Immunological Control of Neoplasia - Chapter 4

Author: Christine Ichim

Chapter 4 Immune Interactions with Chronic Myeloid Leukemia (CML)

In the previous chapters the case was made for the ability of immune response to effect tumour growth and for the ability of the tumour to protect its self from immune attack. This base of knowledge will now be applied to CML in hopes of providing future research directions. CML was chosen due to several factors that suggest an important role of the immune response against it. First, CML cells possess a novel chimeric protein, p210 that allows them to be distinguished immunologically from their untransformed counterparts (600-605). p210 is essential for the existence of CML since treatment with antisense oligonucleotides abrogates leukemogenicity in vitro (606). Furthermore, mice transgenic for bcr-abl, the gene encoding p210, develop a CML-like disease (607-609). The fact that p210 is recognizable by the immune system and is essential for the growth of CML means that CML cells cannot evade immune response by downregulating p210 expression since if they did, they will no longer be leukemic. Second, the progress of CML patients seems to be correlated with immune activity and in one study a decrease of certain immune parameters was predictive of relapse (610). Third, CML patients have suppressed immune function (611) and one of the drugs that boosts this immune function is also highly efficient at deterring the progression of CML (612). Fourth, transfer of immune cells from bone marrow transplant donors into relapsing recipients can induce remission (613-615).

CML is a disease of the bone marrow characterized by an initial chronic phase followed by a lethal blastic phase. The initial signs of CML are leukocytosis and chronic fatigue. The leukocytosis is usually managed with a myelosuppressive drug such as busulfan or hydroxyurea. The fatigue is usually the result of erythropoietic suppression by the malignant clone and when severe is treated by transfusions or administration of erythropoietin. Patients remain in the chronic phase for about 2-4 years before progressing. Myelocyte differentiation occurs in the chronic phase although some differentiated cells do not function as well as progenitors from normal stem cells. For example, the ability of monocytes from a CML chronic phase patient to phagocytose particular matter is markedly impaired. As well, neutrophil mobility is diminished in cells originating from the patient. In spite of these deficiencies, chronic phase CML patients can live normal lives. When the CML develops into the blastic phase, undifferentiated leukemic stem cells enter the circulation in large amounts. Sometimes making up as much as 40% of the total blood cells, therefore in severe CML cases, the blood possesses a whitish hue. These CML blasts end up killing the patient because they do not allow for production of normal immune cells by the bone marrow. In addition, CML blasts invade organs, where they proliferated and eventually cause organ failure (616).

The untransformed counterpart of the CML cell is a hematopoietic stem cell that can produce cells of the myeloid lineage but not of the lymphoid. This cell possesses the CD34 stem cell marker but also MHC 2 molecules. Pluripotent stem cells do not possess MHC 2 molecules until they differentiate, therefore the CML abnormality does not effect the true stem cell. This is an important finding since investigators are attempting to purify nonleukemic stem cells for clinical applications (617). Another point to note is that MHC 2 can activate immune responses. It has been shown previously that in vitro anti-CML responses are restricted by MHC 2, and occur from T cells expressing CD4 (618).

During the chronic phase, the CML stem cells are expanding in the bone marrow, taking over the function of normal stem cells. The interaction between CML cells and normal stem cells is not understood. However, soluble mediators secreted by CML cells in vitro can suppress activation of the normal stem cell, thus giving the leukemic cells a proliferative advantage. Some studies have recently suggested that CML stem cells in chronic phase do not proliferate faster than normal stem cells but acquire an advantage by not undergoing apoptosis, in fact CML cells have increased levels of the antiapoptotic protein bcl-xL (619). Evidence exists that CML cells “turn off” normal stem cells but does not kill them since treating patients with high dose chemotherapy will induce the reactivation of normal stem cells (620). This is important since if the patient did not have a reservoir of untransformed stem cells immunotherapy would be useless and probably kill the patient since it would wipe out all cells capable of generating blood cells. Besides suppressing the normal stem cells, the CML starts acquiring additional mutations that enable it to transform to the blastic phase. These mutations are not completely understood, although progression of CML into blastic phase is associated with an increase in the tyrosine phosporylation of several genes and the constitutive activation of certain components of the cell cycle. A cellular manifestation prior to blast transformations is a sharp increase in circulating basophils (621,622), this is interesting since basophils are potent secreters of the Th2 cytokine IL-4 (623), and histamine, which has been shown to have NK inhibitory activities (624). The role of histamine however is disputed since some investigators have used histamine to increase the activity of LAK cells against AML blasts, with success (625).

From a tumour immunological perspective, the chronic phase CML cells should be immunogenic since they possess the abnormal protein p210. Although this is an intracellular protein, portions of it are presented on both MHC 1 and MHC 2, thus allowing for activation of immune response. An interesting comparison has been made between T cells infiltrating the bone marrow of chronic phase CML patients and tumour infiltrating lymphocytes in that both may be cultured ex vivo and toxicity toward autologous targets increased. Bhatia et al demonstrated that stimulating marrow derived T cells from chronic phase patients with irradiated autologous marrow, in the presence of the immune stimulator IL-2, induced the proliferation of T cells. These T cells were cloned and shown to be MHC 2 restricted, CD4 positive cells, which possessed cytotoxicity. This cytotoxicity was not present when the cloned T cells were cultured with the NK target K562, thus showing that the effect is similar to a traditional T cell response to pathogen and not spontaneous cytotoxicity (626). Besides T cells, LAK and NK cells have been shown to be effective against CML cells both at the chronic and blastic phase. This is shown by experiments that mimic the in vivo bone marrow microenvironment. Klingemann et al prepared in vitro cultures of bone marrow stromal cells, seeded with hematopoietic cells. Upon treating these cultures with IL-2, the hematopoietic activity of the normal stem cells remained uneffected while the amount of leukemic cells in the culture decreased. This is correlated with activation of NK cells (627).

Control of hematopoiesis, both normal and malignant seems partly related to the function of NK cells. First, NK cells possess homing receptors that allow them to share the same anatomical niches as some stem cells (628). Second, activated NK attachment to bone marrow stroma can physically interfere with ability of malignant stem cells to adhere to stroma (629). Thirdly, NK cells secrete hematopoietic promoting cytokines such as GM-CSF and IL-3 and hematopoietically suppressing cytokines such as IFN-( or TNF-" depending on the subset and stage of activation (630). In vivo evidence for the importance of NK cells in regulating hematopoiesis comes from animal s where certain subsets of NK cells suppress hematopoietic reconstitution post bone marrow transplant, whereas other subsets increase the rate of reconstitution (631). Thus, NK cells seem to possess a considerable amount of influence regarding events occurring in the bone marrow microenvironment. It is very probable that NK cells are very important in determining how long the patient remains in chronic phase, when progression to blast phase occurs and the severity of it. NK responses against the CML are inhibited by CML secreted substances such as IL-10 (560). These substances allow chronic phase clones to expand in the presence of NK cells. The importance of NK protection of the host during chronic phase is shown in the clinical study of Pawelec et al where they show that NK activity is suppressed in patients, but is increased upon IFN-" treatment. The patients that have increased levels of NK activity in response to IFN-" are the patients who remain in chronic stage the longest (632).

Since it appears that immune functions have a role in controlling, and in some cases even reversing CML, the next question is how can the immune system be modulated to eradicate CML? The conclusion of this work is the proposal of a combinatory treatment that will describe this.

CML Therapy

The CML patient should be treated at the chronic phase since during this phase many immune functions remain. In the chronic phase the normal stem cells can take over the role of hematopoiesis after the CML ones have been abolished. Initially the treatment should include restoration of nutrition in the patient so the necessary cofactors will exist in the patient’s body to be able to mount an immune response. For example, zinc is a micronutrient that is deficient in leukemic patients, and severity of zinc deficiency correlates with poor prognosis (633). Zinc is important for T cell proliferation and NK activation (634-637). Therefore, zinc supplementation should be part of the preparative regime per immunotherapy. Several other nutrients that are deficient in CML patients should also be restored to normal levels to attain proper immune activation, these include ascorbic acid, retinoic acid, selenium, and alpha tocopherol. Another preimmunotherapy intervention should be the normalization of hormones involved in activation of immune response such as prolactin and DHEA. These normalization procedures will likely to be difficult since they require close monitoring of several compounds in the patient.

Besides restoring components necessary to immune activation, during immunotherapy the patient should somehow be modulated so that free radicals are scavenged effectively. The best method of doing this is not known at the time although administration of N-acetyl cysteine in combination with ascorbic acid and alpha tocopherol has been reported to be effective at reducing oxidative stress on the patient. The need to reduce oxidative stress is that immune activation increases production of various free radical species that can damage host cells and lead to inflammation by activation of endothelial cells. The immune initiated free radicals also act as a negative feedback mechanism downregulating further immune activation. This has been shown by researchers who have coincubated neutrophils with NK cells and saw that NK cell activity was diminished in a noncontact dependent manner. When antioxidants were added to the culture, the inhibitory effect of the neutrophils on NK activation was abrogated. In a personal communication, Dr Arfors from Princeton told the author that in aged and cancer bearing mice a glutathione deficiency is present, which suppresses immune functions such as T cell proliferation and NK activation. Addition of the antioxidant N-acetyl cysteine partly restores the levels of immune cell activation. In the same communication, Dr Arfors referred to studies in which the levels of glutathione in antigen presenting cells decide whether immune response will be Th1 or Th2, with Th2 responses being formed in the presence of low glutathione levels. Physiologically, it would make sense that chronic oxidative stress triggers Th2 responses since these antagonize the oxidative stress producing Th1 responses.

In addition to protecting the patient from overproduction of oxidative stress, CML immunotherapy should guard against production of mediators of chronic inflammation such as PGE-2, glucacorticoids, nitric oxide and adenosine. All of this compounds are immune suppressive or shift the response to the ineffective Th2 response. PGE-2 production can be inhibited by pharmacologically administrable agents such as indomethacin or aspirin, glucocorticoid production can be suppressed by administration of hormones such as prolactin, nitric oxide can be suppressed by administration of inhibitors of the enzyme that makes nitric oxide such as NMMA, and the activity of adenosine can be suppressed by administration of adenosine antagonists that have safely been pharmacologically administered in the past.

During the administration of the immunotherapeutic regime levels of all the parameters described above should be monitored in conjunction with the immune responsiveness. Certain aldehydes are secreted in when the patient is under oxidative stress. These can be measured in any medical laboratory. The levels of the other mediators such as PGE-2, glucocorticoids, NO, and adenosine can be measured from serum, but since microenvironmental effects may be more important than systemic effects, analysis should be made during bone marrow biopsy. Activation of Th1 immune functions can be assessed crudely by measuring plasma levels of a protein called neopterin. Neopterin is produced almost exclusively by activated macrophages, and since macrophages become activated during Th1 responses, neopterin is a semi-accurate indicator. Other measures of immune response such as blood NK activity and T cell proliferation are needed to know when different immunomodulations are needed. As well, cytokines secreted by activated immune cells need to be examined since this provides in depth insight on whether the immune response is becoming Th1 or Th2.

It is the authors’ opinion that actual immunotherapy for CML should consist of a five-stage approach:

First, stem cells from the patient are mobilized into circulation by administration of the cytokine GM-CSF. Besides sending bone marrow stem cells into circulation, GM-CSF activates macrophages in the bone marrow: activated macrophages have antileukemic effects. The mobilized stem cells are then collected and the malignant ones are purified from the normal ones by MHC 2 selection. The normal stem cells are maintained in culture until reinfusion.

Second, patient leukocytes are collected. NK cells purified from the leukocytes are expanded by culturing in the presence of irradiated autologous bone marrow cells and IL-2. The role of the NK cell is to provide cytokines and the proper alterations in the bone marrow microenvironment in order for other immune responses to be activated.

Third, CML specific T cell clones are generated toward autologous irradiated bone marrow cells. The T cell clones should be specific for the CML cells, and this is tested in vitro prior to reinjection of the clone.

Fourth, the NK cells are reinjected into the host every second day for a week. Injections of these NK cells are accompanied by injections of the untransformed stem cells. Following the initial week, the T cell clones are reinjected every second day for another week.

Fifth, the patient is injected once a month, intradermally, with irradiated CML cells obtained from the original purification. Injection is performed in the presence of the macrophage activating adjuvant beta 2 glucan.

The author’s believe that the combination of nutritional/endocrine restoration followed by the above immunotherapeutic procedure should yield favorable results since this approach overcomes several shortcomings in other immunotherapy trials. For example, the fact that immunotherapy is given to patients who are nutritionally deficient is absurd since immune enhancement cannot be obtained in the absence of the needed cofactors necessary for production of the proteins that mediate immune response. Further, some types of immunotherapy, such as IL-2 have been shown to suppress levels of certain nutrients needed for immune activation, yet no attempt has yet been made to combine nutritional supplementation with cytokine therapy.

The treatment proposed attempts to mimic the natural activation of immune responses, and to integrate this with a bone marrow transplant-like conditioning regime. In a normal immune response first the macrophages are activated, followed by NK cells, and lastly the T cell response is initiated. This is a chain of activation from the sensory part of the immune response, to the synthesis and finally resulting in activation of effector cells. In past immunotherapeutic approaches investigators have only concerned themselves with activation of T cells, NK cells and even macrophages. However, no study has tried to integrate activation of the immune cells in the order that they would be activated if a normal response was occurring. To reiterate, the GM-CSF activates the macrophages, the NK cells get reinfused in the first week or treatment, and the T cells get reinfused on the last week.

Reinfusing purified stem cells simultaneously with the NK and T cells attempts to take advantage of the ability of both these cell types to promote nonmalignant hematopoiesis while at the same time kill the malignancy. It is hoped the activated immune cells will put enough pressure on the malignant clone to subside so the usage of myeloablative chemotherapy can be circumvented.

The repeated restimulation of the patient with autologous CML cells is meant to remind the immune system consistently that cells expressing the leukemic antigens are “dangerous” and should be destroyed. The danger signal in the case comes from the fact that the CML cells are coinjected with the adjuvant beta 2 glucan. Similar restimulation procedures have yielded better success than only one immunization in patients with melanoma.

Chapter 1 - Introduction to The Immunotherapy of Cancer
Chapter 2 - Components of Immune Response
Chapter 3 - Immune Suppression in Cancer
Chapter 4 - Immune Interactions with Chronic Myeloid Leukemia (CML)

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