predictions)
  - pMHC-multimer-induced T-cell suppression (anergy/apoptosis markers)
  - Cross-allergen reactivity patterns

---

## 2. MATERIALS & REAGENTS

### 2.1 Sample Collection & Transport

| Item | Specification | Supplier | Notes |
|---|---|---|---|
| EDTA vacutainer tubes | 10 mL | BD Biosciences | For PBMC isolation |
| Preservative-free heparin | 10 IU/mL | BD (green cap) | Alternative to EDTA if needed |
| Room-temperature transport buffer | RPMI 1640 + 2% FBS | In-house prep | Keep at 18–24°C during transit |
| Sample containers | Sterile, leak-proof | Falcon/Eppendorf | Biohazard shipping label |
| Transport time | ≤24 h from phlebotomy | — | Delayed transport reduces viability |

### 2.2 Tissue Processing (Biopsy-derived T cells)

| Reagent | Specification | Supplier | Concentration |
|---|---|---|---|
| Collagenase IV | Grade IV, endotoxin-free | Worthington | 1 mg/mL in PBS |
| Trypsin inhibitor | Soybean | Worthington | 1 mg/mL in PBS |
| DNase I | Grade II | Roche | 20 Kunitz U/mL |
| HBSS | Ca²⁺/Mg²⁺-free | Thermo | pH 7.4 |
| Percoll | GE Healthcare | 1.069–1.077 g/cm³ | Density gradient |

### 2.3 T-Cell Expansion Medium

| Component | Concentration | Source | GMP Grade |
|---|---|---|---|
| RPMI 1640 | Base medium | Thermo | ✓ |
| Fetal bovine serum (FBS) | 10% v/v | Serum source; heat-inactivated, lot-tested | ✓ |
| L-glutamine | 2 mM | Thermo or in-house | ✓ |
| Penicillin/streptomycin | 50 U/mL / 50 µg/mL | Thermo | ✓ (optional, monitor for T-cell effects) |
| Recombinant human IL-2 | 100–200 IU/mL | Prometheus (Peprotech) or Roche | ✓ |
| HEPES buffer | 10 mM | Thermo | ✓ |

**Quality assurance:** Each FBS lot tested for endotoxin (<0.5 EU/mL), sterility, mycoplasma, and T-cell proliferation using standard thymidine incorporation assay.

### 2.4 Epitope Peptides

**Source:** Synthetic peptides, HPLC purified >95% (Genscript, Mimotopes, or equivalent)

**Target epitopes:** selected from the Task 1 netMHCIIpan-4.1 panel (2,287 strong binders, IC50 < 500 nM, across 10 allergen proteins / 8 food categories; 470 unique binding-core registers). For practical synthesis, select the top strong-binding core per allergen per assay allele — prioritizing the DRB1*01:01-restricted cores, which dominate the predicted repertoire (363 of 470 unique cores). Representative highest-affinity cores include soy glycinin FRVIPSEVL and milk α-S1-casein FYQLDAYPS (both IC50 < 6 nM on DRB1*01:01).
  - Allergens covered: milk (β-lactoglobulin, α-S1-casein), wheat (gliadin), egg (ovomucoid), soy (glycinin, β-conglycinin), peanut (Ara h 2), fish (parvalbumin), shellfish (tropomyosin), tree nut (walnut Jug r 1)
  - Format: Lyophilized; reconstitute in DMSO (5 mM stock), store −20°C

**Pools:** Generate allergen-specific peptide pools (all epitopes per allergen combined) at final assay concentration 0.1–10 µg/mL each

| Pool | Epitopes | Total Peptides | Allergen |
|---|---|---|---|
| Pool-Milk | BLG + CSN | 2 | Milk |
| Pool-Wheat | Gliadin | 1 | Wheat |
| Pool-Egg | Ovomucoid | 1 | Egg |
| Pool-Soy | Glycinin + β-Conglycinin | 2 | Soy |
| Pool-Peanut | Ara h 2 | 1 | Peanut |
| Pool-Fish | Parvalbumin | 1 | Fish |
| Pool-Shellfish | Tropomyosin | 1 | Shellfish |

**Storage:** −20°C, desiccant, ≤12 months prior to use

### 2.5 Feeder Cells (Antigen-Presenting Cells, APCs)

| APC Type | Source | Preparation | Notes |
|---|---|---|---|
| Autologous B cells + monocytes | PBMC after T-cell depletion | Negative selection (CD3 microbeads, MACS) | Preferred; MHC-matched |
| Allogeneic PBMC (HLA-matched) | Donor bank (matched to patient HLA-DRB1) | Thawed, rested 4h, γ-irradiated (30 Gy) | Backup if insufficient autologous |
| LCL (lymphoblastoid cell line) | HLA-matched LCL bank | Cultured, γ-irradiated (30 Gy) | Standardized; allows pooling |

**Gamma-irradiation:** 30 Gy (3000 cGy) using ¹³⁷Cs or X-ray; verify dose with dosimeter per ISO 11137.

### 2.6 Readout Reagents

#### 2.6.1 Proliferation Assay

| Reagent | Specification | Supplier | Comments |
|---|---|---|---|
| [³H]-thymidine | 1–5 mCi, 40–100 Ci/mmol | PerkinElmer | Radioactive; requires license |
| Scintillation fluid | Betaplate Scint | PerkinElmer | For plate harvester |
| Cell harvester | Filtermate or equivalent | PerkinElmer | Automated 96-well harvester |

**Alternative (non-radioactive):** BrdU incorporation (Roche ELISA) or CFSE dilution (flow cytometry)

#### 2.6.2 Cytokine Production

| Cytokine | Assay | Supplier | Lower Detection Limit |
|---|---|---|---|
| IFNγ | ELISA or Luminex | BioLegend / R&D Systems | ≥4 pg/mL |
| IL-5 | ELISA or Luminex | BioLegend / R&D Systems | ≥2 pg/mL |
| IL-13 | ELISA or Luminex | BioLegend / R&D Systems | ≥2 pg/mL |
| IL-2 | Luminex (if multiplexed) | BioLegend / R&D Systems | ≥2 pg/mL |
| IL-10, TGF-β | Optional (suppression markers) | BioLegend / R&D Systems | — |

**Multiplexing:** Luminex xMAP preferred if budget permits (simultaneous 3–5 cytokines, 50 µL sample)

#### 2.6.3 Flow Cytometry

**Antibody panel:**

| Marker | Clone | Fluorophore | Supplier | Gating Logic |
|---|---|---|---|---|
| CD3 | UCHT1 or SK7 | BV421 or FITC | BD / BioLegend | Live CD3+ T cells |
| CD4 | RPA-T4 or OKT4 | PE-Cy7 or APC | BD / BioLegend | CD3+ CD4+ (helper T) |
| CD8 | RPA-T8 | BV510 or PerCP | BD / BioLegend | Gate out CD8+ |
| CD25 | BC96 or 2A3 | PE | BioLegend / R&D | Activation (IL-2R α) |
| HLA-DR | L243 or G46-6 | FITC or APC | BD / BioLegend | MHC-II induction |
| PD-1 | EH12.2H7 | PE or BV480 | BioLegend / BD | Exhaustion marker |
| PD-L1 | 29E.2A3 | APC or PE | BioLegend | Checkpoint ligand |
| Live/Dead | 7-AAD or Fixable Viability Dye | 7-AAD or BV510 | BD / BioLegend | Exclude dead cells |

**Tetramer reagent (optional, for TCR cloning):**
  - HLA-DRB1/peptide tetramer (custom, ProImmune or NIH Tetramer Facility)
  - Fluorochrome-conjugated (PE or APC)
  - Used for enrichment of peptide-specific CD4+ T cells via magnetic sorting

---

## 3. PROCEDURES

### 3.1 Sample Collection & Storage

**3.1.1 Peripheral Blood (PBMC Route)**

**Timeline:**
  - Day 0: Phlebotomy (morning, fasting if possible)
  - Day 0: Transport to lab (≤24 h)
  - Day 1: Processing

**Procedure:**
  1. Collect 40–50 mL blood into EDTA vacutainers (4 × 10 mL tubes)
  2. Invert gently 8–10 times to mix (do NOT shake vigorously)
  3. Transport at room temperature (18–24°C) in insulated carrier
  4. Upon arrival, proceed immediately to PBMC isolation (see 3.2.1)

**3.1.2 Esophageal Biopsy (Tissue Route)**

**Timeline:**
  - Day 0: Endoscopy with biopsy (~6–8 biopsies, 2–3 mm each into sterile cup with cold PBS)
  - Day 0: Transport to lab (≤2 h, on ice)
  - Day 1: Tissue digestion

**Procedure:**
  1. Place biopsies into sterile, conical tube with cold HBSS (Ca²⁺/Mg²⁺-free)
  2. Transport on ice in insulated carrier
  3. Upon arrival (within 2 h), proceed to enzymatic digestion (see 3.2.2)

**Note:** Obtain informed consent; comply with IRB protocols. (Expected from EoE biopsy for clinical diagnosis; research use of surplus tissue.)

---

### 3.2 T-Cell Source Preparation

#### 3.2.1 PBMC Isolation (Density Gradient Centrifugation)

**Materials:**
  - Ficoll-Paque (GE Healthcare, 1.077 g/mL)
  - 50 mL conical tubes, centrifuge (refrigerated 4°C, 400–1200g capable)

**Procedure:**
  1. Dilute whole blood 1:1 with PBS (room temperature)
  2. Layer 15 mL diluted blood over 10 mL Ficoll in 50 mL tube (tilt at 45°)
  3. Centrifuge 20 min at 400 g, room temperature, without brake
  4. Remove buffy coat (lymphocyte layer) at interface; transfer to fresh 50 mL tube
  5. Wash twice: resuspend in 50 mL PBS, centrifuge 10 min at 300 g; discard supernatant
  6. Resuspend pellet in RPMI + 10% FBS
  7. Count viable cells (Trypan blue exclusion; expected yield ~3–5 × 10⁸ cells from 40 mL blood)

**QC:** ≥90% viability required; if <90%, repeat with fresh sample

#### 3.2.2 Esophageal Tissue Digestion (Enzymatic Dissociation)

**Materials:**
  - Collagenase IV, Trypsin inhibitor, DNase I (see 2.2)
  - 40 µM nylon mesh filter
  - 6-well tissue culture plates

**Procedure:**
  1. Drain HBSS from biopsy tube; transfer biopsies to 6-well plate
  2. Add 2 mL digestion medium (Collagenase 1 mg/mL + Trypsin inhibitor 1 mg/mL + DNase 20 U/mL in HBSS)
  3. Incubate 30 min at 37°C, 5% CO₂, with gentle agitation (orbital shaker ~50 rpm)
  4. Add 2 mL RPMI + 10% FBS to stop digestion
  5. Pass through 40 µM filter into 50 mL tube
  6. Centrifuge 5 min at 300 g; discard supernatant
  7. Resuspend in RPMI + 10% FBS
  8. Count viable cells (expected yield ~10⁵–10⁶ cells per biopsy set)

**QC:** ≥85% viability; assess CD3+ frequency by flow (should be ≥5% of viable cells)

---

### 3.3 Initial Allergen Stimulation & T-Cell Expansion (Days 1–14)

**Objective:** Prime naive/resting T cells; expand allergen-specific population; generate sufficient cell numbers for functional assays.

**3.3.1 Setup**

| Parameter | Value | Justification |
|---|---|---|
| Starting cell density | 1–2 × 10⁶ cells/mL | Standard for polyclonal expansion |
| Culture vessel | 24-well plate | Adequate surface area, easy handling |
| Culture volume per well | 0.5–1 mL | Sufficient for sampling + expansion |
| Epitope pool concentration | 0.1–1.0 µg/mL final | Physiological dose; escalate if needed |
| Incubation | 37°C, 5% CO₂ | Standard culture conditions |
| Medium refresh | Day 4, 7, 10 | Half-medium replacement to reduce toxin buildup |
| Duration | 14 days | Optimal window for polyclonal expansion |

**3.3.2 Procedure**

**Day 1 (Initial stimulation):**
  1. Isolate T-cell-depleted APC population:
     - Add anti-CD3 microbeads (CD3 Microbeads, Miltenyi) per kit to PBMC suspension
     - Run through MACS LS column; collect flow-through (T-cell-depleted APC, contains B cells + monocytes)
     - Count APC; resuspend at 1 × 10⁶ cells/mL
  
  2. Plate T cells and APCs:
     - Per well of 24-well plate:
       - T cells: 5 × 10⁵ (1 mL of 5 × 10⁵/mL)
       - APC: 5 × 10⁵ (1 mL of 5 × 10⁵/mL) [1:1 T:APC ratio]
     - Total volume: 1 mL per well
  
  3. Add allergen stimulation:
     - Allergen peptide pool (7 pools total, see 2.4) at final concentration 0.1 µg/mL each
     - Positive control: Anti-CD3/CD28 (Dynabeads Human T-Activator, 25 µL / 1 mL per kit instructions)
     - Negative control: No peptide, no beads (baseline)
  
  4. Incubate: 37°C, 5% CO₂

**Days 4, 7, 10 (Medium Refresh):**
  1. Remove 0.5 mL supernatant from each well (save for cytokine analysis if desired)
  2. Add 0.5 mL fresh expansion medium (RPMI + 10% FBS + IL-2 100 IU/mL)
  3. Assess cell density visually; if confluent, split 1:2 with fresh medium

**Day 14 (Expansion Complete):**
  1. Count viable cells per well (Trypan blue)
  2. Assess proliferation: Expected 10–50-fold expansion; if <5-fold, may indicate poor response (flag for data analysis)
  3. Harvest cells: Transfer to 15 mL tube, centrifuge 5 min at 300 g
  4. Resuspend in RPMI + 2% FBS for downstream assays (see 3.4 & 3.5)

**3.3.3 Batch Quality Control**

| QC Parameter | Acceptance Criterion | Method |
|---|---|---|
| Cell viability | ≥80% | Trypan blue exclusion |
| Expansion fold | ≥5-fold | Cell count vs. input |
| Contamination (bacteria/fungi) | Negative | Visual inspection, optional culture |
| Mycoplasma | Negative | PCR (Lonza MycoAlert or equivalent) |

---

### 3.4 Proliferation Assay ([³H]-Thymidine Incorporation)

**Objective:** Quantify dose-dependent T-cell proliferation in response to allergen peptide pools.

**3.4.1 Setup**

**Timeline:** Day 14 (from 3.3.2) → Day 18 (readout), 4-day measurement window

**Plate layout:** 96-well flat-bottom plate (Corning); replicate wells n=3–4 per condition

**Conditions per allergen pool:**
  1. Allergen peptide (dose-response: 0.01, 0.1, 1.0, 10 µg/mL)
  2. Positive control: Anti-CD3 (5 µg/mL; e.g., OKT3)
  3. Negative control: No peptide

**APC source:** Autologous PBMC (T-cell-depleted) from Day 1, γ-irradiated (30 Gy) if stored; otherwise fresh prep

**3.4.2 Procedure**

**Day 14 (Setup):**
  1. Prepare stimulator plates (96-well):
     - Seed T-cell-expanded population: 5 × 10⁴ cells/well (50 µL of 10⁶/mL suspension)
     - Add γ-irradiated APC: 5 × 10⁴ cells/well (50 µL of 10⁶/mL suspension)
     - Final volume before peptide: 100 µL per well
  
  2. Add peptide or control:
     - Allergen peptide pools: Add 50 µL at 2× desired concentration (final 0.01–10 µg/mL)
     - Positive control (anti-CD3): Add 50 µL at 10 µg/mL (final 5 µg/mL)
     - Negative control (vehicle): Add 50 µL PBS
     - Final volume: 150 µL per well
  
  3. Incubate 68–72 h at 37°C, 5% CO₂ (do NOT open plate)

**Day 17 (Labeling):**
  1. Add [³H]-thymidine: 20 µCi/mL stock → 0.5 µCi/well (1 µL of stock added to 40 µL medium per well)
  2. Incubate 18–20 h (overnight, 37°C, 5% CO₂)

**Day 18 (Harvest & Measurement):**
  1. Harvest on automated cell harvester (PerkinElmer Filtermate or Tomtec); transfer to scintillation plates
  2. Add Betaplate Scint fluid (50 µL per well)
  3. Count on TopCount scintillation counter: 1 min per well
  4. Record counts per minute (cpm)

**3.4.3 Data Analysis**

**Stimulation index (SI):**
$$	ext{SI} = rac{	ext{Mean cpm (peptide-stimulated)}}{	ext{Mean cpm (unstimulated control)}}$$

**Interpretation:**
  - SI < 2: No response
  - SI 2–3: Weak response (borderline positive)
  - SI 3–5: Moderate response
  - SI ≥ 5: Strong response (robust allergen reactivity)

**Dose-response curve:**
  - Plot SI vs. peptide concentration (semi-log, concentration on x-axis)
  - Identify peak concentration (typically 0.1–1.0 µg/mL for high-affinity epitopes)
  - EC₅₀ calculation (if 4-parameter logistic fit): Optional, for detailed kinetics

**Example output:**
```
Milk Beta-Lactoglobulin Epitope (Patient P04, high individualized predicted burden):
  Concentration (µg/mL)  |  SI   |  Status
  0.01                   |  1.8  |  No response
  0.1                    |  4.2  |  Moderate
  1.0                    |  7.1  |  Strong ← Peak
  10.0                   |  5.9  |  Strong (plateau)
```

---

### 3.5 Cytokine Production Assay (ELISA / Luminex)

**Objective:** Measure allergen-induced IFNγ, IL-5, IL-13 production; assess polyfunctionality.

**Timeline:** Parallel to proliferation assay (Day 14 stimulation, Day 16 harvest)

**3.5.1 Setup**

| Parameter | Value |
|---|---|
| Cell density | 5 × 10⁵ cells/mL |
| Culture volume | 200 µL per well (96-well plate) |
| Stimulation time | 48–72 h |
| Readout | Supernatant harvest at 48h (early) & 72h (late) |

**Procedure:**
  1. Plate expanded T cells at 5 × 10⁵ cells/mL in 96-well U-bottom plate (100 µL/well)
  2. Add γ-irradiated APC (100 µL of 5 × 10⁵/mL)
  3. Add allergen peptide pools (final 0.1–1.0 µg/mL) or controls (anti-CD3 5 µg/mL, no peptide)
  4. Incubate 48–72 h at 37°C, 5% CO₂
  5. Centrifuge plate 5 min at 300 g; transfer supernatant to fresh 96-well plate (100 µL per well)
  6. Store at −20°C (or measure immediately)

**3.5.2 ELISA Measurement (if Luminex unavailable)**

**Kit:** Use commercial sandwich ELISA (e.g., BD OptEIA, BioLegend)

**Per-cytokine protocol:**
  1. Coat plate with capture antibody overnight at 4°C
  2. Block with 1% BSA in PBS, 1 h room temperature
  3. Add 50–100 µL standards (0–5000 pg/mL recombinant cytokine) & supernatants in duplicate
  4. Incubate 2 h room temperature
  5. Wash; add detection antibody conjugated to HRP (1 h, room temp)
  6. Wash; develop with TMB substrate (15–30 min, stop with 1 M H₂SO₄)
  7. Read OD450 nm on plate reader
  8. Generate standard curve (4-parameter logistic); interpolate unknowns

**3.5.3 Luminex Measurement (Preferred if ≥3 cytokines)**

**Kit:** Luminex xMAP Human Cytokine Panel (BioLegend or R&D Systems)

**Protocol (per manufacturer):**
  1. Prepare magnetic bead suspension (premixed panels include IFNγ, IL-5, IL-13 + others)
  2. Add 25 µL sample supernatant to assay plate
  3. Add 25 µL bead suspension; mix
  4. Incubate 2 h room temperature on orbital shaker (500 rpm)
  5. Wash with wash buffer; add 25 µL detection antibody
  6. Incubate 1 h room temperature
  7. Add 25 µL streptavidin-PE; incubate 30 min
  8. Wash; resuspend in 120 µL sheath fluid
  9. Run on Luminex analyzer (typically Luminex 200 or MAGPIX)
  10. Generate 5-parameter logistic standard curves; report pg/mL

**3.5.4 Data Analysis**

**Results table (example):**

| Allergen Pool | Concentration (µg/mL) | IFNγ (pg/mL) | IL-5 (pg/mL) | IL-13 (pg/mL) | Polyfunctional? |
|---|---|---|---|---|---|
| Milk | 0 | <2 | <2 | <2 | No |
| Milk | 0.1 | 45 | 12 | 8 | Yes (all 3 +) |
| Milk | 1.0 | 127 | 34 | 25 | Yes |
| Wheat | 0.1 | 8 | 2 | 3 | No |
| Positive control (Anti-CD3) | — | 250 | 95 | 60 | Yes |

**Polyfunctionality classification:**
  - **Monofunctional:** Only IFNγ (>10 pg/mL)
  - **Bifunctional:** IFNγ + IL-5 OR IFNγ + IL-13
  - **Polyfunctional:** All three cytokines (IFNγ + IL-5 + IL-13, each >5 pg/mL)

---

### 3.6 Flow Cytometry: Activation Phenotyping

**Objective:** Assess CD25+/HLA-DR+ activation, PD-1/PD-L1 checkpoint expression, CD4/CD8 subset composition.

**Timeline:** Day 16–18 (parallel to cytokine harvest)

**3.6.1 Sample Preparation**

  1. Harvest T-cell culture (as in 3.5, after 48–72h stimulation)
  2. Wash: Centrifuge 5 min at 300 g; discard supernatant
  3. Resuspend in ice-cold PBS (2% FBS)
  4. Count viable cells (Trypan blue); adjust to 5 × 10⁶ cells/mL

**3.6.2 Staining Protocol**

| Step | Reagent | Volume | Time | Condition |
|---|---|---|---|---|
| 1 | Live/Dead dye (1:1000) | 1 µL per 100 µL cells | 15 min | Room temp, dark |
| 2 | Fc block (anti-CD32/CD16) | Per kit | 10 min | Room temp |
| 3 | Antibody cocktail (see panel, 2.6.3) | 20 µL/well | 30 min | 4°C, dark |
| 4 | Wash (PBS + 2% FBS) | 1 mL × 2 | — | 4°C |
| 5 | Fixation (2% paraformaldehyde) | 200 µL | 15 min | Room temp, dark |
| 6 | Wash (PBS) | 1 mL × 2 | — | Room temp |

**3.6.3 Acquisition & Analysis**

  - **Instrument:** Flow cytometer (BD FACSCanto II, Beckman Coulter Navios, or equivalent; ≥10⁴ events minimum)
  - **Gating hierarchy:** Live cells → Single cells → CD3+ → CD4+ → Analyze CD25, HLA-DR, PD-1, PD-L1
  - **Data export:** FCS 3.0 format; analyze using FlowJo or equivalent software
  - **Reporting:** % CD4+CD25+HLA-DR+, % CD4+PD-1+, % CD4+PD-L1+ (if APC-expressing)

**Expected phenotypes:**

| Condition | CD25+HLA-DR+ (%) | PD-1+ (%) | Interpretation |
|---|---|---|---|
| Unstimulated control | <5% | <10% | Resting state |
| Allergen-stimulated | 15–40% | 10–25% | Activated (antigen-specific) |
| pMHC-therapy treated | <10% (reduced) | ↑↑ (increased) | Anergy/suppression (intended effect) |

---

### 3.7 T-Cell Cloning & TCR Sequencing (For Subjects with Robust Responses, ≥3 Clones/Patient)

**Objective:** Isolate monoclonal T-cell populations for TCR analysis and mechanism validation studies.

**3.7.1 Single-Cell Cloning (Limiting Dilution)**

**Timeline:** Day 18–21 (from initial expansion)

**Procedure:**
  1. Harvest allergen-expanded T cells (from 3.4 or 3.5)
  2. Count viable cells; dilute to 0.3 cells/well in expansion medium
  3. Distribute 100 µL per well into 96-well U-bottom plates (~32–40 wells occupied by single cells statistically)
  4. Add 100 µL γ-irradiated APC + IL-2 (100 IU/mL final) per well
  5. Culture 2–3 weeks at 37°C, 5% CO₂; inspect weekly for clonal growth (single T-cell line visible as monolayer in well)
  6. Positive wells (>50% confluent): Transfer to 24-well plate; expand to generate sufficient cells for TCR sequencing (typically ≥10⁶ cells)

**QC:** Perform flow cytometry to confirm single T-cell clone (homogeneous CD4+ population, single TCR Vα/Vβ by tetramer or TCR sequencing)

**3.7.2 TCR Sequencing (CDR3 Identification)**

**Method:** High-throughput TCR sequencing (ImmunoSEQ Adaptive Biotech, 10X Genomics, or equivalent) or conventional cDNA RT-PCR + Sanger sequencing

**Protocol:**
  1. Lyse cloned T cells in TRIzol; extract RNA
  2. Perform RT-PCR with TCR Cα/Cβ-specific primers (generate CDR3-enriched cDNA library)
  3. Sequence libraries (Illumina MiSeq or Sanger capillary); identify dominant TCR α and β clonotypes
  4. Report: TCR Vα/Jα, Vβ/Jβ, CDR3 aa sequence, clonal frequency

**Expected outcome:**
  - ≥3 dominant TCR clonotypes per patient
  - Each clonotype traceable to specific epitope (via functional assay cross-linking epitope + TCR clone)

---

## 4. POSITIVE & NEGATIVE CONTROLS

### 4.1 Positive Controls (Robust Activation)

| Control | Rationale | Expected Output |
|---|---|---|
| Anti-CD3 (OKT3, 5 µg/mL) | TCR-independent activation; validates assay function | SI ≥10; IFNγ ≥100 pg/mL |
| PHA (phytohemagglutinin, 5 µg/mL) | Polyclonal T-cell activator (lectin) | SI ≥8; high cytokine |
| ConA (concanavalin A, 5 µg/mL) | Alternative polyclonal activator | SI ≥6 |

### 4.2 Negative Controls (No Activation)

| Control | Rationale | Expected Output |
|---|---|---|
| No peptide, no stimulator | Baseline T-cell viability/drift | SI ~1; cytokines <LDL |
| Vehicle (PBS, DMSO) | Chemical background | SI ~1 |
| Non-allergen peptide (e.g., influenza epitope in food-allergic patients unlikely to have chronic exposure) | Specificity check | SI ~1–1.5 |
| T cells only (no APC) | APC requirement confirmation | SI ~1 (no response without MHC presentation) |

---

## 5. QUALITY CONTROL & VALIDATION

### 5.1 Assay Performance Criteria

| Parameter | Acceptance Range | Frequency |
|---|---|---|
| Positive control SI | ≥8 (anti-CD3) | Every run |
| Intra-assay CV (³H incorporation) | ≤20% | Every run |
| Inter-assay CV (proliferation) | ≤25% | Per batch (monthly) |
| Cytokine recovery (Luminex) | 80–120% of spiked control | Monthly |
| Cell viability (day of assay) | ≥80% | Every run |
| Mycoplasma (expanded cultures) | Negative | Per experiment (monthly screening) |

### 5.2 Data Integrity

  - **LIMS documentation:** Record all sample IDs, processing dates, technician initials, lot numbers of reagents
  - **Plate maps:** Photograph or PDF save per 96-well plate layout (identifiable by barcode/lane)
  - **Raw data:** Retain scintillation counts, flow FCS files, ELISA plate images indefinitely (regulatory requirement)
  - **Analysis audit trail:** Document all exclusions (e.g., failed positive control, contamination) with justification

---

## 6. TROUBLESHOOTING & COMMON ISSUES

| Problem | Likely Cause | Solution |
|---|---|---|
| Low cell viability after isolation | Prolonged transit time, harsh processing | Reduce handling; process within 24h of phlebotomy |
| Weak allergen response (SI < 2 across all peptides) | Low individualized predicted burden (expected for some genotypes), or poor APC function | Confirm the patient's predicted burden; verify APC viability; try fresh feeder cells or anti-CD28 co-stimulation |
| High background (no peptide SI > 2) | APC contamination, media endotoxin | Use new media lot; verify APC γ-irradiation; test medium for endotoxin |
| Assay failure (positive control SI < 5) | Defective reagents, culture temperature drift | Re-run with fresh antibodies/media; verify incubator calibration |
| Inconsistent replicates (CV > 30%) | Pipetting error, heterogeneous cell distribution | Re-plate; use cell mixer before distribution |

---

## 7. DATA REPORTING & INTERPRETATION

### 7.1 Individual Patient Report

**Example (patient with high individualized predicted burden):**

```
PATIENT: P04 | HLA: DRB1*01:01 / DRB1*03:01 | EPITOPE BURDEN TIER: A (High, 15 epitopes)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

PROLIFERATION ASSAY (Day 18)
Allergen Pool       | Peak SI | Concentration | Status
────────────────────|─────────|───────────────|─────────────
Milk (BLG + CSN)    | 8.2     | 1.0 µg/mL     | ✓ Strong
Wheat (Gliadin)     | 1.9     | 1.0 µg/mL     | ✗ No response
Egg (Ovomucoid)     | 4.1     | 0.1 µg/mL     | ✓ Moderate
Soy (Glycinin)      | 6.7     | 0.1 µg/mL     | ✓ Strong
Peanut (Ara h 2)    | 3.2     | 1.0 µg/mL     | ✓ Moderate
Fish (Parvalbumin)  | 1.1     | 1.0 µg/mL     | ✗ No response
Shellfish (Tropo)   | 5.4     | 1.0 µg/mL     | ✓ Strong
Positive control    | 12.1    | — (Anti-CD3)  | ✓ Pass

CYTOKINE PRODUCTION (48h, Luminex)
Allergen Pool       | IFNγ (pg/mL) | IL-5 (pg/mL) | IL-13 (pg/mL) | Poly.
────────────────────|──────────────|──────────────|───────────────|──────
Milk (1.0 µg/mL)    | 156          | 42           | 31            | Yes
Soy (0.1 µg/mL)     | 103          | 28           | 19            | Yes
Shellfish (1.0)     | 87           | 15           | 11            | Yes

FLOW CYTOMETRY PHENOTYPE (Day 18)
CD3+CD4+ cells expressing:
  CD25+HLA-DR+:    28.5% (activated, antigen-specific)
  PD-1+:           18.3% (moderate exhaustion)

INTERPRETATION:
✓ Patient P04 shows robust food-allergen-specific CD4+ T-cell responses consistent with this patient's high individualized predicted epitope burden.
✓ Multi-allergen reactivity (4/7 allergen pools positive) observed.
✓ Polyfunctional response (IFNγ + IL-5 + IL-13) indicates Th2-polarized phenotype typical of EoE.
✓ Suitable for pMHC-therapy mechanism validation (proceeding to anergy induction testing).
```

### 7.2 Cohort Summary (Burden–Response Across All-Comers)

The cohort is enrolled without HLA selection. The analysis regresses response on each patient's individualized predicted epitope burden; patients are shown here binned by naturally-occurring burden (high/low tertiles arising *within* the all-comers cohort — not an eligibility split) for illustration only.

**Illustrative comparison table:**

| Parameter | High-burden tertile (illustrative; powered n=25/group) | Low-burden tertile (illustrative; powered n=25/group) | p-value |
|---|---|---|---|
| **Proliferation (Mean Peak SI)** | 6.2 ± 2.1 | 1.8 ± 0.6 | <0.001 |
| **IFNγ (pg/mL, mean)** | 94 ± 38 | 12 ± 8 | <0.001 |
| **IL-5 (pg/mL, mean)** | 28 ± 15 | 3 ± 2 | <0.01 |
| **Polyfunctional (%)** | 83% (10/12) | 12% (1/8) | <0.01 |
| **CD25+HLA-DR+ activation (%)** | 31 ± 11% | 6 ± 3% | <0.001 |

**Primary analysis (all patients):** allergen-specific response correlates with individualized predicted epitope burden (Pearson r, full cohort). **Conclusion (illustrative):** response scales with each patient's personalized predicted burden, supporting the epitope-prediction platform — without excluding any patient on HLA grounds.

---

## 8. REFERENCES & REGULATORY NOTES

### Regulatory Alignment
- **21 CFR 312.23(a)(4):** Pharmacology and toxicology data
- **ICH S3A:** "Toxicity and Pharmacokinetic Guidelines" (establishes T-cell ex vivo assay standards)
- **FDA Guidance for Industry: "Immunogenicity Assessment"** (E6(R2), 2018) — relevant for antigen-specific assay validation

### Key Literature
- Mosmann et al. (1986) JI 136: 2348–2357. [Th1/Th2 paradigm; IFNγ vs. IL-4/5]
- Betts et al. (2006) Nat. Med. 12: 972–979. [Polyfunctionality markers]
- Brodin et al. (2015) Cell Systems. [TCR repertoire & cloning]

### Version History
| Version | Date | Changes |
|---|---|---|
| 1.0 | Phase 4 Development | Initial IND-ready protocol (post-Task 1–2) |

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**Document Prepared By:** EoE pMHC Therapy Development Team  
**Distribution:** Phase 4 Study Investigators, IND Regulatory File