GvHD staging, grading, pathophysiology, and novel targets – EBMT webinar summary

This article is a summary of the European Society for Blood and Marrow Transplantation (EBMT) webinar entitled ‘Pathophysiology of acute and chronic GvHD, novel therapeutic strategies in the preclinical phase’ delivered by Ernst Holler on May 5^th, 2020.

Allogeneic transplantation and GvHD

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) involves the infusion of stem cells following administration of high-dose chemotherapy or total body irradiation for either tumor control or to prevent solid transplant rejection. The graft usually comes from a human leukocyte antigen (HLA)-matched donor and contains hematopoietic cells as well as immune cells, including T lymphocytes. Allo-HSCT is an important treatment strategy, which allows administration of myeloablative therapy and the elimination of cancer cells by the transplanted donor T and natural killer (NK) cells in a phenomenon known as the graft-versus-leukemia (GvL) effect. The restoration of hematopoiesis together with the donor’s immune system can lead to a major immune reaction, graft-versus-host disease (GvHD).

Classification of GvHD

After engraftment, patients are at risk of developing acute (a)GvHD or chronic (c)GvHD, depending on the timing of the onset:

• aGvHD
  • Classical GvHD – developed < 100 days from transplantation
  • Late onset – developed > 100 days post-transplant with no prior aGvHD
  • Recurrent – developed > 100 days post-transplant after prior aGvHD
  • Persistent – developed >100 days post-transplant after prior active aGvHD
• cGvHD developed > 100 days
  • De novo with no prior aGvHD
  • Quiescent after prior aGvHD
  • Progressive after prior active aGvHD

In contrast to aGvHD, which mainly affects the skin, gastrointestinal (GI) tract, and liver, cGvHD involves multiple organs around the body. The most commonly affected organs in GvHD are presented in Table 1. In particular, the changes are seen in the epithelial tissue exposed to the environment and/or microbiota with strong regulation of inflammation.

Table 1. Organs affected by acute and chronic GvHD

Classification of GvHD according to the joint guidelines from EBMT, the National Institutes of Health (NIH) and the Center for International Blood and Marrow Transplant Research (CIBMTR):¹

A. aGvHD (classic or delayed onset) with manifestations limited to:

• Skin – inflammatory maculopapular erythematous skin rash
• Liver – elevated bilirubin
• GI tract – anorexia with weight loss, nausea, vomiting, diarrhea, severe pain, GI bleeding and/or ileus

B. cGvHD (classic) manifestations meeting the NIH 2014 diagnostic criteria, localized in:

• Skin, nails, scalp, and body hair
• Mouth
• Eyes
• Esophagus
• Lungs
• Muscles, joints, and fascia
• Genitalia

C. Undefined other cGvHD:

• Atypical signs and symptoms of alloreactivity falling outside the NIH 2014 diagnostic criteria

D. Overlap cGvHD (encompassing manifestations of aGvHD and cGvHD)

Ernst Holler also recommended the use of the eGvHD application, an electronic tool that helps clinicians classify GvHD based on the NIH-EBMT-CIBMTR criteria.

Staging and grading of acute and chronic GvHD

Simple clinical and laboratory parameters are used for the staging and grading of aGvHD, while cGvHD requires a more comprehensive assessment of different organs. The staging and grading of aGvHD are presented in Table 2 and Table 3, respectively, while cGvHD grading is outlined in Table 4.

Table 2. Staging of aGvHD according to the EBMT 2019 criteria²

Table 3. The overall grading of aGvHD according to the EBMT 2019 criteria²

The staging of cGvHD requires a comprehensive assessment of multiple organs. The current staging is based on the NIH scoring system, which takes into account the condition of the skin, mouth, eyes, GI tract, liver, lungs, and genitals.³ The grade is based on the severity of organ involvement, as well as the number of affected organs (Table 4).

Table 4. Grading of cGvHD according to the NIH criteria³

However, despite the guidelines, pitfalls in GvHD staging and grading remain, including:

• Initial uncertainty of diagnosis pending biopsy results
• Failure to fix GvHD stages and grades in real-time based on confidence levels:
  • A retrospective classification has shown up to 30% of false-positive classifications in clinical trials
• Poorly defined manifestations, e.g. chronic GI GvHD beyond esophageal web

Pathophysiology of GvHD

GvHD may develop after allo-HSCT transplantation, transfusions or organ transplant. The conditions required for GvHD:

• The graft must contain immunocompetent cells
• The hostsmust be expressing major and minor antigens that are absent in the donor
• Because of the immunosuppression, the host must be incapable of rejecting donor cells

aGvHD

aGvHD is driven by the recipient’s antigen-presenting cells (APC) expressing major and minor histocompatibility complexes (MHC and MiHA) presenting antigens to the T-cell receptor on donor CD8/CD4 cells. Since peripheral stem cell transplantation (PBMC) grafts contain higher levels of donor T-cells, the risk of developing GvHD, especially cGvHD, is higher with PBMC relative to bone marrow (BM).

Host APC activation is initiated by conditioning-mediated damage to epithelial cells. The damage results in release of damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) from the translocation of commensal pathogens, that in turn lead to the chemotaxis and activation of immune cells and the subsequent production of proinflammatory cytokines including tumor necrosis factor α (TNFα) and interleukin (IL)-1, resulting in further amplification of tissue damage and antigen presentation to donor T-cells. Antigen presentation leads to T cell priming and further tissue damage. The immune responses of cellular and inflammatory effector cells are influenced by proinflammatory signals and interferon (INF)-γ produced by NK and T helper (Th) 1 cells in the microenvironment.

In contrast, the T regulatory cells (Tregs), resident in tissues and found circulating, promote tolerance of antigens and therefore dampen the inflammatory response. In vivo studies have demonstrated that Tregs are able to abrogate GvHD while maintaining the GvL effect.⁴ Other regulatory signals present on host tissues, APCs, and donor cells, include the programmed death-ligand 1 (PD-L1) and other immune checkpoint inhibitors, inhibitors of apoptosis, and Siglec-G.^5,6

aGvHD prophylaxis and treatment require not only elimination/suppression of alloreactive cells but also restoration of immune and tissue tolerance. Based on that idea, a number of different therapeutic approaches targeting those important processes are being investigated (Table 5). GvHD prophylaxis and treatment have been previously covered in our previous editorial articles.

Table 5. Some of the therapeutic approaches for aGvHD prophylaxis and treatment

cGvHD

aGvHD is a risk factor for cGvHD and is caused by continuous alloreactivity. Another important risk factor for developing cGvHD is age ≥ 50 years. The increased risk with age can be explained by the defective thymic selection of newly generated donor T cells.

In cGvHD, there is a switch from host APCs to donor APCs presenting antigens to donor T-cells. In contrast to aGvHD, where Th1 cells play a major role, in cGvHD the alloreactive Th2 cells stimulate B cells to produce allo- and autoreactive antibodies. Those antibodies are then responsible for excess cytokine production. Therefore, B cells play a more prominent role in cGvHD compared to aGvHD. Another important factor in cGvHD is thymic damage, which contributes to the selection of dysfunctional newly formed T cells, resulting in autoreactive donor T cells stimulating B cells.

The transition process from aGvHD to cGvHD can be divided into three phases:⁷

1. Acute inflammation and tissue injury, where innate immunity plays a key role and involves:

• Cytokines
• Toll-like receptor (TLR) agonists
• Neutrophils
• Platelets
• Vascular inflammation

2. Chronic inflammation and dysregulated immunity, where adaptive immunity plays a central role, with key players including:

• Thymic injury and dysfunction
• T cells
• B cells
• NK cells
• APCs
• Regulatory cells
  • Tregs
  • Bregs

3. Aberrant tissue repair and fibrosis, where both innate and adaptive immunity play role, with the contributing molecules and cells including:

• Transforming growth factor β (TGFβ)
• Platelet-derived growth factor α (PDGFα)
• TNFα
• IL-17
• Macrophages
• Fibroblasts

Over the last decade, therapies targeting B cell dysfunction, such as rituximab and ibrutinib, started to play a more prominent role among cGvHD treatment options. However, cGvHD treatment needs improvement as the current rate of both leukemia- and GvHD-free patients is only 20-25%. New treatment options for cGvHD include:

• Inhibition of activated T cells using:
  • Proteasome inhibitors
  • Spleen tyrosine kinase (SYK) inhibitors
  • Metabolic inhibitors e.g. inositol kinase B
• Restoring immunoregulation
  • Adoptive Treg transfer
  • Low dose IL-2
  • Abatacept

Future directions

Role of the microbiota in GvHD

The microbiome plays an important role in GvHD. Patients with diverse microbiota have significantly better short- and long-term outcomes after transplantation compared to patients with lower microbiota diversity.^8,9 This loss of diversity is often caused by the use of broad-spectrum antibiotics early after allo-HSCT. Another factor disrupting the microbiota is diet. Choline-rich diet was shown to result in metabolite aggravating GvHD. Similarly, lactose-rich diet that leads to enterococcal expansion has been recently shown to promote GvHD. Since bacterial metabolites derived from commensal species have immunomodulatory properties, it is not surprising that changes in microbiota diversity can affect GvHD.

Additionally, GvHD leads to gut crypt destruction resulting in loss of  Paneth cells releasing anti-microbial peptides, and decreasing microbiota diversity.¹⁰ Initial studies showed a potential role of fecal microbiota transplantation in restoring tissue tolerance as well as treatment and prophylaxis of GvHD.^11,12 However, such an approach can result in severe infections.

In order to fully understand the role of microbiota damage in GvHD further studies are needed to explore:

• The mechanisms involved, including whether the effect is bacterial strain specific
• The modulation of fecal microbiota transplantation, including defining patients more likely to benefit and whether to transplant a broad range of fecal species or only selected ones
• The optimization of antibiotic regimens to cause the least possible damage to the microbiota

Risk adapted early/pre-emptive treatment

Stratification of patients by risk of developing severe GvHD is also an important aspect, as it would allow intensified treatment in patients with the highest risk and thus reduce tissue damage. Conversely, it could allow treatment de-escalation in those with the lowest risk. Therefore, biomarkers predicting GvHD outcomes at the onset of disease are crucial. The most promising candidates include suppression of tumorigenicity 2 (ST2) and regenerating islet-derived 3α (Reg3α).^13,14 The cGvHD disease is more complicated and therefore, the development of predictive biomarkers is more complex.¹⁵ Although there are some potential candidates they need to be validated in larger multicenter studies.

Conclusion

Both aGvHD and cGvHD are characterized by loss of immunological and tissue tolerance. Effective treatment will need to address all key processes, including T cell suppression, restoration of immunoregulation, and tissue repair. Microbiota diversity is important in the interaction of immune and epithelial cells. Biomarkers could help in patient risk stratification and adapting treatment by disease risk, which in turn would improve GvHD outcome.