HC27+28: Allogenic stem cell transplantation and donor lymphocyte infusion I&II

What is hematopoietic stem cell transplantation?

Hematopoietic stem cell transplantation (SCT) is performed because:

• It is a rescue procedure after a high dose of chemotherapy or irradiation to ensure hematopoiesis
• It is a way to reset the immune system
• It replaces non- or dysfunctional bone marrow
• It is an allogeneic cellular immune therapy for malignant disorders

Disorders treated with autologous SCT are lymphoma and multiple myelomas. SCT is a form of cellular immunotherapy.

Harvesting a stem cell graft

A stem cell graft is harvested as follows:

1. Filgrastim (neupogen) is injected subcutaneously into the patient
2. G-CSF receptors are activated → cells migrate out of the bone marrow into the circulation
3. A certain machine removes the cells which the patient doesn’t need, and returns the other cells back to the patient

Autologous stem cell transplantation

Autologous SCT is performed as follows:

1. The stem cell graft is harvested from the patient
2. Cells are cryopreserved in liquid nitrogen
3. The patient is treated with high dose chemotherapy → results in ablation of the stem cells from the bone marrow
4. The stem cell product is reinfused
   • All stem cell harvests contain a mixture of leukocytes: stem cells, T-cells, B-cells, NK-cells, monocytes and granulocytes

The actual treatment is the chemotherapy → stem cell transplantation only is a rescue procedure.

Allogeneic hematopoietic SCT

The difference between allogeneic and autologous SCT is that in allogeneic SCT the graft of bone marrow cells is taken out of a healthy donor instead of the patient.

Procedure:

Allogeneic SCT is performed as follows:

1. A stem cell graft is harvested from the donor
   • Using G-CSF treatment
2. The patient’s immune system and leukemia cells are destroyed
   • Immune ablation of the patient is necessary to allow acceptance of donor cells → a stem cell graft is highly immunogenic
3. Chemotherapy is used to make space for donor stem cells in the bone marrow
4. Sometimes the stem cell product is modified → is optional
5. A donor stem cell collection is infused into the patient, who doesn’t have an immune system
   • This can include donor T-lymphocytes
6. The immune system is rebuilt with donor cells → the patient has a new hematopoietic system

Appliances:

Allogeneic SCT is used to treat:

• Genetic disorders or failure of hematopoiesis or lymphopoiesis
• Acquired stem cell failure syndromes
• Hematological malignancies
  • Autologous SCT cannot be performed because autologous stem cells harbor the mutation/abnormality of the original stem cell disease

Rejection:

Allogeneic hematopoietic SCT is a very complex procedure because it messes with 2 different immune systems:

• T-cells from the patient can reject the stem cells → host versus graft disease
• T-cells in the graft coming from the donor can react with various cells from the patient → graft versus host disease

There are 2 ways to prevent graft rejection:

• Suppression of T-cells from the patient via chemotherapy or irradiation
• Removal of T-cells from the patient via treatment of antibodies against T-cells
  • Anti-T-cell globulin (ATG)
  • Alemtuzumab (anti-CD52)
    • Anti-CD52 is present on all lymphocytes

T-cells from the graft can induce graft versus host disease (GVHD), which is unwanted, or graft versus leukemia/lymphoma (GVL), which is the aim:

• T-cell depletion of the graft reduces the incidence and severity of GVHD
• T-cell depletion of the graft increases the frequency of recurrence of the malignancy after SCT

Donor T-cells can mediate a potent desired graft versus leukemia/lymphoma (GVL) effect. They mediate GVHD and GVL disease. The balance between GVL and GVHD is a major factor in the outcome of allogeneic SCT.

GVHD develops as follows:

1. After transplantation, normal hematopoiesis and lymphopoiesis in the patient are derived from donor stem cells
2. Donor T-cells which are newly developing in the patient consider the tissues from the patient as “self” → tolerance
3. Donor T-cells present in the graft and which are educated in the donor may recognize the tissues from the patient as foreign → GVHD

GVL is the desired therapeutic effect and develops as follows:

1. Donor T-cells developing in the patient from donor stem cells consider the hematopoietic stem cells from the patient, including the malignancy, as “self” → relapse
2. Donor T-cells educated in the donor recognize hematopoietic tissues from the patient, including malignant cells, as foreign → GVL

The goal of allogeneic SCT is tumor immunity through GVL reactions while minimizing unwanted GVHD side effects.

Minor differences:

Transplantation always occurs in an HLA-matched setting. Even if the donor and patient are HLA matched, there still may be some minor differences in the 2 types of cells → minor histocompatibility antigens (MiHA). These are genetic differences between donor and patient, which determine the potential repertoire of allo-antigens that can be targeted by T-donor cells. For instance, small nucleotide polymorphisms (SNP) may result in an amino-acid polymorphism. In case of an amino-acid polymorphism, the following happens:

1. Proteins are cleaved into small peptides
2. The peptides are expressed on the MHC-molecules and are presented to the T-cell repertoire
3. Non-self polymorphic peptides are recognized by T-cells → immune activation

Graft versus host disease

Cause:

GVHD is usually triggered by chemotherapy or irradiation therapy:

1. Chemotherapy or irradiation therapy leads to tissue damage and upregulation of a various number of factors.
2. Donor T-cells arrive through the transplant and recognize these host APCs and other abnormal surface proteins
3. The donor T-cells become CD4 and CD8 T-cells and attack the tissue → GVHD

In short, immune cells present in the graft from the donor react with various cells from the patient. Regulatory T-cells can dampen this reaction, but in the beginning the transplantation isn’t really capable of stopping the total reaction.

Symptoms:

GVHD is most common in the:

• Skin
  • Blisters
  • Dry skin
  • Redness
  • Tears in the skin
  • Very painful
• Intestines
  • Mucositis
  • Ulcers
  • Painful
  • Bloody diarrhea
• Liver
  • Particularly bile ducts are attacked
  • Apoptotic bodies are found
  • Icterus
    • Caused by accumulation of bilirubin in the circulation
• Lung
  • Only in chronic GVHD cases

Because these tissues are in direct contact with the outside world, they contain dendritic cells → present MiHAs picked up from damaged tissues. Inflamed non-hematopoietic tissues express multiple MiHAs.

Prevention:

GVHD can be prevented with T-cell depletion of the graft:

• Purification of stem cells from the graft
  • Positive CD34 selection
• Removal of T-cells from the graft with antibodies
  • Antibodies against CD3
• Killing T-cells in the graft by adding antibodies and serum
  • Alemtuzumab (campath) in the bag

Strategies to separate GVL from GVHD are:

• Timing of T-cell infusion
• In vitro selection of T-cells preferentially recognizing patient hematopoietic cells

Postponed donor lymphocyte infusion

Balance:

There is a balance between GVHD and relapse:

• If there is a high chance of GVHD, there is a small chance of relapse
• If syngeneic allogenic SCT is used (in case of twins), the chance of GVHD is small but the risk of relapse is very high → there is no allo-immune effect

Thus, the relationship between GVHD and GVL is inverse. There will be an exam question about benign hematological diseases, which are only mentioned in the books.

The balance between GVHD and GVL after allogeneic SCT is influenced by:

• Genetic disparity between donor and recipient
• The T-cell repertoire of the donor → the donor must have T-cells which are capable of recognizing minor transplantation antigens
• Tissue distribution of target antigens
• Inflammatory circumstances
• Immunogenicity of malignant cells

The risk of relapse after allogeneic SCT can be reduced via donor lymphocyte infusion (DLI) 3, 6 or more months after allogeneic SCT. Postponed DLI reduces the risk of GVHD, but has a risk of early relapse of leukemia.

Procedure:

In Leiden, the following is done:

1. A patient with leukemia is conditioned and given a T-cell depleted donor stem cell graft
   • This doesn’t need any immune suppression → GVHD is virtually absent
2. During this time, tissue damage is repaired and HLA-II expression isn’t necessary anymore
3. Recipient dendritic cells are gradually replaced by donor dendritic cells
4. After a couple of months, the patient receives an unmodified DLI → causes the desired GVL reaction with limited to no GVHD

In this case, tumor cells still express HLA class II antigens which still cause the desired GVL disease, but because the tissue is already healed, there is no GVHD. Therefore, the chance of getting GVHD is reduced, but the risk of relapse is not.

Nevertheless, the balance between GVHD and GVL remains hard to maintain with postponed DLI:

• Relapses may occur early after transplantation → early DLI is better
• Early DLI causes severe GVHD → late DLI is better
• If no professional normal or malignant patient APCs are present → no GVL occurs

A future solution may be separation of T-cells causing GVHD or GVL.

Applications:

DLI can be used to treat incomplete immune responses after allogeneic SCT:

• Mixed chimerism in the absence of GVHD requires pre-emptive DLI
  • Not only donor cells are in the bone marrow, but also some old cells of the patient → the recipients own cells need to be removed
  • Infused T-cells only attack the recipient’s healthy cells → the donor cells are genetically the same
  • Infused T-cells can also help against relapse → T-memory cells are made which can find some relapsing leukemic cells
• Minimal residual disease after allogeneic SCT requires DLI
• Profound relapse after allogeneic SCT requires chemotherapy + DLI
  • Chemotherapy is required because of the veto-effect of the leukemic cells → are unable to present antigens to T-cells and inhibit them

In case of insufficient response to DLI, activation can be induced with an additional immune modulatory drug such as interferon-α.

Opportunistic infections

Besides GVHD, several opportunistic infections can occur in case of T-cell depleted and non T-cell depleted allogeneic SCT. This is caused by immunosuppression. Immunosuppressives are given to prevent infections. Examples are:

• Cytomegalovirus (CMV)
• Epstein-Barr virus (EBV)

Cytomegalovirus:

Many people have been infected with CMV → 50-85% of the population is CMV positive. Primary infection of CMV usually occurs during infancy and has very low morbidity rates. Afterwards, the virus remains latent in the body and normally remains under control of CD8 T-cells. In case of allogeneic SCT, these T-cells aren’t present in both the recipient and the donor cells:

• The recipient doesn’t have any T-cells due to chemotherapy and/or ablation
• Donor stem cells take a while to form new T-cells

Because there aren’t any CD8 T-cells, there is no regulation of the CMV levels → can result in reactivation and disease. In this case, the mortality and morbidity is high. Organs become infected and their function is compromised. T-cells are necessary to recognize the virus antigens on HLA-I molecules. It can take up to a year for CMV to reactivate, but if it happens almost every patient dies.

Incidence of reactivation in CMV+ patients 12 months after transplantation is:

• 40-60% in conventional allogeneic SCT
• 95% in T-cell depleted allogeneic SCT

Target organs of CMV disease are:

• Encephalitis in the brain
• Retinitis in the eye
• Pneumonia
• Gastro-enteritis

If the donor and patient are CMV+, the 95% of patients will show a CMV reactivation after T-cell depleted allogeneic SCT. This can be treated, which frequently prevents disease:

• Drugs
  • Valganciclovir
  • Foscarnet
    • If valganciclovir doesn’t work
• Donor lymphocyte infusion
  • The donor must be CMV+
• CMV specific T-cells
  • The donor must be CMV+
  • If drugs aren’t effective

The donor needs to be CMV+ because CMV specific T-cells must be present to prevent further CMV reactivation. Treatment with CMV specific T-cells works as follows:

1. Mononuclear cells are taken out of a CMV+ donor via the leukapheresis procedure
2. Virus derived peptides are added → virus specific T-cells expand
3. CMV specific T-cells are selected using magnetic cell separation
4. CMV specific T-cells are infused into the patient

Epstein-Barr virus:

EBV re-infections work the same way as CMV. Many people get infected with EBV somewhere in their life, and after transplantation or chemotherapy, EBV reactivates because there is no active immune system present.

EBV is mainly present in B-cells. When reactivated, the infected B-cells usually are cleared by the T-cells. These T-cells aren’t present in the patient → EBV integrates itself into the genome of the B-cells, causing uncontrolled proliferation of B-cells. This results in a lymphoma → post-transplant lymphoproliferative disease (PTLD).

Treatment consists of rituximab, an antibody against CD-20, and if this doesn’t work cytotoxic drugs (chemotherapy).