Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Summary

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Abstract

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo.

Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity).

Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.¹ developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant.

Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Protocol

1. Lethal Irradiation

1. Place up to 10 recipient mice into a microisolator cage compatible with the irradiator to be used.
2. Irradiate in 2 equal doses summing total dose (total dose = 9 cGy for BALB.B recipients). Second irradiation should be 3 hr after first. Injection should occur between 4-6 hr after final irradiation. Irradiate mice in either Cs¹³⁷ source or RS2,000, irradiator.
3. Following the second radiation dose, store mice in microisolator cage with acidified water until the time of transplant.

2. Splenocyte Preparation

1. Euthanize one donor mouse according to institutional gui....

Discussion

The protocol for inducing GVHD in mice presented here represents a clinically relevant model of murine GVHD. Originally established by Berger et al. in 1994, the C57Bl/6 into BALB.B strain combination is MHC-matched, with GVHD mortality mediated by CD4 dependent, CD8 T effectors², highly similar to the most common clinical scenario³. It is known that transplanting CD8 T cells alone does not cause GVHD in this model; however, disease progression is significantly worse when both CD4 and CD8 T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Invitrogen	12633-012
Fetal Calf Serum	Invitrogen	10439016
40 μM Cell Strainer	BD Biosciences	352340
CD3e-Biotin	Miltenyi Biotec	130-093-021
Anti-Biotin Microbeads	Miltenyi Biotec	130-091-147
CD8a Microbeads	Miltenyi Biotec	130-049-401	Used to deplete CD8 T cells from spleen.
CD8a Purification Antibody Cocktail	Miltenyi Biotec	130-095-236	Used to purify CD8 T cells from spleen.
D-Luciferin	Caliper Life Sciences	122796