The relationshipe between cancer and

monocytesPrepared by:

Mazen Saeedpharmacology department, Ege university

Monocytes• Monocytes are produced by the bone marrow from precursors called monoblasts, bipotent cells that differentiated from hematopoietic stem cells.

Monocytes types• Monocytes which migrate from the bloodstream to other tissues will then differentiate into tissue resident macrophages or dendritic cells. 

Macrophages• Macrophages are responsible for protecting tissues from foreign substances, but are also suspected to be important in the formation of important organs like the heart and brain.

• They are cells that possess a large smooth nucleus, a large area of cytoplasm, and many internal vesicles for processing foreign material.

Macrophages and cancer• Macrophages contribute to tumor growth and progression. Attracted to oxygen-starved (hypoxic) and necrotic tumor cells they promote chronic inflammation. Inflammatory compounds such as Tumor necrosis factor (TNF)-alpha released by the macrophages activate the gene switch nuclear factor-kappa B. NF-κB then enters the nucleus of a tumor cell and turns on production of proteins that stop apoptosis and promote cell proliferation and inflammation.

• Moreover macrophages serve as a source for many pro-angiogenic factors including vascular endothelial factor (VEGF), tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-1 and IL-6 contributing further to the tumor growth. Macrophages have been shown to infiltrate a number of tumors.

• Their number correlates with poor prognosis in certain cancers including cancers of breast, cervix, bladder and brain.Tumor-associated macrophages (TAMs) are thought to acquire an M2 phenotype, contributing to tumor growth and progression. Recent study findings suggest that by forcing IFN-α expression in tumor-infiltrating macrophages, it is possible to blunt their innate protumoral activity and reprogramme the tumor microenvironment toward more effective dendritic cell activation and immune effector cell cytotoxicity.

Dendritic cells• Dendritic cells (DCs) are antigen-presenting cells, (also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.

Monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system. These are:-

• Phagocytosis, • Antigen presentation, and• Cytokine production

Phagocytosis•  Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material.

•  Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody, termed antibody-mediated cellular cytotoxicity.

Antigen presentation• Microbial fragments that remain after such digestion can serve as antigens. The fragments can be incorporated into MHC molecules and then trafficked to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it leads to activation of T lymphocytes, which then mount a specific immune response against the antigen.

Cytokine production• Other microbial products can directly activate monocytes and this leads to production of pro-inflammatory and, with some delay, of anti-inflammatory cytokines. Typical cytokines produced by monocytes are TNF, IL-1, and IL-12.

Monocytes in human bloodThere are at least three types of monocytes in human blood:

a) The classical monocyte is characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16- monocyte)b) The non-classical monocyte shows low level expression of CD14 and additional co-expression of the CD16 receptor (CD14+CD16++ monocyte).

c) The intermediate monocyte with high level expression of CD14 and low level expression of CD16 (CD14++CD16+ monocytes).

• There appears to be a developmental relationship in that the classical monocytes develop into the intermediate monocytes to then become the non-classical CD14+CD16++ monocytes. Hence the non-classical monocytes may represent a more mature version. After stimulation with microbial products the CD14+CD16++ monocytes produce high amounts of pro-inflammatory cytokines like tumor necrosis factor and interleukin-12.

• Said et al. showed that activated monocytes express high levels of PD-1 which might explain the higher expression of PD-1 in CD14+CD16++ monocytes as compared to CD14++CD16- monocytes. Triggering monocytes-expressed PD-1 by its ligand PD-L1 induces IL-10 production which activates CD4 Th2 cells and inhibits CD4 Th1 cell function.

CD4 • CD4 (cluster of differentiation 4) is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.

• CD4+ T helper cells are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T-helper cells or T4 cells. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells. CD4 cells send the signal and CD8 cells destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following immune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight.

• CD4 continues to be expressed in most neoplasms derived from T helper cells. It is therefore possible to use CD4 immunohistochemistry on tissue biopsy samples to identify most forms of peripheral T cell lymphoma and related malignant conditions.

Tumour Antigens• Tumour antigens are those expressed by tumor cells, and recognizable as being different from self cells. Most currently classified tumor antigens are endogenously synthesized, and as such are presented on MHC class I molecules to CD8+ T cells. Such antigens include products of oncogenes or tumor suppressor genes, mutants of other cellular genes, products of genes that are normally silenced, over-expressed gene products, products of oncogenic virisus, oncofetal antigens (proteins normally expressed only during development of the fetus) glycolipids and glycoproteins. Detailed explanations of these tumor antigens can be found in Abbas and Lichtman, 2005.

• MHC class II restricted antigens currently remain somewhat obscure. Development of new techniques has been successful in identifying some of these antigens, however, additional research is required. (Wang, 2003)

CD8+ T cells and Antitumour Immunity

• Historically, much more attention and funding has been devoted to the role of CD8+ T cells in antitumor immunity, rather than to CD4+ T cells. This can be attributed to a number of things; CD4+ T cells respond only to presentation of antigens by MHC class II, however, most cells express only MHC class I; second, CD8+ T cells, upon being presented with antigen by MHC class I, can directly kill the cancerous cell, through mechanisms which will not be discussed in this article, but which have been well categorized; (See Abbas and Lichtman, 2005) finally, there is simply a more widespread understanding and knowledge of MHC class I tumor antigens, while MHC class II antigens remain somewhat obscure.(Pardol and Toplain, 1998).

• It was believed that CD4+ T cells were not involved directly in antitumour immunity, but rather functioned simply in the priming of CD8+ T cells, through activationantigen presenting cells (APCs) and increased antigen presentation on MHC class I, as well as secretion of excitatory cytokines such as IL-2 (Pardol and Toplain, 1998, Kalams and Walker, 1998, Wang 2001).

• CD4+ T cells (mature T-helper cells) play an important role in modulating immune responses to pathogens and tumor cells, and are important in orchestrating overall immune responses.

CD4+ T cells, a More Central Role

• Today, there is adequate evidence to prove a more central role of the CD4+ T cell in antitumor immunity. In one important experiment, mice were given a tumor vaccine (irradiated tumor cells) several weeks prior to subcutaneous injection of tumour cells Immediately before exposure, some mice were treated with anti-CD4+monoclonal antibodies, thus eliminating all CD4+ T cells. Mice in this group showed an extremely low rate of tumour rejection when compared those not treated with antibodies. (Dranoff et al. 1993)

• Despite the fact that CD4+ T cells were able to enhance the proliferation and differentiation of CD8+ cells, the immune response was extremely reduced. Furthermore, MHC class I deficient tumour cells have been shown to elicit immune responses in mice comparable to those elicited by tumours which have functional MHC class I (Hung, 1998).

• Subsequent depletion of CD4+ cells and NK cells, but not CD8+ cells results in the inability to reject the tumour.

• In 1998, K. Hung demonstrated that vaccinated CD4-/- mice were unable to mount an effective immune response to a certain form of melanoma, while CD8-/- mice, could mount a response, weaker than the response of wild type mice, but nevertheless important. In this experiment, effectiveness was recognised by the number of mice surviving, following the second exposure to the cancer.

• The knockout of CD4+ T cells, on its own, does not prove a central role of these cells in antitumor immunity. It can be argued that, because CD4+ T cells were not present, CD8+ T cells could not mount as effective a response, and thus, the mice died. However, the fact that CD8-/- mice were still able to mount some form of response undisputedly shows a central role of CD4+ T cells in anti-tumour immunity. (Hung, 1998)

Th1 and Th2 CD4+T cells• The same series of experiments, examining the role of CD4+ cells, showed that high levels of IL-4 and IFNγ were present at the site of the tumor, following vaccination, and subsequent tumour challenge. (Hung, 1998)

• IL-4 is the predominant cytokine produced by Th2 cells, while IFNγ is the predominant Th1 cytokine. 

• Earlier work has shown that these two cytokines inhibit the production of each other by inhibiting differentiation down the opposite Th pathway, in normal microbial infections, (Abbas and Lichtman, 2005) yet here they were seen at nearly equal levels.

• Even more interesting was the fact that both these cytokines were required for maximal tumor immunity, and that mice deficient in either showed greatly reduced antitumor immunity. IFN-γ null mice showed virtually no immunity, while IL-4 null mice showed a 50% reduction when compared to immunised wildtype mice.

• The reduction of immunity in IL-4 deficient mice, has been attributed to a decrease in eosinophil production. In mice deficient in IL-5, the cytokine responsible for differentiation of myeloid progenitor cells into eosinophils, less eosinophils are seen at the site of tumour challenge, which is to be expected. (Hung, 1998)

• These mice also show reduced antitumor immunity, suggesting that IL-4 deficient mice, which would produce less IL-5, and subsequently have reduced eosinophil levels, elicit their effect through eosinophils.

IFN-γ• A number of mechanisms have been proposed to explain the role of IFN-γ in antitumor immunity. In conjuncture with TNF (Tumor Necrosis Factors), IFN-γ can have direct cytotoxic effects on tumor cells (Franzen et al., 1986) Increased MHC expression, as a direct result of increased IFN-γ secretion, may result in increased presentation to T cells. (Abbas and Lichtman, 2005) It has also been shown to be involved in the expression of iNOS as well as ROIs.

• iNOS (inducible nitric oxide synthase) is an enzyme responsible for the production of NO, an important molecule used by macrophages to kill infected cells. (Abbas and Lichtman, 2005)

• A decrease in the levels of iNOS, (as seen through immuno histochemical staining) has been observed in IFNγ-/- mice although levels of macrophages at the site of tumor challenge are similar to wild type mice. INOS -/- mice also show decreased immunity, indicating a direct role of CD4+-stimulated iNOS production in protection against tumours. (Hung et al., 1998)

• Similar results have been seen in knock out mice deficient in gp91phox, a protein involved in the production ROIs (Reactive Oxygen Intermediates) which are also an important weapon utilized by macrophages to elicit cell death.

• In 2000, Qin and Blankenstein, showed that IFNγ production was necessary for CD4+ T cell-mediated antitumor immunity.

• A series of experiments showed that it was essential for nonhematopoietic cells at the site of challenge, to express functional IFNγ receptors. Further experiments showed that IFN-γ was responsible for inhibition of tumor induced angiogenesis and could prevent tumor growth through this method. (Qin and Blankenstein, 2000)

MHC Class II and Immunotherapy• Many of the afore mentioned mechanisms by which CD4+ cells play a role in tumor immunity are dependent on phagocytosis of tumors by APCs and subsequent presentation on MHC class II. It is rare that tumor cells will express sufficient MHC class II to directly activate a CD4+ T cell. As such, at least two approaches have been investigated to enhance the activation of CD4+ T cells.

• The simplest approach involves upregulation of adhesion molecules, thus extending

The presentation of antigens by APC. (Chamuleau et al., 2006) A second approach involves increasing the expression of MHC class II in tumor cells.

• This technique has not been used in vivo, but rather involves injection of tumor cells which have been transfected to express MHC class II molecules, in addition to suppression of the invariant chain (Ii, see below) through antisense technology. (Qiu, 1999)

• Mice vaccinated with irradiated strains of these cells show a greater immune response to subsequent challenge by the same tumor, without the upregulation of MHC class II, then do mice vaccinated with irradiated, but otherwise unaltered tumor cells.

• These findings signify a promising area of future research in the development of cancer vaccines.

MHC Class I and Class II pathways

• The down regulation of the invariant chain (Ii) becomes important when considering the two pathways by which antigens are presented by cells.

• Most recognized tumor antigens are endogenously produced, altered gene products of mutated cells. These antigens, however, are normally only presented by MHC class I molecules, to CD8+ T cells, and not expressed on the cell surface bound to MHC class II molecules, which is required for presentation to CD4+ T cells.

•

• Research has shown that the two pathways by which antigens are presented cross over in the endoplasmic reticulum of the cell, in which MHC class I, MHC class II and endogenously synthesized antigenic proteins are all present.

• These antigen proteins are prevented from binding to MHC class II molecules by a protein known as the invariant chain or Ii, which, in a normal cell, remains bound to the MHC class II molecule until leaving the ER.

• Down regulation of this Ii, using antisense technology, has yielded promising results in allowing MHC class I tumor antigens to be expressed on MHC class II molecules at the cell surface. (Qui, 1999)

Upregulation of MHC class II• Due to the extremely polymorphic nature of MHC class II molecules, simple transfection of these proteins does not provide a practical method for use as a cancer vaccine. (Chamuleau et al., 2006)

• Alternately, two other methods have been examined to upregulate the expression of these proteins on MHC class II- cells.

The first is treatment with IFNγ, which can lead to increased MHC class II expression. (Trincheiri and Perussia, 1985, Fransen L, 1986)

A second, more effective approach involves targeting the genes responsible for the synthesis of these proteins, the CIITA or class II transcription activator.

• Selective gene targeting of CIITA has been used ex vivo to allow MHC class II- cells to become MHC class II+. (Xu, et al. 2000)

• Upregulation of CIITA also causes an increased expression of Ii, and as such, must be used in conjuncture with the antisense techniques referred to earlier. (Qui, 1999)

• In some forms of cancer, such as acute myeloid leukemia (AML) the cells may already be MHC class II+, but because of mutation, express low, levels on their surface. It is believed that low levels are seen as a direct result of methylation of the CIITA promoter genes (Morimoto et al., 2004, Chamuleau et al., 2006) and that demethylation of these promoters may restore MHC class II expression. (Chamuleau et al., 2006)