Graft vs Host Disease

Graft-versus-host disease (GVHD) is defined as a syndrome in which cells originating from a donor recognize and mount a deleterious immune response against antigens found in the immunocompromised recipient. The disease presents as a heterogeneous condition involving multiple organs, most commonly the skin, mucosa, gastrointestinal tract, liver and lungs. For patients undergoing allogeneic hematopoietic stem cell transplant (HSCT), GVHD is a significant cause of morbidity and mortality; up to 80% of patients will develop GVHD at approximately 14 -21 days following HSCT (acute), and of those surviving beyond 100 days, between 30 and 70% will develop chronic GVHD. Unsurprisingly, chronic GVHD represents the single leading cause of death in HSCT survivors.

Development of GVHD is dependent on the activation of antigen-specific donor T cells that are able to mount a specific immune response targeting host tissues. This event is potentiated by conditioning of the host prior to HSCT in order to suppress the immune system and ensure effective engraftment.

The focus of current therapy is general immune suppression using corticosteroids, which has dubious efficacy; sustained amelioration of disease symptoms are only seen in around 50% of patients. More promising therapies are based on the prophylactic prevention of GVHD, and use T cell targeting agents such as methotrexate, tacrolimus, and JAK/STAT inhibitors.

Allogeneic Bone Marrow Transplant

Recipient mice are pre-conditioned with a lethal dose of total body irradiation prior to the adoptive transfer of T-cell depleted bone marrow supplemented with splenocytes harvested from an MHC-mismatched donor mouse. Following transplant, the mice rapidly lose weight due to the conditioning regimen and then partially recover by day 14, after which they develop a progressively worsening disease characterized by weight loss and increasing GVHD score. Endpoint survival in untreated mice is approximately 50%.

Study Design Table

Model Description Duration Endpoints
Allogeneic Bone Marrow Transplant GVHD reaction is assessed using a 5-criteria score. Initial recovery from conditioning regimen is followed by progressive exacerbation of disease from day 14 onwards 5-8 Weeks GVHD score, Weight, Survival, Histology

 

Recipient mice pre-conditioned and transplanted with 10,000,000 donor-derived CD3-depleted bone marrow cells supplemented with different numbers of splenocytes(see legend) on day 0. Weights were recorded daily. Percent weight change relative to day 0 was calculated. Statistical evaluation by one-way ANOVA with Holm-Šidák’s multiple comparison post-test. **: p<0.01 comparing indicated groups; #: p<0.05 comparing indicated groups to ‘no splenocytes’ control. AUC: area under the curve; n=15 per group; data i
GVHD Scoring Scale for Disease Evaluation
Recipient mice were pre-conditioned and transplanted with 10,000,000 donor-derived CD3-depleted bone marrow cells supplemented with different numbers of splenocytes (see legend) on day 0. GVHD score was recorded daily. Statistical evaluation by one-way ANOVA with Holm-Šidák’s multiple comparison post-test. *: p<0.05 comparing indicated groups; #: p<0.05 comparing indicated groups to  ‘no splenocytes’ control. AUC: area under the curve; n=15 per group; data is presented as mean ±SEM.
Recipient mice were pre-conditioned and transplanted with 10,000,000 donor-derived CD3-depleted bone marrow cells supplemented with different numbers of splenocytes (see legend) on day 0. Survival was recorded daily. Statistical evaluation by Mantel-Cox test. *: p<0.05 compared to ‘no splenocytes’ control; #: p<0.05 compared to 2,000,000 splenocytes’ group. n=15 per group.
Endoscopy was carried out on a weekly basis from day 0 to day 28. At sacrifice, the colon and jejunum were excised, flushed, weighed, and measured. Statistical evaluation by two-way ANOVA with Holm-Šidák’s multiple comparison post-test (endoscopy scores), or Student’s t-test (diarrhea incidence, colon/jejunum weight:length ratio). *: p<0.05; **: p<0.01; ***: p<0.005; ****: p<0.001 comparing ‘no splenocytes’ and 2,000,000 splenocytes’ groups. n=15 per group; data is presented as mean ±SEM
H&E stained oral mucosa sections from Naïve, Early Disease (14 days after cell transplant), and Severe Disease animals (33-40 days after cell transplant)  were evaluated for lymphocytic infiltration in buccal and lingual mucosa.  Lesions were given a severity score 0-5 (0=not present/normal, 1=minimal, 2=mild, 3=moderate, 4=marked, 5=severe). Black arrows indicate lymphocytic infiltration.  Black arrowheads indicate apoptotic bodies.  OC – oral cavity. SKM – skeletal muscle. M – mucosa
Human PBMC Xenograft Model

Recipient severely immunocompromised (NSG) mice are injected with a pre-determined number of purified single-donor human PBMCs. Following transplant, the mice develop a progressively worsening disease characterized by weight loss and increasing GVHD score. Endpoint survival in untreated mice is determined by the number of cells injected. Cryopreserved PBMCs from identified donors are available for maximizing continuity across multiple studies.

Study Design Table

Model Description Duration Endpoints
Human PBMC Xenograft GVHD reaction is assessed using a 5-criteria score. Progressive exacerbation of disease is seen from day 14 onwards 5-8 Weeks GVHD score, Weight, Survival, Histology

 

Recipient mice were injected with a pre-determined number of purified single-donor human PBMCs (see legend) on day 0. Weights were recorded daily and the percent weight change relative to day 0 was calculated. Statistical evaluation by one-way ANOVA with Holm-Šidák’s multiple comparison post-test. *: p<0.005 comparing indicated groups. AUC: area under the curve; n=15 per group; data is presented as mean ±SEM.
Recipient mice were injected with a pre-determined number of purified single-donor human PBMCs (see legend) on day 0. GVHD score was recorded daily. Statistical evaluation by one-way ANOVA with Holm-Šidák’s multiple comparison post-test. *: p<0.01 comparing indicated groups. AUC: area under the curve; n=15 per group; data is presented as mean ±SEM.
Recipient mice were injected with a pre-determined number of purified single-donor human PBMCs (see legend) on day 0. Survival was recorded daily. Statistical evaluation by Mantel-Cox test. *: p<0.05 comparing indicated groups. n=15 per group.
Chronic Sclerodermatous Model

Recipient C57Bl/6 mice are pre-conditioned with high dose of total body irradiation prior to the adoptive transfer of bone marrow supplemented with splenocytes harvested from an MHC-mismatched donor mouse (LP/J). Following irradiation and transplant, the recipient mice show transient mild weight loss followed by recovery up to approximately day 21 to 28, after which they develop a progressively worsening disease characterized by weight loss, increasing GVHD score, and development of sclerodermatous skin lesions. Animals are monitored up to day 60; endpoint survival in untreated mice is approximately 50%.

Study Design Table

Model Description Duration Endpoints
Allogeneic Bone Marrow Transplant GVHD reaction is assessed using a modified 5-criteria score. Initial recovery from conditioning regimen is followed by progressive exacerbation of disease from day 21-28 onwards 9-10 Weeks GVHD score, Weight, Survival, Histology

 

Timeline schematic showing induction schedule and expected onset of disease. Daily measurements include weight change and GVHD score; endpoint collections include blood, broncho-alveolar lavage, and histopathological analysis of selected tissues (e.g. skin, lungs).
Representative photographs showing the development of sclerodermatous skin lesions over time. Animals are briefly anaesthetized with isoflurane during photography; the animal shown here was photographed immediately after sacrifice on day 45.
Graft vs Leukemia (GVL)

While a HSCT can result in the life-threatening complication of GVHD, it can also provide a beneficial effect to the host known as the Graft versus Leukemia (GVL) effect in which the T-cells in the donor cell population target and kill malignant cells in the host.  This phenomenon can be demonstrated in the allogeneic bone marrow transplant model of GVHD when tumor cells are injected shortly after cell transplant.  When T cells are present in the cell transplant, tumors fail to grow.  Tumor cells will only grow in this model when T-cells are depleted from the cell transplant.  This allows clients to assess whether treatments in this GVHD model have any effect on the GVL phenomenon.   

Study Design Table

Model Description Duration Endpoints
Graft versus Leukemia  Following induction of GVHD by allogeneic cell transplant, tumor cells are implanted, and their growth is observed.   4-8 Weeks Tumor size, tumor bioluminescence, GVHD score, weight, survival, histology

 

Subcutaneous A20 Tumor Growth:  B-cell lymphoma cell line A20 cells form tumors when injected subcutaneously. A20 cells or luciferase-expressing A20 cells (Luc-A20) were injected at either 10,000; 100,000; or 1,000,000  cells into host mice that had received a T-cell depleted bone marrow transplant.  IVIS imaging of tumors show dose dependent increases in tumor bioluminescence.   Tumors fail to grow in host mice that received a bone marrow transplant containing T cells (data not shown).
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