In Vitro Resident Memory CD8 T Cell Differentiation Using Epithelial Organoid-T Cell Co-culture System

Summary

To study CD8 T cell differentiation and function in vitro, CD8 T cells can be isolated from the mouse to be co-cultured long-term with preformed infected murine vaginal epithelial organoids. Here, we describe this process and assess the acquisition of resident memory T cell markers upon co-culture.

Abstract

Barrier mucosal tissues play an important role in the differentiation and function of resident T cells. Among the diverse cell populations within these tissues, epithelial cells are key drivers of T cell differentiation. However, our understanding of the epithelial-derived signals that shape T cell biology in mucosal environments remains limited, largely due to a lack of specific tools and the complexity of available model systems. Here, we describe a murine vaginal epithelial organoid-CD8 T cell co-culture system that models dynamic interactions between infected epithelium and virus-specific CD8 T cells. This protocol enables phenotypic and functional analysis of T cell responses within a physiologically relevant three-dimensional epithelial microenvironment. Using this system, we demonstrate the differentiation of effector CD8 T cells into tissue-resident memory T cells (TRM). This versatile in vitro culture system provides reductionist opportunities to investigate molecular details of epithelial cues behind CD8 T cell differentiation that govern protective immunity and immunopathology in barrier organs.

Introduction

CD8 resident memory T cells (TRMs) represent a unique population of memory CD8 T cells that are retained permanently in the peripheral tissues without routinely circulating through blood and lymphoid tissues. These TRM cells are strategically positioned in barrier organs, including skin, lungs, intestines, and the reproductive tract, where they act as sentinels against pathogens and nascent tumors^1,2. Upon pathogen detection, CD8 TRM cells rapidly release cytotoxic granules to directly eliminate infected cells while simultaneously secreting inflammatory cytokines that alert surrounding tissue cells and recruit....

Protocol

All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Brown University (Protocol number 22-10-0001).

1. Basic setup and preparation of an activation plate

1. One day prior to beginning the experiment, prepare a non-treated tissue culture plate for seeding with naïve CD8 T cells. Prepare a solution of activating antibodies in sterile dPBS at a final concentration of 1 µg/mL for anti-CD3ε (2C11) and 0.167 µg/mL for B7-1 Fc. For a 24-well plate, dispense 600 µL of this solution per well. Incubate overnight at 4 °C until ready to use.
2. Prepare Lymphoc....

Results

Microscopic visualization of CD8 T cell-infected organoid co-culture
Preformed mouse VEOs were infected with LCMV and co-cultured with LCMV-specific CD8 T cells. We have also performed these co-cultures with HSV-2 infection model to establish the versatility of the protocol but are showing microscopic data for LCMV model and have included T cell differentiation data in HSV-2 model below. The LCMV expresses yellow fluorescent protein (YFP), so the infected cells can be visualized using routine epifluo.......

Discussion

CD8 TRMs rely on local environmental cues for differentiation and maintenance. These cues instill tissue-specific programming that allows CD8 TRMs to adapt to their surroundings and perform their immunosurveillance duties as needed. Unfortunately, TRMs failed to survive in vitro once extracted from the tissue, and this has limited their detailed analysis^12,13. In this protocol, we have provided a method by which CD8 T cells can be co-cultured with epithe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Paraformaldehyde Aqueous Solution	Electron Microscopy Sciences	15710-S
24-Well Polystyrene Non-Treated Multiple Well Tissuce Culture Plates	VWR International	10861-558
2-Mercaptoethanol	Gibco	21985023
3 mL Sterile Syringes	Fisher Scientific	14-955-457
5 mL, Snap Cap, Sterile, Round-Bottom Polystyrene Test Tubes	Falcon	352058
60 mm Petri Dishes	Fisher Scientific	FB0875713A
70 μm Sterile Cell Strainers	Fisher Scientific	22-363-548
96-well Non-treated Polystyrene Round Bottom Microplates	Greiner Bio-One	650101
96-Well Polystyrene Non-Treated Multiple Well Tissue Culture Plates	VWR International	10861-562
Albumin bovine/fraction V	Thermo Scientific Chemicals	J6465518
Ammonium Chloride	Fisher Scientific	A661-500
Amphotericin B	Sigma-Aldrich	A9528-100MG
B-27 Supplement	Gibco	17504044
ioTek Cytation 5 Cell Imaging Multimode Reader
DMEM/F12 1:1 Medium	Cytiva	SH30271.FS
Dulbecco's Phosphate-Buffered Salt Solution 1x	Corning	21031CV
Ethylenediaminetetraacetic Acid (0.5 M Solution/pH 8.0)	Fisher Scientific	BP2482-500
Extra Fine Micro Dissecting Scissors	Roboz Surgical Instrument Co.	RS-5882
Fetal Bovine Serum - Charcoal/Dextran Treated	R&D Systems	S11650
Flowjo v10 software	Becton, Dickinson and Company	FlowJo (RRID:SCR_008520)
Graefe Forceps	Roboz Surgical Instrument Co.	RS-5135
HEPES Solution	Cytiva	SH30237.01
L-Glutamine (200 mM)	Gibco	A2916801
Magnet 5mL	Biolegend	480019
Mouse CD8 Naïve T Cell Isolation Kit	Biolegend	480044
Mouse EGF Recombinant Protein	Gibco	315091MG
Non-Essential Amino Acids Solution (100x)	Gibco	11140050
Penicillin-Streptomycin solution	Cytiva	SV30010
Potassium Bicarbonate	MP Biomedicals	0215255780
Purified anti-mouse CD3ε Antibody	Biolegend	100360
Recombinant Mouse B7.1 (CD80)-Fc Chimera (carrier-free)	Biolegend	555406
Recombinant Mouse IL-12 (p70) (carrier-free)	Biolegend	577004
Recombinant Mouse IL-2 (carrier-free)	Biolegend	575406
Reduced Growth Factor Basement Membrane Extract	R&D Systems	BME001-10
RPMI 1640 Media	Cytiva	SH30027.LS
Sodium Azide	Fisher Scientific	S227I-25
Sodium Pyruvate (100 mM)	Gibco	11360070
Trypsin 0.25% protease with porcine trypsin	Cytiva	SH30042.02
Viability Dye (Red 780)	Cytek Biosciences	13-0865-T100