The Role of Polymorphisms in Co-Signalling Molecules’ Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is associated with several humoural and cellular immune abnormalities (Scrivener et al, 2003; Stevenson & Caligaris-Cappio, 2004) that could lead to an inadequate anti-tumour response. The immune surveillance of tumour cells depends on the recognition of antigens presented in the context of human leukocyte receptor HLA class I molecules by cytotoxic T lymphocytes (CTLs), via their T-cell receptors (TCRs) (Rosenberg, 2001). However, antigen alone is insufficient to drive the activation of naïve T-cells (Lafferty et al, 1978), and the two-signal model of T-cell activation was proposed. According to this model, the effective activation of naïve T cells requires second, antigen independent, co-stimulatory signal provided by the interaction between a costimulatory receptor and its ligand on an antigen-presenting cell (Jenkins et al, 1990; Schwartz et al, 1989). The lack of co-stimulation results in T-cell tolerance and anergy. Over the past several years, a large number of molecules have been identified that function as second signals following TCR engagement, and many have been revealed to be negative costimulatory molecules, which dampen T-cell activation and regulate immune tolerance. Some have been shown to be upregulated in the tumour microenvironment and have become potential targets for augmenting anti-tumour immunity (Sharpe, 2009).


Introduction
Chronic lymphocytic leukaemia (CLL) is associated with several humoural and cellular immune abnormalities (Scrivener et al, 2003;Stevenson & Caligaris-Cappio, 2004) that could lead to an inadequate anti-tumour response.The immune surveillance of tumour cells depends on the recognition of antigens presented in the context of human leukocyte receptor HLA class I molecules by cytotoxic T lymphocytes (CTLs), via their T-cell receptors (TCRs) (Rosenberg, 2001).However, antigen alone is insufficient to drive the activation of naïve T-cells (Lafferty et al, 1978), and the two-signal model of T-cell activation was proposed.According to this model, the effective activation of naïve T cells requires second, antigen independent, co-stimulatory signal provided by the interaction between a costimulatory receptor and its ligand on an antigen-presenting cell (Jenkins et al, 1990;Schwartz et al, 1989).The lack of co-stimulation results in T-cell tolerance and anergy.Over the past several years, a large number of molecules have been identified that function as second signals following TCR engagement, and many have been revealed to be negative costimulatory molecules, which dampen T-cell activation and regulate immune tolerance.Some have been shown to be upregulated in the tumour microenvironment and have become potential targets for augmenting anti-tumour immunity (Sharpe, 2009).
Polymorphisms in genes can influence the level of protein expression (Anjos et al, 2002;Kouki et al, 2000;Wang et al, 2002b;Oki et al, 2011).Therefore, genetic variation in genes encoding co-signalling molecules may also alter the antitumour response and influence cancer susceptibility, particularly susceptibility to CLL. ligands CD80 (B7.1) and CD86 (B7.2),CD28 transduces a signal that enhances the activation and proliferation of T cells and IL-2 production (Frauwirth & Thompson, 2002;Carreno & Collins, 2002).In addition, a higher level of secretion of other cytokines such as IFN-γ, GM-CSF, IL-4, IL-8 and IL-13, can be observed after CD28 ligation.Moreover, CD28 signalling promotes cell survival via Bcl-x L transcriptions and prevents anergy (Boise et al, 1995).It has been shown that mice deficient in CD28 or both of its ligands (B7.1 and B7.2) have severely impaired CD4 + T cell proliferation (Shahinian et al, 1993;Borriello et al, 1997) and lymphokine secretion after stimulation with concanavalin A (Con A) or superantigen (Mittrucker et al, 1996).Furthermore, CD28-deficient mice exhibit lower levels of certain isotypes of immunoglobulins, and germinal centres are not formed in response to immunisation (Ferguson et al, 1996).The requirement for CD28 for the co-stimulation of CD8 + T cells is more controversial; it was postulated that CD8 + T cells are less CD28 dependent than CD4 + cells (Green et al, 1995).
The CD28 is located on the q33 region of chromosome 2.The gene encoding CD28 consist of four exons, of which exon 1 encodes the leader peptide, exon 2 the ligand binding domain, exon 3 the transmembrane segment, and exon 4 the cytoplasmic tail.Within the gene encoding the CD28 molecule several polymorphic sites have been identified.Three of these sites are non-synonymous gene polymorphisms: the CD28c.73G>A(rs3181099) single nucleotide polymorphism (SNP) in exon 1, which leads to change from Gly 25 to Arg; the CD28c.224G>A(rs35290181) SNP in exon 2 which changes Ser 75 to Asn, and the CD28c.272G>A(rs75899942) SNP that is also in the second exon, which changes Gly91 to Asp.Moreover, 4 synonymous SNP were found, all in the third exon.
It has been reported that variations in non-coding regions can regulate gene expression by altering the motif of functional DNA binding sites, thereby affecting their affinity to transcription factors.
In the intronic region of CD28 gene eleven SNPs have been described, but CD28c.17+3T>C(rs3116496) is the best studied in the context of susceptibility to autoimmune and neoplastic disease.This polymorphism is located near the splice receptor site and might influence the mRNA splicing efficiency and thus the expression of the CD28 molecule (Ahmed et al, 2001).Another widely investigated SNP in the CD28 gene is CD28 -372G>A (rs35593994), which is situated in the promoter (Teutsch et al, 2004).The potential functional effect of this SNP remains to be elucidated, but a search for transcription factor binding sites suggested that the CD28-372G>A [A] allele differs from the CD28-372G>A [G] allele by the presence of a binding site for the CCAAT enhancer-binding protein and the lack of a binding site for growth factor independence 1 (Teutsch et al, 2004).Only one microsatellite polymorphism was described in the CD28 gene and in comparison with many other microsatellites, it presented a low degree of polymorphism.The most common allele occurred at frequencies higher than 0.8, and the gene diversity is close to 0.3 (Pincerati et al, 2010).

Inducible co-stimulator (ICOS)
The second co-stimulatory molecule is ICOS, which appears on T lymphocytes rapidly upon activation (Hutloff et al, 1999) and on unpolarised as well as Th1, Th2, Th17, and Treg subpopulations of CD4 + cells (McAdam et al, 2000;Tan et al, 2008;Nakae et al, 2007;Akiba et al, 2005;Burmeister et al, 2008).This co-stimulatory molecule binds the B7-related protein www.intechopen.com The Role of Polymorphisms in Co-Signalling Molecules' Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia 181 B7RP-1 (Yoshinaga et al, 1999).Like CD28, ICOS provides a signal for T-cell activation and differentiation, and in one model, animals lacking this molecule had a reduced CD4 + T-cell response (Dong et al, 2003).
While CD28 and ICOS have overlapping functions in early T-cell activation, ICOS augments the T-cell effector function, in particular the production of IL-4, IL-5, IL-10, IFN-, and IFN-γ (Beier et al, 2000), but not IL-2 (Hutloff et al, 1999).In addition, ICOS is important for the generation of chemokine receptor 5 (CXCR5) + follicular helper T cells (T FH ), a unique T-cell subset that regulates germinal centre formation and humoural immunity (Nurieva et al, 2008).
ICOS knockout mice have reduced CD4 + T-cell responses, an increased risk of experimental autoimmune encephalomyelitis (Dong et al, 2001), and defects in immunoglobulin class switching and germinal centre formation (McAdam et al, 2001).
In human, the homozygous loss of the ICOS gene is the cause of the ICOS deficiency (ICOSD) form of common variable immunodeficiency (CVID).ICOSD patients suffer from recurrent bacterial infections of the respiratory and digestive tracts, which are characteristic of humoural immunodeficiency, but do not have other complicating features, such as splenomegaly, autoimmune phenomena, or sarcoid-like granulomas, or clinical signs of overt T-cell immunodeficiency (Grimbacher et al, 2003).A severe disturbance of T celldependent B-cell maturation occurs in the secondary lymphoid tissue; B cells exhibit a naive IgD + /IgM + phenotype, and the numbers of IgM memory and switched memory B cells are substantially reduced in individuals with ICOSD (Grimbacher et al, 2003).
The ICOS gene also located in the 2q33 region contains five exons.Exons 1-4 are parallel to those of CD28, while exon 5 encodes an additional fragment of the cytoplasmic tail.In the ICOS gene, two microsatellites in the fourth intron and 31 single-nucleotide polymorphisms (SNPs) (http://www.hapmap.org)have been found.None of the described ICOS SNPs leads to changes in the amino acid sequence, although a few have been demonstrated to be functional variants (Kaartinen et al, 2007;Haimila et al, 2009;Castelli et al, 2007;Shilling et al, 2005).
The ICOSc.1624C>T (rs10932037) polymorphism has been shown to influence the ICOS mRNA level (Kaartinen et al, 2007).The authors described that activated CD4 + T cells from ICOSc.1624C>T [CC] homozygous people had higher actual levels of ICOS mRNA than cells from [TC] heterozygous people 1 h and 3 h after activation, following which this difference disappeared.
The ICOSc.1624C>T (rs10932037), ICOSc.1624C>T(rs10932037), and ICOSc.2373G>C(rs10183087) SNPs, which are located in the 3' untranslated region (UTR) of the ICOS gene, influence the functions of the ICOS gene (Castelli et al, 2007).Three major haplotypes, which were associated with different levels of expression of ICOS in CD3 + cells and IL-10 secretion have been identified.The AA genotype, characterised by presence of ICOSc.1624C>T[CC], ICOSc.602A>C[AA], and ICOSc.2373G>C[GG] was shown to be associated with the lowest percentage of CD3+ activated cells expressing ICOS and the highest levels of IL-10 secretion.
The ICOSISV1+173T>C (rs10932029) polymorphism, which is located close to the CTLA-4 gene, has been reported to affect the expression of the CTLA-4 isoforms (Kaartinen et al, 2007;Brown et al, 2007).
Two microsatellite polymorphisms have been described in the fourth intron of the ICOS gene.The first is a GT repeats at position 1554, the location of an Sp1 binding site, and the second, a T repeats, is at a position near the splice donor site (Ihara et al, 2001).

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)
CTLA-4 has been well established as negative regulator of T-cell function (Walunas et al, 1994;Walunas et al, 1996).CTLA-4 is rapidly expressed on T cells following activation and is highly up-regulated by CD28 engagement.CTLA-4 shares the B7 ligands with CD28.
CTLA-4 binding with its ligands antagonises early T-cell activation, leading to decreased IL-2 production, the inhibition of cell-cycle progression, decreased cyclin expression, and the modulation of TCR signalling (Luhder et al, 2000).CTLA-4-deficient mice develop a severe lymphoproliferative disease and die within 3-4 weeks (Tivol et al, 1995;Waterhouse et al, 1995).CTLA-4 is also important in the function of regulatory cells, which suppress effector T-cell activation and function (Tang et al, 2004;Tai et al, 2005).
Many mechanistic models have been postulated for the function of CTLA-4.These models include competition with the co-stimulatory CD28 molecule by more effectively binding their common ligands, the inhibition of downstream TCR signalling by the phosphates SH2 domain, the inhibition of lipid-raft and microcluster formation, and the negative regulation of the immune response by extrinsic components such as TGF- and the tryptophandegrading enzyme indoleamine 2,3-dioxygenase (IDO) (Rudd et al, 2009).
The CTLA-4 gene is located between CD28 and ICOS genes.It is similar to CD28 gene and consists of four exons, of which exon 1 encodes the leader peptide, exon 2 the ligand binding domain, exon 3 the transmembrane segment, and exon 4 the cytoplasmic tail.The functional significance of the polymorphisms in the CTLA-4 gene have been widely explored and described.The most studied is the CTLA-4c.49A>G(rs231775) transition.This nonsynonymous SNP causes an amino acid change from threonine to alanine.It influences Tcell activation and could have a role in antitumour immunity.The presence of the [AA] genotype as opposed to the [GG] genotype has been shown to be associated with significantly lower levels of activation of T lymphocytes and lower proliferation.The protein product encoded by the CTLA-4c.49A>G[AA]genotype, CTLA-4 17 Thr, had a higher capacity to bind B7.1 and a stronger inhibitory effect on T-cell activation compared with CTLA-4 17 Ala (Sun et al, 2008).It was also postulated that the CTLA-4c.49A>Gpolymorphism in the leader sequence of the protein alters the inhibitory function of the molecule by influencing the rate of endocytosis or surface trafficking (Kouki et al, 2000) and the glycosylation of CTLA-4 (Anjos et al, 2002).
The CTLA-4g.319C>T (rs5742909) SNP located in the promoter region also has documented functional significance.The CTLA-4g.319C>T[T] allele has been associated with a higher promoter activity (Wang et al, 2002b), probably due to the creation of a lymphoid enhancer factor-1 (LEF1) binding site in the CTLA-4 promoter (Chistiakov et al, 2006).This allele has also been associated with significantly increased mRNA and surface expression of CTLA-4 in stimulated and non-stimulated cells (Ligers et al, 2001;Anjos et al, 2002).

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The Role of Polymorphisms in Co-Signalling Molecules' Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia 183 The CTLA-4g.1661A>C (rs4553808) SNP, also located in the promoter, appears to be involved in the transcription-associated binding activity of nuclear factor (NF-1) and C/EBP and might cause abnormal alternative spicing and affect the expression of CTLA-4 (Wang et al, 2008).
The CTLA-4g.*6230G>A (CT60) (rs3087243) polymorphism situated in the 3' UTR was shown to be associated with variations in the mRNA level of soluble CTLA-4, an isoform lacking the transmembrane domain, that is generated by the alternative splicing of the primary transcript (Ueda et al, 2003).Our recent results indicate that the CT60 polymorphism together with the Jo31 SNP (CTLA-4g.10223G>T,rs11571302, also located in the 3' region) is associated with the levels of membrane and cytoplasmic CTLA-4 in CD4 + T lymphocytes from multiple sclerosis patients (Karabon et al, 2009) and with the altered levels of soluble CTLA-4 in the serum of Graves' disease patients (Daroszewski et al, 2009).
Another widely investigated genetic marker situated in the 3' UTR region of CTLA-4 gene is a microsatellite polymorphism CTLA-4g.*642AT(8_33).The number of dinucleotide (AT) repeats at position 642 in the 3'UTR region has been shown to be associated with the stability of the mRNA transcripts (Wang et al, 2002a).

Programmed death-1 (PD-1)
Similar to CTLA-4, PD-1 has been described as a negative regulator of T-and B-cell function.PD-1 is an inducible molecule expressed on activated T-and B-cells (Greenwald et al, 2005).In reactive lymph nodes, PD-1 was mainly expressed in follicular T cells (Dorfman et al, 2006).PD-1 binding limits T-cell functions, including T-cell proliferation, apoptosis and interferon- production (Freeman et al, 2000).
Knockout PD-1 mice develop different autoimmune diseases depending on the genetic background: BALBc mice develop autoimmune cardiomyopathy (Nishimura et al, 2001); C57BL mice develop progressive arthritis and lupus-like glomerulonephritis (Nishimura et al, 1999); and NOD mice develop autoimmune diabetes (Wang et al, 2005).PD-1 also has a critical role in murine experimental encephalomyelitis (Salama et al, 2003).
PD-1 has two ligands, which belong to the B7 superfamily: PD-L1 (B7-H) and PD-L2 (B7-DC).The mRNA expression patterns of PD-L1 and PD-L2 are different.PD-L1 is broadly expressed in different human and mouse cells, such as leukocytes, non-haematopoietic cells and non-lymphoid tissue (Freeman et al, 2000), while PD-L2 is present exclusively on dendritic cells and monocytes (Latchman et al, 2001;Liang et al, 2006).The differential expression patterns of PD-L1/PD-L2 and CD80/CD86 are crucial differences between CTLA-4 and PD-1, and this fact raises the hypothesis that CTLA-4 has a key role in the early stages of tolerance induction, while PD-1 functions late for the maintenance of long-term tolerance.The expression of PD-1 ligands limits T-cell function within tissue-specific sites, while CTLA-4 limits T cells in lymphoid structures because CD80 and CD86 are expressed on antigen-presenting cells.
The PD-1 gene is also located on the long arm of chromosome 2, but in the 37.3 region.Similar to the ICOS gene, it consists of 5 exons: exon 1 encodes leader peptide, exon 2 extracellular IgV-like domain, exon 3 the transmembrane domain, exon 4 and 5 the intracellular domain.So far, more than 30 SNPs have been identified in the PD-1 gene.These polymorphisms have been investigated mainly in context of susceptibility to autoimmune disease, such as rheumatoid arthritis (RA) (Kong et al, 2005), type I diabetes (Flores et al, 2010), ankylosis spondylitis (AS) (Lee et al, 2006), and systemic lupus erytrhomatosus (SLE) (Velazquez-Cruz et al, 2007), but only a few studies have been devoted to determining the functional significance of these genetic variations.Among the PD-1 gene polymorphisms, seven namely PD-1.1, PD-1.2, PD-1.3, PD-1.4,PD-1.5, PD-1.6 and PD-1.9, have been studied the most frequently.Two SNPs (PD-1.5 (57785C>T -rs2227981) and PD-1.9 (7625C>T -rs2227982)) occur in exon 5.The C>T transition in the PD-1.9SNP causes a change in amino acid from valine to alanine, while PD1.5 is a synonymous coding variant.
The data describing the functional roles of the PD-1 gene polymorphisms are limited.It has been shown that the PD-1.3 (7146G>A) polymorphism has functional significance, and the presence of PD-1.3.The [A] allele has been associated with a significantly lower expression of the PD-1 receptor in SLE patients, their relatives and healthy individuals (Kristjansdottir et al, 2010).
Patients homozygous for PD-1.3[AA], but not heterozygous for PD-1.3[AG], had reduced basal and induced PD-1 expression on activated CD4 + T cells.In an autologous mixed lymphocyte reactions (AMLRs), activated CD4 + cells from SLE patients had defective PD-1 induction, and this abnormality was more pronounced in homozygotes than heterozygotes.Moreover, the A allele conferred decreased transcriptional activity in transfected Jurkat cells (Bertsias et al, 2009).
The 7209C>T SNP located in intron 4 of the PD-1 gene was also found to be associated with protein expression.Using a luciferase reporter assay, it was shown that the PD-1 7209C>T[T] allele creates a negative cis-element for gene transcription (Zheng et al, 2010).
The promoter polymorphism PD-1-606G>A (rs360488323) alters the promoter activity.The significantly higher promoter activity was observed with the construct with the PD-1-606G>A [G] allele than with the PD-1-606G>A [A] allele (Ishizaki et al, 2010).

B and T lymphocyte attenuator (BTLA)
Although BTLA shares only 9-13% amino acid identity with CTLA-4 and PD-1, it is structurally similar to them.The presence of two ITIM motifs in its cytoplasmic region suggests, that it has an inhibitory function.In mice, it is expressed at a very low level on resting T cells, and it is induced during activation.Interestingly, after T-cell differentiation, only T helper type Th1, but not Th2, cells express BTLA, and its expression is independent of interleukin 12 (IL-12) or IFN-, suggesting a specific role for BTLA in Th-1 cells (Watanabe et al, 2003).However, BTLA transcripts have been detected in primary B cells and B-cell lines, which indicates its role in the regulation of the B-cell response.In comparison with other co-inhibitory molecules, BTLA is more widely expressed than CTLA-4, which is expressed only on T-cells, but has more limited expression than PD-1, which is expressed on T, B and myeloid cells.

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The Role of Polymorphisms in Co-Signalling Molecules' Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia 185 Blocking BTLA prevents proliferation and cytokine production by T cells.BTLA-deficient mice exhibit a moderate increase in specific antibody responses and increased susceptibility to experimentally induced autoimmune encephalomyelitis (EAC) (Watanabe et al, 2003).
In humans, BTLA is highly expressed on CD14 + monocytes and CD19 + B cells, constituently on CD4 + and CD8 + lymphocytes and weakly on CD56 + NK-cells.Among normal B cells, the highest level of BTLA-expression is found in naïve B cells.Of normal T cells, high levels of BTLA expression are found in T follicular helper (T FH ) cells (M'Hidi et al, 2009).When PBMCs were stimulated 2 days with LPS, the expression of BTLA on CD14 + monocytes and CD19 + B cells decreased to some extent, while the expression on the other cell types, CD4 + and CD8 + lymphocytes and CD56 + NK cells, is upregulated.
BTLA binds the herpes virus entry mediator (HVEM).Interestingly HVEM is a member of the TNFR family, and its interaction with BTLA is the first demonstration of crosstalk between CD28 and the TNFR family.HVEM is expressed on resting T cells, B cells, macrophages and immature dendritic cells, and its expression is downregulated on activated T cells (Sedy et al, 2005;Gonzalez et al, 2005) Unlike the other co-signalling molecules described, the BTLA gene is located on chromosome 3 in q13.2 region.However, like ICOS it has 5 exons (Garapati VP & Lefranc MP, 2007).Because BTLA was relatively recently described in the literature, there are only a few studies that address BTLA gene polymorphisms, and most have investigated its role in susceptibility to autoimmune diseases, such as RA (Lin et al, 2006;Oki et al, 2011), SLE and type 1 diabetes mellitus (Inuo et al, 2009).The non-synonymous BTLAc.800G>ASNP (rs9288952) which leads to a Pro 219 to Leu exchange, has been associated with susceptibility to RA (Lin et al, 2006).
Another study has described a functional polymorphism, BTLAc.590A>C(rs76844316) (Oki et al, 2011).This polymorphism is located in forth exon of the BTLA gene gene and leads to the exchange of asparagine to threonine in the intracellular domain.It was found that the C allele is associated with decreased inhibitory activity of BTLA in ConA-and anti-CD3induced IL-2 production, although the surface expression level is similar in transfectants of both the A and C alleles.It was postulated that the change in amino acids interferes with BTLA signalling and downregulates the association of an unidentified kinase that phosphorylates BTLA or SHP1/SHP2 (Oki et al, 2011).

Polymorphisms in co-signalling genes and susceptibility to cancer
Because the significance of co-signalling molecules in the regulation of immune response has been clearly documented, polymorphisms in the genes encoding those molecules have been widely investigated, previously as susceptibility determinants for autoimmune disease and recently for cancer.Among others, CTLA-4 gene polymorphisms have been investigated the most intensively.It was found that the CTLA-4c.49A>G[A]allele was associated with an increased risk of many types of cancers, including oesophageal cancer, gastric cardia cancer (Sun et al, 2008), non-Hodgkin's lymphoma (Piras et al, 2005), breast cancer (Ghaderi et al, 2004;Sun et al, 2008), renal cancer (Cozar et al, 2007) and lung cancer, esophagus and gastric cardia cancer (Sun et al, 2008).Wong et al (2006) reported that although the CTLA-4c.49A>G[AA]genotype did not increase the risk of oral squamous cell cancer, it correlated significantly with a younger age at onset and poorer survival.Notably, the CTLA-4c.49A>G[GG] genotype was found to be prevalent in mucosa-associated lymphoid tissue lymphoma (Cheng et al, 2006) and in multiple myeloma (Karabon et al, 2011c).There was no association between the CTLA-4c.49A>GSNP and colorectal cancer (Solerio et al, 2005;Hadinia et al, 2007), B-cell chronic lymphocytic leukaemia (Suwalska et al, 2008), cervical squamous cell carcinoma (Su et al, 2007), malignant melanoma (Bouwhuis et al, 2009), or non-malignant melanoma (Welsh et al, 2009).
The CTLA-4g.319C>T polymorphism was shown to be associated with female-related cancers such as sporadic breast cancer (Wong et al, 2006) cervical cancer (Su et al, 2007;Pawlak et al, 2010) and lung cancer in women (Karabon et al. 2011).However, this polymorphism was not associated with lung cancer (without stratification by gender) (Sun et al, 2008) or other cancers, such as colon cancer (Cozar et al, 2007), colorectal cancer (Dilmec et al, 2008) or multiple myeloma (Karabon et al, 2011c).
A limited number of studies have been devoted to the association between the CT60 and Jo31 SNPs and cancers.No association was found between CT60 and Jo31 and lung cancers (Karabon et al, 2011b;Sun et al, 2008), cervical squamous cell carcinoma (Su et al, 2007;Pawlak et al, 2010) or malignant melanoma (Bouwhuis et al, 2009).However, the CT60 [AA] homozygosity correlated with an increased risk of renal cell cancer and with tumour grade (Cozar et al, 2007), while the presence of the A allele is associated with increased susceptibility to non-melanoma skin cancer (Welsh et al, 2009).In contrast, the CT60[G] alleles were found to be prevalent in patients with sporadic breast cancer (Wong et al, 2006) and in multiple myeloma patients (Karabon et al, 2011c).
Only one study indicates a possible predisposing role for the CTLA-4g.1661A>G[G] allele in susceptibility to oral squamous cell carcinoma (OSCC) (Kammerer et al, 2010).
In summary, the latest meta-analysis, which summarised data from 48 studies, confirmed that the presence of the G allele in CTLA-4c.49A>Gpolymorphisms decreased the risk of cancer compared with that with the homozygous CTLA-4c.49A>G[AA]genotype.Interestingly, the CTLA-4c.49A>G[AG+GG] genotype was associated with a decreased risk of cancer in Asians, but not among Europeans, while the CTLA-4g.319C>T[T]allele was associated with an increased risk among Europeans but not Asians.The meta-analysis did not confirm the role of the CT60 SNP as a cancer risk factor (Zhang et al, 2011).
Polymorphisms in the CD28 gene have not been as widely investigated.The CD28c.17+3T>C SNP was not associated in an indirect way with non-solid tumour cancer, while several conditioner associations were established.Our study revealed a lack of association between the CD28c.17+3T>Cpolymorphism and CSCC, while we found an association with well-differentiated tumours (Pawlak et al, 2010).No association with the CD28c.17+3T>Cpolymorphism was found in a previous study with cervical cancer, but Guzman showed an epistatic effect between CD28 and IFNG genes in susceptibility to cervical cancer (Guzman et al, 2008).Recently a Chinese study and a Swedish study confirmed the CD28c.17+3T>Cpolymorphism as an independent risk factor for the development of that cancer (Ivansson et al, 2010;Chen et al, 2011).The CD28c.17+3T>C SNP is not susceptibility locus for gastric mucosa-associated lymphoid tissue (MALT) lymphoma (Cheng et al, 2006), colorectal cancer (Dilmec et al, 2008) or, www.intechopen.comThe Role of Polymorphisms in Co-Signalling Molecules' Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia 187 together with other tag polymorphisms in the CD28 gene, malignant melanoma (Bouwhuis et al, 2009).Two polymorphic sites, rs3181100 and rs3181113, were shown to not be associated with OSCC (Kammerer et al, 2010).
ICOS gene polymorphisms have been widely examined for their potential role as susceptibility locus for melanoma (Bouwhuis et al, 2009), but none of the tested tag polymorphisms was associated with this disease.The ICOSc.602A>C and ICOSc.1624C>Tpolymorphisms are not related to the risk for MALT (Cheng et al, 2006).Similarly, the distribution of alleles and genotypes of ICOSc.602A>C and ICOSc.1599C>Tpolymorphisms were no different between OSCC patients and controls (Kammerer et al, 2010).
The PD-1 gene polymorphism mentioned in the previous subsection (2.4) has been widely explored as a susceptibility locus for autoimmune diseases (Kong et al, 2005;Flores et al, 2010;Lee et al, 2006;Velazquez-Cruz et al, 2007).Only a few studies have been devoted to the relationship between PD-1 polymorphisms and cancer.Recently, it has been shown that polymorphisms (PD-1.1, and PD-1.5) alone and as a part of haplotype confers susceptibility to breast cancer in Chinese population (Hua et al, 2011).In contrast, in an Iranian population, neither PD1.3 nor PD-1.5 was associated with the risk of breast cancer (Haghshenas et al, 2011).
Polymorphisms in the gene of another co-signalling molecule BTLA have been investigated mostly in context of autoimmunity.Only one Chinese study was performed to investigate the relationship between BTLA polymorphisms and breast cancer (Fu et al, 2010).A strong association was found between three polymorphisms, rs9288952, rs2705535 and rs1844089, and the risk of breast cancer.Moreover, associations were also found with tumour size, the oestrogen receptor, the progesterone receptor, C-erbB-2 and the P53 status.
The more important polymorphisms in CD28, CTLA-4, ICOS, PD-1 and BTLA genes and their associations with susceptibility to cancer are displaying on Figure 1.

Expression of co-signalling molecules in B-cell chronic lymphocytic leukaemia
One of the mechanisms by which neoplastic cells escape elimination by host cells is the downregulation of the co-stimulatory pathway.Actually, a decreased expression of costimulatory molecules and the overexpression of co-inhibitory molecules in peripheral blood (PB) T cells have been reported in patients with several neoplastic diseases.
The downregulated expression of the CD28 antigen on peripheral blood T lymphocytes was reported in patients with solid tumours such as: gastric carcinoma, cervical cancer and malignant melanoma (reviewed by (Bocko et al, 2002)), and in patients with multiple myeloma (Brown et al, 1998;Robillard et al, 1998) and hairy-cell leukaemia (van de Corp et al, 1999) Considering the pivotal role of the co-signalling pathway in the antitumour response, several studies have been devoted to examining the expression level of co-signaling molecules in patients with CLL.The investigation by Rossi et al. (Rossi et al, 1996), which was confirmed by Van den Hove et al. (1997) showed significantly lower expression of CD28 on T-cell subsets of chronic lymphocytic leukaemia, and this lower expression was more pronounced in the CD8 + subset than in the CD4 + subset.Scrivener et al. (2003) reported a decreased proportion of CD2 + /CD28 + cells in unstimulated and stimulated PB from CLL patients.
Results from our lab indicated abnormal kinetics and levels of CD28 expression on T cells in CLL patients.The mean percentages of CD4 + and CD8 + cells expressing CD28 were significantly lower in CLL patients than in controls.Moreover, after anti-CD3 and rIL-2 stimulation, the mean percentages of those cells decreased rapidly, and the return to the basal level took longer than it did in healthy individuals (Frydecka et al, 2004) In contrast to the results above, we observed a markedly increased expression of CTLA-4 on unstimulated CD4 + and CD8 + T cells in CLL patients than in controls.The pattern and kinetics of CTLA-4 expression on CD4 + and CD8 + cells in CLL patients after stimulation also differed from that observed in normal subjects.In CLL patient samples, the highest proportion of T cells co-expressing CTLA-4 was found after 24 h of culture as compared to 72 h in samples from normal individuals, and the basal levels were achieved after 5 days compared to 4 days in normal individual samples (Frydecka et al, 2004).The dysregulated expression of both the co-stimulatory CD28 and the inhibitory CTLA-4 molecules in peripheral blood T cells might contribute to the T cell-mediated anti-tumour responses in CLL.
Our group also observed a higher expression of both intracellular and surface CTLA-4 in malignant B cells from CLL patients compared with the normal population of CD19 + /CD5 + cells, and the level of its expression in leukaemic cells positively correlated with the progression of the disease.The upregulated CTLA-4 expression in CLL cells was also previously described by (Pistillo et al, 2003) in 3 of 4 studied patients.Furthermore, we observed positive correlations between the frequency of CD19 + /CD5 + /CTLA-4 + cells with the frequency of leukaemic B cells co-expressing the inhibitory protein p27KIP1 and the early G1 phase regulator cyclin D2.We also found a negative association between CD19 + /CD5 + /CTLA-4 + lymphocytes and CD19 + /CD5 + positive for cyclin D3, which is expressed in the late G1 phase of cell cycle progression.These findings led us to hypothesise that CTLA-4 might contribute to the arrest of leukaemic cells in the G0/G1 phase of the cell cycle (Kosmaczewska et al, 2005).Similar to our results, it has been shown that both BTLA and PD-1 are strongly expressed on malignant B cells from chronic lymphocytic leukaemia/small lymphocytic leukaemia (CLL/SLL) compared with other small-cell lymphomas, such as follicular lymphoma, mantle cell lymphoma and marginal zone lymphoma (M'Hidi et al, 2009;Xerri et al, 2008).An explanation for why the expression of both BTLA and PD1 is increased in CLL/SLL was proposed by M' Hidi et al., (2009).According to this hypothesis, CLL is considered a monoclonal expansion of antigen selected B lymphocytes with varying degrees of autospecificity.The upregulation of inhibitory receptors on CLL precursor cells may result from an attempt by the immune system to prevent autoimmune disorders.To this extent, the simultaneous expression of BTLA and HVEM in CLL cells suggests the triggering of an inefficient autocrine inhibitory loop.This hypothesis is strongly supported by the study of Costello et al (2003) who described the upregulated expression of HVEM in human B-cell malignancies.

Polymorphisms in co-signalling molecules' genes and susceptibility to Bcell chronic leukaemia
Despite the strong familial basis to CLL, with the risk in first-degree relatives being increased approximately sevenfold, the inherited genetic basis of the disease is yet largely unknown, and the major disease-causing locus has not been established.Therefore, a model of genetic predisposition based on the inheritance of multiple risk variants has been proposed (Houlston & Catovsky, 2008).
Our group focused on the co-signalling pathway, because the development of CLL could be regarded as a failure of immunological surveillance.Therefore genes involved in the regulation of the immunological response might be predisposing loci for disease development.We found that among the three polymorphisms studied in the CTLA-4 gene (CTLA-4c.49A>G,CTLA-4g.319C>T and CT60) only one, CTLA-4g.319C>T, which is located in the promoter region, confers susceptibility to CLL.We have shown that the presence of the [T] allele or a [T]-positive phenotype increases the risk of the disease about twofold.Moreover, the [T]-positive phenotype correlates with the progression of disease (about 30% of patients with this phenotype increased their Rai stage during the 24 months follow-up compared with 12% of the [CC] patients) (Suwalska et al, 2008).Interestingly, we observed that the intracellular distribution of CTLA-4 was markedly higher in CLL patients possessing CTLA-4g.*642AT(8_33)[AT 8 ] repeat allele compared to patients possessing longer alleles.That allele was shown by Wang et al., (2002) to be associated with higher mRNA transcription than longer alleles (Kosmaczewska et al. 2005).
Moreover, we found an association between the CD28 gene polymorphism with the incidence of CLL.The presence of the CD28c.17+3T>C[C] allele and the [C] phenotype confers an approximately twofold increased risk of CLL in the Polish population.Additionally, the CD28c.17+3T>Cpolymorphism tended to associate with a higher frequency of Rai stage progression (Suwalska et al, 2008).
We also studied a polymorphism of the ICOS gene.We found a relationship between micro satellite gene c.1544+4GT(8_15) polymorphism and susceptibility to disease.The long alleles (>11 repeats) were associated with protection from disease, while short alleles (< 10) predispose to CLL (Suwalska et al, 2008).Further studies on functional the ICOS SNP: ICOSISV1+173C>T[TT], ICOSc.602A>C,ICOSc.1624C>T, and ICOSc.2373G>Cshowed that these SNPs do not modulate the risk of CLL in the Polish population.However, we noted that ICOSISV1+173T>C[TT] alone, ICOSc.602A>C[AA]alone, and together as part of the genotype AA defined by Casteli et al ( 2007 [GG] increased in the Rai stage during the 60 months of follow-up, compared to more than 40% of the patients possessing other genotypes (Karabon et al, 2011a).
Polymorphisms in the 2q33 region were also investigated by (Monne et al, 2004;Piras et al, 2005) in non-Hodgkin's lymphoma.In both studies the group of patients was very heterogenous and patients with small lymphocytic leukaemia/chronic lymphocytic leukaemia, marginal zone lymphoma, follicular lymphoma, mantle-cell lymphoma, large Bwww.intechopen.com The Role of Polymorphisms in Co-Signalling Molecules' Genes in Susceptibility to B-Cell Chronic Lymphocytic Leukaemia 191 cell lymphoma and T-cell lymphoma were included in these studies.The results obtained by the Sardinian group differed from ours, wherein the CTLA-4c.49A>G and the microsatellite CTLA-4g.*642AT(8_33)polymorphism alone and as a part of the CTLA-4c.49A>G/CTLA-4g.319C>T/CTLA-4g.*642AT(8_33)haplotype were related to the risk of NHL.No independent association was found between CD28 or ICOS gene polymorphisms and NHL in that study.
The explanation for the different results might be the fact that the patients and controls originated from a Sardinian population, which is genetically distinct from other European populations.Moreover, the Sardinian study was performed on a patient group comprising different subtypes of non-Hodgkin`s lymphoma, with only 29 (of a total of 100) patients with CLL/small lymphocytic lymphoma.
Recently, we have focused on BTLA gene polymorphisms.Our preliminary (not published) results indicate that the BTLA+800A>G (rs9288952) non-synonymous polymorphism is not associated with susceptibility to CLL in a Polish population.
To the best of our knowledge, there have been no studies on PD-1 gene polymorphisms and CLL risk.
The described association between polymorphisms in CD28, CTLA-4 and ICOS gene and their associations with susceptibility or course of CLL are displaying on Figure 1.

Conclusions
Considering the pivotal role of co-inhibitory molecules in tumourgenesis and, genetic predisposition to various rates of gene transcription, translation and amino acid sequence caused by polymorphisms, investigation for genetic markers predisposing to the development and influencing prognosis of cancer, in particular CLL is eligible and important.