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 circulating immune effectors to contain pathogen dissemination^3,4. The effectiveness of CD8 TRMs in antipathogen immunity as well as cancer immunotherapy has already been demonstrated^5,6. Accordingly, positioning an abundant quantity of functionally competent CD8 TRM in barrier mucosal tissues is a crucial goal for vaccines and immunotherapeutic strategies.

The formation and maintenance of CD8 TRM in peripheral organs is critically dependent on local tissue-specific programming. These local environmental cues include unique tissue-derived cytokines and metabolic agents, as well as intercellular interactions that collectively enforce the tissue-specific TRM identity^7,8,9. This tissue programming allows TRM to adapt to tissue-specific demands in the execution of their immunosurveillance duties. However, our understanding of the nature of these interactions is incomplete, owing to the lack of robust model systems that faithfully capture these processes. Small animal models, while being very informative, often suffer from a highly interconnected, complex web of interactions that thwart rapid high-throughput studies.

Here, we describe an in vitro alternative to the mouse model that will allow investigation into the molecular details of epithelial cell-derived cues behind CD8 TRM differentiation. By co-culturing preformed vaginal epithelial organoids (VEOs) with activated CD8 T cells, we generated CD8 TRM in vitro. A detailed description of the CD8 T cell activation and co-culture process will allow wider adoption of this process across tissues, leading to a greater understanding of cellular interactions shaping CD8 T cell memory in frontline tissues.

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 Lymphocyte Enrichment Buffer in sterile dPBS by adding 2% charcoal-stripped FBS and 1 mM EDTA to sterile dPBS. Store at 4 °C until ready to use.
3. Prepare ammonium-chloride-potassium red blood cell (ACK RBC) lysis buffer in ddH₂O by adding 10 mM potassium bicarbonate, 155 mM ammonium chloride, and 0.1 mM EDTA. Filter sterilize and keep at room temperature.
4. Prepare the appropriate amount of harvest media in RPMI by adding 5% charcoal-stripped FBS. Store at 4 °C until ready to use. For each mouse to be harvested, 50 mL of harvest media is recommended.
   NOTE: Penicillin/streptomycin and amphotericin B are optional, but a final concentration of 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.2 µg/mL amphotericin B may help prevent bacterial and fungal contamination of T cell culture during tissue isolation if that presents an issue.
5. Prepare the T cell medium in RPMI by adding 10% charcoal-stripped FBS, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 1x non-essential amino acids, and 1 mM sodium pyruvate. For a 24-well plate, use 2 mL of T cell medium per well and 50 mL of T cell medium for a full plate. Store at 4 °C until ready to use.
6. Prepare dissection tools (0.4 mm micro dissecting scissors and 0.8 mm fine curved micro dissecting serrated forceps) by autoclaving in a sterilization pouch or soaking in 70% ethanol.

2. Harvesting and dissociation of adult mouse secondary lymphoid organs

1. Fast cool a centrifuge to 4 °C with appropriate adapters to hold 15 mL conical tubes.
2. Prepare ice for transporting tissues and lymphocytes during harvest and incubation periods.
3. Prepare a sterile 15 mL conical tube with at least 5 mL of harvest media per mouse to be dissected.
4. Anesthetize and euthanize an adult CD8 T cell transgenic mouse (~8 weeks old) according to institutional protocol in a laminar flow hood.
5. Pin the limbs of the mouse down onto a clean polystyrene foam surface using any size needles, and spray the torso with 70% ethanol to prevent fur from obstructing the incision and sticking to tools.
6. Using clean micro dissecting scissors and forceps, make a vertical abdominal incision and separate the skin from the thin layer of connective tissue underneath. Pin the skin onto the polystyrene foam surface using any size needles, and cut through any remaining connective tissue to expose the organs within the torso.
7. Carefully locate and excise the spleen, removing excess fat using the curved forceps, and transfer it into the 15 mL conical tube containing harvest media. Leave the spleen on ice and isolate as many lymph nodes as possible (e.g., iliac and inguinal) to combine with the spleen in the same conical tube on ice.
   NOTE: The iliac lymph nodes are located on either side of the caudal vena cava in the lower abdominal region underneath the gastrointestinal tract, and the inguinal lymph nodes are located near the branching of the superficial epigastric vein on either side of the torso's lower abdominal skin. Isolate more lymph nodes to improve the yield of CD8 T cells following enrichment; however, isolating the spleen alone is generally sufficient to provide several million CD8 T cells.
8. Transfer the spleen and lymph nodes to a scored 60 mm dish and, using the flat end of a 3 mL syringe plunger, mechanically disassociate the spleen and lymph nodes by grinding the tissue until no large fragments remain.
9. Collect the suspension by passing it through a sterile 70 µm cell strainer into a fresh sterile 15 mL conical tube. Wash the 60 mm dish with 5 mL of harvest media to ensure complete transfer of disaggregated cells and filter into the same 15 mL conical tube.
10. Centrifuge the suspension at 584 × g for 5 min at 4 °C. Discard the supernatant and rack the tube by running the bottom quickly and forcefully across the grid of a centrifuge tube holder to loosen the pellet.
11. Lyse erythrocytes by resuspending the loosened pellet in 2 mL of ACK RBC lysis buffer. Vortex and incubate at room temperature for 2 min.
12. After 2 min, neutralize the lysis buffer by adding 2 mL of harvest media. Aspirate the suspension using a 5 mL serological pipette and filter through a sterile 70 µm filter back into the same 15 mL conical tube.
    NOTE: This filtration is important to ensure the removal of all clumps.
13. Centrifuge the suspension at 584 × g for 5 min at 4 °C. Discard the supernatant and resuspend the pellet in 950 µL of Lymphocyte Enrichment Buffer.

3. CD8 T cell enrichment and activation

1. Proceed to enrich naïve CD8 T cells from lymphocytes using a commercially available isolation kit. Follow the manufacturer's protocol.
2. Once CD8 T cells have been enriched, transfer the enriched suspension to a new sterile 15 mL conical tube and fill the volume up to 10 mL with harvest media.
3. Count using a hemocytometer.
   NOTE: From one adult mouse, it is possible to isolate at least 5-10 million naïve CD8 T cells at >95% purity.
4. Plate at least 500,000 cells in 2 mL of T cell medium per well of a 24-well plate. Mix T cells thoroughly prior to plating to ensure a homogeneous single-cell suspension. Supplement the T cell medium with a final concentration of 100 units/mL IL-2, 2.5 ng/mL IL-12, and 55 µM beta-mercaptoethanol.
5. Remove the previously antibody-coated tissue culture plate from the 4 °C and carefully aspirate the coating solution without touching the bottom of the wells.
6. Immediately plate T cells. Incubate at 37 °C with 5% CO₂ for 2 days.

4. Removing activation signals and resting activated CD8 T cells

1. Observe activated wells under a light microscope. After 2 days of activation, look for small clusters of T cells throughout the wells. Larger clusters are indicative of more activation. If there are no or few clusters, incubate the plate at 37 °C with 5% CO₂ for 1 more day before proceeding with resting.
2. Prepare ice for storing T cell suspension during incubation periods and when not in use.
3. Resuspend T cells using a P1000 pipette by pipetting up and down several times in each corner of the well. When T cells have lifted, transfer the contents of the well to a sterile 50 mL conical tube and count.
   NOTE: Washing each well with 1 mL of dPBS is optional, but pooling dPBS wash with the T cell suspension may increase T cell yield.
4. Re-plate the T cells at the same concentration as in Step 3.4, 250,000 cells/mL at 2 mL per well. Prepare the appropriate amount of T cell medium supplemented with 100 units/mL IL-2 and 55 µM beta-mercaptoethanol.
5. Centrifuge the suspension at 584 × g for 5 min at 4 °C, discard supernatant, and resuspend the pellet in the appropriate volume of T cell medium prepared in the previous step for the number of wells to be plated. Mix thoroughly to ensure a homogeneous single-cell suspension.
6. Plate 2 mL of T cell suspension (500,000 cells/well) in a new non-treated tissue culture plate without antibody coating. Incubate at 37 °C with 5% CO₂ for 2 days.

5. Infecting preformed murine vaginal epithelial organoids with virus

NOTE: We have used both Herpes Simplex virus-2 (HSV-2) and Lymphocytic choriomeningitis virus (LCMV) to perform viral infection of the organoids. Both are considered Biosafety Level-2 (BSL-2) agents. Carry out all procedures involving infectious agents inside a certified BSL-2 biosafety cabinet. Clean work areas with 70% ethanol both prior to and following use, and discard all materials according to institutional biosafety guidelines. For this protocol, we established VEOs from vaginal epithelial cells harvested from the vaginal epithelium of C57BL/6 female mice as described before¹⁰.

1. Plate VEOs at a density of 10,000 cells in 20 µL of basement membrane extract (BME) per well of a 24-well plate 7-10 days prior to infection.
2. One day prior to co-culture, set aside one well of VEOs to be trypsinized and counted to obtain an accurate multiplicity of infection (MOI). Prewarm 0.25% Trypsin-EDTA in a 37 °C water bath for 5 min.
   NOTE: 0.25% Trypsin-EDTA will not be used during infection; it is only necessary for disassociating the VEOs during MOI calculations. Coating plasticware (i.e., pipette tips, conical tubes) with 2.5% BSA in dPBS is optional at this step but may help reduce organoid and cell loss for more accurate counting.
3. Aspirate cell culture media from the designated well and wash with dPBS.
4. Dispense 600 µL of prewarmed 0.25% Trypsin-EDTA into the well and resuspend thoroughly with a BSA-coated P1000 pipette to mechanically disrupt the BME. Transfer the contents of the well into a BSA-coated sterile 15 mL conical tube.
5. Incubate in a 37 °C water bath for 5 min. After 5 min, add 10 mL 10% FBS in RPMI to quench the trypsinization.
6. Centrifuge the suspension at 500 x g for 5 min at 4 °C. Discard the supernatant and resuspend using a BSA-coated pipette tip in 100-300 µL of T cell-organoid culture medium or plain DMEM/F12.
   NOTE: The volume depends on the size of the pellet. If the pellet is barely visible, resuspend in a lower volume.
7. Count using a hemocytometer. Calculate the appropriate amount of virus needed to achieve 0.1 MOI using the known viral titer and cell density per well of VEOs according to the following equation:
   
8. Aspirate cell culture media from the VEO wells to be infected and wash with 500 µL of prewarmed (37 °C) dPBS. Incubate the wells with dPBS for 5 min at 37 °C.
9. After 5 min, discard the dPBS and add 500 µL of organoid media per well containing enough viral particles to achieve a MOI of 0.1. Incubate at 37 °C with 5% CO₂ for 1 h with gentle manual swirling of the tissue culture plate every 20-30 min.
10. After the 1 h incubation, aspirate the viral organoid media and wash with 500 µL of prewarmed (37 °C) dPBS. Aspirate dPBS and replace with fresh organoid media.

6. Co-culturing CD8 T cells with virus-infected, preformed murine vaginal epithelial organoids

1. Prior to co-culture (ideally 4-24 h in advance), transfer the BME from the -80 °C to 4 °C for thawing. Anticipate the appropriate amount of BME to thaw based on the co-culture experimental design: plan to plate 8 μL of BME per well of a 96-well plate and 20 μL of BME per well of a 24-well plate.
   NOTE: BME is viscous, and a longer thawing period at 4 °C will reduce the viscosity and improve maneuverability.
2. Prepare 2.5% BSA in dPBS and filter sterilize. Use this BSA solution to coat every plastic surface that comes into contact with the VEOs (i.e., pipette tips, conical tubes).
   NOTE: This coating will prevent VEOs from sticking to the plastic surface and being lost during handling.
3. Check VEOs under a light microscope for size and overall health. Confirm that VEOs are 7-10 days post culture and on average ~100 microns in diameter. Healthy VEOs will present few or no single cells and feature a uniform, round morphology.
   NOTE: Replate VEOs 1:1 in co-culture if grown in a 24-well plate and replating in a 24-well plate (i.e., one well of VEOs will reseed one well of T cell-organoid co-culture). If replating in a 96-well plate from a 24-well plate, replate VEOs 1:2 (i.e., one well of VEOs will reseed two wells of T cell-organoid co-culture).
4. Place the necessary number of sterile 96- or 24-well tissue culture plates to be used for the co-culture in the 37 °C incubator.
   NOTE: A warmed plate will expedite BME adhesion and solidification.
5. Prepare T cell-organoid culture medium in DMEM/F12 by adding 2% B-27 supplement, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 1x Non-essential amino acids, 1 mM sodium pyruvate, and 0.2 µg/mL amphotericin B. Store in 4 °C until ready to use.
6. Aspirate cell culture media from VEO wells and wash each well with 500 µL of cold (2-8 °C) sterile dPBS.
7. Aspirate dPBS and add 200-500 µL of cold (2-8 °C) organoid harvesting solution (at least 10x volume of organoid BME droplet). Pipette up and down with a BSA-coated P200 pipette to mechanically disrupt the BME.
8. Incubate plate with gentle shaking (~100 rpm) on an orbital shaker at 4 °C for 20 min. If a cold orbital shaker is unavailable, incubate the plate on ice with manual shaking every 5 min. If BME has not depolymerized after 20 min, lengthen the incubation time as necessary.
9. Transfer the contents of the wells using a BSA-coated pipette tip into a BSA-coated sterile 15 mL or 50 mL conical tube on ice. Centrifuge the suspension at 500 × g for 5 min at 4 °C.
10. Discard the supernatant and wash VEOs with 5-10 mL of cold (2-8 °C) dPBS (at least 10x volume of combined organoid BME droplets). Centrifuge the suspension at 500 × g for 5 min at 4 °C.
11. Discard the dPBS and resuspend the pellet in 1 mL of T cell-organoid culture medium using a BSA-coated P1000 pipette tip. Leave the VEO suspension on ice until T cells are ready to be co-cultured.
12. Begin collecting the T cells for co-culture. Check resting T cells under a light microscope for size and overall health. Without activating signals, T cells will slow their proliferation but should still have formed some clusters. Confirm that there are no cell debris or fragments, and T cells have an enlarged morphology. Resuspend T cells using a P1000 pipette by pipetting up and down several times in each corner of the well and count the total number of live CD8 T cells.
    NOTE: Per well of a 96-well plate, seed 100,000 T cells for each co-culture in 8 µL of BME. Per well of a 24-well plate, seed 200,000 T cells for each co-culture in 20 µL of BME.
13. Centrifuge the suspension at 584 × g for 5 min at 4 °C. Discard the supernatant and resuspend the pellet in 10 mL of T cell-organoid culture medium. Combine T cells with VEOs by transferring an appropriate number of T cells for the wells to be plated into the conical tube containing the VEOs.
14. Centrifuge at 584 × g for 5 min at 4 °C. Discard the supernatant and resuspend VEOs and T cells in the appropriate amount of BME. Mix thoroughly using a BSA-coated pipette tip, and plate onto prewarmed tissue culture plate, 20 µL per well of a 24-well plate or 8 µL per well of a 96-well plate.
    NOTE: Keep BME on ice or in a cooling block at 4 °C during plating. Placing pipette tips in the 4 °C fridge for 30 min prior to plating may help prevent premature solidification of the BME.
15. Place the plate upside down in the 37 °C incubator and wait 30 min for complete BME adhesion and solidification.
16. During this incubation period, prepare the complete T cell-organoid culture medium by adding 100 ng/mL murine EGF, 55 µM beta-mercaptoethanol, and 10 units/mL murine IL-2 to the premade T cell-organoid culture medium.
17. After the incubation period, carefully add 500 µL of complete T cell-organoid culture medium per well of a 24-well plate or 250 µL of complete T cell-organoid culture medium per well of a 96-well plate. Dispense media against the sides of the well to avoid disrupting the BME droplet. Change the media every 2 days until harvested for analysis.

7. Collecting co-cultured CD8 T cells for flow cytometry analysis

1. Once co-cultures have reached at least day 7 in culture, harvest for analysis. If T cells do not express TRM-like markers in co-culture by day 7, optimize the harvest date between day 7-14, depending on phenotypic acquisition and VEO and T cell viability.
2. Prepare fluorescent activated cell sorting (FACS) buffer for flow cytometry staining and analysis by adding 0.2% w/v bovine serum albumin and 0.1% w/v sodium azide to dPBS.
   NOTE: Sodium azide is an acutely toxic chemical. Follow institutional recommendations in handling and disposal of all waste in compliance with institutional biosafety regulations.
3. Prepare a fluorescent antibody cocktail in FACS buffer for surface staining, depending on which markers are desired for analysis. For each well, account for at least 50 µL of antibody cocktail.
4. Prepare a fluorescent viability dye in dPBS. For each well, account for at least 50 µL of viability dye.
5. Carefully remove the media in each well and thoroughly resuspend in cold (2-8 °C) FACS buffer, pipetting up and down 10-20x to mechanically disrupt the BME. Transfer the contents of each well to a labelled 96-well round-bottom plate.
6. Centrifuge at 859 × g for 2 min at 4 °C. Lower the deceleration ramp for all centrifugation steps onward to prevent sample loss from pellet remixing.
7. Remove the supernatant by inverting the plate and expelling the liquid with a single, firm downward motion. Resuspend in 50 µL of antibody cocktail. Pipette up and down at least 5x using a multichannel pipette to ensure thorough resuspension of the cell pellet.
8. Incubate for 30 min at 4 °C or room temperature as necessary, protected from light. At the end of the incubation period, add 150 µL of dPBS to each well using a multichannel pipette and centrifuge at 859 × g for 2 min at 4 °C.
9. Decant the supernatant by turning the plate upside down and flicking it downward once in a continuous motion. Resuspend in 50 µL of viability dye. Pipette up and down at least 5x using a multichannel pipette to ensure thorough resuspension of the cell pellet.
10. Incubate for 15 min at room temperature, protected from light. At the end of the incubation period, add 150 µL of FACS buffer to each well using a multichannel pipette and centrifuge at 859 × g for 2 min at 4 °C.
11. Decant the supernatant as before, and resuspend in 150 µL of FACS buffer if samples will be run on a flow cytometer immediately or fix in 150 µL of 0.5% paraformaldehyde and leave at 4 °C. Pipette up and down at least 5x using a multichannel pipette to ensure thorough resuspension of the cell pellet. Filter fixed samples to prevent instrument clogging and run on a flow cytometer within 12-36 h to prevent loss of fluorescence.
    NOTE: Paraformaldehyde is considered a hazardous chemical. Follow institutional biosafety regulations in handling and disposal.

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