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Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma
Human Immunology xxx (2014) xxx–xxx
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Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma Sasijit Vejbaesya a,⇑, Pradermchai Sae-Tam a, Archrob Khuhapinant b, Duangporn Srinak a a b
Department of Transfusion Medicine, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand Department of Medicine, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand
a r t i c l e
i n f o
Article history: Received 1 October 2013 Accepted 6 April 2014 Available online xxxx Keywords: KIR Leukemia DLBCL Thai
a b s t r a c t Natural killer (NK) cells are key components of the innate immune system that have been implicated in the immune response against tumor cells. Killer cell immunoglobulin-like receptors (KIR) regulate NK cell activity by interaction with specific human leukocyte antigen (HLA) class I. In this study, KIR gene polymorphisms and their HLA ligands were investigated in Thai patients with chronic myelogenous leukemia (CML) (n = 60), acute myelogenous leukemia (AML) (n = 60), acute lymphoblastic leukemia (ALL) (n = 55), and diffuse large B-cell lymphoma (DLBCL) (n = 60) compared with 150 healthy controls. The frequency of KIR3DL1 with HLA-Bw4 was significantly lower in DLBCL patients than in controls (P = 0.0006, Pc = 0.02), whereas no significant differences were seen in KIR gene frequencies and their ligands between leukemia patients and controls. This study suggest a role of inhibitory KIR with its ligand in the protection against DLBCL. Ó 2014 American Society for Histocompatibility and Immunogenetics All rights reserved.
1. Introduction Leukemia and lymphoma are common hematological malignancies. In lymphoma, diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL). The immune surveillance of tumor cells depends to a large extent on T cell or natural killer cell (NK) reactivity. HLA class I molecules are important in the interaction with T- and NK- specific receptors. Previous studies have shown that malignant cells can escape from elimination by cytotoxic T cells by a reduction or loss of some HLA class I molecules, which in turn exposes them to the attack from NK cells [1,2]. Natural killer cells are the key components of the innate immune response and have the ability to kill tumor and virusinfected cells [3,4]. The killer cell immunoglobulin-like receptos (KIR) are cell surface receptors of the immunoglobulin superfamily that are expressed on NK cell and subsets of activated or memory T lymphocytes in human. KIR receptors are divided into functional inhibitor groups that prevent target cell lysis and activators that incite cell lysis. KIR with long cytoplasmic tail (L) transmit inhibitory signals after binding their recognate ligand via immunotyrosine inhibitory motifs (ITIM), while those with short cyto⇑ Corresponding author. Address: Department of Transfusion Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Fax: +66 2 4128419. E-mail address: [email protected] (S. Vejbaesya).
plasmic tails (S) send activating signals via adapter molecules DAP12. KIR are encoded by a family of highly polymorphic genes clustered in the leukocyte receptor complex on chromosome 19q13.4. KIR exhibits extensive genotypic diversity due to gene number and component [5]. Several KIR receptors use HLA molecules as their ligand. The ligand for KIR2DL1 and 2DS1 is the C2 group (HLA-Cw alleles with lysine at position 80). The ligand for KIR2DL2 and 2DL3 and 2DS2 is the C1 group (HLA-Cw alleles with asparagine at position 80). The ligand for KIR3DL1 is Bw4 [5]. HLA-Bw4 can be divided into two groups on the basis of whether isoleucine or threonine is present at position 80. Receptor binding and lysis inhibition data suggest that Bw4 80Ile may be more effective ligands for KIR3DL1 than Bw4 80Thr [6]. Epidemiological studies implicate HLA-Bw4 ligand for KIR3DS1 [7]. The effects of KIRs on host immune responses are mediated by specific interaction of these receptors and HLA class I ligands, which result in either activating or inhibition of NK cell cytotoxicity. In hematological malignancies, the associations of KIR genes and HLA ligands were reported in leukemia, however the results are controversial [17–23] and to our knowledge, the association of KIR genes with NHL has not been reported. The present study was undertaken to investigate the association of KIR genes and their ligands with leukemia and DLBCL patients in Thai population.
http://dx.doi.org/10.1016/j.humimm.2014.04.004 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics All rights reserved.
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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S. Vejbaesya et al. / Human Immunology xxx (2014) xxx–xxx
at 94 °C, 10 cycles of 94 °C 10 s, 65 °C 60 s, then 20 cycles of 94 °C 10 s, 61 °C 50 s, 72 °C 30 s. The products were photographed from standard 1% agarose electrophoresis gels containing ethidium bromide.
2. Materials and methods 2.1. Study subjects The subjects studied consisted of four groups of Thai patients diagnosed at hematology clinic, Department of Medicine, Siriraj Hospital, Bangkok. The first group included 60 patients with chronic myelogenous leukemia (CML) (30 males and 30 females, mean age 42 years, range 19–79 years,). The second group included 60 patients with acute myelogenous leukemia (AML) (23 males and 37 females, mean age 36 years, range 16–61 years). The third group included 55 patients with acute lymphoblastic leukemia (ALL), (31 males and 24 females, mean age 25 years, range 11–65 years). The forth group included 60 patients with DLBCL (30 males and 30 females, mean age 49 years, range 20–76 years). All the patients were HIV negative. A control group included 150 healthy Thai individuals from blood donors at Siriraj Hospital. All subjects were interviewed about their ancestry and were from the central Thai ethnic background. Written informed consent was provided by all the subjects and the study was approved by the ethics committee of Siriraj hospital, Mahidol University, Thailand. 2.2. HLA and KIR genotyping HLA-C and Bw4 typing were done by PCR-SSP (polymerase chain reaction with sequence specific primer [8]. Validation of the method was done by using Micro-SSP (one lambda, Canoga Park, CA) in randomly selected samples and external quality program. Typing Individuals were categorized as HLA-C1 and HLA-C2 on the basis of their genotyping data. HLA-Bw4 epitope typing was done by PCR-SSO [9]. HLA-Bw4 was only considered on the HLA-B locus. The presence or absence of KIR genes was detected using PCRSSP (polymerase chain reaction with sequence specific primer) [10,11]. Typing method was applied in previous studies and in external quality control program [12,13]. In brief, the reactions of 10 ll were set up to include 0.1 lg test DNA, buffer IV, 0.2 mM dNTP, 1.08 mM magnesium chloride, 0.3 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) and 0.5 lM specific primer mix (except for 3DL1, 2DS4 which were at final concentration of 1 uM). Internal controls (50 -CAGTGCCTTCCCAACCATTCCCTTA-30 , r 50 -ATCCACTCACGG ATTTCTGTTGTGTTTC-30 ) specific for a 485 basepair human growth hormone fragment were included at 0.067 lM in each reaction. All amplifications were performed in duplicate in Perkin Elmer 9700 (PE Biosystem, California, USA) under thermal cycling conditions as follows 5 min denaturing step
2.3. Statistical analysis Carrier frequencies for KIR genes, and HLA ligands were determined by direct counting. In addition, KIR genotypes and KIR genes with their ligands were determined. The differences in frequencies between patients and controls were done by Pearson’s Chi-square test. Fisher exact tests were performed when relevant. The odds ratios (OR) and 95%CI were also calculated. P values less than 0.05 were considered to be significant. The Bonferroni correction for multiple comparisons was also applied. Statistical data were calculated by using the SPSS version13.0 program. A power calculations were also performed using the PS software program and 80% power was used to calculate sample size. 3. Results The distribution of KIR genes and genotypes in patients and controls is shown in Table 1. Framework genes KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1 were present in all samples. KIR genes with high frequency or more than 90% are KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4. There were no significant differences in the frequencies of KIR genes between each group of the patients and controls. In addition, the frequencies of KIR AA, Bx genotypes were not significantly different between each group of the patients and controls. KIR AA was determined when only activating gene 2DS4 was present, while the remaining were considered as Bx. [14] The frequencies of HLA-Bw4 and HLA-C ligands are shown in Table 2. The frequency of HLA-C2 was significantly lower (23.3% vs. 38%, P = 0.04, OR = 0.5), whereas the frequency of HLA-C1C1 was significantly higher (76.7% vs. 62%, P = 0.04, OR = 2.01) in AML patients than in controls. The frequency of Bw4 was significantly lower in DLBCL patients than in the controls (43.3% vs. 66%, P = 0.003, OR = 0.39). However, the statistical differences for these ligands were lost after P correction. When Bw4 epitopes (Bw4 80T and Bw4 80I) were analyzed, only the frequency of Bw4 80T was significantly lower in DLBCL patients than in controls (16.7% vs. 33.3%, P = 0.02, OR = 0.4). There were no significant differences in the frequencies of HLA-C1, C2 and Bw4 in CML and ALL patients compared with controls. The presence of KIR genes with their HLA ligands is shown in Table 3. The frequency of KIR2DL1 with C2 was significantly lower in AML patients than in controls (21.7% vs. 36%, P = 0.04, OR = 0.49), however this was not significant after P
Table 1 Frequencies of KIR genes and genotypes in patients and controls. CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Control N = 150, n (%)
KIR genes 2DL1 2DL2 2DL3 2DL5 3DL1 2DS1 2DS2 2DS3 2DS4 2DS5 3DS1
57(95) 24(40) 57(95) 31(51.7) 59(98.3) 25(41.7) 25(41.7) 12(20) 54(90) 22(36.7) 23(38.3)
57(95) 22(36.7) 57(95) 32(53.3) 57(95) 28(46.7) 22(36.7) 18(30) 56(93.3) 17(28.3) 27(45)
53(96.4) 23(41.8) 54(98.2) 25(45.5) 54(98.2) 18(32.7) 23(41.8) 13(23.6) 51(92.7) 16(29.1) 17(30.9)
58(96.7) 20(33.3) 57(95) 29(48.3) 54(90) 27(45) 21(35) 13(21.7) 54(90) 22(36.7) 25(41.7)
144(96) 59(39.3) 140(93.3) 72(48) 143(95.3) 57(38) 58(38.7) 41(27.3) 143(95.3) 49(32.7) 56(37.3)
KIR genotypes AA Bx
25(41.7) 35(58.3)
24(40) 36(60)
22(40) 33(60)
23(38.3) 37(61.7)
64(42.7) 86(57.3)
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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S. Vejbaesya et al. / Human Immunology xxx (2014) xxx–xxx Table 2 Frequencies of KIR ligands in patients and controls.
a b c
KIR ligands
CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Control N = 150, n (%)
C1 C2 C1/C1 C1/C2 C2/C2 Bw4
55(91.7) 23(38.3) 37(61.7) 18(30) 5(8.3) 33(55)
58(96.7) 14(23.3)a 46(76.7)b 12(20) 2(3.3) 34(56.7)
54(98.2) 17(30.9) 38(69) 16(29.1) 1(1.8) 33(60)
56(93.3) 20(33.3) 40(66.7) 16(26.7) 4(6.7) 26(43.3)c
141(94) 57(38) 93(62) 48(32) 9(6) 99(66)
AML vs. controls, P = 0.04, Pc = 1.2, OR = 0.5, 95% CI: 0.24–1.03. AML vs. controls, P = 0.04, Pc = 1.2, OR = 2.01, 95% CI: 0.97–4.23. DLBCL vs. controls, P = 0.003, Pc = 0.09, OR = 0.39, 95% CI: 0.20–0.76.
Table 3 Frequencies of KIR genes and their ligands in patients and controls.
a b
KIR genes and their ligands
CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Controls N = 150, n (%)
2DL1 + C2 2DL2 + C1 2DL3 + C1 3DL1 + Bw4 2DS1 + C2 2DS2 + C1 3DS1 + Bw4
21(35) 23(38.3) 52(86.7) 32(53.3) 14(23.3) 23(38.3) 12(20)
13(21.7)a 19(31.7) 54(90) 34(56.7) 5(8.3) 19(31.7) 14(23.3)
15(27.3) 22(40) 52(94.5) 32(58.2) 7(12.7) 21(38.2) 9(16.4)
18(30) 18(30) 53(88.3) 22(36.7)b 7(11.7) 19(31.7) 10(16.7)
54(36) 56(37.3) 132(88) 94(62.7) 21(14) 55(36.7) 36(24)
AML vs. controls, P = 0.04, Pc = 1.4, OR = 0.49, 95% CI: 0.23–1.04. DLBCL vs. controls, P = 0.0006, Pc = 0.02, OR = 0.34, 95% CI: 0.18–0.67.
Table 4 Association of KIR genes and ligands with leukemia in different populations. Population
Predisposing
Belgium Turkey China(Han) Poland Canada Belgium Poland
KIR2DL2/2DS2 + C1 (myeloid leukemia) Bw4(CML) KIR2DS4(CML)
Protective KIR2DL2/2DS2 + C1(CML) Full length KIR2DS4(CML) Activating KIR (childhood ALL)
KIR3DL1 + BW4 (CLL)
correction. The frequency of KIR3DL1 with HLA-Bw4 was significantly lower in DLBCL patients than in controls (36.7% vs. 62.7%, P = 0.0006, Pc = 0.02, OR = 0.34). There was no significant difference in the interaction of other KIR and HLA ligands between patients and controls. 4. Discussion NK cells are thought to be important in eliminating tumor cells especially leukemia, lymphoma and metastatic tumor cells [15,16]. The associations KIR genes and leukemia were reported in different groups, but the results are controversial (Table 4). In addition, most of the studies were performed in Caucasians. In myeloid malignancies, a study on the Belgium Caucasian population found a significant increase in KIR2DL2 and 2DS2 with HLA-C1 in myeloid leukemia patients [17], however a study on the Turkish population found a significant decrease in KIR2DL2 and KIR2DS2 with HLA-C1 in CML patients [18]. This group also found a significant increase in HLA-Bw4 in CML patients. In Asian population, a study on the Chinese Han population found significant association of KIR2DS4 with susceptibility to CML [19], however this study did not differentiate full length and deletion allele of KIR2DS4. In contrast, a study on the Polish population found protective effect of full length KIR2DS4 allele with CML [20]. In lymphoid malignancies, most of the studies found no association of KIR genes with ALL [17,18,21]. However, a study on the Canadian Caucasian
KIR3DL1 + Bw4 (CLL)
References [17] [18] [19] [20] [22] [17] [23]
population found significant association of activating KIR with reduced risk for childhood ALL [22]. In CLL, a study on the Belgium Caucasian population found association of KIR3DL1 + HLA-Bw4 with susceptibility to the disease [17], in contrast, a study on the Polish population with much larger sample size found association of KIR3DL1 + HLA-Bw4 with protection against the disease [23]. In our study, we could not confirm association of KIR gene with CML, AML and ALL. Although a decrease in KIR2DL1 + HLA-C2 was found in AML, the significance is weak and the difference was not significant after P correction. The difference in the findings of this study from those of previous studies in leukemia can be attributed to several factors. First, the frequencies of KIR genes or ligands are different between populations. In Thais, the frequency of HLAC1 was higher and that of HLA-C2 was lower than in Caucasians [12]. Second, there are variables in patients selected such as the age of onset, the severity of the disease. Third, the sample size might affect statistical significance. Further studies in larger sample size for each group of leukemia are needed. Interestingly, our study found a protective role of KIR3DL1 + HLA-Bw4 in DLBCL. In addition, Bw4 80T was significantly reduced in DLBCL The result was similar to the association found in CLL [23]. Although there is evidence that Bw4 80I showed stronger interaction with KIR receptor than Bw4 80T [6], it is possible that Bw4 80T may also be important. However, in this study, HLA-A- Bw4 was not included. Although HLA-B epitopes are regarded as important KIR3DL1 ligands, it was suggested that HLA-A might be important
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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S. Vejbaesya et al. / Human Immunology xxx (2014) xxx–xxx
for NK cell function [24]. Previous study has shown that the binding of KIR3DL1 to Bw4 ligand depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism [25]. Further study on the role of KIR3DL1 subtypes and the interactions with HLA-A and HLA-B should be performed. Both CLL and DLBCL are lymphoid malignancies which are subsets of NHL. In Thais, CLL is very rare compared to Caucasians, whereas DLBCL is the most common type of NHL. HLA-Bw4 is ligand for both KIR3DL1 and KIR3DS1. We also observed a decrease in KIR3DS1 + HLA-Bw4, but the difference was not significant. This might be from a low frequency of KIR3DS1 in the controls. KIR3DS1 was found to be protective against Hodgkin’s lymphoma [26]. In contrast, this gene has been associated with increase risk in HPV induced cervical cancer [27] and EBV-associated nasopharyngeal carcinoma [28]. Although the susceptibility effects of inhibitory KIR-HLA on some cancers were reported [29,30], a protective role of inhibitory KIR-HLA was observed in DLBCL in this study and in cervical neoplasia [27]. It is known that etiologic cause of NHL and cervical neoplasia are associated with infectious agent [31], Therefore, KIR genes might be important in the pathogenesis of virus induced malignancies, however, the role of inhibitory or activating KIR are still not clear. In summary, KIR3DL1-HLA-Bw4 was found to be associated with the protection against DLBCL, however the association should be confirmed in other populations. There were no associations of KIR genes and ligands with CML, AML and ALL in Thai patients. Acknowledgments The present study was supported by Siriraj Research Development Fund. References [1] Demanet C, Mulder A, Deneys V, Worsham MJ, Maes P, Claas FH, et al. Downregulation of HLA-A and HLA-Bw6, but not HLA-Bw4, allospecificities in leukemic cells: an escape mechanism from CTL and NK attack? Blood 2004;103:22–30. [2] Brouwer RE, van der Heiden P, Schreuder GM, Mulder A, Datema G, Anholts JD, et al. Loss or downregulation of HLA class I expression at the allelic level in acute leukemia is infrequent but functionally relevant, and can be restored by interferon. Hum Immunol 2002;63:200–10. [3] Khakoo SI, Carrington M. KIR and disease: a model system or system of models? Immunol Rev 2006;214:186. [4] Purdy AK, Campbell KS. Natural killer cells and cancer: regulation by the killer cell Ig-like receptors(KIR). Cancer Biol Ther 2009;8:13–22. [5] Middleton D, Curran M, Marwett L. Natural killer cells and their receptors. Transpl Immunol 2002;10:147–220. [6] Celia M, Longo A, Ferrara GB, Strominger JL, Colonna M. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J Exp Med 1994;180:1235–42. [7] Martin MP, Gao X, Lee JH. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 2002;31:429–34. [8] Bunce M, O Neill CM, Barnardo MCNM, Krausa P, Browning MJ, Morris PJ, et al. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 1995;46:355–67. [9] Middleton D, Williams F, Cullen C, Mallon E. Modification of a HLA-B PCR-SSOP typing system leading to improved allele determination. Tissue Antigens 1995;45:232–6.
[10] Norman PJ, Carington CVF, Byng M, Maxcell LD, Curran MD, Stephens HAF, et al. Natural killer cell immunoglonulin-like receptor (KIR) locus profiles in African and South Asian populations. Genes Immun 2002;3:86–95. [11] Kulkarini S, Martin MP, Carrington M. KIR genotyping by multiplex PCR-SSP. Methods Mol Biol 2010;612:365–75. [12] Hollenbach JA, Meenagh A, Sleator C, Alaez C, Bengoche M, Canoss A, et al. Report from the killer immunoglobulin-like receptor (KIR) anthropology component of the 15th International histocompatibility workshop: world variation in the KIR loci and further evidence for the co-evolution of KIR and HLA. Tissue Antigens 2010;76:9–17. [13] Vejbaesya S, Nonnoi Y, Tanwandee T, Srinak D. Killer Immunoglobulin like receptors and response to antiviral treatment in Thai patients with chronic hepatitis C virus genotype 3a. J Med Virol 2011;83:1733–7. [14] Mcqueen KL, Dorighi KM, Guethlein LA, Wong R, Sanjanwala NB, Perham P. Donor-recipient combinations of group A and B KIR haplotypes and HLA class I ligand affect the outcome of HLA-matched, sibling donor hematopoietic cell transplantation. Hum Immunol 2007;68:309–23. [15] Verheyden S, Demanet C. NK cell receptors and their ligands in leukemia. Leukemia 2008;22:249–57. [16] Gluck WL, Hurst D, Yuen A, Levine AM, Dayton MA, Goehermen JP. Phase I studies of interleukin (IL)-2 and rituximab in B-cell non-hodgkin’s lymphoma: IL-2 mediated natural killer cell expansion correlations with clinical response. Clin Cancer Res 2004;10:2253–64. [17] Verheyden S, Demanet C. Susceptibility to myeloid and lymphoid leukemia mediated by distinct inhibitory KIR-HLA ligand interactions. Leukemia 2006;20:1437–8. [18] Middleton D, Diler AS, Meenagh A, Sleator C, Gourraud PA. Killer immunoglobulin-like receptors (KIR2DL2 and/or KIR2DS2) in presence of their ligand (HLA-C1 group) protect against chronic myeloid leukaemia. Tissue Antigens 2009;73:553–60. [19] Zhang Y, Wang B, Ye S, Liu S, Liu M, Shen C, et al. Killer cell immunoglobulinlike receptor gene polymorphisms in patients with leukemia: possible association with susceptibility to the disease. Leuk Res 2010;34:55–8. [20] Giebel S, Nowak I, Wojnar J, Krawczyk-Kullis M, Holowiecki J, Kyrcz-Kezemiem S, et al. Association of KIR2DS4 and its variant KIR1D with leukemia. Leukemia 2008;22:2129–30. [21] Babor F, Manser A, Schönberg K, Enczmann J, Borkhardt A, Meisel R, et al. Lack of association between KIR genes and acute lymphoblastic leukemia in children. Blood 2012;120:2770–3301. [22] Almalte Z, Samarani S, Iannello A, Debbeche O, Duval M, Infante-Rivard C, et al. Novel associations between activating killer-cell immunoglobulin-like receptor genes and childhood leukemia. Blood 2011;118:1323–8. [23] Karabon L, Jedynak A, Giebel S, Wołowiec D, Kielbinski M, Woszczyk D, et al. KIR/HLA gene combinations influence susceptibility to B-cell chronic lymphocytic leukemia and the clinical course of disease. Tissue Antigens 2011;78:129–38. [24] Augusto DG, Zehnder-Alves L, Pincerati MR, Martin MP, Carrington M, et al. Diversity of the KIR gene cluster in an urban Brazilian population. Immunogenetics 2012;64:143–52. [25] Thananchai H, Gillespie G, Gillespie G, Martin MP, Bashirova A, Yawata N, et al. Cutting edge: allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B. J Immunol 2007;178:33–7. [26] Besson C, Roetynck S, Williams F, Orsi L, Amiel C, Lependeven C. Association of killer cell immunoglobulin-like receptor genes with Hodgkin’s lymphoma in a familial study. PLoS One 2007;2:406. [27] Carrington M, Wang S, Martin MP, Gao X, Schiffman M, Cheng J, et al. Hierarchy of resistance to cervical neoplasia mediated by combinations of killer immunoglobulin-like receptor and human leukocyte antigen loci. J Exp Med 2005;201:1069–75. [28] Butsch Kovacic M, Martin M, Gao X, Fuksenko T, Chen CJ, Cheng YJ, et al. Variation of the killer cell immunoglobulin-like receptors and HLA-C genes in nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 2005;14(11 Pt 1):2673–7. [29] Naumova E, Mihaylova A, Ivanova M, Mihailova S. Impact of KIR/HLA ligand combination on immune responses in malignant melanoma. Cancer Immunol Immunother 2007;56:95–100. [30] Gati A, Da Rocha S, Guerra N, Escudier B, Moretta A, Chouaib S, et al. Analysis of the natural killer mediated immune response in metastatic renal cell carcinoma patients. Int J Cancer 2004;109:393–401. [31] Roschewski M, Wilson WH. EBV-associated lymphomas in adults. Best Pract Res Clin Haematol 2012;25:75–89.
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
Contents lists available at ScienceDirect
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journal homepage: www.elsevier.com/locate/humimm
Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma Sasijit Vejbaesya a,⇑, Pradermchai Sae-Tam a, Archrob Khuhapinant b, Duangporn Srinak a a b
Department of Transfusion Medicine, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand Department of Medicine, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand
a r t i c l e
i n f o
Article history: Received 1 October 2013 Accepted 6 April 2014 Available online xxxx Keywords: KIR Leukemia DLBCL Thai
a b s t r a c t Natural killer (NK) cells are key components of the innate immune system that have been implicated in the immune response against tumor cells. Killer cell immunoglobulin-like receptors (KIR) regulate NK cell activity by interaction with specific human leukocyte antigen (HLA) class I. In this study, KIR gene polymorphisms and their HLA ligands were investigated in Thai patients with chronic myelogenous leukemia (CML) (n = 60), acute myelogenous leukemia (AML) (n = 60), acute lymphoblastic leukemia (ALL) (n = 55), and diffuse large B-cell lymphoma (DLBCL) (n = 60) compared with 150 healthy controls. The frequency of KIR3DL1 with HLA-Bw4 was significantly lower in DLBCL patients than in controls (P = 0.0006, Pc = 0.02), whereas no significant differences were seen in KIR gene frequencies and their ligands between leukemia patients and controls. This study suggest a role of inhibitory KIR with its ligand in the protection against DLBCL. Ó 2014 American Society for Histocompatibility and Immunogenetics All rights reserved.
1. Introduction Leukemia and lymphoma are common hematological malignancies. In lymphoma, diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL). The immune surveillance of tumor cells depends to a large extent on T cell or natural killer cell (NK) reactivity. HLA class I molecules are important in the interaction with T- and NK- specific receptors. Previous studies have shown that malignant cells can escape from elimination by cytotoxic T cells by a reduction or loss of some HLA class I molecules, which in turn exposes them to the attack from NK cells [1,2]. Natural killer cells are the key components of the innate immune response and have the ability to kill tumor and virusinfected cells [3,4]. The killer cell immunoglobulin-like receptos (KIR) are cell surface receptors of the immunoglobulin superfamily that are expressed on NK cell and subsets of activated or memory T lymphocytes in human. KIR receptors are divided into functional inhibitor groups that prevent target cell lysis and activators that incite cell lysis. KIR with long cytoplasmic tail (L) transmit inhibitory signals after binding their recognate ligand via immunotyrosine inhibitory motifs (ITIM), while those with short cyto⇑ Corresponding author. Address: Department of Transfusion Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok 10700, Thailand. Fax: +66 2 4128419. E-mail address: [email protected] (S. Vejbaesya).
plasmic tails (S) send activating signals via adapter molecules DAP12. KIR are encoded by a family of highly polymorphic genes clustered in the leukocyte receptor complex on chromosome 19q13.4. KIR exhibits extensive genotypic diversity due to gene number and component [5]. Several KIR receptors use HLA molecules as their ligand. The ligand for KIR2DL1 and 2DS1 is the C2 group (HLA-Cw alleles with lysine at position 80). The ligand for KIR2DL2 and 2DL3 and 2DS2 is the C1 group (HLA-Cw alleles with asparagine at position 80). The ligand for KIR3DL1 is Bw4 [5]. HLA-Bw4 can be divided into two groups on the basis of whether isoleucine or threonine is present at position 80. Receptor binding and lysis inhibition data suggest that Bw4 80Ile may be more effective ligands for KIR3DL1 than Bw4 80Thr [6]. Epidemiological studies implicate HLA-Bw4 ligand for KIR3DS1 [7]. The effects of KIRs on host immune responses are mediated by specific interaction of these receptors and HLA class I ligands, which result in either activating or inhibition of NK cell cytotoxicity. In hematological malignancies, the associations of KIR genes and HLA ligands were reported in leukemia, however the results are controversial [17–23] and to our knowledge, the association of KIR genes with NHL has not been reported. The present study was undertaken to investigate the association of KIR genes and their ligands with leukemia and DLBCL patients in Thai population.
http://dx.doi.org/10.1016/j.humimm.2014.04.004 0198-8859/Ó 2014 American Society for Histocompatibility and Immunogenetics All rights reserved.
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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at 94 °C, 10 cycles of 94 °C 10 s, 65 °C 60 s, then 20 cycles of 94 °C 10 s, 61 °C 50 s, 72 °C 30 s. The products were photographed from standard 1% agarose electrophoresis gels containing ethidium bromide.
2. Materials and methods 2.1. Study subjects The subjects studied consisted of four groups of Thai patients diagnosed at hematology clinic, Department of Medicine, Siriraj Hospital, Bangkok. The first group included 60 patients with chronic myelogenous leukemia (CML) (30 males and 30 females, mean age 42 years, range 19–79 years,). The second group included 60 patients with acute myelogenous leukemia (AML) (23 males and 37 females, mean age 36 years, range 16–61 years). The third group included 55 patients with acute lymphoblastic leukemia (ALL), (31 males and 24 females, mean age 25 years, range 11–65 years). The forth group included 60 patients with DLBCL (30 males and 30 females, mean age 49 years, range 20–76 years). All the patients were HIV negative. A control group included 150 healthy Thai individuals from blood donors at Siriraj Hospital. All subjects were interviewed about their ancestry and were from the central Thai ethnic background. Written informed consent was provided by all the subjects and the study was approved by the ethics committee of Siriraj hospital, Mahidol University, Thailand. 2.2. HLA and KIR genotyping HLA-C and Bw4 typing were done by PCR-SSP (polymerase chain reaction with sequence specific primer [8]. Validation of the method was done by using Micro-SSP (one lambda, Canoga Park, CA) in randomly selected samples and external quality program. Typing Individuals were categorized as HLA-C1 and HLA-C2 on the basis of their genotyping data. HLA-Bw4 epitope typing was done by PCR-SSO [9]. HLA-Bw4 was only considered on the HLA-B locus. The presence or absence of KIR genes was detected using PCRSSP (polymerase chain reaction with sequence specific primer) [10,11]. Typing method was applied in previous studies and in external quality control program [12,13]. In brief, the reactions of 10 ll were set up to include 0.1 lg test DNA, buffer IV, 0.2 mM dNTP, 1.08 mM magnesium chloride, 0.3 U Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) and 0.5 lM specific primer mix (except for 3DL1, 2DS4 which were at final concentration of 1 uM). Internal controls (50 -CAGTGCCTTCCCAACCATTCCCTTA-30 , r 50 -ATCCACTCACGG ATTTCTGTTGTGTTTC-30 ) specific for a 485 basepair human growth hormone fragment were included at 0.067 lM in each reaction. All amplifications were performed in duplicate in Perkin Elmer 9700 (PE Biosystem, California, USA) under thermal cycling conditions as follows 5 min denaturing step
2.3. Statistical analysis Carrier frequencies for KIR genes, and HLA ligands were determined by direct counting. In addition, KIR genotypes and KIR genes with their ligands were determined. The differences in frequencies between patients and controls were done by Pearson’s Chi-square test. Fisher exact tests were performed when relevant. The odds ratios (OR) and 95%CI were also calculated. P values less than 0.05 were considered to be significant. The Bonferroni correction for multiple comparisons was also applied. Statistical data were calculated by using the SPSS version13.0 program. A power calculations were also performed using the PS software program and 80% power was used to calculate sample size. 3. Results The distribution of KIR genes and genotypes in patients and controls is shown in Table 1. Framework genes KIR2DL4, KIR3DL2, KIR3DL3 and KIR3DP1 were present in all samples. KIR genes with high frequency or more than 90% are KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4. There were no significant differences in the frequencies of KIR genes between each group of the patients and controls. In addition, the frequencies of KIR AA, Bx genotypes were not significantly different between each group of the patients and controls. KIR AA was determined when only activating gene 2DS4 was present, while the remaining were considered as Bx. [14] The frequencies of HLA-Bw4 and HLA-C ligands are shown in Table 2. The frequency of HLA-C2 was significantly lower (23.3% vs. 38%, P = 0.04, OR = 0.5), whereas the frequency of HLA-C1C1 was significantly higher (76.7% vs. 62%, P = 0.04, OR = 2.01) in AML patients than in controls. The frequency of Bw4 was significantly lower in DLBCL patients than in the controls (43.3% vs. 66%, P = 0.003, OR = 0.39). However, the statistical differences for these ligands were lost after P correction. When Bw4 epitopes (Bw4 80T and Bw4 80I) were analyzed, only the frequency of Bw4 80T was significantly lower in DLBCL patients than in controls (16.7% vs. 33.3%, P = 0.02, OR = 0.4). There were no significant differences in the frequencies of HLA-C1, C2 and Bw4 in CML and ALL patients compared with controls. The presence of KIR genes with their HLA ligands is shown in Table 3. The frequency of KIR2DL1 with C2 was significantly lower in AML patients than in controls (21.7% vs. 36%, P = 0.04, OR = 0.49), however this was not significant after P
Table 1 Frequencies of KIR genes and genotypes in patients and controls. CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Control N = 150, n (%)
KIR genes 2DL1 2DL2 2DL3 2DL5 3DL1 2DS1 2DS2 2DS3 2DS4 2DS5 3DS1
57(95) 24(40) 57(95) 31(51.7) 59(98.3) 25(41.7) 25(41.7) 12(20) 54(90) 22(36.7) 23(38.3)
57(95) 22(36.7) 57(95) 32(53.3) 57(95) 28(46.7) 22(36.7) 18(30) 56(93.3) 17(28.3) 27(45)
53(96.4) 23(41.8) 54(98.2) 25(45.5) 54(98.2) 18(32.7) 23(41.8) 13(23.6) 51(92.7) 16(29.1) 17(30.9)
58(96.7) 20(33.3) 57(95) 29(48.3) 54(90) 27(45) 21(35) 13(21.7) 54(90) 22(36.7) 25(41.7)
144(96) 59(39.3) 140(93.3) 72(48) 143(95.3) 57(38) 58(38.7) 41(27.3) 143(95.3) 49(32.7) 56(37.3)
KIR genotypes AA Bx
25(41.7) 35(58.3)
24(40) 36(60)
22(40) 33(60)
23(38.3) 37(61.7)
64(42.7) 86(57.3)
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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S. Vejbaesya et al. / Human Immunology xxx (2014) xxx–xxx Table 2 Frequencies of KIR ligands in patients and controls.
a b c
KIR ligands
CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Control N = 150, n (%)
C1 C2 C1/C1 C1/C2 C2/C2 Bw4
55(91.7) 23(38.3) 37(61.7) 18(30) 5(8.3) 33(55)
58(96.7) 14(23.3)a 46(76.7)b 12(20) 2(3.3) 34(56.7)
54(98.2) 17(30.9) 38(69) 16(29.1) 1(1.8) 33(60)
56(93.3) 20(33.3) 40(66.7) 16(26.7) 4(6.7) 26(43.3)c
141(94) 57(38) 93(62) 48(32) 9(6) 99(66)
AML vs. controls, P = 0.04, Pc = 1.2, OR = 0.5, 95% CI: 0.24–1.03. AML vs. controls, P = 0.04, Pc = 1.2, OR = 2.01, 95% CI: 0.97–4.23. DLBCL vs. controls, P = 0.003, Pc = 0.09, OR = 0.39, 95% CI: 0.20–0.76.
Table 3 Frequencies of KIR genes and their ligands in patients and controls.
a b
KIR genes and their ligands
CML N = 60, n (%)
AML N = 60, n (%)
ALL N = 55, n (%)
DLBCL N = 60, n (%)
Controls N = 150, n (%)
2DL1 + C2 2DL2 + C1 2DL3 + C1 3DL1 + Bw4 2DS1 + C2 2DS2 + C1 3DS1 + Bw4
21(35) 23(38.3) 52(86.7) 32(53.3) 14(23.3) 23(38.3) 12(20)
13(21.7)a 19(31.7) 54(90) 34(56.7) 5(8.3) 19(31.7) 14(23.3)
15(27.3) 22(40) 52(94.5) 32(58.2) 7(12.7) 21(38.2) 9(16.4)
18(30) 18(30) 53(88.3) 22(36.7)b 7(11.7) 19(31.7) 10(16.7)
54(36) 56(37.3) 132(88) 94(62.7) 21(14) 55(36.7) 36(24)
AML vs. controls, P = 0.04, Pc = 1.4, OR = 0.49, 95% CI: 0.23–1.04. DLBCL vs. controls, P = 0.0006, Pc = 0.02, OR = 0.34, 95% CI: 0.18–0.67.
Table 4 Association of KIR genes and ligands with leukemia in different populations. Population
Predisposing
Belgium Turkey China(Han) Poland Canada Belgium Poland
KIR2DL2/2DS2 + C1 (myeloid leukemia) Bw4(CML) KIR2DS4(CML)
Protective KIR2DL2/2DS2 + C1(CML) Full length KIR2DS4(CML) Activating KIR (childhood ALL)
KIR3DL1 + BW4 (CLL)
correction. The frequency of KIR3DL1 with HLA-Bw4 was significantly lower in DLBCL patients than in controls (36.7% vs. 62.7%, P = 0.0006, Pc = 0.02, OR = 0.34). There was no significant difference in the interaction of other KIR and HLA ligands between patients and controls. 4. Discussion NK cells are thought to be important in eliminating tumor cells especially leukemia, lymphoma and metastatic tumor cells [15,16]. The associations KIR genes and leukemia were reported in different groups, but the results are controversial (Table 4). In addition, most of the studies were performed in Caucasians. In myeloid malignancies, a study on the Belgium Caucasian population found a significant increase in KIR2DL2 and 2DS2 with HLA-C1 in myeloid leukemia patients [17], however a study on the Turkish population found a significant decrease in KIR2DL2 and KIR2DS2 with HLA-C1 in CML patients [18]. This group also found a significant increase in HLA-Bw4 in CML patients. In Asian population, a study on the Chinese Han population found significant association of KIR2DS4 with susceptibility to CML [19], however this study did not differentiate full length and deletion allele of KIR2DS4. In contrast, a study on the Polish population found protective effect of full length KIR2DS4 allele with CML [20]. In lymphoid malignancies, most of the studies found no association of KIR genes with ALL [17,18,21]. However, a study on the Canadian Caucasian
KIR3DL1 + Bw4 (CLL)
References [17] [18] [19] [20] [22] [17] [23]
population found significant association of activating KIR with reduced risk for childhood ALL [22]. In CLL, a study on the Belgium Caucasian population found association of KIR3DL1 + HLA-Bw4 with susceptibility to the disease [17], in contrast, a study on the Polish population with much larger sample size found association of KIR3DL1 + HLA-Bw4 with protection against the disease [23]. In our study, we could not confirm association of KIR gene with CML, AML and ALL. Although a decrease in KIR2DL1 + HLA-C2 was found in AML, the significance is weak and the difference was not significant after P correction. The difference in the findings of this study from those of previous studies in leukemia can be attributed to several factors. First, the frequencies of KIR genes or ligands are different between populations. In Thais, the frequency of HLAC1 was higher and that of HLA-C2 was lower than in Caucasians [12]. Second, there are variables in patients selected such as the age of onset, the severity of the disease. Third, the sample size might affect statistical significance. Further studies in larger sample size for each group of leukemia are needed. Interestingly, our study found a protective role of KIR3DL1 + HLA-Bw4 in DLBCL. In addition, Bw4 80T was significantly reduced in DLBCL The result was similar to the association found in CLL [23]. Although there is evidence that Bw4 80I showed stronger interaction with KIR receptor than Bw4 80T [6], it is possible that Bw4 80T may also be important. However, in this study, HLA-A- Bw4 was not included. Although HLA-B epitopes are regarded as important KIR3DL1 ligands, it was suggested that HLA-A might be important
Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004
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for NK cell function [24]. Previous study has shown that the binding of KIR3DL1 to Bw4 ligand depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism [25]. Further study on the role of KIR3DL1 subtypes and the interactions with HLA-A and HLA-B should be performed. Both CLL and DLBCL are lymphoid malignancies which are subsets of NHL. In Thais, CLL is very rare compared to Caucasians, whereas DLBCL is the most common type of NHL. HLA-Bw4 is ligand for both KIR3DL1 and KIR3DS1. We also observed a decrease in KIR3DS1 + HLA-Bw4, but the difference was not significant. This might be from a low frequency of KIR3DS1 in the controls. KIR3DS1 was found to be protective against Hodgkin’s lymphoma [26]. In contrast, this gene has been associated with increase risk in HPV induced cervical cancer [27] and EBV-associated nasopharyngeal carcinoma [28]. Although the susceptibility effects of inhibitory KIR-HLA on some cancers were reported [29,30], a protective role of inhibitory KIR-HLA was observed in DLBCL in this study and in cervical neoplasia [27]. It is known that etiologic cause of NHL and cervical neoplasia are associated with infectious agent [31], Therefore, KIR genes might be important in the pathogenesis of virus induced malignancies, however, the role of inhibitory or activating KIR are still not clear. In summary, KIR3DL1-HLA-Bw4 was found to be associated with the protection against DLBCL, however the association should be confirmed in other populations. There were no associations of KIR genes and ligands with CML, AML and ALL in Thai patients. Acknowledgments The present study was supported by Siriraj Research Development Fund. References [1] Demanet C, Mulder A, Deneys V, Worsham MJ, Maes P, Claas FH, et al. Downregulation of HLA-A and HLA-Bw6, but not HLA-Bw4, allospecificities in leukemic cells: an escape mechanism from CTL and NK attack? Blood 2004;103:22–30. [2] Brouwer RE, van der Heiden P, Schreuder GM, Mulder A, Datema G, Anholts JD, et al. Loss or downregulation of HLA class I expression at the allelic level in acute leukemia is infrequent but functionally relevant, and can be restored by interferon. Hum Immunol 2002;63:200–10. [3] Khakoo SI, Carrington M. KIR and disease: a model system or system of models? Immunol Rev 2006;214:186. [4] Purdy AK, Campbell KS. Natural killer cells and cancer: regulation by the killer cell Ig-like receptors(KIR). Cancer Biol Ther 2009;8:13–22. [5] Middleton D, Curran M, Marwett L. Natural killer cells and their receptors. Transpl Immunol 2002;10:147–220. [6] Celia M, Longo A, Ferrara GB, Strominger JL, Colonna M. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J Exp Med 1994;180:1235–42. [7] Martin MP, Gao X, Lee JH. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 2002;31:429–34. [8] Bunce M, O Neill CM, Barnardo MCNM, Krausa P, Browning MJ, Morris PJ, et al. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 1995;46:355–67. [9] Middleton D, Williams F, Cullen C, Mallon E. Modification of a HLA-B PCR-SSOP typing system leading to improved allele determination. Tissue Antigens 1995;45:232–6.
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Please cite this article in press as: Vejbaesya S et al. Killer cell immunoglobulin-like receptors in Thai patients with leukemia and diffuse large B-cell lymphoma. Hum Immunol (2014), http://dx.doi.org/10.1016/j.humimm.2014.04.004