Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Summary

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Abstract

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Introduction

Allogeneic hematopoietic stem cell transplant (HSCT) has become routine treatment for patients suffering from hematological malignancies such as leukemia with poor prognosis. A significant complication of HSCT is acute graft-versus-host disease (GVHD). A 2012 study reported that acute GVHD developed in 39% of HSCT patients receiving transplants from sibling donors and 59% of patients receiving transplants from unrelated donors¹. Acute GVHD occurs when donor-derived T cells attack recipient’s organs. The only successful therapy for GVHD is treatment with highly immunosuppressive drugs², which are highly toxic and in....

Protocol

All mouse experiments were performed in compliance, and with approval from, the University of Kansas Medical Center Institutional Animal Care and Use Committee. All healthy human blood samples were obtained under informed consent and with approval from the Institutional Review Board at the University of Kansas Medical Center.

1. Irradiation of NSG Mice

1. One day prior to PBMC injection, irradiate 8–12-week old NSG mice (either sex can be used). In a sterile biosafety cabinet, place mice in a sterilized pie cage or microisolator. Irradiate mice in a Cs¹³⁷ source or small animal irradiator (e.g., RS 2000) with a tot....

Results

Sublethally irradiated 8-12-week old NSG mice of both sexes that received human PBMC started displaying clinical signs of GVHD around day 10 post injection compared to negative control mice that received PBS only (Figure 1A). XenoGVHD mice had a median survival of 23.5 days (Figure 1B). With digital PCR, CD3 epsilon positive human T cells could be detected in the lung and liver samples of mice that received human PBMC. Tissue sam.......

Discussion

Disease progression is generally consistent in the xenoGVHD model, even with injection of PBMC from different donors, so multiple experiments can be combined. The key steps required to maintain this consistency are proper i.v. injection technique, blinding and consistent scoring. A study by Nervi et al.²⁵ demonstrated that compared to intravenous tail vein injection, retro-orbital injections of PBMC resulted in more consistent engraftment and more severe GVHD. Leon-Rico et al.^26<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL eppendorf tubes	Fisher	05-408-129
10 mL serological pipet	VWR International	89130-898
10mL BD Vacutainers - Green capped with Sodium Heparin	Becton Dickinson	366480
250 µL Ranin pipette tips	Rainin	17001118	Do not use other pipettes or pipet tips for droplet generation
50 mL conical tube	VWR International	89039-656
96-Well ddPCR plate	Bio-Rad	12001925
ACK (Ammonium-Chloride-Potassium) Lysing Buffer	Lonza	10-548E	Optional
Alcohol Wipes	Fisher Scientific	6818
Anesthesia Chamber	World Precision Instruments	EZ-178	Provided by animal facility
Anesthesia Machine	Parkland Scientific	PM1002	Provided by animal facility
BD Vacutainer Safety-Lok Blood Collection Set	Becton Dickinson	367281
DG8 Cartridges and Gaskets for QX100/QX200 Droplet Generator	Bio-Rad	1864007
DNAse and RNAse free Molecular Grade H2O	Life Technologies	1811318
Ethyl alcohol, Pure,200 proof, for molecular biology	Sigma-Aldrich	E7023-500ML
Fetal Bovine Serum	Atlanta Biologicals	S11150
Ficoll	Fisher Scientific	45001750
Insulin Syringe	Fisher Scientific	329424
Isoflurane	Sigma-Aldrich	CDS019936	Provided by animal facility
Liquid nitrogen	N/A	N/A
Mouse Irradiator Pie Cage	Braintree Scientific, Inc.	MPC 1	Holds up to 11 mice
Nexcare Gentle Paper Tape (a.k.a. 3M Micropore Surgical Tape / 3/4")	Fisher Scientific	19-027-761
P1000 pipetman	MidSci	A-1000
P200 pipetman	MidSci	A-200
Pierceable Foil Heat Seal	Bio-Rad	1814040
Pipetaid Gilson Macroman	Fisher Scientific	F110756
Pipet-Lite Multi Pipette L8-200XLS+	Rainin	17013805	Do not use other pipettes or pipet tips for droplet generation
Qiagen DNeasy Blood and Tissue Kit	Qiagen	69506
qPCR plates	VWR International	89218-292
QX200 Droplet Digital PCR System	Bio-Rad	12001925	Includes droplet generator, droplet reader, laptop computer, software, associated component consumables, for EvaGreen or probe-based digital PCR applications
QX200 Droplet Generation Oil for EvaGreen	Bio-Rad	1864006
QX200 ddPCR EvaGreen Supermix	Bio-Rad	1864033
RNase and DNase-free plate seal	Thermo Scientific	12565491
RPMI Advanced 1640	Life Technologies	12633012
Sterile Gauze Pads (2" x 2", 12-Ply)	Fisher Scientific	67522
Sterile Phosphate Buffered Saline	Fisher Scientific	21040CV
Sterile reservoir	VWR International	89094-662
Surgial Scissors	Kent Scientific	INS600393-4
Surgical Forceps	Kent Scientific	INS650914-4